SBC 418 (424): ANIMAL BIOTECHNOLOGY

LECTURE NOTES

BY ORINDA

Course Outline

Mammalian cell lines: Monkey kidney cells, 293T, Hela cells, Vilo cells; Embryonated egg culture for viral propagation; Cell and tissue culture: media preparation, culture techniques for protozoa, viruses, helminthes, bacteria, fungi; Embryo transfer; Production recombinant proteins in bacteria and mammalian cell lines: insulin, human growth hormone, vaccines; Development of transgenic farm animals; Future prospects of recombinant techniques in understanding diseases, human gene therapy, and genetic engineering in animals and Ethical consideration in regard to DNA manipulation in food animals.

TOPIC 1: INTRODUCTION TO BIOTECHNOLOGY

Years ago, animal, plant and microbes have been used by humans for nutrition and development

of products for consumption such as bread, brewing alcohol and cheese production, although the

phenomenon of fermentation was not understood thoroughly. Technological advancement has

also allowed humans to exploit plant, animal and microbial wealth to provide products of

commercial or pharmaceutical importance. All these activities (research and development) fall

under the big umbrella of biotechnology.

BIOTECHNOLOGY

The term biotechnology is a fusion of biology and technology. It is basically the controlled use

of biological agents, such as micro organisms or cellular components for human beneficial use.

In simpler word, biotechnology is the summation of activities involving technological tools and

living organism in such a way that it will enhance the efficiency of the production. The ultimate

goal of biotechnology is to improve the product yield from living organism either by employing

principles of bio-engineering/bio-process technology or by genetically modifying the organisms.

In broad terms, biotechnology is the manipulation of living organisms or their components to

perform practical tasks or provide useful products. It is the integrated use of biochemistry,

microbiology and engineering sciences in order to exploit microorganisms, cultured tissues/cells,

to their best. Man has continued his quest for improving the natural capabilities of micro

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organisms and making them capable of novel processes and to create them for highly valuable

cause, for human welfare.

Differences between traditional and modern

Traditional biotechnology Modern biotechnology:

1. Slow, many generations of selective breeding before new traits appear.2. Almost always combines genetic material from closely related species.3. Cannot manipulate the DNA sequence of genes.

1. Massive genetic changes in a single generation.2. Able to recombine DNA from very different species in one organism.3. Can produce new genes never before seen on earth.

Subfields of biotechnology

Red Biotechnology: applies to Medical biotechnology, designing of organisms to produce antibiotics and to cure diseases through genetic engineering and manipulations.

White Biotechnology (also known as grey biotechnology): is applied to industrial biotechnology

Green Biotechnology: is biotechnology applied to agricultural processes. This aims at production of more environment friendly solutions than conventional traditional industrial biotechnology.

Bioinformatics: addresses biological problems with the aid of computational techniques.

Blue Biotechnology: describes marine and aquatic applications of biotechnology.

Biotechnological Applications

Biotechnology has influenced human life in many ways by inventions to make his life more

comfortable. Recent decades have seen a vast increase in our knowledge of the genetic basis of

human disease and disease susceptibility. This has largely been as a consequence of the

abundance of DNA sequence information coupled with powerful methods of engineering defined

genetic modifications, particularly in mice. It is generally recognized that large animals such as

the pig are more relevant to biomedical research than rodents, being anatomically and

physiologically closer to humans. Livestock species are already used in biomedical research, for 2

example in experimental surgery, organ transplantation, and imaging techniques. However very

few large animal models of human disease are currently available. These are restricted to either

artificially (e.g. chemically or surgically) induced disease, or spontaneous disease predispositions

which are often ill-defined. Advanced reproductive and transgenic techniques are now being

extended to large animal species and it is timely to use these to benefit human health. If

biomedical researchers can be provided with physiologically-relevant models of serious human

diseases, the development of preventive, diagnostic and therapeutic strategies will be

significantly advanced. The use of animals as a source of organs and tissues for

xenotransplantation can overcome the growing shortage of human organ donors.

Now, the extent of biotechnological application is more sophisticated. Researchers can

manipulate living organisms and transfer genetic material between organisms, generating

transgenics (plants/animals). Modern techniques allow production of new and improved foods.

Insect resistant crops have been developed using recent advances in biotechnology. In the field

of medicine, it has resulted in development of newer antibiotics, vaccines for various diseases

such as cancer, AIDS, hereditary diseases such as Huntington’s chorea etc. Biotechnology is also

being applied in the area of pollution control, mining and energy production (biofuel

production). Genetically engineered micro-organism and plants are used to clean up toxic wastes

from industrial effluents and oil spills. It has also found applications in forestry and aquaculture

industries. Overall, biotechnology has significantly impacted and improved quality of life and

there are many exciting opportunities in biotechnology sector.

The brief description of application of biotechnology in different field is as follows-

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Agricultural sciences

Genetic Engineering has allowed us to produce genetically modified plants with diversified

properties such as resistance against pest, drought and abiotic stress. It has enabled us to produce

ediable plants with short life-span or ability to grow in different season to increase the number of

crops in a year to ultimately increase the food production. Horticulture has used biotechnology

tools to produce plants with multiple color, shades, aroma to increase the production of natural

colors and scent. A detail description of other biotechnology application in plant sciences is

discussed in next lecture.

Animal sciences

One of the early applications of biotechnology in animal science is developing method to

separate cheese and other food products from milk by enzyme and microbes. Genetic

engineering in conjugation with cell biology and biochemistry has developed multiple products

of animal origin. Transgenic animal strains with desired phenotype such as high milk yielding

animals, fishes and hens with more fat content. A detail description of other biotechnology

application in animal sciences is discussed later.

Medical Sciences

Biotechnology helped identification of drug like molecules, antibiotics and other medicines. At

present a number of antibiotics are being produced by fermentation or in cell based systems.

Apart from antibiotic, vaccine, diagnostic kits and other immunotherapy are gift of

biotechnological advancement. Development of artificial limb, arms, heart and medical

procedures to perform open-heart operation, dialysis, artificial insemination, test-tube baby and

other medical procedures.

Four Broad Categories of Biotechnology Based on applications, biotechnology is any industrial or commercial use or alteration of

organisms, cells, or biological molecules to achieve specific practical goals. On the basis of this

definition, four broad categories include:

1. DNA Recombination in nature and in the lab

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2. Biotechnology and Medicine (Health)

3. Biotechnology and Forensics

4. Biotechnology and Agriculture

Recombinant Biotechnology

The spectacular progress and enormous understanding over the past two decades in biological

processes at both molecular and cellular level is revolutionized by the advent of recombinant

DNA technology or Genetic engineering. This field of science is broadly spawned under modern

biotechnology, which is precisely the usage of living organisms to produce improved and

valuable products for human consumption. Biotechnology is truly multidisciplinary in nature and

it encompasses several disciplines of basic sciences and engineering. The science disciplines

which are included under biotechnology are: Microbiology Chemistry Biochemistry

GeneticsMolecular biology Immunology Cell and tissue culture Physiology. On the engineering

side, it leans heavily on chemical and biochemical engineering since large scale cultivation of

microorganisms and cells, their down stream processing etc. are based on them.

Development of recombinant biotechnology date back to 1953, when double helical structure of

DNA was elucidated by Watson and crick and the genetic code was cracked by Nirenberg. Cohen

and Boyer in 1973 invented the technique to cut and paste DNA sequences i.e. the concept of

restriction enzymes came into the picture. Since then recombinant DNA technology has rapidly

progressed and expanded. It has sparked a new age in disparate fields. It is the benediction of

recombinant DNA technology, that now it is possible to put two genes together, to clone the

genes for polypeptides like human insulin, growth factors, hormones, interferons, blood

clotting factors and viral coat proteins (for vaccines) in bacteria in a way that protein can be

expressed and the resulting recombinant protein can be extracted from the cell cultures.

Regions of DNA called genes contain information that leads to synthesis of specific proteins,

which are strings of amino acids. Each of the protein is unique in context of its function and the

reaction it catalyses. If now one is able to express a natural gene from any organism in a very

simple bacterium such as Escherichia coli, a bacterium living in intestines that has become the

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model organism for biotechnology and brought a turning point in the field. Now, one can induce

this bacterium to make a lot of protein that is coded by the gene regardless of the nature and

source of donor organism. The techniques used include:

Gene isolation that codes for a particular specific protein

Cloning of this gene into an appropriate production host

Improvement of production yields via improving expression by using better promoters,

tighter and controlled regulation

Optimization of media and growth conditions at fermentor scale.

Commercial implications of recombinant DNA technology

In 1977, the first human protein (somatostatin) was produced in E. coli and in 1982; first

recombinant protein (human insulin) was released in the markets. In 1985, Kary Mullis

conceived the idea of polymerase chain reaction (PCR), which has given recombinant DNA

technology a new face and uplift. Molecular ecology, biomolecular archaeology and DNA

forensics and fingerprinting are new disciplines that have become possible as a direct

consequence of invention of PCR. The principles of commercial implications of recombinant

DNA technology are that large number of proteins that exist in minute amounts in nature can be

mass-produced, if required. Moreover, the yields of the desired products can be increased with

improved efficiency from nanogram levels to milligram levels. More recent advances in mid

eighties and early nineties have made possible to transform even distantly related DNA in

another organism i.e. to genetically modify any organism for production of some desired

proteins. Such genetically modified organisms (GMOs) are called Transgenics.

Biotechnology and Medicine (Health)

Biotechnology in medicine and pharmacology has been developed in the following areas:- 1. Therapeutics 2. Vaccines, antibodies and drugs 3. New methods of drug delivery 4. Molecular diagnostics AND 5. Molecular Markers

A. Therapeutics

1. Gene Therapy (GT)Human Genomic Project has changed medicine. Many scientists and physicians think that many medical benefits could flow from knowing the location and sequences of all the genes. Such

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knowledge would facilitate locating genes that are associated with diseases or disease susceptibility. This makes possible the development of “Gene Therapy”.

Definition Gene Therapy is: The introduction of a normal gene, via genetically engineered cell, into individuals who do not possess a functional copy of this gene, for therapeutic purposes.

The insertion of usually genetically altered genes into cells especially to replace defective genes in the treatment of genetic disorders or to provide a specialized disease-fighting function

Experimental treatment of a genetic disorder by replacing, supplementing, or manipulating the expression of abnormal genes with normally functioning genes

It is an approach to treating disease by either modifying the expressions of an individual's genes or correction of abnormal genes

Gene therapy is the use of DNA as a pharmaceutical agent to treat disease

Types of GT

It is classified into two types:

Germ line gene therapy

Germ line gene therapy is where germ cells (sperm or eggs) are modified by the introduction of

functional genes. Therefore, the change is heritable and will be passed on to the later generations.

This is theoretically highly effective in treating genetic disorders but this option is not considered

at present for application in human beings for a variety of ethical reasons.

Somatic gene therapy

Somatic gene therapy is where the gene is introduced only in somatic cells but it is not herited as

germline is not involved. Somatic gene therapy is further divided into two groups:

augmentation therapy

The first one where the functional gene is introduced in addition to the defective gene

endogenously that is the modified cell contains both the defective as well as the normal

(introduced) copies of the gene. This is called as augmentation therapy.

targeted gene transfer

The second is targeted gene transfer, which uses homologous recombination to replace the

endogenous gene with the introduced functional gene.

Steps in gene therapy

The first step in gene therapy is:

To transform the cell with a specific gene

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Introducing the gene into specific cells within the body (in vivo)

Removing cells from the body

Introducing the gene and then returning the cells (ex vivo).

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In Vivo Gene Therapy: The in vivo gene therapy involves use of a vector that carries the gene.

The most common types of vectors are viral vectors. Usually the integration of gene into the

genome is random and is only transient and is quite possible that indispensable genes may be

inactivated or oncogenes may be activated during this phenomenon. Non viral systems of gene

delivery are safer comparatively and it includes liposome mediated delivery, electroporation,

microinjection etc. Moreover, they do not possess the risk of immune response and are able to

survive transport though the body to reach the target cell. Nucleic acid probes can be used to

detect variety of plant and animal diseases even before the onset of symptoms. The nucleic acid

sequences of pathogen labeled with some markers can be used as probes. Monoclonal antibodies

act as an extremely useful tool for rapid and accurate detection and diagnosis of diseases. The

advent of hybridoma technology provided methods for the production of specific antibodies

targeted against a unique epitope of the immunizing antigen.

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B. Vaccines (Disease Prevention)

Vaccine is the use of biological preparation for immunizations. Vaccines represent an invaluable

contribution of biotechnology and provide protection against various diseases.

Features of an ideal vaccine

An ideal vaccine formulation should consist of following features:-

It should not be toxic

It should be safe with minimum side effects

It should be eco-friendly

It should produce long lasting effective humoral and cell mediated immunities.

It should be simple to administer

It should be cheap to be affordable to all the classes of people.

Types of vaccines

Conventional vaccines: Conventional vaccines consist of whole pathogenic organisms which

are either killed or live but its virulence is greatly reduced (attenuation). It suffers various

limitations although it is relatively easy to produce at low cost. It carries a risk of disease due to

the occasional presence of active virus particles or reversion of virulence after one round of

replication in the vaccinated individuals.

Purified antigen vaccines: Purified antigen vaccines are based on isolation of antigen from the

concerned pathogen. Thus, these non recombinant vaccines do not possess the risk of

pathogenicity, since it does not involve whole organism. But the cost is higher due to

cumbersome steps involved in purification of antigen and subsequently vaccine preparation.

Toxoids: Many bacteria produce exotoxins, which are highly immunogenic. The toxin, although

is inactivated by heat, formaldehyde and other chemicals, most exotoxins, when treated in this

way loose their toxicity but still retain its immunogenicity. These are called toxoids and are used

as efficient vaccines. For many pathogenic diseases like tetanus, diphtheria, toxiods are

available. Precipitation of toxoids with alum enhances the immunogenicity.

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Recombinant vaccine: A recombinant vaccine contains either a protein or a gene encoding

pathogen’s protein that is immunogenic and critical to the pathogen function. The vaccines based

on recombinant protein are called as subunit vaccines. The genes encoding such proteins can be

identified and isolated form a pathogen and then expressed in E. coli or any other host for large

scale production of the protein. Generally, the whole protein molecule is not necessary for

immunogenicity, the immunogenicity is usually confirmed only by a small portion of the protein

molecule. Segments containing these immunogenic residues are effective in immunization and

can provide immunity against the deadly pathogen. Recombinant protein or polypeptide vaccines

are safe since whole organisms are not involved. They are highly efficacious. But the cost is high

and transportation may pose a problem since protein has to be stored at low temperatures, as heat

can destabilize the protein. Thus, their storage and transportation to remote areas nay be

problematic and a liming factor in their use.

DNA vaccines: Recently vaccines based on DNA are being developed. The gene encoding the

relevant immunogenic protein is isolated, cloned and then integrated into a suitable expression

vector. This is introduced, into the individuals to be immunized. This can generate both humoral

and cell-mediated response. Usually the DNA is injected intramuscularly which leads to its

uptake and expression of DNA in the muscle cells. Another approach is the use of vectors like

vaccinia, adenoviruses, etc for gene delivery. Another approach is to remove cells from the body

of an individual into which the concerned immunogen, encoding gene is introduced and

expressed. These cells are again introduced into the body.

C. Disease Diagnosis

An accurate diagnosis of the diseases is critical for its effective management and cure. Following

are some novel disease diagnostic approaches that have been developed by biotechnology which

are efficient, specific precise and rapid.

DNA/ RNA Probes

Probes are small (15-30 bases long) nucleotide (DNA/RNA) sequences used to detect the

presence of complementary sequences in nucleic acid samples. The probes may be DNA/RNA or

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either radioactively or non-radioactively labeled. Use of probes for disease diagnosis is

advantageous over conventional diagnostic tools like in the following ways:-

1. High specificity, rapid and much simpler

2. No culturing is required therefore is applicable to those pathogens also which cannot

be cultured.

3. Can detect infections even in a very latent stage where antibodies are yet not

generated.

4. Probe can be easily prepared

5. A single species-species probe can identify all the serotypes of pathogen.

Monoclonal Antibodies

Antibodies are proteins synthesized in blood against antigens and are collected from the blood

serum. The antibodies, which are heterogenous and non specific in action are called polyclonal

antibodies. If a specific lymphocyte, after isolation and culture in vitro becomes capable of

producing a single type of antibody bearing specificity against specific antigen, it is known as

monoclonal antibody (Mabs) . When Monoclonal antibodies are used as enzymes using the

technique of enzyme engineering, then they are called abzymes. Thus Mabs are homogeneous,

specific Abs and from one type of B cell OR antibodies that are identical because they were

produced by one type of immune cell and are all clones of a single parent cell (B-lympocyte).

Production of Mabs through Hybridoma Technology

In monoclonal antibody technology, tumor cells that can replicate endlessly are fused with

mammalian spleen B cells that produce an antibody. The result of this cell fusion is a

"hybridoma," which will continually produce antibodies. When myeloma cells were fused with

antibody-producing mammalian spleen cells,the resulting hybridomas, produced large amounts

of monoclonal antibody. This product of cell fusion combined the desired qualities of the two

different types of cells: the ability to grow continually, and the ability to produce large amounts

of pure antibody.

The steps involved in the production of monoclonal antibodies12

These are as follows:

1. Finding an appropriate cell-line as a fusion partner for the plasma cells.

2. The use of an efficient means to fuse the two parental cell types.

3. The use of a selective system to remove unfused parental cell types.

4. The identification of those hybrid cells which secret the desired antibody.

OR

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A mouse is immunized by injection of an antigen X to stimulate the production of antibodies

targeted against X. The antibody forming cells are isolated from the mouse's spleen.

Explanation of the above diagram: To produce monoclonal antibodies, one removes B-cells

from the spleen or lymph nodes of an animal that has been challenged several times with the

antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow

indefinitely in culture (myeloma is a B-cell cancer) and that have lost the ability to produce

antibodies. This fusion is done by making the cell membranes more permeable by the use of

polyethylene glycol, electroporation or, of historical importance, infection with some virus. The

fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and

indefinitely. Large amounts of antibodies can therefore be produced. The hybridomas are

sufficiently diluted to ensure clonality and grown. The antibodies from the different clones are

then tested for their ability to bind to the antigen (for example with a test such as ELISA) or

immuno-dot blot, and the most sensitive one is picked out.

In the above process, one uses myeloma cell lines that have lost their ability to produce their own

antibodies or antibody chain, so as to not contaminate the target antibody. Furthermore, one

employs only myeloma cells that have lost a specific enzyme (hypoxanthine-guanine

phosphoribosyltransferase, HGPRT) and therefore cannot grow under certain conditions (namely

in the presence of HAT medium). These cells are preselected by the use of 8-azaguanine media

prior to the fusion. Cells that possess the HGPRT enzyme will be killed by the 8-azaguanine.

During the fusion process many cells can fuse. Myeloma with myeloma, spleen cell with spleen

cell, 3 cells of different types etc... The desired fusions are between healthy B-cells producing

antibodies against the antigen of interest and myeloma cells. These are relatively rare, but when

one succeeds, then the healthy partner supplies the needed enzyme and the fused cell can survive

in HAT medium. This is the trick to detect the successfully fused cells. The medium must be

enriched during selection to favour hybridoma growth. This can be achieved by the use of a layer

of feeder cells or supplement media such as briclone.

Monoclonal antibodies can be produced in cell culture or in live animals. When the hybridoma

cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an

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antibody-rich fluid called ascites fluid. Production in cell culture is usually preferred as the

ascites technique may be very painful to the animal and if replacement techniques exist, may be

considered unethical. Fermentation chambers have been used to produce antibodies on a larger

scale. Nowadays, bioengineering allow production of antibodies in plants.Once a monoclonal

antibody is made, it can be used as a specific probe to track down and purify the specific protein

that induced its formation.

Practical aspects of monoclonal antibody production

1. Immunization: The purpose of immunizing animals, like rats, is to induce a good immune

response to the antigen of interest, this immune response being indicated by the presence of a

high titre of serum antibodies.

2. Cell fusion and selection of hybridomas: These processes rely on a good tissue culture

technique. Myeloma cells and hybridomas grow in suspension in various tissue culture media,

which are usually supplemented with 10-20% foetal calf serum as a source of growth factors.

Note: The decision to proceed to cell fusion will only be taken when the following conditions

are satisfied: (i) Immunized mice are available, and have been shown to make antibody of the

required specificity. (ii) An assay suitable for screening the products of the fusion is available.

(iii) Healthy myeloma cells have been in culture for at least a week. (iv) The experimenter has

available a continuous period of at least four weeks to devote to the care and maintenance

of the fusion.

If the above conditions are met, the immunized mouse (or mice) destined to provide the

stimulated lymphocytes are given their final inoculation with antigen. The fusion should be

performed 2-4 days after this inoculation. The spleen is removed aseptically from the mouse

and gently dissociated into a single cell suspension. The suspension contain antibody –

forming cells in a mixture of other cell types.

3. Screening the products of hybridoma fusions

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The purpose of this is to identify those wells which contain hybridoma colonies which are

secreting antibodies of the required specificity. There are three ways of screening fusions.

These are: (i) The antigen may be immobilized on a solid support, such as nitrocellulose sheet

or a PVC microtitre well, and the hybridoma supernatants applied to the immobilized antigen.

After an incubation period, the antibodies bound to the support are identified by use of a

labelled secondary reagent. (ii) The antibodies present in the supernatants are immobilized on

a solid support, and labelled antigen is applied to the support. After incubation and washing,

the location of the labelled antigen is revealed by a suitable detection method. (iii) It is

possible to screen for monoclonal antibodies using a functional test, such as inhibition of an

enzyme activity or of a measurable response of cultured cells.

4. Growth and cloning

Twelve to fourteen (12-14) days after fusion, the hybridoma colonies identified by the

screening assay as producers of useful antibody must be transferred from their small culture

wells into larger vessels. The essential aim of cloning is to isolate single cells from the

hybridoma culture and allow these cells to form colony (clone) by division.

Summary: Mab production steps (i) Identify antigen (Ag) of interest and challenge an animal

with the Ag several times (primed animal) (ii) Remove B-cells from the spleen or lymph node of

primed animal (iii) Fuse the B–cells with myeloma tumor cells to make them grow indefinitely

in culture. Fusion is achieved by using polyethylene Glycol (PEG) or electroporation to make the

cell membrane more permeable. The fused cells are called hybridomas or hybrid cells-being

cancer cells and multiply rapidly and indefinitely. Using a selective medium=HAT

(hypoxanthine; aminopterine and thymidine to remove infused parental cell types. (iv)

Identification of those hybrid cells which secret the desired antibody and (v) The resulting hybrid

cells are the cloned and propagated for the production of desired antibody. The produced

antibodies are purified using methods such as affinity chromatography.

