paper no. : animal cell biotechnology module :06 basics of

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Animal Cell biotechnology

Basics of Animal Cell Biotechnology

Paper No. : 09Animal Cell Biotechnology

Module :06 Basics of Animal Cell Biotechnology

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director

The Energy and Resources Institute (TERI), New Delhi

Paper Coordinator: Dr. Minakshi, Professor & Head, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

Content Writer: Dr. Minakshi, Professor & Head, LalaLajpatRai University of Veterinary & Animal Sciences, Hisar

Paper Reviewer: Dr. Hari Mohan, Assistant Professor, Maharshi Dayanand University, Rohtak

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry

JamiaHamdard University, New Delhi

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Description of Module

Subject Name Biotechnology

Paper Name Animal Cell Biotechnology

Module Name/Title Basics of Animal Cell Biotechnology

Module Id 06

Pre-requisites

Objectives 1. To provide basic knowledge about the introduction to cell lines.

2. To explain, why we needed cell lines and their sub culturing.

3. To describe the history of culture media animal cell culture.

4. To provide knowledge about the applications of animal cell biotechnology

through cell culture in various fields.

Keywords Applications, cell lines, cell culture systems, gene farming, gene therapy

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1. Introduction

The idea of animal tissue culture in animal cell biotechnology comes first during 1903,

during the investigation of dividing cell techniques in vitro inside a test tube. Animal cell

culture was first time successfully established in 1907 by Ross Harrison. Till, the early of

1950, several developments has been take place which provide animal cell culture as a

technique for researcher. Firstly, the development of antibiotics and its use in animal cell

culture provide a great opportunity for doing contaminants free cell culture system that is

faced in the previous culture system. Secondly, the establishment of many techniques, for

example cells from the culture vessel is removed using typsin to obtain continuously growing

and healthy cell lines, e.g. HeLa cells. Thirdly, researchers were proficient to synthesize a

new consistent chemically modified culture medium which helps it the cell lines distant

easier to revive and grow. The technique of animal tissue culture was initially limited to cold

blooded animals. However, subsequent studies expends to the warm blooded animals into its

sphere. Since from many years explants used in from various, and tissue culture tools have

indeed become the backbone of animal cell biotechnology. The advanced biotechnological

tools have a great influence on animal cell biotechnology as well as on animal cell culture.

The basic techniques of animal cell biotechnology have wide variety of application in the

field of toxicity test, cancer study, virology, virology, drug selection and establishment,

genetic engineering etc.

2. History of animal cell culture

1878- After the death of an individual physiological systems can be sustained in a living healty

system Claude Bernard.

1885- In a saline culture embryonic chick cells are maintained by Roux.

1897- Loeb showed