Summary of Mabs Applications

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Once monoclonal antibodies for a given substance have been produced, they can be

used to detect the presence and quantity of this substance, for instance in a Western blot test

(to detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a

cell).

They are also very useful in immunohistochemistry which detect antigen in fixed tissue

sections.

Monoclonal antibodies can also be used to purify a substance with techniques called

immunoprecipitation and affinity chromatography.

Mabs as diagnostic tools: (1) Detection or diagnosis of diseases and contaminants in food

and environment for instance in a western blot test or immunofluorescence test (2)

Immunohistochemistry for the detection Ags in fixed tissue sections and (3) purification of

substances with techniques called immunoprecipitation and affinity chromatography

Mabs as therapeutic tools: (1) In the management of rejection of transplantation aimed at

suppressing T-cell activity and (2) In delivery of drugs for example “magic bullet” or

immunotoxin in cancer treatment.

3. Biotechnology and Forensics

The chemical structure of everyone’s DNA is the same. The basic difference between two

individual’s DNA is the order of base pairs. Using these sequences, every person could be

identified solely by the sequence of their base pairs. Scientists usually use a small numbers of

sequences of DNA that are known to very among individuals. In medicine, DNA finger

printing has application in genetic counselling, proof of parentage, identification of criminals in

thefts etc. Since a person inherits his or her VNTRs [variable numbers of tandem repeats, which

are, dispersed islands throughout the genome and are made up of a variable numbers of end to

end duplications of identical or almost identical sequences of 2-80 each. VNTRs are

polymorphic due to difference in numbers of repeat units at a given locus or position in a

chromosome] from his or her parents. Thus, analysis of VNTR patterns can be used to establish

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paternity and maternity. DNA can be isolated from blood, hair, skin cells etc, and can be

compared with that of a suspected criminal for a particular VNTR pattern.

Biotechnology Tools

In order to study genes, methods have been developed to “purify” or isolate genes or DNA for

study. To achieve this goal, various tools are in existence and include:

Plasmid: Plasmid is a small, circular piece of DNA located in the cytoplasm of many bacteria;

normally does not carry genes required for the normal functioning of the bacterium but may

carry genes that assist bacterial survival in certain environments, such as a gene for antibiotic

resistance. Many types of bacteria contain plasmids ranging in size from 1000 to 100,000

nucleotides (ATCG) long. Plasmids can be passed between bacteria and yeast – moving genes

between a prokaryotic and eukaryotic cell! Plasmids are useful because:

Plasmids (small rings of DNA) replicate in the cytoplasm of the bacterial cell.

Bacterium’s chromosome contains all of the genes necessary for basic survival, plasmid

not required for survival.

Plasmids: enhance a bacterium’s chance of survival. Some carry genes that enable

bacteria to grow in the presence of an antibiotic.

Restriction enzyme: An enzyme normally isolated from a bacterium that cuts double-stranded

DNA at a specific nucleotide sequence; the nucleotide sequence that is cut differs for different

restriction enzymes. Restriction enzymes are major tools of recombinant DNA technology

derived from a group of bacteria.

Cloning Vector: An agent used to transfer DNA in genetic engineering, such as a plasmid that

moves recombinant DNA from a test tube back into a cell, or a virus that transfers recombinant

DNA by infection.

DNA probes: A sequence of nucleotides that is complementary to the nucleotide sequence in a

gene under study; used to locate a given gene within a DNA library.

DNA libraries: A library is a collection of cells that host fragments of DNA from a particular

organism.

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GENOMIC LIBRARY: a library that contains the organism’s entire set of genetic

material, or “genome”.

cDNA (complementary DNA) LIBRARY: is produced from mRNA’s, and represents

only genes that are expressed.

GEL Electrophoresis: Used to separate and identify segments of DNA. A technique in which

molecules (such as DNA fragments) are placed on restricted tracks in a thin sheet of gelatinous

material and exposed to an electrical field; the molecules then migrate at a rate determined by

certain characteristics, such as length. In GEL Electrophoresis:

DNA samples are pipetted into wells in a gel made of agarose

The gel has electrodes connected to each end. One electrode is made positive, one

negative; therefore, current will flow between the electrodes through the gel.

The phosphate groups in backbone of DNA are negatively charged.

When the electrical current flows through the gel, the negatively charged DNA molecules

flow toward the positively charged electrode.

Smaller DNA molecules travel faster than larger.

Gel is placed on a special nylon paper. Electrical current drives DNA out of gel onto

nylon.

The DNA in the gel may form one continuous streak of every possible size of DNA

fragment.

Nylon paper with DNA is bathed in a solution of radioactive or fluorescent DNA probes

of the DNA segments of interest.

The probes base pair to the DNA and create bands that are labeled.

Fluorescent dye identifies alleles or segments of DNA.

DNA sequencing: A method to determine the nucleotide sequence of a gene, a segment of a

gene, or the entire genome.

DNA sequencing is the gold standard for the study of DNA.

The method will copy DNA in a test tube using modified nucleotides that will block

further DNA synthesis.

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DNA fingerprinting: As revealed in this DNA fingerprint, the DNA band created by the blood

on the defendant’s clothing matches the DNA bands of the victim’s blood.

Polymerase Chain Reaction (PCR): a method of producing virtually unlimited numbers of

copies of a specific piece of DNA, starting with as little as one copy of the desired DNA. With

each cycle of PCR, the amount of DNA doubles. This solved the problem of small DNA samples

OR amplifying any gene of interest.

IN VITRO FERTILIZATION AND EMBRYO TRANSFER

Union of egg cell and sperm outside the body in a culture vessel is known as in vitro fertilization. This involves collection of healthy ova and sperms from healthy females and males, and their fusion under in vitro conditions. The resulting zygote may be cultured in vitro for a period of time, which is then implanted in the uterus of healthy female. This technique of in vitro fertilization and embryo transfer are done to obtain desirable genotypes and in cases of infertility. In vitro fertilized embryos at 16 celled stage have been successfully transferred into the uterus. The babies produced using this approach is termed as Test tube babies. The first test tube baby, named Loise joy Brown, was born on 25th July, 1978. However, this has few ethical and social issues related which may need resolution. Although high degree of expertise is required and the cost of production of each progeny is more, the gains will be attractive and it will be possible to obtain relatively rare genotype.

Recombinant DNA: is DNA that has been altered by the recombination of genes from a different organism, typically from a different species. large amounts of recombinant dna can be grown in bacteria, viruses, or yeast and then transferred to other species. DNA recombination in nature – natural recombinant DNA.

Transformation: a method of acquiring new genes, whereby DNA from one bacterium (normally released after the death of the bacterium) becomes incorporated into the DNA of another, living, bacterium. A phenomenon in which external genetic material is assimilated by a cell. Transformation in Bacteria

Transformation enables bacteria to pick up DNA from the environment. The DNA may be part of the chromosome from another bacterium or from another

species. The DNA fragment is incorporated into bacterial chromosome. Transformation may also occur when bacteria pick up tiny circular DNA molecules called

plasmids.

Transgenic animals carry and express genetic information not normally found in that species (Singleton, 1999). Aims of producing transgenic animals: Biomedical, veterinary, biological and genetic research; Agriculture: enhancing growth and development; Increase disease resistance; Produce foreign proteins; Xenotransplantation and Gene therapy

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TOPIC 2: MAMMALIAN CELL CULTURE AND CELL LINES

2.1 HISTORY OF ANIMAL CELL CULTURE

It was Jolly, who (1903) showed for the first time that the cells can survive and divide  in vitro.

Ross Harrison, (1907) was able to show the development of nerve fibres from frog embryo

tissue, cultured in a blood clot. Later, Alexis Carriel (1912) used tissue and embryo extracts as

cultural media to keep the fragments of chick embryo heart alive. In the late 1940s, Enders,

Weller and Robbins grew poliomyelitis virus in culture which paved way for testing many

chemicals and antibiotics that affect multiplication of virus in living host cells.

The significance of animal cell culture was increased when viruses were used to produce

vaccines on animal cell cultures in late 1940s. For about 50 years, mainly tissue explants rather

than cells were used for culture techniques, although later after 1950s, mainly dispersed cells in

culture were utilized. In 1966, Alec Issacs discovered Interferon by infecting cells in tissue

culture with viruses. He took filtrates from virus infected cells and grew fresh cells in the filtered

medium. When the virus was reintroduced in the medium, the cells did not get infected. He

proposed that cells infected with the virus secreted a molecule which coated onto uninfected cells

and interfered with the viral entry. This molecule was called “Interferon”.

Chinese Hamster Ovary (CHO) cell lines were developed during 1980s. Recombinant

erythropoietin was produced on CHO cell lines by AMGEN (U.S.A.). It is used to prevent

anaemia in patients with kidney failure who require dialysis. After this discovery, the Food and

Drug Administration (U.S.A) granted the approval for manufacturing erythropoietin on CHO cell

lines. In 1982, Thilly and co-workers used the conventional conditions of medium, serum, and

O2 with suitable beads as carriers and grew certain mammalian cell lines to densities as high as

5x106 cells/ml. A lot of progress has been also made in the area of stem cell technology which

will have their use in the possible replacement of damaged and dead cells. In 1996, Wilmut and

co-workers successfully produced a transgenic sheep named Dolly through nuclear transfer

technique. Thereafter, many such animals (like sheep, goat, pigs, fishes, birds etc.) were

21

produced. Recently in 2002, Clonaid, a human genome society of France claimed to produce a

cloned human baby named EVE.

Animal cell cultures have been used to generate valuable products based on their own genetic

information or due to genes transferred to them (Transgenes) using recombinant DNA

technology.

2.2 PRINCIPLES OF ANIMAL CELL CULTURES AND CELL LINES

Animal cells can grow in simple glass or plastic containers in nutritive media but they grow

only to limited generations. The cells exhibit contact inhibition. In culture the cancer cells

apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape,

they loose contact inhibition and pile over each other.

Difference in the in vitro and in vivo growth pattern of cells

There is a difference in the in vitro and in vivo growth pattern of cells. In vitro for example -

(i) there is an absence of cell-cell interaction and cell matrix interaction,

(ii) there is a lack of three-dimensional architectural appearance

(iii) changed hormonal and nutritional environment and they way of adherence to glass or

plastic container in which they grow, cell proliferation and shape of cell results in

alterations.

Requirements for Animal Cell Culture

Among the essential requirements for animal cell culture are special incubators to maintain the

levels of oxygen, carbon dioxide, temperature, humidity as present in the animal’s body. The

synthetic media with vitamins, amino acids and fetal calf serum.

1. Physical environment and nutrient media

The physical environment includes the optimum pH, temperature, osmolality and gaseous

environment, supporting surface and protecting the cells from chemical, physical, and

22

mechanical stresses. Nutrient media is the mixture of inorganic salts and other nutrients capable

of sustaining cell survival in vitro. In nutrient medium, serum is essential for animal cell culture

because it contains growth factors which promote cell proliferation. It is obtained as exuded

liquid from blood undergoing coagulation and filtered using Millipore filters.

Parameters: Under physical environment and nutrient media, the following parameters are

essential for successful animal cell culture:

Temperature

In most of the mammalian cell cultures, the temperature is maintained at 370C in the incubators

as the body temperature of Homo sapiens is 370C.

Culture media

The culture media is prepared in such a way that it provides:- 1) The optimum conditions of

factors like pH, osmotic pressure, etc. 2) It should contain chemical constituents which the cells

or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and

other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions,

trace elements, vitamins, cofactors, and ions. Glucose is added as energy source-it’s

concentration varying depending on the requirement. Phenol Red is added as a pH indicator of

the medium.

Two types of media

There are two types of media used for culture of animal cells and tissues- the natural media and

the synthetic media.

a) Natural Media

The natural media are the natural sources of nutrient sufficient for growth and proliferation of

animal cells and tissues. The Natural Media used to promote cell growth fall in three categories.

i) Coagulant, such as plasma clots. It is now commercially available in the form of liquid plasma

23

kept in silicon ampoules or lyophilized plasma. Plasma can also be prepared in the laboratory

taking out blood from male fowl and adding heparin to prevent blood coagulation. ii) Biological

fluids such as serum. Serum is one of the very important components of animal cell culture

which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts

containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors

which promotes cell proliferation, cell attachment and adhesion factors. Serum is obtained from

human adult blood, placental, cord blood, horse blood, calf blood. The other forms of biological

fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture

filtrate, aqueous humour, from eyes etc. iii) Tissue extracts for example Embryo

extracts- Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow

etc are also used for culture of animal cells. 

b) Synthetic media

Syntheic media are prepared artificially by adding several organic and inorganic nutrients,

vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of synthetic media

can be prepared for a variety of cells and tissues to be cultured.

Types of synthetic media: Synthetic media are of two types- Serum containing media (media

containing serum) and serum- free media (media with out serum). Examples of some media are:

minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc. 

Advantages of serum in culture medium are: 

i) serum binds and neutralizes toxins, 

(ii) serum contains a complete set of essential growth factors, hormones, attachment and

spreading factors, binding and transport proteins, 

(iii) it contains the protease inhibitors, 

(iv) it increases the buffering capacity, 

(v) it provides trace elements.

Disadvantages of serum in culture medium are

(i) it is not chemically defined and therefore it’s composition varies a lot, 

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(ii) it is sometimes source of contamination by viruses, mycoplasma, prions etc, 

(iii) it increases the difficulties and cost of down stream processing, 

(iv) it is the most expensive component of the culture medium.

PREPARATION OF CULTURE MEDIA

Preparation of culture media seeks to prepare desired medium for the given Animal cell culture.

Principle: All the Animal cells can be grown in a liquid culture medium consisting of a mixture of vitamins, salts, glucose, amino acids and growth factors. Moreover, Calf serum is an easily available source of growth and attachment factors. Antibiotics are added to prevent the growth of bacteria. Under these conditions cells will grow at physiological pH (7.4) and at body temperature (37ºC) to form a monolayer on the culture vessels.

Materials required: Medium, Adult bovine serum, Membrane filter (Millipore 0.45μ), SterilizeDouble distilled water 1000 ml, 1 litre measuring cylinder, 100 ml measuring cylinder, 1 litre filtration flask, Medium storage bottles and Other Glasswares

Method: Sterilize the laminar air flow by UV irradiation for 45 minutes before using it. 1. Take 500ml of sterile double distilled water in a 1000 ml measuring cylinder. 2. Transfer the contents of the powdered medium into 1 litre measuring cylinder add 3.7 gms of NaHCO3 in the absence of CO2 incubator. 3. Mix thoroughly to dissolve the powdered medium, and add penicillin /streptomycin/gentamycin. 4. Fill the cylinder with1 litre double distilled water mix and transfer to sterile 2 litre flask and mix. Pinkish red color of the medium indicates normal pH range. 5. Assemble the filter sterilization set-up and carry out the filtration under negative pressure. 6. Prepare 400 ml of medium containing 10% Adult bovine serum using 100 ml measuring cylinder and store in a 500 ml sera lab bottle. 7. Transfer the remaining medium without serum into big glass bottles. 8. Store the medium in refrigerator, dispose the used membrane and immerse the used glassware in water for washing. 9. Different types of medium is used for various kind of Experiments. 10. The components of different types of medium is given in the following tables.

pH

Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. The

optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular

enzymes and binding of hormones and growth factors to cell surface receptors in the cell

cultures. The regulation of pH is done using a variety of buffering systems. Most media use a

bicarbonate-CO2 system as its major component.

Osmolality

25

A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the

growth media determine the osmolality of the medium. All commercial media are formulated in

such a way that their final osmolality is around 300 mOsm.

2. Cryopreservation and aseptic conditions

Cryo preservation is storing of cells at very low temperature (-1800C to -196 0C) using liquid

nitrogen. DMSO is a cryopreservative molecule which prevents damage to cells. In order to

maintain the aseptic conditions in a cell culture, a LAF hood is used. Based on the nature of cells

and organism the tissue culture hoods are grouped into three types: Class I, Class II, and Class

III.

3. CO2 incubators and microscope

CO2 incubators are used and designed to mimic the environmental conditions of the living cells

while an inverted microscope is used for visualizing cell cultures in situ. For most animal cell

cultures low speed centrifuges are needed. NOTE: Neuronal cells constitute the nervous

system. In culture the neuronal cells cannot divide and grow. The cells that form connective

tissue (skin) is called fibroblast. The fibroblast can divide and grow in culture to some

generations after which they die. All normal animal cells are mortal.

2.3 TYPES OF ORGAN AND CELL CULTURES

2.3.1 ORGAN CULTURE AND HISTOTYPIC CULTURES

Organ Culture

In this type of culture, the whole organs or small fragments of the organs with their special and

intrinsic properties intact are used in culture. In the organ culture, the cells are integrated as a

single unit which helps to retain the cell to cell interactions found in the native tissues or organs.

Due to the preservation of structural integrity of the original tissue, the associated cells continue

to exchange signals through cell adhesion or communications. Due to the lack of a vascular

system in the organ culture, the nutrient supply and gas exchange of the cells become limited. In

26

order to overcome this problem, the organ cultures are placed at the interface between the liquid

and gaseous phases. Sometimes, the cells are exposed to high O2 concentration which may also

lead to oxygen induced toxicity. Due to the inadequate supply of the nutrients and oxygen, some

degree of necrosis at the central part of the organ may occur. In general, the organ cultures do not

grow except some amount of proliferation that may occur on the outer cell layers.

Techniques and Procedure for organ culture

In order to optimize the nutrient and gas exchanges, the tissues are kept at gas limited interface

using the support material which ranges from semisolid gel of agar, clotted plasma, micropore

filter, lens paper, or strips of Perspex or plexiglass. The organ cultures can also be grown on top

of a stainless steel grid. Another popular choice for growing organ cultures is the filter-well

inserts. Filter-well inserts with different materials like ceramic, collagen, nitrocellulose are now

commercially available. Filter well inserts have been successfully used to develop functionally

integrated thyroid epithelium, stratified epidermis, intestinal epithelium, and renal epithelium.

The procedure for organ cultures has the following steps:

The organ tissue is collected after the dissection. The size of the tissue is reduced to less than 1mm in thickness.

The tissue is placed on a gas medium interface support.

Incubation in a CO2 incubator.

M199 or CMRL 1066 medium is used and changed frequently.

The techniques of histology, autoradiography, and immunochemistry are used to study the organ cultures.

The advantages of organ culture: The organ cultures can be used to study the behavior of an

integrated tissue in the laboratory. It provides an opportunity to understand the biochemical and

molecular functions of an organ/tissue.

27

Limitations of organ culture: It is a difficult and expensive technique. The variations are high

with low reproducibility. For each experiment, a new or fresh organ is needed as organ cultures

are not propagated.

Histotypic culture

The cell lines grown in three dimensional matrix to high density represent histotypic cultures.

Using histotypic culture, it is possible to use dispersed monolayers to regenerate tissue like

structures. It the growth and propagation of cell lines in three-dimensional matrix to high cell

density that contributes to this. The techniques used in histotypic cultures are:

a) Gel and sponge technique- In this method, the gel (collagen) or sponges (gelatin) are used

which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels

and sponges while growing. b) Hollow fibers technique- In this method, hollow fibers are used

which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers

with a bed of plastic capillary fibers have been developed to be used for histotypic type of

cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like

structures. c) Spheroids – The re-association of dissociated cultured cells leads to the formation

of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into

specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic

or homotypic aggregates have the ability to sort themselves out and form groups which form

tissue like architecture. However, there is a limitation of diffusion of nutrients and gases in these

cultures. d) Multicellular tumour spheroids- These are used as an in vitro proliferating models

for studies on tumour cells. The multicellular tumour spheroids have a three dimensional

structure which helps in performing experimental studies related to drug therapy, penetration of

drugs besides using them for studying regulation of cell proliferation, immune response, cell

death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of

necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with

trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in

magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates of cells representing spheroids

are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids

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are used for many purposes. They are used as models for a vascular tumour growth. They are

used to study gene expression in a three-dimensional configuration of cells. They are also used to

study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain

diseases like rheumatoid arthritis.

Organotypic cultures

These cultures are used to develop certain tissues or tissue models for example skin equivalents

have been created by culturing dermis, epidermis and intervening layer of collagen

simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic

culture involves the combination of cells in a specific ratio to create a component of an organ.

2.3.3 CELL CULTURE AND CELL LINES

On the basis of morphology (shape & appearance) or on their functional characteristics they are

divided into three.

Epithelial like- Attached to a substrate and appears flattened and polygonal in shape

Lymphoblast like- Cells do not attach, remain in suspension with a spherical shape

Fibroblast like- Cells attached to a substrate, appear elongated and bipolar

CELL CULTURE

Cell (or tissue) is the maintenance of growth of cells under laboratory conditions in suitable

culture medium. Cell culture is developed from a single cell and therefore consisting of cells

with a uniform genetic make-up. Generally stem cells are used in this culture. Cell culture is

therefore the multifaceted process through which cells are isolated from animal and their

subsequent growth under controlled artificial conditions, generally outside their natural

environment. In this procedure cells are directly isolated from body or disaggregated by

enzymatic or mechanical procedure or they may be derived from cell lines or cell strains . The

historical development and methods of cell culture are closely interrelated to those of tissue

culture and organ culture. The in vitro propagation of cells has become a common practice in

many laboratories for a huge numbers of applications. The ranges of cell types grown are vast.

29

Generally the cells are sensitive to a wide range of compounds and it is therefore necessary to

ensure that they come into contact only with those under study and not with extraneous

materials. Adherent mammalian cells require a suitable surface for attachment.

Types of cell culture

Primary cell culture

Primary cell culture is the primary step of cell culturing in which the cell is first isolated from

tissue and then proliferated under the appropriate conditions until they consume all available

contents for their growth. Now the cell is ready for subculturing by transferring them to new

growth medium that furnish more opportunity for continued growth. The maintenance of growth

of cells dissociated from the parental tissue (such as kidney, liver) using the mechanical or

enzymatic methods, in culture medium using suitable glass or plastic containers is called Primary

Cell Culture. Thus primary cell culture is the original growth of cells under laboratory conditions

in suitable culture medium. Under primary cell culture, cells are dissociated form tissues by

mechanical means and by enzymatic digestion using proteolytic enzymes. Cells can grow as

adherent cells (anchorage dependent) or as suspension cultures (anchorage independent).

Primary cells have a finite life span and a very heterogeneous population. Cells such as

macrophages and neurons do not divide in vitro so can be used as primary cultures.

Two types of primary cell culture

The primary cell culture could be of two types depending upon the kind of cells in culture.

Anchorage Dependent /Adherent cellsCells shown to require attachment for growth are set to be Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. They grow adhering to the cell culture.

Suspension Culture/Anchorage Independent cells Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes.

Secondary cell culture30

When a primary culture is sub-cultured, it becomes known as secondary culture or cell line.

Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture

vessel.

Subculturing or splitting cells

Subculturing or splitting cells is required to periodically provide fresh nutrients and growing

space for continuously growing cell lines. The process involves removing the growth media,

washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures are

called secondary cultures.

CELL LINES

Cell line is a permanently established cell culture that will proliferate indefinitely given

appropriate fresh medium and space. Sub culturing of primary cells leads to the generation of

cell lines. Cell lines have limited life span and they are passaged several times before they

become senescent. Therefore a continuous cell culture is one that is apparently capable of an

unlimited number of population doublings, often referred to as an immortal cell culture. Such

cells may or may not express the characteristics of in vitro neoplastic or malignant

transformation. Continuous cell lines are usually aneuploid and often have a chromosome

number between the diploid and tetraploid values. There is also considerable variation in

chromosome number and constitution among cells in the population (heteroploidy).

Some important properties of Continuous cell lines:

Reduced serum requirement

Reduced density limitation of growth

Growth in semisolid media

Aneuploidy

Several normal cells do not give rise to continuous cell lines. The classical example are normal

human fibroblasts that remain euploid throughout their life span and at crisis (usually around 50

generations) will stop dividing, although they may remain viable for up to 18 months thereafter.

Human glia and chick fibroblasts behave similarly. Epidermal cells, on the other hand, have

shown gradually increasing life spans with improvements in culture techniques. 31

Terms that is associated with the cell lines

The following terms are associated with the cell lines.  

Split ratio: The divisor of the dilution ratio of a cell culture at subculture. 

Passage number: It is the number of times that the culture has been cultured, 

Generation number: It refers to the number of doublings that a cell population has undergone.

These parameters help us to distinguish the cancer cells in culture from the normal cells

because the cancer cells in culture, change shape (more rounded), loose contact inhibition, pile

on each other due to overgrowth and uncontrolled growth.

Types of cell line

A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited

culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines

are categorized into two types:

Finite cell Lines

The cell lines which have a limited life span and go through a limited number of cell generations

(usually 20-80 population doublings) are known as Finite cell lines. These cell lines exhibit the

property of contact inhibition, density limitation and anchorage dependence. The growth rate is

slow and doubling time is around 24-96 hours.

Continuous Cell Lines 

A cell line is a permanently established cell culture that will proliferate indefinitely in

appropriate fresh medium and space. Cell lines differ from cell strains in that they have

absconded the Hayflick limit and become immortalised.

32

The Hayflick limit (or Hayflick Phenomenon) is the number of times a normal cell population

will divide before it stops, presumably because the telomeres reach a critical length. A cell line

arises from a primary culture at the time of the first successful subculture. The terms finite or

continuous are used as prefixes if the status of the culture is known. Cell Strain: By applying

cloning, the positive population of cell lines are selected, therefore this cell lines now becomes a

cell strain. A cell strain often acquires additional genetic changes resulting to the initiation of the

parent line. Cell lines transformed under laboratory conditions or in vitro culture conditions give

rise to continuous cell lines. The cell lines show the property of ploidy (aneupliody or

heteroploidy), absence of contact inhibition and anchorage dependence. The growth rate is rapid

and doubling time is 12-24 hours. Continuous cell lines are therefore transformed immortal and

tumorigenic cell cultures. Characteristics of continuous cell lines:

show the property of ploidy (aneupliody or heteroploidy

Smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio

Fast growth

Grow more in suspension conditions

Ability to grow up to higher cell density

Stop expressing tissue specific genes

Continuous cell lines grow in monolayer or suspension form described below.

Monolayer cultures: When the bottom of the culture vessel is covered with a continuous layer

of cells, usually one cell in thickness, they are referred to as monolayer cultures. Suspension

cultures: Majority of continuous cell lines grow as monolayers. Some of the cells which are

non-adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in

suspension, can be propagated in suspension.

Advantages in propagation of cells by suspension culture

The following are certain advantages in propagation of cells by suspension culture method.

The process of propagation is much faster.

The frequent replacement of the medium is not required.

33

Suspension cultures have a short lag period,

Treatment with trypsin is not required, 

A homogenous suspension of cells is obtained, 

The maintenance of suspension cultures is easy and bulk production of the cells is easily

achieved.

Scale-up is also very convenient.

Characterizing cell lines

Cell lines are characterized by their growth rate and karyotyping as illustrated in the figure

below.

Figure 1: The salient features of cell culture with evolution of  a cell line

Growth Rate: A growth curve of a particular cell line is established taking into consideration

the population doubling time, a lag time, and a saturation density of a particular cell line. A

growth curve consists of:

1) Lag Phase: The time the cell population takes to recover from such sub culture, attach

to the culture vessel and spread.

2) Log Phase: In this phase the cell number begins to increase exponentially.

34

3) Plateau Phase: During this phase, the growth rate slows or stops due to exhaustion of

growth medium or confluency.

Karyotyping: Karyotyping is important as it determines the species of origin and determine the

extent of gross chromosomal changes in the line. The cell lines with abnormal karyotype are also

used if they continue to perform normal function. Karyotype is affected by the growth conditions

used, the way in which the cells are subcultured and whether or not the cells are frozen.

Difference in the in vitro and in vivo growth pattern of cells

There is a difference in the in vitro and in vivo growth pattern of cells. In vitro for example -

there is an absence of cell-cell interaction and cell matrix interaction,

there is a lack of three-dimensional architectural appearance

changed hormonal and nutritional environment and they way of adherence to glass or

plastic container in which they grow, cell proliferation and shape of cell results in

alterations.

TABLE 1: SOME ANIMAL CELL LINES AND THE PRODUCTS OBTAINED FROM THEM

Cell line Product

Human tumour Angiogenic factor

Human leucocytes Interferon

Mouse fibroblasts Interferon

Human Kidney Urokinase

Transformed human kidney cell line, TCL-598

Single chain urokinase-type plasminogen activator (scu-PA)

Human kidney cell (293) Human protein (HPC)

Dog kidney Canine distemper vaccine

Cow kidney Foot and Mouth disease (FMD) vaccine

Chick embryo fluid Vaccines for influenza, measles and mumps

Duck embryo fluid Vaccines for rabies and rubella

35

Chinese hamster ovary (CHO) cells

1. Tissue-type plasminogen activator (t-PA)

2. B-and gamma interferons

3. Factor VIII

Table 2: Properties of finite and continuous cell lines

Properties Finite Continuous(transformed)

Ploidy Euploid, Diploid Aneuploid, Hetroploid Transformation Normal Immortal growth control

altered and tumerigenic Anchorage dependence Yes No Contact inhibition Yes No Density limitation of cell proliferation

Yes Reduced or lost

Mode of growth Monolayer Monolayer or suspension Maintainance Cyclic Steady state possible Serum Requirement High Low Cloning efficiency Low High Markers Tissue specific Chromosomal, enzymatic,

antigenic Special function (e.g. virus susceptibility and differentiation)

May be retained Often lost

Growth rate Slow(TD of 24-96 h) Rapid(TD of 12-24 h) Yield Low High Control parameter Generation time, tissue

specific marker Stain characteristics

SCALE-UP OF ANIMAL CELL CULTURE

Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling

up. Laboratory procedures are normally scaled up via intermediate models of increasing size.

The larger the plant, the greater the running costs, as skilled people are required to monitor and

maintain the machinery. The first pre-requisite for any large scale cell culture system and its

36

scaling up is the establishment of a cell bank. Master cell banks (MCB) are first established and

they are used to develop Master Working Cell Banks (MWCB). The MWCB should be sufficient

to feed the production system at a particular scale for the predicted life of the product. The cell

stability is an important criteria so MWCB needs to be repeatedly subcultured and each

generation should be checked for changes. A close attention should be paid to the volume of

cultured cells as the volume should be large enough to produce a product in amounts which is

economically viable. The volume is maintained by

increasing the culture volume,

increasing the concentration of cells in a reactor by continuous perfusion of fresh

medium, so that the cells keep on increasing in number without the dilution of the

medium. 

A fully automated bioreactor maintains the physicochemical and biological factors to optimum

level and maintains the cells in suspension medium. The most suitable bioreactor used is a

compact-loop bioreactor consisting of marine impellers. The animal cells unlike bacterial cells,

grow very slowly. The main carbon and energy sources are glucose and glutamine. Lactate and

ammonia are their metabolic products that affect growth and productivity of cells. So, the on-line

monitoring of glucose, glutamate, and ammonia is carried out by on line flow injection analysis

(FIA) using gas chromatography (GC), high performance liquid chromatography (HPLC) etc.

Methods for Batch Cultures Scale-up

In batch cultures, the following methods are used in Scale-up of animal cell culture.

Roller Bottles

The Roller bottles provide total curved surface area of the micro carrier beads for growth. The

continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire

cell monolayer in culture. The roller bottles are well attached inside a specialized CO2

incubators. The attachments rotate the bottles along the long axis which helps to expose the

entire cell monolayer to the medium during the one full rotation. This system has the advantage

37

over the static monolayer culture: (a) it provides increase in the surface area, (b) provides

constant gentle agitation of the medium, (c) provides increased ratio of surface area of medium

to its volume, which allows gas exchange at an increased rate through the thin film of the

medium over the cells.

Diagram showing the roller bottle cell culture 

Micro Carrier Beads

Micro carrier beads are small spherical particles with diameter 90-300 micrometers, made up of

dextran or glass. Micro Carrier beads increase the number of adherent cells per flask. These

dextran or glass-based beads come in a range of densities and sizes. The cells grow at a very high

density which rapidly exhausts the medium and therefore the medium has to be replaced for the

optimum cell growth. At the recommended concentration when the microcarriers are suspended

they provide 0.24 m2 area for every 100 ml of culture flask.

Spinner cultures

The spinner flask, was originally developed to provide the gentle stirring of microcarriers but are

now used for scaling up the production of suspension cells. The flat surface glass flask is fitted

with a Teflon paddle that continuously turns and agitates the medium. This stirring of the

medium improves gas exchange in the cells in culture. The spinner flask used at commercial

scale consists of one or more side arms for taking out samples and decantation as well.

GENERATION OF CELL LINES

38

For the generation and maintenance of Cell line some basic conditions are required. These are

described as follows.

pH: Most cell lines grow well at pH 7.4. Although the optimum pH for cell growth varies

relatively little among different cell strains, some normal fibroblast lines perform best at pH 7.4

to pH 7.7, and transformed cells may do better at pH 7.0 to pH 7.4.

Buffering: Culture media must be buffered under two sets of conditions: a) Open dishes, where

the evolution of CO2 causes the pH to rise b) Overproduction of CO2 and lactic acid in

transformed cell lines at high cell concentrations, when the pH will fall.

Temperature: The temperature recommended for most human and warm-blooded animal cell

lines is 37°C, closely to body heat, but generally set a little lower for safety, because overheating

may become major problem than under heating.

Media: Although many cell lines are still propagated in medium supplemented with serum, in

many instances cultures may now be propagated in serum-free media. Media that have been

produced commercially will have been tested for their capability of sustaining the growth of one

or more cell lines. However under certain circumstance we can use our own media.

Growth curve: A growth curve gives three parameters of measurement: (1) the lag phase before

cell proliferation is initiated after subculture, indicating whether the cells are having to adapt to

different conditions; (2) the doubling time in the middle of the exponential growth phase,

indicating the growth promoting capacity of the medium; and (3) the maximum cell

concentration attainable indicating whether there are limiting concentrations of certain nutrients.

In cell lines whose growth is not sensitive to density (e.g., continuous cell lines), the terminal cell

density indicates the total yield possible and usually reflects the total amino acid or glucose

concentration.

Transfected cell lines

The generation of stably-transfected cell lines is essential for a wide range of applications including :

• Cell line can be used for gene function studies • Drug discovery assays or the production of recombinant proteins can be carried out by cell lines. • In contrast to transient expression, stable expression of cell line allows long term, as well as defined and reproducible expression of the gene of interest.

39

In drug discovery, stably transfected cell lines expresses a target of interest, such as a G-protein

coupled receptor (GPCR) or a reporter gene, form the basis for most cell-based compound

screening campaigns. In establishing new assays for high throughput screening, creation of the

appropriate cell line is a bottleneck. Typically, a stable cell line is created by transfection with a

plasmid encoding the target of interest or reporter gene construct, and an additional gene which

allows for chemical selection of successfully transfected cells (usually an antibiotic resistance

gene). Through a lengthy selection process and subsequent limiting dilution to obtain clones, the

desired stable cell line is generated. This process takes approximately 2-3 months, usually

yielding 5-10 usable clones and allowing little control over the end result throughout the process.

Transfection Method

Stable expression can be influenced by the transfection method used. The choice of transfection

method determines which cell type can be targeted for stable integration. While biochemical

transfection reagents can be used to transfer DNA into standard cell lines, efficient delivery of

DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with

viral methods or Nucleofection. Unfortunately, viral methods suffer from several limitations,

such as time consuming production of vectors and safety concerns.

Experimental outlines for the Generation of cell lines

Procedure

Outline

Important Information

Design experiment and

choose cell type,

expression vector and

transaction method

Make sure that transfection method

and expression vector are suitable

for the cell type

40

Determine the appropriate

cell number per plate

(only for limiting dilution)

and G418 concentration

Cells differ in their susceptibility to

G418. The activated concentration

of stock G418 can vary from batch

to batch

Transfect expression

vector into cells

Amount of expression vector per

expression is dependent on

transfection method and cell type.

Plant transfect cells and

cultivate cells into

medium without G418

Do not add G418 to culture medium

immediately after transfection as

this may drastically increase

mortallity

Dilute cell into culture

plate and start selection

24-48 hour post

transfection.

Feed every 2-3 days (for

batch culture) or 10 days

(for limiting dilution)

Choose culture condition [batch

culture limiting dilution] depending

upon the experimental design

Refresh selection medium is

important to avoid false positive

cells.

Analyze stably transfected

cell

Make sure the chosen cell is

suitable for your application.

CHARACTERIZATION OF CELL LINES

41

Characterization of a cell line is vital for determining its functionality and in proving its

authenticity as pure cell line. Special attention must be paid to the possibility that the cell line

has become cross-contaminated with an existing continuous cell line or misidentified because of

mislabeling or confusion in handling DNA profiling. This has now become the major standard

procedure for cell line identification, and a standard procedure with universal application.

The various important factors for cell line characterization are:

(1) It leads to authentication or confirmation that the cell line is not cross-contaminated or

misidentified (2) It is confirmation of the species of origin (3) It is used for correlation with the

tissue of origin, which comprises the following characteristics: a) Identification of the lineage to

which the cell belongs b) Position of the cells within that lineage (i.e., the stem, precursor, or

differentiated status) (4) For determination whether the cell line is transformed or not: a)

Whether the cell line is finite or continuous? b) Whether the cell line expresses properties

associated with malignancy? (5) It indicates whether the cell line is prone to genetic instability

and phenotypic variation (6) Identification of specific cell lines within a group from the same

origin, selected cell strains, or hybrid cell lines, all of which require demonstration of features

unique to that cell line or cell strain

Decisive factors for characterization of cell lines and corresponding methods

Decisive factor Method

DNA profile PCR of microsatellite repeats

Karyotype Chromosome spread with banding

Isoenzyme analysis Agar gel electrophoresis

Genome analysis Microarray

Gene expression analysis Microarray

Proteomics Microarray

Cell surface antigen Immunohistochemistry

42

Parameters of Characterization

The nature of the technique used for characterization depends on the type of work being carried

out. Some of the parameters are: 1. In case molecular technology, DNA profiling or analysis of

gene expression are most useful. 2. A cytology laboratory may prefer to use chromosome

analysis coupled with FISH (fluorescence in situ hybridization) and chromosome painting.

Chromosomal analysis also known as karyotyping, is one of the best traditional methods for

distinguishing among species. Chromosome banding patterns can be used to distinguish

individual chromosomes. Chromosome painting, explicitly using combinations of specific

molecular probes that hybridize to individual chromosomes, adds further resolution and

specificity to this technique. These probes identify individual chromosome pairs and are species

specific. Chromosome painting is a good method for distinguishing between human and mouse

chromosomes in potential cross-contaminations. 3. A laboratory with immunological capability

may prefer to use MHC (Major Histo compatibility complex) analysis (e.g., HLA typing)

coupled with lineage specific markers. 4. Lineage or Tissue markers: The progression of cells

down a particular differentiation pathway towards a specific differentiated cell type and can be

considered as a lineage, and as cells progress down this path they acquire lineage markers

specific to the lineage and distinct from markers expressed by the stem cells. These markers

often reflect the embryological origin of the cells from a particular germ layer.

Lineage markers are helpful in establishing the relationship of a particular cell line to its tissue

of origin. There are some lineage markers which are described as follows:

a) Cell surface antigen: These markers are particularly useful in sorting hematopoietic

cells and have also been effective in discriminating epithelium from mesenchymally

derived stroma with antibodies such as anti- and anti-HMFG 1 and, distinguishing among

epithelial lineages, and identifying neuroectodermally derived cells (e.g., with anti-

A2B5).

43

b) Intermediate filament proteins: These are among the most widely used lineage or

tissue markers. Glial fibrillary acidic protein (GFAP) for astrocytes and desmin for

muscle are the most specific, whereas cytokeratin marks epithelial cells and mesothelium.

c) Differentiated products and functions: Haemoglobin for erythroid cells, myosin or

tropomyosin for muscle, melanin for melanocytes, and serum albumin for hepatocytes are

examples of specific cell type markers, but like all differentiation markers, they depend

on the complete expression of the differentiated phenotype. Transport of inorganic ions,

and the resultant transfer of water, is characteristic of absorptive and secretary epithelia.

Polarized transport can also be demonstrated in epithelial and endothelial cells using

Boyden chambers or filter well inserts. Other tissue-specific functions that can be

expressed in vitro include muscle contraction and depolarization of nerve cell membrane.

d) Enzymes: Three parameters are available in enzymatic characterization: The

constitutive level (in the absence of inducers or repressors) ; The induced or adaptive

level (the response to inducers and repressors) and Isoenzyme polymorphisms

Enzymatic markers used for cell line

Enzyme Cell types Inducer Repressor

Alkaline ephosphatase Type II pneumocyte

(in lung alveolus)

Dexamethasone,

Oncostain, IL-6

TGF-β

Alkaline Phosphatase Enterocytes Dexamethanose, NaBt

collagen, Matrigel

Angiotensin-

converting enzyme

Endothelium Collagen, Matrigel

Creatine Kinase BB Neurons, neuroendocrine

cells, SCLC

Creatine Kinase MM Muscle cells IGF-II FGF-1,2,7

DOPA- decarboxylase Neuron, SCLC

Glutamyl synthetase Astroglia (brain) Glutamine

44

Neuron specific

enolase

Neuron, neuroendocrine

cell

Hydrocortisone

Non-specific esterase Macrophage PMA, Vitamin D3

e) Regulation: The level of expression of many differentiated products is under the regulatory

control of environmental influences, such as nutrients, hormones, the matrix, and adjacent cell.

Hence the measurement of specific lineage markers may require preincubation of the cells in, for

example, a hormone such as hydrocortisone, specific growth factors, or growth of the cells on

extracellular matrix of the correct type.

f) Lineage fidelity: Lineage markers are more properly regarded as tissue or cell type markers,

as they are often more characteristic of the function of the cell than its embryonic origin.

5. Unique Markers: Unique markers include specific chromosomal aberrations (e.g., deletions,

translocations, polysomy), major histocompatibility (MHC) group antigens (e.g., HLA in

humans), which are highly polymorphic, and DNA fingerprinting or SLTR DNA profiling.

Enzymic deficiencies, such as thymidine kinase deficiency (TK−) and drug resistance such as

vinblastine resistance (usually coupled to the expression of the P-glycoprotein by one of the mdr

genes that code for the efflux protein) are not truly unique, but they may be used to distinguish

among cell lines from the same tissues but different donors.

6. Transformation: The transformation status forms a major element in cell line

characterization and is dealt with separately.

a) Cell Morphology: Observation of morphology is the simplest and most direct technique used

to identify cells. Most of these are related to the plasticity of cellular morphology in

response to different culture conditions. For example, epithelial cells growing in the center

of a confluent sheet are usually regular, polygonal, and with a clearly defined birefringent

edge, whereas the same cells growing at the edge of a patch may be more irregular and

distended and, if transformed, may break away from the patch and become fibroblast-like in

shape.

45

b) Microscopy: The inverted microscope is one of the most important tools in the tissue culture

laboratory, but it is often used incorrectly. As the thickness of the closed culture vessel makes

observation difficult from above, because of the long working distance, the culture vessel is

placed on the stage, illuminated from above, and observed from below. As the thickness of the

wall of the culture vessel still limits the working distance, the maximum objective magnification

is usually limited to 40X. The use of phase-contrast optics, where an annular light path is masked

by a corresponding dark ring in the objective and only diffracted light is visible, enables

unstained cells to be viewed with higher contrast than is available by normal illumination.

Because this means that the intensity of the light is increased, an infrared filter should be

incorporated for prolonged observation of cells. It is useful to keep a set of photographs at

different cell densities for each cell line, prepared shortly after acquisition and at intervals

thereafter, as a record in case a morphological change is subsequently suspected. Photographs of

cell lines in regular use should be displayed above the inverted microscope. Photographic records

can be supplemented with photographs of stained preparations and digital output from DNA

profiling and stored with the cell line record in a database or stored separately and linked to the

cell line database.

c) Staining: A polychromatic blood stain, such as Giemsa, provides a convenient method of

preparing a stained culture. Giemsa stain is usually combined with May–Grunwald stain when

staining blood, but not when staining cultured cells. Alone, it stains the nucleus pink or magenta,

the nucleoli dark blue, and the cytoplasm pale gray-blue. It stains cells fixed in alcohol or

formaldehyde but will not work correctly unless the preparation is completely anhydrous.

Chromosome Content: Chromosome content or karyotype is one of the most characteristic and

best-defined criteria for identifying cell lines and relating them to the species and sex from which

they were derived. Chromosome analysis can also distinguish between normal and transformed

cells because the chromosome number is more stable in normal cells (except in mice, where the

chromosome complement of normal cells can change quite rapidly after explantation into

culture).

46

Chromosome Banding: This group of techniques was devised to enable individual chromosome

pairs to be identified when there is little morphological difference between them. For Giemsa

banding, the chromosomal proteins are partially digested by crude trypsin, producing a banded

appearance on subsequent staining. Trypsinization is not required for quinacrine banding. The

banding pattern is characteristic for each chromosome pair. Other methods for banding are:

a) Using trypsin and EDTA rather than trypsin alone

b) Q-banding, which stains the cells in 5% (w/v) quinacrine dihydrochloride in 45%

acetic acid, followed by rinsing Giemsa banding the slide, and mounting it in deionized

water at pH 4.5

c) C-banding, which emphasizes the centromeric regions

Techniques have been developed for discriminating between human and mouse chromosomes,

principally to aid the karyotypic analysis of human-mouse hybrids. These methods include

fluorescent staining with Hoechst 33258, which causes mouse centromeres to fluoresce more

brightly than human centromeres.

Chromosome painting: Chromosome paints are available commercially from a number of

sources. The hybridization and detection protocols vary with each commercial source, but a

general scheme is available. Karyotypic analysis is carried out classically by chromosome

banding, using dyes that differentially stain the chromosomes. Thus each chromosome is

identified by its banding pattern. However, traditional banding techniques cannot characterize

many complex chromosomal aberrations. New karyotyping methods based on chromosome

painting techniques—namely spectral karyotyping (SKY) and multicolour fluorescence in situ

hybridization (M-FISH)—have been developed. These techniques allow the simultaneous

visualization of all 23 human chromosomes in different colours.

Chromosome Analysis: The following are methods by which the chromosome complement may

be analyzed: (1) Chromosome count: Count the chromosome number per spread for between 50

and 100 spreads. (The chromosomes need not be banded.) (2) Karyotype: Digitally photograph

about 10 or 20 good spreads of banded chromosomes. Image analysis can be used to sort

chromosome images automatically to generate karyotypes. Chromosome counting and 47

karyotyping allow species identification of the cells and, when banding is used, distinguish

individual cell line variations and marker chromosomes. However, karyotyping is time-

consuming, and chromosome counting with a quick check on gross chromosome morphology

may be sufficient to confirm or exclude a suspected cross-contamination.

DNA Analysis: DNA content can be measured by propidium iodide fluorescence with a CCD

camera or flow cytometry, although the generation of the necessary single-cell suspension will,

of course, destroy the topography of the specimen. DNA can be estimated in homogenates with

Hoechst 33258 and other DNA fluorochromes such as DAPI, propidium iodide, or Pico Green

(Molecular Probes). Analysis of DNA content is particularly useful in the characterization of

transformed cells that are often aneuploid and heteroploid.

DNA Hybridization: Hybridization of specific molecular probes to unique DNA sequences

(Southern blotting) can provide information about species specific regions, amplified regions of

the DNA, or altered base sequences that are characteristic to that cell line. Thus strain-specific

gene amplifications, such as amplification of the dihydrofolate reductase (DHFR) gene, may be

detected in cell lines selected for resistance to methotrexate; amplification of the MDR gene in

vinblastine-resistant cells overexpression of a specific oncogene, or oncogenes in transformed

cell lines or deletion, or loss, of heterozygosity in suppressor genes. Although DNA aberrations

can be detected in restriction digests of extracts of whole DNA, this is limited by the amount of

DNA required. It is more common to use the polymerase chain reaction (PCR) with a primer

specific to the sequence of interest, enabling detection in relatively small numbers of cells.

Alternatively, specific probes can be used to detect specific DNA sequences by in situ 48

hybridization having the advantage of displaying topographical differences and heterogeneity

within a cell population.

DNA fingerprinting: DNA fingerprints appear to be quite stable in culture, and cell lines from

the same origin, but maintained separately in different laboratories for many years, still retain the

same or very similar DNA fingerprints. DNA fingerprinting is a very powerful tool in

determining the origin of a cell line, if the original cell line, or DNA from it or from the donor

individual, has been retained. This emphasizes the need to retain a blood, tissue, or DNA sample

when tissue is isolated for primary culture. Furthermore, if a cross-contamination or

misidentification is suspected, this can be investigated by fingerprinting the cells and all potential

contaminant.

Antigenic Markers: Immunostaining and ELISA assays are among the most useful techniques

available for cell line characterization facilitated by the abundance of antibodies and kits which

is commercially available. Antibody is essential to be certain of its specificity by using

appropriate control material. This is true for monoclonal antibodies and polyclonal antisera alike;

a monoclonal antibody is highly specific for a particular epitope.

Immunostaining: Antibody localization is determined by fluorescence, wherein the antibody is

conjugated to a fluorochrome, such as fluorescein or rhodamine, or by the deposition of a

precipitated product from the activity of horseradish peroxidase or alkaline phosphatase

conjugated to the antibody. Various methods have been used to enhance the sensitivity of

detection of these methods, particularly the peroxidase linked methods. In the peroxidase–anti-

peroxidase (PAP) technique, a further amplifying tier is added by reaction with peroxidase

conjugated to anti-peroxidase antibody from the same species as the primary antibody. Even

greater sensitivity has been obtained by using a biotin-conjugated second antibody with

streptavidin conjugated to peroxidase or alkaline phosphatase or gold-conjugated second

antibody with subsequent silver intensification.

Differentiation: Many of the characteristics described under antigenic markers or enzyme

activities may also be regarded as markers of differentiation, and as such they can help to

49

correlate cell lines with their tissue of origin as well as define their phenotypic status. Although

sometimes constitutively expressed (e.g., melanin in B16 melanoma or Factor VIII in endothelial

cells), expression of differentiated lineage markers may need to be induced before detection is

possible.

Interesting facts

• EDTA, a chelator of divalent cations, is added to trypsin solutions to enhance activity.

• The calcium and magnesium in the extracellular matrix, which aids in cell-cell adhesion, also

obliterates the peptide bonds that trypsin acts on.

• The EDTA is added to remove the calcium and magnesium from the cell surface which allows

trypsin to hydrolyze specific peptide bonds. This activity can be arrested by adding a serum

media mixture or a trypsin inhibitor (from soybean, for example) in serum-free systems.

CONTAMINATION IN CELL CULTURE

Contamination is the presence of a minor and unwanted constituent (contaminant) in material,

physical body, natural environment, at a workplace, etc. In biological sciences accidental

introduction of foreign material (contamination) can seriously distort the results of experiments

where small samples are used. In cases where the contaminant is a living microorganism, it can

often multiply and take over the experiment, especially cultures, and render them useless.

Source of Contamination: Maintaining asepsis is one of the most difficult challenges to work

with living cells. There are several potential routes to contamination including failure in the

sterilization procedures for solutions, glassware and pipettes, turbulence and particulates (dust

and spores) in the air in the room, poorly maintained incubators and refrigerators, faulty laminar-

flow hoods, the importation of contaminated cell lines or biopsies, and lapses in sterile

technique.

Monitoring for Contamination

Potential sources of contamination are enumerated along with the precautions that should be

taken to avoid them. Even in the best laboratories contaminations do arise, so the following

procedure is generally recommended: (1) Contamination by eye and with a microscope at each

50

handling of a culture should be checked properly. (2) If it is suspected that a culture is

contaminated and the fact cannot be confirmed in situ, the hood or bench should be kept clear

except suspected culture and Pasteur pipettes. Because of the potential risk to other cultures, this

should be better to do after all your other culture work is finished. A sample should be removed

from the culture and placed on a microscope slide. Slide should be checked with a microscope,

preferably by phase contrast. If it is confirmed that the culture is contaminated, pipettes should

be discarded, hood or bench should be swabbed with 70% alcohol containing a phenolic

disinfectant. The hood or bench should not be used until the next day. (3) Nature of the

contamination should be recorded. (4) If the contamination is new and is not widespread, the

culture, the medium bottle used to feed it, and any other reagent (e.g., trypsin) that has been used

in conjunction with the culture should be discarded properly into disinfectant, preferably in a

fume hood and outside the tissue culture area. (5) If the contamination is new and widespread all

media, stock solutions, trypsin, and so forth in current use should be discarded immediately.

(6) If the same kind of contamination has occurred before check stock solutions for

contamination (a) by incubation alone or in nutrient broth (b) by plating out the solution on

nutrient agar. If (a) and (b) prove negative, but contamination is still suspected, 100 mL of

solution should be incubated, filtered it through a 0.2-μm filter, and plated out filter on nutrient

agar with an uninoculated control. (7) If the contamination is widespread, multispecific, and

repeated then one should check (a) the laboratory’s sterilization procedures (e.g., the

temperatures of ovens and autoclaves, particularly in the center of the load, the duration of the

sterilization cycle), (b) the packaging and storage practices, (e.g., unsealed glassware should be

resterilized every 24 h), and (c) the integrity of the aseptic room and laminar-flow hood filters.

(8) One should not be attempting to decontaminate cultures unless they are irreplaceable.

Visible Microbial Contamination: Characteristic features of microbial contamination are as

follows: (1) A sudden change in pH, usually a decrease with most bacterial infections, very little

change with yeast until the contamination is heavy, and sometimes an increase in pH with fungal

contamination. (2) Cloudiness in the medium, sometimes with a slight film or scum on the

surface or spots on the growth surface that dissipate when the flask is moved (3) Under a 10X

objective, spaces between cells will appear granular and may shimmer with bacterial

51

contamination. Yeasts appear as separate round or ovoid particles that may bud off smaller

particles. Fungi produce thin filamentous mycelia and, sometimes, denser clumps of spores

which may be blue or green. With toxic infection, some deterioration of the cells will be

apparent. (4) Under a 100X objective, it may be possible to resolve individual bacteria and

distinguish between rods and cocci. At this magnification, the shimmering that is visible in some

infections will be seen to be caused by mobility of bacteria. Some bacteria form clumps or

associate with the cultured cells. 5) With a slide preparation, the morphology of the bacteria can

be resolved with a 100× objective, but this is not usually necessary. Microbial infection may be

confused with precipitates of media constituents (particularly protein) or with cell debris, but can

be distinguished by their regular morphology. Precipitates may be crystalline or globular and

irregular and are not usually as uniform in size. Clumps of bacteria may be confused with

precipitated protein, but, particularly if shaken, many single or strings of bacteria will be seen. If

you are in doubt, plate out a sample of medium on nutrient agar.

Ways of Disposal of Contaminated Cultures

The following procedures are generally used for disposal of contaminated culture:

• It is important to ensure that all contaminated material is disposed of correctly. Culture vessels

should be removed from the culture area, unopened if possible, and autoclaved.

• Open items, such as Petri dishes with the lids in place, and pipettes or other items that have

come in contact with a contaminated culture should be immersed in hypochlorite disinfectant

(Petri dishes can be opened while submerged).

• If only one of a series of similar cultures is contaminated, it is necessary to discard the bottle of

medium that was used with it, but if the contamination is widespread, then all medium as well as

all other stock solutions and reagents, used with these cells, should be discarded into

hypochlorite.

Eradication of Contamination

Eradication of Contamination in cell culture is a challenging job during safe culturing. There are

different way for different organism, some example are given as follows:

52

Bacteria, Fungi, and Yeasts: The most reliable method of eliminating a microbial

contamination is to discard the culture and the medium and reagents used with it as treating a

culture may be unsuccessful or lead to the development of an antibiotic-resistant microorganism.

This procedure is optimal; however, the majority of cell lines do not form spheroids in this

manner. Alternatively, aggregates may be formed from cell suspensions in stationary flasks,

previously base-coated with agar. Aggregates may be left in the original flasks or transferred

individually (by pipette) to multi well plates, where continued growth over weeks will yield

spheroids of maximum size, about 1000 μm. Decontamination should be attempted only in

extreme situations, under quarantine, and with expert supervision. If unsuccessful, the culture

and associated reagents should be autoclaved as soon as failure becomes obvious. The general

rule remains that contaminated cultures are discarded and that decontamination is not attempted

unless it is absolutely vital to retain the cell strain. In any event, complete decontamination is

difficult to achieve, particularly with yeast, and attempts to do so may produce hardier,

antibiotic-resistant strains.

Eradication of Viral Contamination: There are no reliable methods for eliminating viruses

from a culture at present; disposal or tolerances are the only options.

Cross Contamination: During the development of tissue culture, a number of cell strains have

evolved with very short doubling times and high plating efficiencies. Although these properties

make such cell lines valuable experimental material, they also make them potentially hazardous

for cross-infecting other cell lines.

CELL LINE DIFFERENTIATION

Differentiation in cell line is the process which leads to the expression of phenotypic properties

and characteristic of the functionally mature cell in vivo. It is the phenomenon in which less

specialized cell develops or matures to become more distinct in form and function. This may be

irreversible when there is cessation of DNA synthesis in the erythroblast nucleus, neuron, or

mature keratinocyte. The process may be reversible, when the dedifferentiation of mature

hepatocytes into precursors happens during liver regeneration. Some of the properties of the

differentiated cells are adaptive, such as albumin synthesis in differentiated hepatocytes, which is 53

often lost in culture but can be reinduced. Differentiation is the combination of constitutive

(stably expressed without induction) and adaptive (subject to positive and negative regulation of

expression) properties found in the mature cell.

Terminal differentiation: Terminal differentiation is another type of differentiation in which a

cell has progressed down a particular lineage to a point at which the mature phenotype is fully

expressed and beyond which the cell cannot progress. This stage may be reversible in some cells,

such as fibrocytes, that can revert to a less differentiated phenotype, or even a stem cell, and

resume proliferation. It may be irreversible in cells like erythrocytes, neurons, skeletal muscle, or

keratinized squamous cells. The growth of cells on floating collagen has been used to improve

the survival of epithelial cells and promote terminal differentiation

Pluripotent cell: A cell that is able to differentiate into all cell types of the adult organism is

known as pluripotent. Such cells are called embryonic stem cells in animals and meristematic

cells in higher plants.

Totipotent cell: A cell that is able to differentiate into all cell types, including the placental

tissue, is known as totipotent. In mammals, only the zygote and subsequent blastomeres are

totipotent.

Control of Differentiation

Differentiation is controlled by various parameters. There are five major parameters that control

differentiation.

1. Cell- cell Interaction: Cell-cell interactions are of two types, Homotypic and heterotypic.

They can be detailed as follows: Homotypic cell-cell interaction: Homologous cell interaction

occurs at high cell density. It may involve gap junctional communication in which metabolites,

second messengers such as cyclic AMP, diacylglycerol (DAG), Ca2+, or electrical charge may

be involved. This interaction harmonizes the expression of differentiation within a population of

similar cells, rather than initiating its expression. Homotypic cell–cell adhesion molecules,

(CAMs ) or cadherins, which are calcium-dependent, provide another mechanism by which

contacting cells may interact. These adhesion molecules promote interaction primarily between

54

like cells via identical, reciprocally acting,extracellular domains, and they appear to have signal

transduction potential via phosphorylation of the intracellular domains. Heterotypic cell-cell

interaction: Heterologous cell interaction such as between mesodermally and endodermally or

ectodermally derived cells is responsible for initiating and promoting differentiation. During and

immediately after gastrulation in the embryo, and later during organogenesis, mutual interaction

between cells originating in different germ layers promotes differentiation.

2. Cell–Matrix Interactions: Animal cells are not surrounded by cell walls. Animal cells are

surrounded by a plasma membrane which is complex mixture of glycoproteins and proteoglycans

surface that is highly specific for each tissue, and even for parts of a tissue. Recreation of this

complex microenvironment, involving cell–cell and cell–matrix interactions has been shown to

be vital in the expression of the mature keratinocyte phenotype in the reconstruction of skin

equivalents and the maintenance of the stem cell compartment. Collagen has been found to be

essential for the functional expression of many epithelial cells and for endothelium to mature into

capillaries. Small polypeptides containing this sequence effectively block matrix-induced

differentiation, implying that the intact matrix molecule is required. Defined matrices are

required; although fibronectin, laminin, collagen, and numerous other matrix constituents are

commercially available, the specificity probably lies largely in the proteoglycan moiety, within

which there is the potential for wide variability, particularly in the number, type, and distribution

of the sulfated glycosaminoglycan, such as heparan sulfate. The extracellular matrix may also

play important role in the modulation of growth factor activity. One type of extracellular matrix

is exemplified by the thin, sheet-like basal laminae, previously called basement membranes,

upon which layers of epithelial cells rest. In addition to supporting sheets of epithelial cells, basal

laminae surround muscle cells, adipose cells, and peripheral nerves. Extracellular matrix is most

abundant in connective tissues.

3. Polarity and cell shape: Various studies shows that growth of the cells on collagen gel and

the subsequent release of the gel from the bottom of the dish with a spatula or bent Pasteur

pipette are required for full maturation of cell. This process allows shrinkage of the gel and

modification in the shape of the cell from flattened to cuboidal or even columnar shape.

Following the shape change and also possibly due to contact to medium through the gel, the cells

55

develop polarity which is visible by electron microscopy. When the nucleus becomes

asymmetrically distributed nearer to the bottom of the cell an active Golgi complex is formed

and secretion is observed towards the apical surface.

4. Oxygen Tension: Gas exchange enhances when positioning the cells at the air–liquid

interface, particularly facilitating oxygen uptake without raising the partial pressure and risking

free radical toxicity. It is also possible that the thin film above mimics the physicochemical

conditions in vivo (surface tension, lack of nutrients) as well as oxygenation.

Equilibrium between cell proliferation and differentiation

Cells in culture can be thought to be in a state of equilibrium between cell proliferation and

differentiation. Normal culture conditions i.e. in low cell density, mitogens in the medium will

favor cell proliferation, whereas high cell density and addition of differentiation factors will

induce differentiation. The position of the equilibrium will depend on culture conditions.

Dedifferentiation of the culture may be due to the effect of growth factors or cytokines inducing

a more proliferative phenotype, reprogramming of gene expression, or overgrowth of a precursor

cell type. The relationship between differentiation and cell proliferation may become relaxed but

it is not lost. For example, B16 melanoma cells still produce more pigment at a high cell density

and at a low rate of cell proliferation than at a low cell density and a high rate of cell

proliferation

Differentiation and Proliferation56

As differentiation progresses, cell division is reduced and ultimately ceases. In most cell systems,

cell proliferation is incompatible with the expression of differentiated properties. Tumor cells

can sometimes break this restriction, and in melanoma, for example, melanin continues to be

synthesized while the cells are proliferating. Even in these situation, synthesis of the

differentiated product increases when division stops.

Differentiation from stem cells

It may be useful to think of a cell culture as being an equilibrium between stem cells,

undifferentiated precursor cells, and mature differentiated cells and to suppose that the

equilibrium may shift according to the environmental conditions. Diamond nanoparticles have

also been used to modify the substrate for the proliferation and differentiation of neural stem

cells and the configuration of the growth surface can also be altered by photoetching. Treatment

of the substrate with denatured collagen improves the attachment of many types of cells, such as

epithelial cells, and the nondenatured gel may be necessary for the expression of differentiated

functions

THREE DIMENSIONAL CELL CULTURE

Three-Dimensional (3-D) cell cultures have been widely used in biomedical research since the early decades of this century. The potential of 3-D cell cultures is currently being exploited in various areas of biomedical research. One reason for the recent progress in research on multi cell systems may be the increasing interaction between researchers working in different fields of biomedical science and using similar 3-D culture techniques. Such a research effort mirrors the common need for improved and more refined in vitro models as a link between cell-free systems or single cells and organs or whole organisms in vivo. One major advantage of 3-D cell cultures is their well-defined geometry, which makes it possible to directly relate structure to function and which enables theoretical analyses such as diffusion fields. Subsequently, the most promising data on these cultures may be obtained by using techniques allowing for spatial resolution. Combining such approaches with molecular analysis has clearly confirmed that, in comparison with conventional cultures, cells in 3-D cultures more closely resemble the in vivo situation with regard to cell shape and cellular environment.

These parameters (shape and environment) can determine gene expression and the biological behaviour of the cells. In contrast to 2D monolayer, 3D cell culture models are modular, adaptable biomedical systems consisting range in complexity from a single cell type

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(monotypic), representing the minimum unit of the differentiated tissue in vivo to complex co-culture models that recapitulate both the 3D architecture and the multicellular complexity of the parental tissue. There will always be a number of questions that can only be answered by investigations using single cells or cell-free systems. At the same time, 3-D cultures cannot completely replace the testing of biological mechanisms for their relevance in vivo, e.g., in knockout animals

Effect of Cell Density: Cell–cell interaction is manifested at the simplest level when a cell culture reaches confluence and the constituent cells begin to interact more strongly with each other because of contact mediated signaling, formation of junctional complexes and increased potential for exchange of homocrine factors. The first noticeable effect is cessation of cell motility (contact inhibition) and withdrawal from cell cycle (density limitation of cell proliferation) in normal cells and reduced cell proliferation and increased apoptosis in transformed cells.

Reciprocal Interaction: When different cells interact in their population, they have tendency to show reciprocal effect on their respective phenotypes, and the resultant phenotypic changes lead to new interactions. Cell interaction is therefore not just a single event, but a continuing cascade of events. Similarly exogenous signals do not initiate a single event, as may be the case with homogeneous populations, but initiate a new cascade, as a result of the exogenously modified phenotype of one or both partners.

Choice of Model for Three Dimensional Cultures: There are two major way to approach these goals.

One is to accept the cellular distribution within the tissue, explant it and maintain it as an organ culture.

The second is to purify and propagate individual cell lineages, study them alone under conditions of homologous cell interaction, recombine them, and study their mutual interactions

Types of Three Dimensional Cultures

The three main types of three dimensional cultures are described below:

1. ORGAN CULTUREOrgan culture in which whole organs or representative parts are maintained as small fragments in culture and retain their intrinsic distribution, numerical and spatial, of participating cells. Organ culture seeks to retain the original structural relationship of cells of the same or different types, and hence their interactive function in order to study the effect of exogenous stimuli on further development. Organ culture seeks to retain the original structural relationship of cells of the same or different types and hence their interactive function, in order to study the effect of exogenous stimuli on further development. Gas and Nutrient Exchange: A major deficiency in tissue architecture in organ culture is the absence of a vascular system, limiting the size (by diffusion) and potentially the polarity of the

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cells within the organ culture. When cells are cultured as a solid mass of tissue, gaseous diffusion and the exchange of nutrients and metabolites is from the periphery, and the rate of this diffusion limits the size of the tissue. The dimensions of individual cells cultured in suspension or as a monolayer are such that diffusion is not limiting, but survival of cells in aggregates beyond about 250 μm in diameter (∼5000 cell diameters) starts to become limited by diffusion, and at or above 1.0 mm in diameter (∼2.5 × 105 cell diameters) central necrosis is often apparent. To alleviate this problem, organ cultures are usually placed at the interface between the liquid and gaseous phases, to facilitate gas exchange while retaining access to nutrients. This is achieved by most system by positioning the explant in a filter well insert on a raft or gel exposed to the air, but explants anchored to a solid substrate can also be aerated by rocking the culture, exposing it alternately to a liquid medium and a gas phase or by using a roller bottle or rotating tube rack. Anchorage to a solid substrate can lead to the development of an outgrowth of cells from the explant and resultant alterations in geometry even though this effect can be minimized by using a hydrophobic surface. One of the advantages of culture at the gas–liquid interface is that the explant retains a spherical geometry if the liquid is maintained at the correct level. If the liquid is too deep, gas exchange is impaired whereas if it is too shallow, surface tension will tend to flatten the explants and promote outgrowth. Permeation of oxygen increases by using increasing O2 concentrations up to pure oxygen or by using hyperbaric oxygen. As increasing the O2 tension will not facilitate CO2 release or nutrient metabolite exchange, the benefits of increased oxygen may be overridden by other limiting factors.

Structural Integrity: The maintenance of structural integrity is the main reason for adopting organ culture as an in vitro technique in preference to cell culture. Whereas cell culture utilizes cells dissociated by mechanical or enzymatic techniques or spontaneous migration, organ culture deliberately maintains the cellular associations found in the tissue.

Growth and Differentiation: There is a relationship between growth and differentiation such that differentiated cells no longer proliferate. It is also possible that cessation of growth, regardless of cell density, may contribute to the induction of differentiation, if only by providing a permissive phenotypic state that is receptive to exogenous inducers of differentiation. Because of density limitation of cell proliferation and the physical restrictions imposed by organ culture geometry, most organ cultures do not grow or if they do proliferation is limited to the outer cell layers. Hence the status of the culture is permissive to differentiation and the appropriate cellular interactions and soluble inducers are provided as an ideal environment for differentiation to occur.

Limitations of Organ Culture • Organ cultures depend largely on histological techniques and they do not impart themselves readily into biochemical and molecular analyses. • Biochemical monitoring requires reproducibility between samples, which is less easily achieved in organ culture than in propagated cell lines, because of sampling variation in preparing an organ culture, minor differences in handling and geometry, and variations in the ratios of cell types among cultures.

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• Organ cultures are also more difficult to prepare than replicate cultures from a propagated cell line and do not have the advantage of a characterized reference stock to which they may be related. • Organ culture is essentially a technique for studying the behaviour of integrated tissues rather than isolated cells. • Organ culture has contributed a great deal to our understanding of developmental biology and tissue interactions and that it will continue to do so in the absence of adequate synthetic systems.

Figure 1: Organ Culture Small fragment of tissue on a filter laid on top of a stainless steel grid over the central well of an organ culture dish

2. HISTOTYPIC CULTURE

Histotypic culture is defined as high-density cell culture with the cell density approaching that of the tissue in vivo. Various attempts have been made to regenerate tissue-like architecture from dispersed monolayer cultures. As cells reach a high density, medium nutrients will become limiting. To avoid this, the ratio of medium volume to cell number should remain approximately as it was in low density culture. This can be achieved by seeding cells on a small coverslip in the center of a large non–tissue-culture grade dish or by use of filter well inserts, which give the opportunity for the formation of both high-density polarized cultures and heterotypic combinations of cell types to create organotypic cultures. A high medium-to-cell ratio can also be maintained by perfusion.

Gel and Sponge Techniques: Use of three-dimensional sponges and gels has increased extensively with the development of tissue engineering. Two commonly used gel in this technique.

• Collagen gel: Collagen gel (native collagen, as distinct from denatured collagen coating) provides a matrix for the morphogenesis of primitive epithelial structures. Many different types of cell can be shown to penetrate such matrices and establish a tissue-like histology. • Matrigel: Matrigel is a commercial product derived from the extracellular matrix of the Engelbreth–Holm–Swarm (EHS) mouse sarcoma which has been used for coating plastic

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but can also be used in gel form. It is composed of laminin, collagen, fibronectin, and proteoglycans with a number of bound growth factors, although it can be obtained in a growth factor-depleted form. It has been used as a substrate for epithelial morphogenesis formation of capillaries from endothelial cells and in the study of malignant invasion.

3. ORGANOTYPIC CULTURE

High density three-dimensional culture involving the recombination of different cell lineages may be referred to as organotypic culture, a term that used to distinguish these reconstruction techniques from organ culture where the original cells are not dissociated. The key event that distinguishes these constructs from histotypic culture is the introduction of heterotypic cell interaction including diffusible paracrine effects and signaling implicating the extracellular matrix. The relationship of the cells allows the generation of a structured microenvironment, cell polarity and enhanced differentiation. Creation of organotypic culture by mixing cells randomly and allowing them to interact and sort, as can happen spontaneously particularly with embryonic cells or the construct may be design to keep the interacting cells separate so that their interactions may be studied.

Tissue Equivalents: The advent of filter well technology boosted by its commercial availability, has produced a rapid expansion in the study of organotypic culture methods.

Tissue Engineering: Just as organotypic culture needs cell interaction, constructs for tissue engineering often require similar interactions, as in the interaction between endothelium and smooth muscle in blood vessel reconstruction. In addition to biological interactions, some constructs require physical forces; skeletal muscle needs tensile stress, bone and cartilage needs compressive stress, and vascular endothelium in a blood vessel construct needs pulsatile flow.

ROLE OF MATRIX IN CELL GROWTH

Role of matrix in cell growth: Matrix is an insoluble, dynamic gel in the cytoplasm, believed to be involved in cell shape determination and locomotive mechanism, across a solid substrate. It consists of polymeric microtubules, actin microfilaments and intermediate filaments interacting with a number of other proteins.

Extracellular matrix (ECM): The extracellular matrix (ECM) is a part of three connective tissue layers (endomysium, perimysium, and epimysium) surrounding muscle fibres. Extracellular matrix is composed of proteins including collagens and proteoglycans.

Component of extra cellular matrixECM is comprised variously of collagen, laminin, fibronectin, hyaluronan and proteoglycans such as beta glycan, decorin, perlecan, and syndecan-1, some of which bind to growth factors or cytokines. • Proteoglycans in extracellular matrix form a cross-linked meshwork with fibrous proteins

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• Some proteins bind multiple other proteins and glycosaminoglycans (fibronectin).

• Integrin is a family of proteins that mediate signalling between cell interior and extracellular matrix

• Mass of interactions between cells and matrix not only anchors cells to matrix but also provides paths that direct migration of cells in developing tissue and (through integrin) conveys information in b

Cell matrix

ECMs are composed of proteins such as collagen and elastin that serve as scaffolds for cells as well as networks of various adhesion ligands and growth factors, which promote cell signalling. ECM is complex in both structure and composition.

Role of matrix: There are some important roles that matrix play in biological system which is described as follows. • Matrixes are generally used for providing support • It involve in segregating tissues from one another • It takes part in regulation of intercellular communication • Extracellular Matrix cells have been found to cause regrowth and healing of tissue. • Some time it acts as fibrosis • The use of ECM constituents can be highly beneficial in enhancing cell survival, proliferation, or differentiation, but unless recombinant molecules are used • In human foetuses, the extracellular matrix works with stem cells to grow and regrow all parts of the human body and foetus can regrow anything that gets damaged in the womb • In case of injury repair and tissue engineering, the extracellular matrix serves two main functions a) It prevents the immune system by triggering from the injury and responding with inflammation and scar tissue b) It facilitates the surrounding cells to repair the tissue instead of forming scar tissue

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Molecular components: Components of the ECM are produced intracellularly by resident cells and secreted into the ECM through exocytosis. Once secreted, they then aggregate with the existing matrix. The ECM is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs).

Proteoglycans: Since we know that, GAGs are carbohydrate polymers and are usually attached to extracellular matrix proteins to form proteoglycans (exception-hyaluronic acid). Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+) which attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help to trap and store growth factors within the ECM. There are the different types of proteoglycan found within the extracellular matrix.

1. Heparin sulphate: Heparin sulphate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (PG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. HS binds to a variety of protein ligands and involve in regulation of a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation and tumour metastasis. In the extracellular matrix, particularly basement membranes, the multi-domain proteins perlecan, agrin and collagen XVIII are the main proteins to which heparin sulphate is attached. 2. Chondroitin sulphate: Chondroitin sulfates help to provide the tensile strength of cartilage, tendons, ligaments and walls of the aorta. They have also been known to affect neuroplasticity. 3. Keratan sulphate: Keratan sulfates have variable sulfate content and unlike many other GAGs, do not contain uronic acid. They are present in the cornea, cartilage, bones and the horns of animals.

Non-proteoglycan polysaccharide: There are various non-proteoglycan polysaccharides. 1. Hyaluronic acid: Hyaluronic acid (or hyaluronan at physiological pH) is a polysaccharide containing alternating residues of D-glucuronic acid and N-acetyl glucosamine. Unlike other glycosaminoglycan (GAGs) it is not found as a proteoglycan. Like cellulose and chitin, it is synthesized at the plasma membrane by a transmembrane hyaluronan synthase. Hyaluronan is the only GAG that occurs as a single long polysaccharide chain. Hyaluronate is also an essential component of the extracellular matrix of cartilage and tendons, to which it contributes tensile strength and elasticity as a result of its strong interactions with other components of the matrix. A number of proteoglycans interact with hyaluronan to form large complexes in the extracellular matrix. A well-characterized example is aggrecan, the major proteoglycan of cartilage. Hyaluronic acid acts as an environmental sign that regulates cell behaviour during embryonic development, healing processes, inflammation and tumour development. It interacts with a specific trans-membrane receptor, CD44.

2. Matrix Structural Proteins: Extracellular matrices are composed of tough fibrous proteins embedded in a gel-like polysaccharide ground substance-a design basically similar to that of plant cell walls.

Collagen: In ECM of most animals, collagens are the abundantly found structural protein. In fact, collagen is the most abundant protein in the human body and accounts for 90% of bone

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matrix protein content. Collagens are present in the ECM as fibrillar proteins and give structural support to resident cells. Collagens are a large family of proteins containing at least 27 different members. They are characterized by the formation of triple helices in which three polypeptide chains are wound tightly around one another in a rope-like structure. The different collagen polypeptides can assemble into 42 different trimers. The triple helix domains of the collagens consist of repeats of the amino acid sequence Gly-X-Y.

Elastin: In contrast to collagens, Elastins give elasticity to tissues, allowing them to stretch when needed and then return to their original state. This is useful in blood vessels, the lungs, in skin, and the ligaments. Elastins are synthesized by fibroblasts and smooth muscle cells. Elastins are highly insoluble, and tropoelastins are secreted inside a chaperone molecule, which releases the precursor molecule upon contact with a fiber of mature elastin. Tropoelastins are then deaminated to become incorporated into the elastin strand. Diseases such as cutis laxa and Williams syndrome are associated with deficient or absence of elastin fibers in the ECM.

Matrix adhesion proteins: Matrix adhesion proteins, the final class of extracellular matrix constituents are responsible for linking the components of the matrix to one another and to the surfaces of cells. They interact with collagen and proteoglycans to specify matrix organization and are the major binding sites for integrins

Commercially available matrices

Commercially available matrices such as Matrigel™ (Becton Dickinson) from the Engel breth–Holm–Swarm (EHS) sarcoma, contain laminin, fibronectin, and proteoglycans, with laminin predominating. Other matrix products include Pronectin F (Protein Polymer Technologies), laminin, fibronectin, vitronectin entactin (UBI), heparan sulfate, EHS Natrix (BD Biosciences), ECL (US Biological), and Cell-tak (BD Biosciences). Some of these products are purified, whereas others are a mixture of matrix products that have been poorly characterized and may also contain bound growth factors. If cell adhesion for survival is the main objective, and defined substrates are inadequate, the use of these matrices is acceptable, but if mechanistic studies are being carried out, they can only be an intermediate stage on the road to a completely defined substrate.

APPLICATIONS OF CELL LINES

The animal cell cultures are used for a diverse range of research and development. These areas are: a) production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses. b) Cancer research, which requires the study of uncontrolled cell division in cultures. c) Cell fusion techniques. d) Genetic manipulation, which is easy to carry out in cells or organ cultures. e) Production of monoclonal antibodies requires cell lines in culture. f) Production of pharmaceutical drugs using cell lines. g) Chromosome analysis of cells derived from womb. h) Study of the effects of toxins and pollutants using cell lines. i) Use of artificial skin. j) Study the function of the nerve cells. 

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Somatic Cell Fusion: One of the applications of animal cell culture is the production of hybrid cells by the fusion of different cell types. These hybrid cells are used for a the following purposes: (i) study of the control of gene expression and differentiation,(ii) study of the problem of ‘ malignancy’,(iii) viral application,(iv) gene mapping and (v) production of hybridomas for antibody production. In 1960s, in France for the first time, the hybrid cells were successfully produced from mixed cultures of two different cell lines of mouse. Cells growing in culture are induced by some of the viruses such as ‘Sendai virus’ to fuse and form hybrids. This virus induces two different cells first to form heterokaryons. During mitosis, chromosomes of heterokaryon move towards the two poles and later on fuse to form hybrids. It is important to remove the surface carbohydrates to bring about cell fusion. Some other chemicals like polyethylene glycol also induce somatic cell fusion.

Commercial Proteins: Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. 

Tissue Plasminogen activator

The production of Tissue Plasminogen activator (t-pa)

Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.

Blood Factor VIII

Haemophilia A is a blood disorder which is a sex-linked genetic disease in humans. The patients suffering from Haemophilia A lack factor VIII, which plays an important role in the clotting of blood. This factor VIII is secreted by a gene present on X-chromosome but this gene undergoes mutations in people suffering from Haemophilia. Current therapy for this disease is the transfusion of blood factor VIII into patients. Using rDNA technology, Factor VIII has been produced from mammalian cell culture e.g. Hamster kidney cell.

Erythropoietin (EPO)

The EPO is a glycoprotein consisting of 165 amino acids and is formed in the foetal liver and kidneys of the adults. It causes proliferation and differentiation of progenitor cells into the

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erythrocytes (erythroblasts) in the bone marrow. Erythropoietin is hormone-like in nature and is released by the kidney under hypoxic or anoxic conditions caused by anaemia.

Amgen Inc. holds US patent for preparation of, eErythropoietin, by recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic conditions caused by anaemia. r-HUEPO- recombinant human erythro- protein has been effectively used to treat anemia associated with AIDS, renal failure etc.

Monoclonal Antibodies

Once monoclonal antibodies for a given substance have been produced, they can be used to:

detect the presence and quantity of this substance, for instance in a Western blot test (to

detect a protein on a membrane) or an immunofluorescence test (to detect a substance in

a cell).

They are also very useful in immunohistochemistry which detect antigen in fixed tissue

sections.

Monoclonal antibodies can also be used to purify a substance with techniques called

immunoprecipitation and affinity chromatography.

Mabs as diagnostic tools: (1) Detection or diagnosis of diseases and contaminants in food

and environment for instance in a western blot test or immunofluorescence test (2)

Immunohistochemistry for the detection Ags in fixed tissue sections and (3) purification of

substances with techniques called immunoprecipitation and affinity chromatography

Mabs as therapeutic tools: (1) In the management of rejection of transplantation aimed at

suppressing T-cell activity and (2) In delivery of drugs for example “magic bullet” or

immunotoxin in cancer treatment.

Cell based therapy

The animal cell culture techniques are used in replacing the damaged and dead cells with normal and healthy cells using the stem cell technology. This therapy is called Cell-Based

therapy which involves the use of stem cell technology involving the replacement of damaged

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and dead cells with normal and healthy cells. This is used to treat blood cancer, and other neuro-degenerative diseases etc.

EXAMPLES OF CELL LINES

1. VERO CELLS

Vero cells are lineages of cells used in cell cultures. The 'Vero' lineage was isolated from kidney epithelial cells extracted from an African green monkey (Chlorocebus sp.; formerly called Cercopithecus aethiops, this group of monkeys has been split into several different species). The lineage was developed on 27 March 1962, by Yasumura and Kawakita at the Chiba University inChiba, Japan. The original cell line was named "Vero" after an abbreviation of verda reno, which means "green kidney" in Esperanto, while vero itself means "truth" also in Esperanto.

Characteristics

Continuous: can be replicated through many cycles of division and not become senescent. Aneuploidy: have an abnormal number of chromosomes. The typical chromosome mode of the Vero cell line is 59 rather than 58

Interferon-deficient; unlike normal mammalian cells, they do not secrete interferon alpha or beta when infected by viruses. However, they still have the Interferon-alpha/beta receptor, so they respond normally when interferon from another source is added to the culture. Chromosome 12 of Vero cells has a homozygous ~9-Mb deletion, causing the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitors CDKN2A and CDKN2B in the genome. 

Lineages

Vero (ATCC No. CCL-81): Isolated from C. aethiops kidney on 27 Mar 1962, Vero 76 (ATCC No. CRL-1587): Isolated from Vero in 1968, it grows to a lower saturation density (cells per unit area) than the original Vero. It is useful for detecting and counting hemorrhagic fever viruses by plaque assays. Vero E6, also known as Vero C1008 (ATCC No. CRL-1586): This line is a clone from Vero 76. Vero E6 cells show some contact inhibition, so are suitable for propagating viruses that replicate slowly.

Applications

Vero cells are used for many purposes, including: screening for the toxin of Escherichia coli, first named "Vero toxin" after this cell line, and later called "Shiga-like toxin" due to its similarity to Shiga toxin isolated from Shigella dysenteriae; as host cells for growing virus; for example, to measure replication in the presence or absence of a research pharmaceutical, the testing for the presence of rabies virus, or the growth of viral stocks for research purposes; as host cells for eukaryotic parasites, specially of the trypanosomatids; Vero cells are one of the most common mammalian continuous cell lines used in research. This anchorage-dependent cell

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line has been used extensively in virology studies, but has also been used in many other applications, including:

the propagation and study of intracellular bacteria (e.g., Rickettsia spp.; ) and parasites (e.g., Neospora),

assessment of the effects of chemicals, toxins and other substances on mammalian cells at the molecular level. I

production of both live (rotavirus, smallpox) and inactivated (poliovirus) viral vaccines, and other viruses, including Rabies virus, Reovirus and Japanese encephalitis virus

ExamplePreparation of live, attenuated human influenza virus vaccines : the preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. preliminary studies indicated that an african green monkey kidney cell line (vero) is a suitable system for the primary isolation and cultivation of influenza a viruses . it has now been demonstrated that vero cells are suitable for isolation and productive replication of influenza b viruses and determine the biological and genetic properties of both influenza a and b viruses in vero cells

Propagation of Vero cell culture from frozen stocks

For long term storage, Vero cells are kept either in liquid nitrogen or at -80°C. This protocol describes how to start growing Vero cells obtained from frozen stock. After recovery from frozen stock, Vero cells usually take 2-3 passages to reach their regular growth rate, and this should be taken into account if planning to use the cells for experiments, infections, etc. It is important to note that Vero cells are anchorage-dependent cells and therefore cannot be grown in suspension.

Critical Parameters and Troubleshooting in Vero Cell Culture

Mycoplasma contamination

One of the most important issues when growing any cell culture is contamination. To reduce the risk of contamination, always work with the cells in a sterile, laminar flow hood, make sure all equipment and solutions that come into contact with the cells are sterile, and use proper sterile technique when working in the hood. Many researchers will maintain their cells with a low level of antibiotic added to the medium, most commonly a penicillin/streptomycin mixture. This can also help to reduce extraneous bacterial contamination; however, depending on the application for which the Vero cells will be used, adding antibiotics may not be recommended (for example, if the cells are to be infected with bacteria).

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One of the most common sources of contamination in cell cultures is Mycoplasma. These bacteria are very small (< 1μm), and therefore contamination withMycoplasma may not be visible with the naked eye, making these organisms difficult to detect. SeveralMycoplasma detection kits are commercially available and procedures for detection and treatment of Mycoplasma contamination are also available. Vero cells should be regularly tested (once per month) for the presence of Mycoplasmacontamination. If cells are found to be infected, the recommended course of action is to discard the cells and start new cultures from uninfected frozen stocks. There are also several options for treating Mycoplasma-infected cells (see Uphoff and Drexler 2002), including commercially available kits.

Culture media

While DMEM is a very common culture medium, a variety of other media can also be successfully used with Vero cells. Depending on the application, it may be desired or necessary to count the number of cells (i.e., if a specific number of cells need to be analyzed, plated, etc.) The concentration of cells in suspension (following trypsin treatment) can be determined using a hemacytometer. See Phelan 2007 for a complete protocol for determining the number of viable cells in solution. When the Vero cells will be used for bacterial infections, the dividing host cells can dilute the infections, which, depending on the system, can confound the analysis of the results. One technique that has been employed to address this issue is gamma irradiation. At appropriate levels, irradiation does not kill the Vero cells, but does prevent cell division, allowing bacterial growth in Vero cells without the complicating factor of host cell division and growth..

Certain applications, such as vaccine production, may require the scaling-up of Vero cell cultures. There are two growth systems used for the scaling-up of anchorage-dependent cell lines: roller bottles and microcarriers. Roller bottles are cylindrical vessels, and the cells grow on the inner surface of the tube. The bottles slowly revolve to continually bathe the cells in growth medium. The surface area available for cell attachment can be even further increased by growing the cells on microcarrier beads. The beads, usually around 0.2mm, can be made of dextran, cellulose, gelatin, glass or silica, and can considerably increase the surface area available for Vero cell growth.

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2. CV-1 MONKEY KIDNEY CELL LINEThe CV-1 cell line, derived from cercopithecus aethiops monkey kidneys, acquired in the course of serial passaging an epitheloid morphology and heteroploid karyotype. as compared with vero and gmk cell lines, the CV-1 line proved to be more susceptible to a broad spectrum of arboviruses belonging to the families togaviridae and bunyaviridae. Most of them reached high titres and are markedly cytopathic in CV-1 cells so that all tests based on the cytopathic effect could be carried out. The cv-1 cell line can thus be recommended as an extra-ordinarily suitable substrate for the diagnosis and study of arboviruses.

3. HELA CELL LINE

A HeLa cell, Hela or hela cell, is an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. The line was derived from cervical cancer cells taken on February 8, 1951, from Henrietta Lacks, a patient who eventually died of her cancer on October 4, 1951. The cell line was found to be remarkably durable and prolific — which has led to its contamination of many other cell lines used in research.

Gey named the sample HeLa, after the initial letters of Henrietta Lacks' name. As the first human cells grown in a lab that were "immortal" (they do not die after a few cell divisions), they could be used for conducting many experiments. Demand for the HeLa cells quickly grew. Since they were put into mass production, Henrietta's cells have been used by scientists around the globe for "research into cancer, AIDS, the effects of radiation and toxic substances, gene mapping, and countless other scientific pursuits".  HeLa cells have been used to test human sensitivity to tape, glue, cosmetics, and many other products. 

Characteristics

Telomerase: These cells proliferate abnormally rapidly, even compared to other cancer cells. Like many other cancer cells, HeLa cells have an active version of telomerase during cell division, which prevents the incremental shortening of telomeres that is implicated in aging and eventual cell death. In this way the cells circumvent the Hayflick Limit, which is the limited number of cell divisions that most normal cells can later undergo before becoming senescent.

Chromosome number: HeLa cells are rapidly dividing cancer cells, and the number of chromosomes varied during cancer formation and cell culture. The current estimate (excluding very tiny fragments) is a "hypertriploid chromosome number (3n+)" which means 76 to 80 total chromosomes (rather than the normal diploid number of 46) with 22–25 clonally abnormal chromosomes, known as HeLa signature chromosomes. The signature chromosomes can be derived from multiple original chromosomes making challenging summary counts based on original numbering. Researchers have also noted how stable these aberrant karyotypes can be.

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Contamination

Because of their adaptation to growth in tissue culture plates, HeLa cells are sometimes difficult

to control. They have proven to be a persistent laboratory "weed" that contaminates other

cell cultures in the same laboratory, interfering with biological research and forcing

researchers to declare many results invalid. It has been demonstrated that a substantial

fraction of in vitro cell lines — estimates range from 10% to 20% — are contaminated with

HeLa cells.   Recent data suggest that cross-contaminations are still a major ongoing problem

with modern cell cultures.

 

STEM CELL TECHNOLOGY

Stem cells retain the capacity to self renew as well as to produce progeny with a restricted mitotic potential and restricted range of distinct types of differentiated cell they give rise to. The

formation of blood cells also called haematopoiesis is the classical example of concept of stem cells. Indirect assay methods were developed to identify the haematopoietic stem cells. The process of haematopoeis is occurs in the spleen and bone marrow in mouse. In human beings about 100,000 haematopoietic stem cells produce one billion RBC, one billion platelets, one million T-cells, one million B cells per kg body weight per day.

Stem Cell Research

Stem cells are the raw material from which all of the body’s mature, differentiated cells are made.  Stem cells give rise to brain cells, nerve cells, heart cells, pancreatic cells, etc. They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses. They can replicate themselves over and over for a very long time.Understanding how stem cells develop into healthy and diseased cells will assist the search for cures. There are two types of Stem Cells:

a) Embryonic (also called “pluripotent”) stem cells

Embryonic stem cells are capable of developing into all the cell types of the body. There are two Sources of Embryonic Stem Cells-

1) Excess fertilized eggs from IVF (in-vitro fertilization) clinics can be used as a source of embryonic stem cells. Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. These surplus embryos can be used to produce stem cells. Regenerative medical research is finding modern methodology to develop these cells into new, healthy tissue to treat severe and often fatal illnesses.

2) Therapeutic Cloning (Somatic Cell Nuclear Transfer)

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In the Somatic Cell Nuclear Transfer, the nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). No sperm is involved in this process, and the embryo are not created to be implanted in a woman’s uterus. The resulting stem cells can be induced to develop into specialized cells that are useful to treat dangerous illnesses.

b) Adult stem cells

Adult stem cells are less versatile and more difficult to identify, isolate, and purify. The stem cells are extracted from a 5-7 days old blastocyst. Stem cells can divide in culture to form more of their own kind, thereby creating a stem cell line. Later these are induced to generate healthy tissue needed by patients.

Stem Cell Therapy: The Importance of Stem Cell TherapyStem cells allow us to study how organisms grow and develop over time. Stem cells can replace diseased or damaged cells that cannot heal or renew themselves. To develop and research and find new drugs and medicines, stem cells can be used to test these chemical and drugs. Stem cells can helps us to understand what is called the “genetic machinery”. All ready tremendous efforts are going on to treat diseases like Parkinson’s Disease, Leukemia (Bone Marrow Transplants) diabetes, multiple sclerosis, etc using stem cell therapy. Another area of importance is to regenerate tissues to be used as skin grafts to treat patients suffering severe burns.

The Controversy regarding Stem cell Therapy: The supporters of Stem cell therapy argue that embryonic stem cell research (ESCR) fulfills the ethical obligation to alleviate human suffering. The end justifies the means. If the research is directed towards making the human species disease and pain free, any kind of research including Stem cell research should be allowed and pursued. They further argue that as excess IVF embryos will be discarded anyway, they should better be used in research. As far as Stem cell nuclear transfer SCNT (Therapeutic Cloning) is concerned, it produces cells in a petri dish, not out of pregnancy. Those who oppose the stem cell therapy accuse the researchers involved in this as murders. Their argument is that extracting the stem cells from a human blastocyst leads to the destruction or killing of the embryo, which amounts to murdering, or killing a potential human life. Further there is risk of commercial exploitation of the couples who willingly or unknowingly become participants in ESCR. Some opponents also predict that in the future this will lead to reproductive cloning.

It is not easy to decide or resolve the serious ethical issues surrounding this area of research. Often question is asked as to at what stage of embryonic development the blastocyst should be regarded as a “person with life”. Often the blastocyst used in the stem cell research is microscopically so small with no nervous system, that the supporters of the stem cell therapy do not consider it as live. There is also a conflict between embryonic stem cell research science and religion, as most of the major world religions do not support using embryos for research. There have been major hurdles in creating and formatting a human public policy on this issue due to sensitive nature of the problem.

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TOPIC 3: EMBRYONATED EGG CULTURE TECHNIQUE FOR VIRUSES

Purpose of Virus Cultivation: The primary purposes of viral cultivation are: 1.To isolate and identify viruses in clinical specimens. 2. To prepare viruses for vaccines. 3. To do detailed research on viral structure, multiplication cycles, genetics and effects on host cells.

Virus Cultivation Systems: Tissue culture system, Embryonated eggs system and Whole animal systems.

Embryonated eggs system

Goodpasture and Burnet in 1931 first used the embryonated hen’s egg for the cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg being used. Eggs provide a suitable means for:

• the primary isolation and identification of viruses• the maintenance of stock cultures • the production of vaccines

Terms most often refer to eggs: •  Embryonated: having an embryo, •  Unembryonated: not having an embryo •  De-embryonated: having lost an embryo Embryonated egg is referred to an advanced stage of development and not merely

after fertilization.

Chick embryo Virus growth in an embryonated egg may result in the death of the embryo (e.g. encephalitis virus), the production of plaques on the chorioallantoic membrane (e.g. herpes, smallpox, vaccinia), the development of hemagglutinins in embryonic fluids or tissues (e.g. influenza) or the development of infective virus (e.g. poliovirus type 2). Inoculation may be into the allantoic or amniotic cavities into the yolk sac or on to the chorioallantoic membrane, the precise route used depending on the particular virus being cultivated. It is the method of choice for the isolation of the influenza and mumps viruses. The alantoic route, which is technically the simplest, is used mainly for the passage of influenza viruses that have already been established in embryo. Yolk sac inoculation is of particular value for the propagation of rickettsia. Whereas inoculation on to the chorioallantoic and characterization of the poxviruses and herpes simplex.

Advantages An embryo is an early developmental stage of animals marked by rapid differentiation of

cells. Birds undergo their embryonic period within the closed protective case of an egg, which

makes an incubating bird egg a nearly perfect system for viral propagation. It is an intact and self-supporting unit, complete with its own sterile environment and

nourishment. It furnishes several embryonic tissues that readily support viral multiplication Defense mechanisms are not involved in embryonated eggs Cost- much less, Maintenance-easier, Less labor and Readily available

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Inoculation of Virus: Chicken, duck, and turkey eggs are the most common choices for inoculation. The egg used for cultivation must be sterile and the shell should be intact and healthy. Rigorous sterile techniques must be used to prevent contamination by bacteria and fungi from the air and the outer surface of the shell.

Detection of viral growth: Viruses multiplying in embryos may or may not cause effects visible to the naked eye. The signs of viral growth include:

Death of the embryo Defects in embryonic development Localized areas of damage in the membranes, resulting in discrete opaque spots called

pocks

If a virus does not produce obvious changes in the developing embryonic tissue, virologists have other methods of detection. Embryonic fluids and tissues can be prepared for direct examination with an electron microscope. Certain viruses can also be detected by:

their ability to agglutinate red blood cells their reaction with an antibody of known specificity

Parts of Embryonated Egg: The air sac is important to the developing embryo for respiration and for pressure adjustments. The shell and shell membrane function both as a barrier and as an exchange system for gases and liquid molecules. The chorioallantoic sac and its contents (allantoic fluid) remove waste products produced by the developing embryo. This Membrane and its contents increase in size as the embryo grows. The yolk sac is the source of nourishment for the developing Embryo. As the embryo develops, the yolk sac decreases in size until it is completely absorbed into the digestive system of the mature embryo. The amnion is a thin membrane that encloses the embryo and protects it from physical damage. It also serves as an exchange system and is best seen in the younger embryos. An embryonated egg offers various sites for the cultivation of viruses;

1. Chorioallantoic membrane (CAM)2. Amniotic Cavity3. Allantoic Cavity4. Yolk sac

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Figure 1a, b & c: An Embryonated egg showing sites of inoculation

The chosen route of inoculation and age of the embryo are determined by the given virus selectivity for a certain membrane or developmental stage of the embryo. For example Infectious bronchitis virus is propagated in the yolk sac of a 5-6 day old embryo. Whereas Rous-sarcoma virus is inoculated on the chorioallantoic membrane of a 9-11 day old embryo and will produce pocks 5-10 days post-infection.

Candling of Egg: It is the process of holding a strong light above or below the egg to observe the embryo. A candling lamp consists of a strong electric bulb covered by a plastic or aluminum container that has a handle and an aperture.

Figure 2a & 2b: Candling of egg to observe embryo

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Chorioallantoic membrane (CAM): This method has been widely used in veterinary virology. Many viruses grow readily or can be adapted to grow on the CAM. Viruses produce visible foci or ‘pocks’, inclusion bodies, oedema or other abnormalities. Each infectious virus particle forms one pock. Viruses which can be grown include: Herpes viruses and poxviruses

Amniotic Cavity: The virus is introduced directly into the amniotic fluid that bathes the developing embryo. The  volume  of  fluid  in  the  infected  amniotic  sac  is  small (1-2 ml). The amniotic route is recommended for the primary isolation of human viruses: mumps virus, and influenza A, B and C viruses. It has little application in veterinary virology. Newly isolated influenza viruses may require several passages before they adapt to growth by other routes, such as allantoic.

Allantoic Cavity: Many viruses such as Newcastle disease virus can grow readily. Other viruses such as influenza, may require repeated amniotic passages before becoming adapted to the egg and grown in the allantoic cavity. Allantoic inoculation is a quick and easy method that yields large amounts (8–15 ml) of virus-infected egg fluids.

Yolk sac: It is also a simplest method for growth and multiplication of virus. Mostly mammalian viruses are isolated using this method. Immune interference mechanism can be detected in most of avian viruses. This method is also used for the cultivation of some bacteria like Chlamydiae and Rickettsiae.

TOPIC 4: MAMMALIAN CELL CULTURE TECHNIQUES FOR PROTOZOA, VIRUSES, HELMINTHES, BACTERIA, FUNGI.

PROTOZOAThe in vitro culture of protozoan parasites involves highly complex procedures, which are subject to many variables. These parasites have very complex life cycles and, depending on the life cycle stage, may require different culture parameters. However, in vitro cultivation is important for many reasons, some of which include: diagnosis, antigen and antibody production, assessment of parasite immune modulating capabilities, drug screening, improvements in chemotherapy, differentiation of clinical isolates, determination of strain differences, vaccine production, development of attenuated strains, and the continued supply of viable organisms for studying host-parasite interactions.

Certainly protozoan parasites have complex life cycles. They may have different morphological stages within the life cycle and may have both cold-blooded and warm-blooded animals as intermediate or definitive hosts within the life cycle. In vitro culture of organisms at any one of these stages within the life cycle involves a tremendous number of variables, including parasite stage, host site, host temperature, host immune responses, parasite species and/or strain, and parasite-protective mechanisms. To simulate the host environment in an in vitro culture system can be extremely demanding, assuming one can actually determine all the relevant variables.

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Often the organisms are very fastidious in their growth requirements, and different lots of media and/or medium components may be toxic. Something as simple as the type of glass used for the culture container can have tremendous influence over the success or failure of culture trials. Many culture medium components require filter sterilization and have relatively short shelf lives. Another requirement for many medium formulations includes the addition of human or animal sera, which are expensive and highly variable and may contain factors that are detrimental to parasite growth. Much research has been devoted to the development of defined medium formulations, although, even with the elimination of serum, various other components may not have been totally defined. In some cases, growth factors have been identified and substituted for serum or serum components.

In vitro cultivation of parasitic protozoa that cause human disease is invaluable, as it provides not only information on the development of the parasite but also avenues for new approaches to the containment and/or eradication of the parasite. In vitro cultivation is important for a number of reasons, as follows: (i) as an important adjunct to diagnosis; (ii) to produce antigens used to prepare monoclonal and polyclonal antibodies against the organisms for use in immunologic tests; (iii) to identify specific proteins that may enhance the invasive properties of the parasite and in turn the development of monoclonal antibodies that will help neutralize parasitic invasion; (iv) to assess functional antibodies and cell-mediated protective systems against the parasites, assessments that can only be made in a cost-effective manner using in vitro culture; (v) to screen drugs, in vitro, in order to identify potential therapeutic agents; (vi) to differentiate susceptible from resistant isolates so that advances in chemotherapy can be made; (vii) to differentiate clinical isolates using techniques such as isoenzyme electrophoresis, monoclonal antibody techniques, and/or DNA probe techniques; (viii) to elucidate isolate and strain differences which will be a useful tool for molecular epidemiology; and (ix) to produce vaccines, as relatively large numbers of parasites at specific stages can be produced in culture. (x) In addition, continuous culture over long periods of time may cause attenuation of strains, and therefore, attenuated strains have potential in the development of suitable vaccines. (xi) In vitro cultivation also provides a system to assess vaccine efficacy, since it can only be done by using intact parasites that can be obtained in large quantities and without the contaminating influences of host components. Finally, culture can be used (xii) to provide the parasite inoculum used for experimental animal disease models; (xiii) to study the biochemistry, physiology, and metabolism of the parasites as well as determine their nutritional requirements; (xiv) to understand the ultrastructural organization of the parasite; and (xv) to provide a system suitable for the assay of lymphokines and other cytokines that may block invasion of the parasite.

VIRUSES

Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication. The primary purpose of virus cultivation is:

1. To isolate and identify viruses in clinical samples.2. To do research on viral structure, replication, genetics and effects on host cell.

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3. To prepare viruses for vaccine production.

Viral culture is a laboratory test in which samples are placed with a cell type that the virus being tested for is able to infect. If the cells show changes, known as cytopathic effects, then the culture is positive.Traditional viral culture has been generally superseded by shell vial culture, in which the sample is centrifuged onto a single layer of cells and viral growth is measured by antigen detection methods. This greatly reduces the time to detection for slow growing viruses such as cytomegalovirus, for which the method was developed.[2] In addition, the centrifugation step in shell viral culture enhances the sensitivity of this method because after centrifugation, the viral particles of the sample are in close proximity to the cells.

Human and monkey cells are used in both traditional viral culture and shell vial culture. Human virus types that can be identified by viral culture include adenovirus, cytomegalovirus, enteroviruses, herpes simplex virus, influenza virus, parainfluenza virus, rhinovirus, respiratory syncytial virus, varicella zoster virus, measles and mumps. For these, the final identification method is generally by immunofluorescence, with exception of cytomegalovirus and rhinovirus, whose identification in a viral culture are determined by cytopathic effects. Viral culture may be particularly useful in cases with a broad viral differential as this technique provides a relatively unbiased approach to the identification of viral pathogens.  Viral culture utilizes a series of primary cell lines (Human Fibroblast, Rhesus Monkey Kidney) and continuous cell lines (A549 Human Lung Carcinoma) selected for their ability to support the replication of a wide variety of clinically relevant viruses.  Specimens are inoculated onto these cell culture monolayers and monitored by light microscopy for cytopathic effect (CPE), the visible cellular changes that occur in response to viral infection. 

Based on the specimen source, the time to CPE, the quality of the CPE, and the cell line(s) showing CPE, a preliminary identification can be made.  The presence of a specific virus is confirmed by immunofluorescent staining using virus-specific, fluorescently-labeled antibodies.  Utilizing viral culture, our laboratory is able to isolate many important pathogenic viruses (including HSV, VZV, cytomegalovirus (CMV), adenovirus, enterovirus, influenza virus, RSV, parainfluenza virus, and rhinovirus) from essentially any source (respiratory, urine, stool, amniotic fluid, tissue, etc.). To expedite the identification of slowly replicating viruses (for example, CMV) we also perform shell vial assays, a modification of traditional viral culture where cells are stained prior to the development of CPE.

HELMINTHS

Three types of culture media may be used for cultivating parasites:

Xenic culture - It refers to culture of parasites grown in association with unknown microbiota, for example stool specimens cultured for E. histolytica in National Institute of Health medium. It is used for primary growth of parasites.[7]

Monoxenic culture - If the parasites are grown with a single known bacterium, the culture is referred to as monoxenic, for example corneal biopsy specimens cultured with Escherichia coli

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as a means of recovering species of Acanthamoeba. It can be used for primary growth as well as a transitional phase in isolation.[7]

Axenic culture - It is a pure culture without any bacterial associate or any other metabolizing cells. It is mainly used as isolation medium for the parasites, but can be used for primary growth also, for example TYI-S-33 medium in case of T. vaginalis.[7]

General principles: Although the province of parasitic cultivation is very diverse, there are certain principles which are applicable at large to the subject:

1. Parasitic helminths are more difficult to cultivate than protozoa. The complexity of helminth body configuration and metabolism, and inability to meet essential environmental conditions account for failure to complete their life-cycles under artificial conditions.

2. Cell cultures are used for the obligate intracellular parasites, for example Plasmodium spp. and coccidia.

3. Various kinds of nutrients such as blood, serum, haem, egg, peptone, minerals and carbohydrates are used in the culture media.

4. Temperature required for optimum growth is usually 37°C though lower temperatures may be required in few cases, e.g. 25°C for Leishmania promastigotes.

5. Incubation condition is aerobic with some exceptions like microaerophilic conditions for amoebae and Giardia and 5% CO2 for Plasmodium spp.

6. Identification tools include parasite's characteristic morphology, direct fluorescent antibody assay, polymerase chain reaction, enzyme immunoassay, etc.

7. Positive controls need to be run in parallel to keep a check on the medium and the method used.

BACTERIA Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

Broth cultures: One method of bacterial culture is liquid culture, in which the desired bacteria are suspended in a liquid nutrient medium, such as Luria Broth, in an upright flask. This allows a scientist to grow up large amounts of bacteria for a variety of downstream applications. Liquid cultures are ideal for preparation of an antimicrobial assay in which the experimenter inoculates

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liquid broth with bacteria and lets it grow overnight (they may use a shaker for uniform growth). Then they would take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).

Agar plates: Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals, or lower for environmental cultures). After the desired level of growth is achieved, agar plates can be stored upside down in a refrigerator for an extended period of time to keep bacteria for future experiments. There are a variety of additives that can be added to agar before it is poured into a plate and allowed to solidify. Some types of bacteria can only grow in the presence of certain additives. This can also be used when creating engineered strains of a bacteria that contain an antibiotic-resistance gene. When the selected antibiotic is added to the agar, only bacterial cells containing the gene insert conferring resistance will be able to grow. This allows the researcher to select only the colonies that were successfully transformed.

Stab cultures: Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed into the center of the agar. Bacteria grow in the punctured area. Stab cultures are most commonly used for short-term storage or shipment of cultures.

FUNGI

Isolation and culture of the fungal strains: The primmorphs were observed daily on inverted microscope, and the fungal hyphae that appeared on the surface were picked and incubated in tubes with DY medium (Dextrose 1% and Yeast Nitrogen base 1% (Fluka) in natural seawater). After 24h, 100 µl of each suspension was disseminated in DY agar (DY medium with 2% agar) to verify the purity of the culture. The fungi were carefully selected according to morphologic characteristics (7, 13), transferred to 125 ml Erlenmeyer flasks with 60 ml of DY medium and incubated at 20ºC. After 15 days, the cultures were sampled for DNA extraction and identification.

To obtain cell-associated fungi, primmorphs were cultivated for 21 days and then dissociated with CMFSW+E. The cell suspension was counted using a Neubauer chamber, and attached in low density to sterile glass coverslips using a cytocentrifuge (80 x g / 5 min, 1.5 x 104 cells per spot. Citospin 248, FANEM). The coverslips were allowed to dry in a sterile laminar flow hood for 3h and then transferred to six-well plates filled with DY medium, with the cell surface facing the overlaying medium. For each plate, one well with a coverslip prepared with CMFSW+E was used as control. Culture observation was carried out using phase contrast inverted microscopy (Eclipse TE300, NIKON), until the mycelia became visible with the naked eye. Morphologically different strains were collected and transferred to the DY agar to verify the purity of the culture, and then cultivated as described above. To differentiate between strains associated with the cells and those incidentally present in the sponge surfaces and canals, fungal cultures were established

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from sponge whole tissues. Sponge fragments were washed with sterile CMFSW+E (5 min) in order to release peripheral cells and eventual transient fungi, gently compressed and the resulting material was collected in a flask with sterile seawater. Then, 100 µl aliquots of this suspension were disseminated in DY and isolates were purified by successive selection and cultivated following the procedure described above.

TOPIC 5: PRODUCTION OF RECOMBINANT PROTEINS IN BACTERIA AND MAMMALIAN CELL LINES: INSULIN, HUMAN GROWTH HORMONE, VACCINES

The pioneering work of Stanley Cohen and Herbert Boyer, who invented the technique of DNA cloning, signaled the birth of genetic engineering, which allowed genes to transfer among different biological species with ease. Their discovery led to the development of several recombinant proteins with therapeutic applications such as insulin and growth hormone. Genes encoding human insulin and growth hormone were cloned and expressed in E. coli in 1978 and 1979 respectively. The first licensed drug produced using recombinant DNA technology was human insulin, which was developed by Genentech and licensed as well as marketed by Eli Lilly in 1982.

Today, there are more than 300 biopharmaceutical products including therapeutic proteins and antibodies in the market. Therapeutic monoclonal antibodies have captured the major market share followed by the hormones and growth factors. Biopharmaceuticals approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) from 2004 to 2013 are largely derived from the following expression systems: mammalian cell (56%); E. Coli (24%); S. Cerevisiae (13%); Transgenic animals & plants (3%) and insect cells (4%).

1. RECOMBINANT INSULIN

Since the early 1920s, diabetic patients were treated with insulin, which was purified from bovine or porcine pancreas. The incidence of diabetes is increasing at an alarming rate and it has been speculated that the number of diabetic patients worldwide would increase to approximately 300 million by the year 2025. Consequently, the requirement for insulin will increase manifold (approximately more than 16000 kg/ year) and the productivity of current insulin expression system would not be sufficient to meet the future market demands. Efficient expression systems for insulin production are also needed and novel route for insulin administration such as oral or inhalation are to be developed.

Expression systems

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Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin. Nowadays, recombinant human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae expression systems.

E. coli expression system: Using E. coli expression system, the insulin precursors (IP) are produced as inclusion bodies and fully functional polypeptides are obtained finally by solubilization and refolding procedures as illustrated below.

Yeast based expression system: Yeast based expression system yield soluble IP which is secreted into the culture supernatant. Saccharomyces cerevisiae is the most preferred and predominant yeast for large scale commercial production of insulin, however several other alternate yeast strains have been explored for insulin production.

Other expression systems: Besides, E.coli and yeast, mammalian cells, transgenic animals and plant expression systems are also employed as a host for large-scale production of recombinant insulin.

Structure and function of insulin

The human insulin is comprised of 51 amino acids and has a molecular weight of 5808 Da. It is produced by beta cells of the pancreas and plays a key role in regulating carbohydrate and fat metabolism in the body. Insulin is synthesized as a single polypeptide known as preproinsulin in pancreatic beta cells. Preproinsulin harbours a 24-residue signal peptide, which directs the nascent polypeptide to the endoplasmic reticulum. The signal peptide is cleaved as the polypeptide is translocated into the human of the endoplasmic reticulum resulting in the formation of proinsulin. In the Endoplasmic reticulum, the proinsulin is folded in proper confirmation with the formation of 3 disulphide bonds. Folded proinsulin is then transported to the trans-Golgi network, where it is converted into active insulin by cellular endopeptidases called as prohormone convertases (PC1 and PC2) and exoprotease carboxypeptidase E. The endopeptidases cleaves at two positions, resulting in the release of a fragment termed as C-peptide. The mature insulin, thus formed consists of an A-chain with 21 aminoacids and a B-chain containing 30 aminoacids and both polypeptides linked together by two disulphide bonds. Besides, the A-chain has an intrachain disulphide bond

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E. coli expression system for production of insulin

E. coli is a preferred microorganism for large-scale production of recombinant proteins.

Disadvantages

: several disadvantages limit its use for production of recombinant biopharmaceuticals. Various post-translational modifications (PTMs) such as glycosylation, phosphorylation, proteolytic processing and formations of disulfide bonds which are very crucial for biological activity, do not occur in E. coli .

Solution

N-linked glycosylation is the most common posttranslational modification of proteins in eukaryotes. It has been discovered that the bacterium Campylobacter jejuni possess the capability to glycosylate the proteins and it was also shown that a functionally active N-glycosylation pathway could be transferred to E. coli . Although the structure of bacterial N-glycan is different from that observed in eukaryotes, engineering of Campylobacter N-linked glycosylation pathway into E. coli, provides an opportunity to express heterologous proteins in glycosylated form in E. coli. Expression of Pglb oligosaccharyltransferase or (OTase) from C. jejuni in E. coli showed a significant increase in glycopepetide yield. Recently efforts has been made to produce glycosylated proteins with substrates other than native and non-native to E. coli and C.jejuni.

The codon usage of the heterologous protein also plays a major role in determining the expression level of recombinant protein. If the codon usage of heterologous protein differs significantly from the average codon usage of the E. coli host, it could result in very low expression. Usually, the frequency of the codon usage reflects the abundance of their corresponding tRNA. Therefore, significant differences in codon usage could result in premature termination of translation, misincorporation of aminoacids and inhibition of protein synthesis.

Expression of heterologous proteins in E. coli can be improved by replacing codons that are rarely found in highly expressed E. coli genes with more favorable major codons. Similarly, co-expression of the genes encoding for a number of the tRNA for rare codon, may enhance the expression of heterologous proteins in E. coli. 

There are some commercial E. coli strains available that encodes for tRNA for rare codons such as BL21 (DE3) CodonPlus-RIL, BL21 (DE3) CodonPlus-RP (Stratagene, USA) and Rosetta (DE3). BL21 (DE3) CodonPlus-RIL harbors tRNA genes for rare codons like AGG, AGA (arginine), AUA (isoleucine) and CUA (leucine). Similarly, Rosetta (DE3) strain harbors tRNA genes for rare codons like AGG, AGA (arginine), CGG (arginine), AUA (isoleucine), CUA (leucine), CCC (proline) and GGA (glycine). These rare codons have been associated with low

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expression of proteins in E. coli, hence application of these genetically engineered E. coli host strains may improve the expression level of heterologous proteins and thus might result in higher yield of desired protein.

The use of protease-deficient E. coli strains, which carry mutations that eliminate the production of proteases may also improve the yield of recombinant protein by reducing proteolytic degradation. E. coli strain BL-21, is deficient in two proteases encoded by the lon (cytoplasmic) and ompT (periplasmic) genes. Rather than the external parameters, targeted methods such as modifications in protease or secretion pathways can provide the insight into biology of recombinant proteins.

In E. coli, complex and large therapeutic proteins can be secreted in periplasm as it provides an oxidizing environment and help in forming disulphide bonds, which facilitate the proper folding of recombinant proteins and likely to yield reliable N- terminus of expressed protein. Periplasm has advantages over cytoplasm in less protein concentration and proteolytic activity, improve the production titer, and enhance the solubility of recombinant protein. Altogether, with these advanced modifications and developments ease the process of target protein production thus accelerating the drug development.

Heterologous proteins generally accumulate in E. coli as inclusion bodies, which comprise of insoluble misfolded aggregates of proteins. Use of molecular chaperones may increase the protein solubility and assist in proper folding of recombinant protein. Some of the chaperones prevent aggregation of protein and some assist in refolding and solubilization of misfolded proteins. The most important chaperones in E. coli are GroEL, GroES, DnaK, DnaJ, GrpE and Trigger factor. These chaperones may be used singly, or in combination to enhance the protein solubility in E. coli.

Production of rapid-acting insulin analogue

Recombinant human insulin was first produced in E. coli by Genentech in 1978, using a approach that required the expression of chemically synthesized cDNA encoding for the insulin A and B chains separately in E. coli . After expressing independently, the two chains are purified and co-incubated under optimum reaction conditions that promoted the generation of intact and bioactive insulin by disulphide bond formation.

The first commercial recombinant insulin was developed for therapeutic use in human by this two-chain combination procedure. Another approach involves the expression of a single chemically synthesized cDNA encoding for human proinsulin in E. coli followed by purification and subsequent excision of C-peptide by proteolytic digestion. This approach was more efficient and convenient for large scale production of therapeutic insulin as compared to the two chain combination approach and has been used commercially since 1986. Eli Lilly followed this

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technology to produce Humulin, the first recombinant insulin approved in 1982, for the treatment of diabetic patients.

The above first generation recombinant insulins have an amino acid sequence identical to native human insulin and are preferred over animal derived insulin products. However, advancement in the field of genetic engineering and development of technology to chemically synthesize genes with altered nucleotide sequence, facilitated the development of insulin analogues with altered amino acid sequence. It had been observed that native insulin in commercial preparations usually exist in oligomeric form, as zinc-containing hexamer due to very high concentration, but in blood, biologically active insulin is in monomeric form. Hence, this oligomeric complex should dissociate so that insulin can be absorbed from the site of injection into the blood. Due to this, subcutaneously injected recombinant insulin usually have a slow onset with peak plasma concentration after 2 hours of injection and longer duration of action that last for 6–8 hours. Hence, in order to develop a fast- acting insulin analogue, it was required to modify the amino acids residues whose side chains are involved in dimer or oligomer formation. It has been shown that amino acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and 23-28 play critical role in oligomerization. Lispro, developed by Eli Lilly, was the first fast acting insulin analogue to obtain regulatory approval in 1996, for therapeutic use. Insulin Lispro is engineered in such a way that it has similar amino acid sequence as the native insulin but has an inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted in reduced hydrophobic interactions and thus prevented dimer formations. For commercial production of insulin Lispro, a synthetic cDNA encoding for Lys B28- Pro B29 human proinsulin was expressed in E. coli and insulin Lispro was excised proteolytically from the proinsulin by treating with trypsin and carboxypeptidase. Another rapid-acting insulin analogue, produced in E. coli is Glulisine (Apidra) which was developed by Aventis Pharmaceuticals and approved by US regulatory authorities in 2004. Insulin Glulisine have been generated by replacing B3 asparagine by a lysine and B29 lysine replaced by glutamic acid.

Production of long-acting insulin analogues

To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was developed by Aventis Pharmaceuticals and approved in 2000. Insulin Glargine was generated by replacing the C-terminal asparagine of the A-chain with a glycine residue and the C-terminal of the B- chain was modified by adding two arginine residues. These modifications resulted in increase of the isoelectric point (pI) from 5.4 to neutral values. Glargine was produced as proinsulin and expressed in E. coli and was finally formulated at pH 4 in soluble form. However, after subcutaneous administration, it precipitated due to neutral pH in the subcutaneous tissue. Resolubilization of insulin occur slowly, resulting in longer duration for its release in the blood.

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Recombinant DNA is a technology scientists developed that made it possible to insert a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene.

Scientists build the human insulin gene in the laboratory. Then they remove a loop of bacterial DNA known as a plasmid and…insert the human insulin gene into the plasmid (Fig 2)

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Researchers return the plasmid to the bacteria and…put the “recombinant” bacteria in large fermentation tanks. There, the recombinant bacteria use the gene to begin producing human insulin.

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Scientists harvest the insulin from the bacteria and…urify the substance for use as a medicine for people.

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Yeast expression system for the production of insulin

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Yeast is a preferred host for expression of various heterologous proteins that require post-translational modifications for its biological activity. Yeast cell has the ability to carry out numerous post-translational modifications such as phosphorylation, O-linked glycosylation, N-linked glycosylation, acetylation and acylation.

Advantages

Recombinant proteins are expressed in soluble form in yeast and properly folded in functionally active form. Production of biopharmaceuticals using yeast expression system is also very cost effective and is amenable to scale up using large bioreactors.

Disadvantage

One major concern for producing therapeutic glycoprotein for human application is that yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and hyper–immunogenicity and thus renders the therapeutic glycoprotein less effective. Various attempts have been made to humanize yeast N-glycosylation pathways in order to produce therapeutic glycoproteins with humanized N-glycosylation structure.

Production

The therapeutic proteins produced in yeast are specifically from Saccharomyces cerevisiae and include hormones (insulin, insulin analogues, non-glycosylated human growth hormone somatotropin, glucagon), vaccines (hepatitis B virus surface antigen), uprate oxidase from Aspergillus flavus, granulocyte-macrophage colony stimulating factor, albumin, hirudin of Hirudo medicinalis and human platelets derived growth factor.

Like E. coli, yeast derived recombinant biopharmaceuticals majorly intended as therapeutics for infectious diseases or endocrine, metabolic disorders. Alternate yeast strains, besides S. cerevisiae, are being explored for large-scale production of biopharmaceuticals. Specifically, Pichia pastoris has the ability to attain high cell densities by its robust methanol-inducible alcohol oxidase 1 (AOX1) promoter and simple developmental approaches contribute to high quality and quantity of recombinant proteins production.

In comparison to Saccharomyces cerevisiae, Pichia pastoris provides a major advantage in the glycosylation of secreted proteins because it does not hyperglycosylate the heterologous proteins. Both yeast strains have a majority of N-linked glycosylation of the high-mannose type, but the length of the oligosaccharides chain added to proteins in Pichia (around 8–14 mannose residues per side chain) is much shorter than those expressed in Saccharomyces cerevisiae (approximately 50–150 mannose residues per side chain), suggesting that glycoproteins produced in Pichia pastoris may be more suitable for therapeutic use in humans. Moreover, very high level of

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expression of heterologous proteins can be attained in Pichia pastoris, that might constitute about 30% of total cellular protein which is very high as compared to S. cerevisiae .

Transgenic plants as host for insulin production

Transgenic plants have been utilized to produce recombinant proteins because of their advantage of cost effectiveness, high quality protein processing, absence of human pathogens, and ease of production and presence of eukaryotic machinery for posttranslational modifications. Initially, the human growth hormone was the recombinant protein product extracted from transgenic tobacco plant. After that, numerous different products have developed from plants such as Hepatitis-B-Virus surface antigen, antibodies, industrial proteins and milk proteins.

Oilseeds of plant Arabidopsis thaliana expression

Recombinant human insulin has been successfully expressed and produced in oilseeds of plant Arabidopsis thaliana. This technology involved the targeted expression of insulin in subcellular organelles known as oilbodies that allowed very high level of expression with easy recovery of recombinant insulin. Oilbodies are storage organelles inside the oilseeds, which comprises of hydrophobic triacylglycerol core encapsulated by phospholipid membrane and an outer wall of proteins known as oleosins. Genetically engineered oil seeds have been generated with recombinant protein specifically targeted to oilbodies as oleosin fusion. Then the oilbodies are easily separated from other seed components by liquid-liquid phase separation, which reduced the number of chromatography steps required to obtain purified insulin. It has been observed that insulin accumulated to high level in transgenic seed (0.13% of total seed protein). Recombinant insulin was cleaved from the oleosin fusion partner and matured with trypsin digestion following oil body purification to yield a biologically active insulin. This study clearly demonstrated that expression of insulin as oleosin fusion protein in plant allow accumulation of large amount of recombinant insulin within the seed and also provide simple downstream purification by centrifugation i.e. oilbody purification. Subsequent maturation to obtain biologically active insulin can be accomplished using standard enzymatic methods currently used for commercial production of insulin from E. coliand yeast. Oilseeds also act as a natural cellular warehouse, where recombinant insulin can be stockpiled until required.

tobacco and lettuce chloroplasts expression system

In another approach, transgenic plants have been generated, in which, tobacco and lettuce chloroplasts were transformed with human proinsulin comprised of A, B and C-chains fused with the cholera toxin B subunit. It has been observed that, old tobacco leaves accumulated proinsulin upto 47% of total leaf protein and similarly, old lettuce leaves amassed proinsulin up to 53% of total leaf protein. Proinsulin stored in leaves of lettuce was found to be very stable as up to 40% of proinsulin was detected even in senescent and dried leaves. Proinsulin from tobacco leaves

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was extracted with 98% purity and cleaved by Furin protease to release insulin peptides. Oral delivery of unprocessed proinsulin encapsulated in plant cell or by injection into mice revealed lowering of blood glucose levels similar to commercially available insulins. Based on the yield (3 mg of proinsulin/gm of leaves), it was estimated that one acre of tobacco plantation could yield upto 20 million daily doses of insulin per year. C-peptide of proinsulin, which is not present in current commercially available insulin and insulin analogues derived from E. coli and S. cereviciae, would be a great advantage in long-term treatment of diabetic complications such as stimulation of nerve and renal functions. Very high level of expression of biologically active proinsulin in tobacco and lettuce leaves and long-term stability in dried leaves offers a reliable low-cost technology for both injectable as well as oral delivery of proinsulin.

2. HUMAN GROWTH HORMONE

Human growth hormone (hGH) also known as somatotropin is a single-chain polypeptide containing 191 amino acid residues and is synthesized in the pituitary gland. hGH participates in a wide range of biological functions such as metabolism of proteins, carbohydrates and lipids as well as in growth, development and immunity. Growth hormone deficiency in human occurs both in children and adults.

Human growth hormone is responsible for your physical and mental growth. It also affects some of the body’s internal organ development. This is the reason why when the body does not produce enough HGH, the person usually encounters several health conditions. One of the most common conditions people with growth hormone deficiency have to deal with is dwarfism. The routine treatment for this condition is administration of recombinant human growth hormone (rhGH) made by prokaryotes.

Structure

The major isoform of the human growth hormone is a protein of 191 amino acids and a molecular weight of 22,124 daltons. The structure includes four helices necessary for functional interaction with the GH receptor. It appears that, in structure, GH is evolutionarily homologous to prolactin and chorionic somatomammotropin. Despite marked structural similarities between growth hormone from different species, only human and Old World monkey growth hormones have significant effects on the human growth hormone receptor. Several molecular isoforms of GH exist in the pituitary gland and are released to blood. In particular, a variant of approximately 20 kDa originated by an alternative splicing is present in a rather constant 1:9 ratio,[9] while recently an additional variant of ~ 23-24 kDa has also been reported in post-exercise states at

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higher proportions.[10] This variant has not been identified, but it has been suggested to coincide with a 22 kDa glycosylated variant of 23 kDa identified in the pituitary gland. Furthermore, these variants circulate partially bound to a protein (growth hormone-binding protein, GHBP), which is the truncated part of the growth hormone receptor, and an acid-labile subunit (ALS).

Clinical significance

Excess: The principal clinical problems of GH excess include headaches, impair vision by pressure on the optic nerves, or cause deficiency of other pituitary hormones by displacement. Prolonged GH excess thickens the bones of the jaw, fingers and toes. Resulting heaviness of the jaw and increased size of digits is referred to as acromegaly. Accompanying problems can include sweating, pressure on nerves (e.g., carpal tunnel syndrome), muscle weakness, excess sex hormone-binding globulin (SHBG), insulin resistance or even a rare form of type 2 diabetes, and reduced sexual function.

Deficiency: The effects of growth hormone deficiency vary depending on the age at which they occur. In children, growth failure and short stature are the major manifestations of GH deficiency, with common causes including genetic conditions and congenital malformations. It can also cause delayed sexual maturity. In adults, deficiency is rare, with the most common cause a pituitary adenoma, and others including a continuation of a childhood problem, other structural lesions or trauma, and very 

therapeutic applications

It has therapeutic applications in the treatment of dwarfism, bone fractures, skin burns, bleeding ulcers and AIDS and has been studied in a variety of medical conditions and genetic syndromes such as children with Down's syndrome, Noonan's syndrome and Prader-Willi syndrome. A recombinant form of hGH called somatropin (INN) is used as a prescription drug to treat children's growth disorders and adult growth hormone deficiency. Growth hormone (GH) has important effects in stimulating the metabolism of bone, cartilage and muscle and somatic growth during childhood. Cardiomyopathy, decrease in myocardial mass with systolic and diastolic dysfunction both at rest and during exercise, has been reported in GH deficiency (GHD) adults. It was reported that GHD adolescents had reduced diastolic filling, cardiac size and disordered cardiac function.

rhGH Production

Until the mid-1980s, the only source of hGH was from human cadaver tissue. The use of pituitary-derived hGH was prohibited when its association with Creutz feldt-Jakob disease was proved. Recombinant DNA technology has facilitated a safe and abundant production of rhGH in various heterologous systems, without risk of transfer of human pathogens, eliminating the requirement for pituitary-derived preparation. Advancement in recombinant DNA technology has made possible the expression of proteins in host cells, such as E. coli. Recombinant hGH

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(rhGH) is now largely used to treat GH deficient short-stature children to final height and as therapy of adults with GHD, acceleration of wound healing, as well as an increase in insulin-like growth factor (IGF)-1 levels in the blood from low to normal. rhGH increases IGF-1, osteocalcin, type I pro-collagen pro-peptide (PICP) and bone density, when administered to children with GHD. Preliminarily studies also suggest that human GH combined with lactulose could prevent and cure severe hepatitis complicated by multiple organ dysfunction.

prokaryotic expression systems

Since nonglycosylated hGH is a biologically active protein, prokaryotic expression systems are preferred in the production of recombinant hGH. In order to achieve optimal productivity, the growth and production phase must be separated; therefore, recombinant organisms with inducible promoters are preferred.

  Escherichia coli (E. coli): Escherichia coli (E. coli) is one of the most widely used hosts for production of heterologous proteins. The strong, inducible promoter systems such as lPL, lPR, trc and T7, commonly used in recombinant E. coli, are advantageous for the over production of recombinant proteins at high cell density fermentation.

This is through cloning the HGH gene and inserting a cDNA gene into E-coli. In order for the gene to be translated and purified, a signal sequence is also added.

Isolation, transfer and production of hGH in E. coli

Genes like the one for HGH contain coded instructions for protein production. Inside cells, this information is first re-coded from DNA, which provides long-term information storage, to a messenger RNA (mRNA) molecule, which provides specific instructions for HGH protein production. The process begins by taking pituitary gland tissue and isolating the mRNA encoded by the HGH gene. Next, they used the mRNA as a template to create complementary DNA (cDNA). This DNA contains the coded instructions for making the HGH protein.

After creating the cDNA, it is added to a plasmid, a small loop of DNA taken from a bacterial cell. Next, the plasmid inserted into bacteria. When the bacteria are grown in culture, the cells use the transferred HGH gene to produce and isolate HGH much more quickly and with less effort and expense than was possible with human pituitary gland tissue. And, because the protein is produced by bacteria, contamination by components of cadaver tissue is not possible.

3. VACCINES

A vaccine is a biological preparation that provides active acquired immunity against a certain disease. Usually a vaccine consists of a biological agent that represents the disease-causing microorganism. It is often made from a weakened or killed form of the microorganism, its toxins or one of its surface protein antigens. An individual that has been vaccinated produces

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antibodies against the protein antigen that protect him/her from contracting the disease upon attack from the pathogenic microorganism.

Approaches for Vaccine Development

There are many approaches to design vaccines against a pathogenic microorganism. These choices are dictated by the nature of pathogen and the infection as well as practical considerations about the use of the vaccine. Some of the options include live attenuated vaccines, inactivated vaccines, DNA vaccines and recombinant subunit vaccines. See schematic below for an overview of the various approaches used to make a vaccine. This technical note discusses the basics of research and production of recombinant vaccines.

Recombinant Vaccine: Vaccine generated using recombinant DNA technology. While there are various types of vaccines made possible by recombinant DNA technology, recombinant vaccines can be classified into two major categories: DNA vaccines and Recombinant (protein subunit) vaccines

DNA vaccines

These vaccines usually consist of synthetic DNA containing the gene that encodes the disease-agent protein. Usually, the plasmid DNA used as vaccine is propagated in bacteria such as E. coli and they are isolated and purified for injection. This “naked” DNA is usually injected intramuscularly or intradermally. The principle behind a DNA vaccine is that the antigen can be expressed directly by host cells in a way that simulates viral infection and invokes an immune response from the host. This is similar to GenScript's DNA Immunization Technology which is a powerful tool that aids in custom antibody production against membrane proteins, other problematic antigens, as well as for early DNA vaccine development studies. DNA immunization technique allows antigen production to occur in vivo, bypassing the need to produce and purify protein antigen in vitro. Schematic below illustrates concept of DNA vaccine.

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Recombinant (protein subunit) Vaccines

These are subunit vaccines containing only a fraction of the pathogenic organism. Often time these are synthetic peptides that represent the protein component that induces an immune response. But they can also consist of protein subunits (antigens) expressed in a heterologous expression system (E. coli, yeast, insect etc.) using recombinant protein expression technologies. Most of the vaccines under investigation today are based on such purified recombinant proteins or subunits of antigens. One of the best examples of recombinant protein vaccine currently in use in humans is the vaccine against Hepatitis B Virus (HBV).

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TOPIC 6: DEVELOPMENT OF TRANSGENIC FARM ANIMALS

Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression.The nucleus of all cells in every living organism contains genes made up of DNA. These genes store information that regulates how our bodies form and function. Genes can be altered artificially, so that some characteristics of an animal are changed. For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo. Animals that have their DNA manipulated in this way are knows as transgenic animals.

A transgenic animal is one whose genome has been changed to carry genes from other species. The Federation of European Laboratory Animal Associations defines the term as an animal in which there has been a deliberate modification of its genome (the genetic makeup of an organism responsible for inherited characteristics). In general, it is an animal that has a foreign gene or exogene which is also referred to as transgene from another animal cell that has been inserted to

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its genome so it can change its properties and later on pass it to its offsprings. The exogene is an artificial DNA created using recombinant DNA methodology.

Different from a cloned animal: A transgenic animal is different from a cloned animal since

the latter comes from an identical to the organism it was derived from. Cloning methods (overview of nuclear transfer)

Nuclei extracted from cultured cells (embryo, fetus or adult) Nuclei inserted into egg cells which have their original nucleus removed (nuclear

transfer) Egg cells with the transplanted nuclei then implanted into a foster mother for

development

Cloning Applications: Produce superior livestock- Facilitate “pharm” animal production; Human therapeutic proteins; Facilitate xenotransplantation; Safer source of organ and tissue transplants; Provide defense against bio-terrorism; Hematechand the DynportVaccine Corporation developing cloned cows to produce antibodies to botulinumtoxin in milk and Save/recover endangered species and Resurrect” treasured pets

The first successful transgenesis was performed on a mouse and it can be processed by plasmid

vectors, pronuclear injection,ballistic DNA injection, viral vectors and protoplast fusion.

Transgenic animals are useful as disease models and producers of substances for human welfare.

The two most common reasons These animals being produced because of the following reasons:

Some transgenic animals are produced for specific economic traits. For example, transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema.

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Other transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied). For example, Harvard scientists made a major scientific breakthrough when they received a U.S. patent (the company DuPont holds exclusive rights to its use) for a genetically engineered mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers.22

Production of transgenic animals

The various objectives for which transgenics are produced are:

Gene transferred and expressed into cultured cell line to obtain a biochemical product.

Genetic modification of recipient to improve the quality of product produced.

Large scale production of the proteins encoded by these genes in milk, urine or blood. This approach is called Molecular Farming or Gene Farming.

To introduce functional copies of the defective gene in patients to cure genetic diseases (Gene Therapy).

The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign genes “must be transmitted through the germ line, so that every cell, including germ cells, of the animal contain the same modified genetic material.” (Germ cells are cells whose function is to transmit genes to an organism’s offspring.)

Methods: The insertion of a foreign gene (transgene) into an animal is successful only if the gene is inherited by offspring. The success rate for transgenesis is very low and successful transgenic animals need to be cloned or mated. To date, there are three basic methods of producing transgenic animals: DNA microinjection; Retrovirus-mediated gene transfer and Embryonic stem cell-mediated gene transfer.

Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. However, the success rate of producing transgenic animals individually by these methods is very low and it may be more efficient to use cloning techniques to increase their numbers. For example, gene transfer studies revealed that

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only 0.6% of transgenic pigs were born with a desired gene after 7,000 eggs were injected with a specific transgene.

1. DNA Microinjection: This method involves:

transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell.

the manipulated cell, which first must be cultured in vitro (in a lab, not in a live animal) to develop to a specific embryonic phase, is then transferred to the recipient female

2. Retrovirus-Mediated Gene Transfer: A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves:

retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution

chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born

3.  Embryonic Stem Cell-Mediated Gene Transfer: This method involves:

isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos

the desired gene is inserted into these cells

cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal

Unlike the other two methods, which require live transgenic offspring to test for the presence of the desired transgene, this method allows testing for transgenes at the cell stage.

Contribution of transgenic animals to human welfare

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The benefits of these animals to human welfare can be grouped into areas: Agriculture, Medicine and Industry. Applications of animal transgenesis may be divided into three major categories:

to obtain information on gene function and regulation as well as on human diseases,

to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy,

to improve animal products for human consumption. All these applications are directly or not related to human health. 

The examples below are not intended to be complete but only to provide a sampling of the benefits.

1. Agricultural Applications

Breeding: Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields.

Quality: Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them and sheep that grow more wool18. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product.

Disease resistance: Work is ongoing to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.

2. Medical Applications

Xenotransplantation: Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein.

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Nutritional supplements and pharmaceuticals: Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.

In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie’s milk contains the human gene alpha-lactalbumin.

Human gene therapy: Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans

3.  Industrial Applications

In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.1

Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.

4. Other Applications

Transgenic Mosquitoes (GM Mosquitoes)

Malaria: In the recent years, researchers have been able to create genetically modified mosquitoes to control the spread of the malaria. Malaria is estimated to cause 0.7 to 2.7 million deaths per year especially in the tropical countries. Mosquitoes are the vectors for spreading the malaria as they are the carriers of the parasites, Plasmodium which actually causes the malaria.

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To control the spread of malaria, transgenic mosquitoes have been created that express antiparasitic gene in their midgut epithelium thus making them incapable of spreading the disease. When the malarial parasite Plasmodium falciparum enters the body of its host (mosquito), it migrates from the insect’s gut to the salivary glands. The researchers inserted a synthetic gene that interferes with this migration by encoding a peptide that prevents the parasite from traversing the insect gut.

Dengue Fever: To control the spread of Dengue fever, genetically modified male mosquitoes containing a lethal gene have been developed. Dengue fever is a virus-induced disease that is spread by mosquitoes in tropical countries. Between 50 -100 million people are affected by Dengue fever every year and 40,000 people die from it. There is no cure or treatment for Dengue fever except proper rest and drinking plenty of fluids.

The GM mosquitoes, also known as OX513A, were created in Oxford University. Later a British Company, Oxitech, developed them for field use. These male GM mosquitoes carried an extra gene, or inserted bacterium or had gene altered so that either their offspring are sterile or simply die. The male mosquitoes when mate with the natural females, the larvae produced die due to the accumulation of an enzyme that is toxic enough to kill them. Aedes aegypti female mosquitoes, the single most important carrier of dengue fever, were reduced by 80% in a 2010 trial of these GM mosquitoes in the Cayman Islands where almost 3.3 million sterile transgenic male Aedes aegypti mosquitoes were released. When these GM mosquitoes mate, their offspring die or if the males don’t find a female, they die anyway. Similar field trials have been carried out in Malaysia as well.

ETHICAL CONSIDERATIONS IN DNA MANIPULATION OF FOOD ANIMALS (TRANSGENESIS)

Despite the practice to be in existence for years now, transgenic animals still remains to be a divisive issue. There are some serious issues related to genetic modification of animals using animal genetic engineering techniques. One is not sure of the consequences of these genetic modifications and the further interaction with the environment. Proper clinical trials are also necessary before one can use it for commercial purposes. In the recent past people have raised objections on some of the methods used e.g. the transfer of a human genes into food animals, use of organisms containing human genes as animal feed. Some religious groups have expressed their concern about the transfer of genes from animals whose flesh is forbidden for use as food into the animals that they normally eat. Transfer of animal genes into food plants that may be objectionable to the vegetarians.

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Although there are groups which believe in the benefits of this scientific breakthrough, there are also sectors which are against the use of transgenic animals. Thoughtful ethical decision-making cannot be ignored by the biotechnology industry, scientists, policy-makers, and the public. These ethical issues include questions such as:

Should there be universal protocols for transgenesis?

Should such protocols demand that only the most promising research be permitted?

Is human welfare the only consideration? What about the welfare of other life forms?

Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering?

Will transgenic animals radically change the direction of evolution, which may result in drastic consequences for nature and humans alike?

Should patents be allowed on transgenic animals, which may hamper the free exchange of scientific research?

Besides this, there are several other aspects of this issue have to be sorted out.

What will be the consequences, if a modified animal will breed with other domestic or wild animals thereby transferring the introduced genes to these populations?

What are the health risks to human on consumption of genetically modified animals and their products?

With the production of disease resistant animals, what will be the effect on ecology?

There is also wide spread concern about the risks of human recipients getting infected with animal viral diseases after a xenotransplantation., which might infect the population at large.

There are also concerns about the risk that drug resistance gene markers used in genetic engineering procedures might inadvertently be transferred and expressed.

The need of the hour is to formulate clear guidelines which should be followed while using genetic engineering techniques in bio-medical research. e.g. products from transgenic organisms should be clearly marked to give choice to people who follow dietary restrictions due to religious beliefs. In fact all the ethical and moral issues raised by some aspects of biotechnology should be addressed by open discussion and dialogue.

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Cons of Transgenic Animals

The use of transgenic animals is unethical. Opponents for modifying genes of animals to

create transgenic offspring say that doing so is clearly against moral ethics. Animal advocates, in

particular, are the ones not in favor of this practice. They strongly affirm that animals have rights

and altering DNA make-up is definitely a violation of these rights. They also are against the use

of hundreds of animals in clinical trial research and transgenic animals are no different,

according to them.

They can be unsafe for human consumption: Critics posit that there is no assurance on the

safety of the products coming from transgenic animals since not all experiments and researches

are successful. They are also concerned about the long-term implications on use of these drugs

and vaccines produces by these 

They are victims themselves: Opponents and animal welfare advocates are against the

proliferation of transgenic animal applications. For them, there might be some truth to the fact

that these animals can help in science and medicine. However, they also point out that many

lives of animals are endangered and even ended just for one successful feat. Aside from being

subjects of experiments, wherein success rates can be low, host animals can also be harmed in

the process.

They are added expense to the government: Just like cloning experiments, transgenesis is an

expensive process and needs funding. Although pharmaceutical companies spend for their own,

the government will have to spend money once these products cause harmful effects and the

method backfires. They say that if meat from transgenic animals will be sold in the market and

suddenly result diseases or medical problems, the government will have to find a solution and

that entails expenses.

TOPIC 6: IN VITRO FERTILIZATION AND EMBRYO TRANSFER

Union of egg cell and sperm outside the body in a culture vessel is known as in vitro fertilization. This involves collection of healthy ova and sperms from healthy females and males, and their

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fusion under in vitro conditions. The resulting zygote may be cultured in vitro for a period of time, which is then implanted in the uterus of healthy female. This technique of in vitro fertilization and embryo transfer are done to obtain desirable genotypes and in cases of infertility. In vitro fertilized embryos at 16 celled stage have been successfully transferred into the uterus. The babies produced using this approach is termed as Test tube babies. The first test tube baby, named Loise joy Brown, was born on 25th July, 1978. However, this has few ethical and social issues related which may need resolution. Although high degree of expertise is required and the cost of production of each progeny is more, the gains will be attractive and it will be possible to obtain relatively rare genotype.

Recombinant DNA: is DNA that has been altered by the recombination of genes from a different organism, typically from a different species. large amounts of recombinant dna can be grown in bacteria, viruses, or yeast and then transferred to other species. DNA recombination in nature – natural recombinant DNA.

Transformation: a method of acquiring new genes, whereby DNA from one bacterium (normally released after the death of the bacterium) becomes incorporated into the DNA of another, living, bacterium. A phenomenon in which external genetic material is assimilated by a cell.Transformation in Bacteria

Transformation enables bacteria to pick up DNA from the environment. The DNA may be part of the chromosome from another bacterium or from another

species. The DNA fragment is incorporated into bacterial chromosome. Transformation may also occur when bacteria pick up tiny circular DNA molecules called

plasmids.

TOPIC 7: FUTURE PROSPECTS OF RECOMBINANT TECHNIQUES IN UNDERSTANDING AND MANAGING DISEASES

Recombinant DNA technology comprises altering genetic material outside an organism to obtain enhanced and desired characteristics in living organisms or as their products. This technology involves the insertion of DNA fragments from a variety of sources, having a desirable gene sequence via appropriate vector. Manipulation in organism’s genome is carried out either through the introduction of one or several new genes and regulatory elements or by decreasing or blocking the expression of endogenous genes through recombining genes and elements. Enzymatic cleavage is applied to obtain different DNA fragments using restriction endo-nucleases for specific target sequence DNA sites followed by DNA ligase activity to join the fragments to fix the desired gene in vector. The vector is then introduced into a host organism, which is grown to produce

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multiple copies of the incorporated DNA fragment in culture, and finally clones containing a relevant DNA fragment are selected and harvested.

Applications of Recombinant DNA Technology

Food and Agriculture: Recombinant DNA technology has major uses which made the manufacturing of novel enzymes possible which are suitable in conditions for specified food-processing. Several important enzymes including lipases and amylases are available for the specific productions because of their particular roles and applications in food industries.

Health and Diseases: Recombinant DNA technology has wide spectrum of applications in treating diseases and improving health conditions. The following sections describe the important breakthroughs of recombinant DNA technology for the improvement of human health:

Gene Therapy

Production of Antibodies and Their DerivativesPlant systems have been recently used for the expression and development of different antibodies and their derivatives. Most importantly, out of many antibodies and antibody derivatives, seven have reached to the satisfactory stages of requirements. Transgenic tobacco plants can be used for the production of chimeric secretory IgA/G known as CaroRx, CaroRx. Oral pathogen responsible for decay of a tooth known as Streptococcus mutants, can be recognized by this antibody. A monoclonal antibody called T84.66 can affectively function to recognize antigen carcinoembryonic, which is still considered an affectively characterized marker in cancers of epithelia.

Investigation of the Drug MetabolismComplex system of drug metabolizing enzymes involved in the drug metabolism is crucial to be investigated for the proper efficacy and effects of drugs. Recombinant DNA approaches have recently contributed its role through heterologous expression, where the enzyme’s genetic information is expressed in vitro or in vivo, through the transfer of gene.

Development of Vaccines and Recombinant HormonesComparatively conventional vaccines have lower efficacy and specificity than recombinant vaccine. A fear free and painless technique to transfer adenovirus vectors encoding pathogen antigens is through nasal transfer which is also a rapid and protection sustaining method against mucosal pathogens. This acts as a drug vaccine where an anti-influenza state can be induced through a transgene expression in the airway.

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Chinese MedicinesAs an important component of alternative medicine, traditional chines medicines play a crucial role in diagnostics and therapeutics. These medicines associated with theories which are congruent with gene therapy principle up to some extent. These drugs might be the sources of a carriage of therapeutic genes and as coadministrated drugs. Transgenic root system has valuable potential for additional genes introduction along with the Ri plasmid. It is mostly carried with modified genes in A. rhizogenes vector systems to enhance characteristics for specific use. The cultures became a valuable tool to study the biochemical properties and the gene expression profile of metabolic pathways. The intermediates and key enzymes involved in the biosynthesis of secondary metabolites can be elucidated by the turned cultures.

Medically Important Compounds in BerriesImprovement in nutritional values of strawberries has been carried through rolC gene. This gene increases the sugar content and antioxidant activity. Glycosylation of anthocyanins requires two enzymes glycosyl-transferase and transferase. Some nutrition related genes for different components in strawberry including proanthocyanidin, l-ascorbate, flavonoid, polyphenols, and flavonoid are important for improving the component of interest through genetic transformation. In case of raspberry, bHLH and FRUITE4 genes control the anthocyanin components whereas ERubLRSQ072H02 is related to flavonol. By specific transformation, these genes can enhance the production and improve the quality. All these mentioned compounds have medical values..

EnvironmentGenetic engineering has wide applications in solving the environmental issues. The release of genetically engineered microbes, for example, Pseudomonas fluorescens strain designated HK44, for bioremediation purposes in the field was first practiced by University of Tennessee and Oak Ridge National Laboratory by working in collaboration [99, 100]. The engineered strain contained naphthalene catabolic plasmid pUTK21 [101] and a transposon-based bioluminescence-producing lux gene fused within a promoter that resulted in improved naphthalene degradation and a coincident bioluminescent response [102]. HK44 serves as a reporter for naphthalene bioavailability and biodegradation whereas its bioluminescence signaling ability makes it able to be used as an online tool for in situ monitoring of bioremediation processes. The production of bioluminescent signal is detectable using fiber optics and photon counting modules.

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Genetic engineering has been widely used for the detection and absorption of contaminants in drinking water and other samples. For example, AtPHR1 gene introduction into garden plants Torenia, Petunia, and Verbenachanged their ability for Pi absorption. The AtPHR1 transgenic plants with enhanced Pi absorption ability can possibly facilitate effective phytoremediation in polluted aquatic environments. A fragment of the AtPHR1 gene was inserted into binary vector pBinPLUS, which contains an enhanced cauliflower mosaic virus 35S promoter. This plasmid was named pSPB1898 and was used for transformation in Petunia and Verbena using Agrobacterium tumefaciens. AtPHR1 is effective in other plant species, such as Torenia,Petunia, and Verbena but posttranscriptional modification of the endogenous AtPHR1 counterpart might be inhibited by overexpression of AtPHR1.

Energy ApplicationsSeveral microorganisms, specifically cyanobacteria, mediate hydrogen production, which is environmental friendly energy source. The specific production is maintained by utilizing the required enzymes properly as these enzymes play a key role in the product formation. But advanced approaches like genetic engineering, alteration in nutrient and growth conditions, combined culture, metabolic engineering, and cell-free technology have shown positive results to increase the hydrogen production in cyanobacteria and other biofuels. The commercialization of this energy source will keep the environment clean which is not possible by using conventional energy sources releasing CO2 and other hazardous chemicals. Also cyanobacteria can be engineered to make them able to convert of CO2 into reduced fuel compounds. This will make the carbon energy sources harmless to environment. This approach has been successful for vast range of commodity chemicals, mostly energy carriers, such as short chain and medium chain alcohols. The conductive biofilms of Geobacter sulfurreducens are potential sources in the field in renewable energy, bioremediation, and bioelectronics.

Current Challenges and Future Prospects

The fact that microbial cells are mostly used in the production of recombinant pharmaceutical indicates that several obstacles come into their way restricting them from producing functional proteins efficiently but these are handled with alterations in the cellular systems. Common obstacles which must be dealt with are posttranslational modifications, cell stress responses activation, and instability of proteolytic activities, low solubility, and resistance in expressing new genes. Mutations occurring in humans at genetic levels cause deficiencies in proteins production, which can be altered/treated by incorporation of external genes to fill the gaps and reach the normal levels. The use of Escherichia coli in recombinant DNA technology acts as a biological framework that allows the producers to work in controlled ways to technically produce the required molecules through affordable processes.

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Recombinant DNA research shows great promise in further understanding of yeast biology by making possible the analysis and manipulation of yeast genes, not only in the test tube but also in yeast cells. Most importantly, it is now possible to return to yeast by transformation with DNA and cloning the genes using a variety of selectable marker systems developed for this purpose. These technological advancements have combined to make feasible truly molecular as well as classical genetic manipulation and analysis in yeast. The biological problems that have been most effectively addressed by recombinant DNA technology are ones that have the structure and organization of individual genes as their central issue. Recombinant DNA technology is recently passing thorough development which has brought tremendous changes in the research lines and opened directions for advanced and interesting ways of research for biosynthetic pathways though genetic manipulation. Actinomycetes are being used for pharmaceutical productions, for example, some useful compounds in health sciences and the manipulation of biosynthetic pathways for a novel drugs generation. These contribute to the production of a major part of biosynthetic compounds and thus have received immense considerations in recombinant drugs designing. Their compounds in clinical trials are more applicable as they have shown high level activity against various types of bacteria and other pathogenic microorganisms. These compounds have also shown antitumor activity and immunosuppressant activity.

Recombinant DNA tech as a tool of gene therapy is a source of prevention and cure against acquired genetic disorders collectively. DNA vaccines development is a new approach to provide immunity against several diseases. In this process, the DNA delivered contains genes that code for pathogenic proteins. Human gene therapy is mostly aimed to treat cancer in clinical trials. Research has focused mainly on high transfection efficacy related to gene delivery system designing. Transfection for cancer gene therapy with minimal toxicity, such as in case of brain cancer, breast cancer, lung cancer, and prostate cancer, is still under investigation. Also renal transplantation, Gaucher disease, hemophilia, Alport syndrome, renal fibrosis, and some other diseases are under consideration for gene therapy.

BIOETHICS IN ANIMAL GENETIC ENGINEERING

There are some serious issues related to genetic modification of animals using animal genetic engineering techniques. One is not sure of the consequences of these genetic modifications and

the further interaction with the environment. Proper clinical trials are also necessary before one can use it for commercial purposes. In the recent past people have raised objections on some of the methods used e.g. the transfer of a human genes into food animals, use of organisms containing human genes as animal feed. Some religious groups have expressed their concern about the transfer of genes from animals whose flesh is forbidden for use as food into the animals that they normally eat. Transfer of animal genes into food plants that may be objectionable to the vegetarians. Besides this, there are several other aspects of this issue have to be sorted out.

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a) What will be the consequences, if a modified animal will breed with other domestic or wild animals thereby transferring the introduced genes to these populations?

b) What are the health risks to human on consumption of genetically modified animals and their products?

c) With the production of disease resistant animals, what will be the effect on ecology?

d) There is also wide spread concern about the risks of human recipients getting infected with animal viral diseases after a xenotransplantation., which might infect the population at large.

e) There are also concerns about the risk that drug resistance gene markers used in genetic engineering procedures might inadvertently be transferred and expressed.

The need of the hour is to formulate clear guidelines which should be followed while using genetic engineering techniques in bio-medical research. e.g. products from transgenic organisms should be clearly marked to give choice to people who follow dietary restrictions due to religious beliefs. In fact all the ethical and moral issues raised by some aspects of biotechnology should be addressed by open discussion and dialogue.

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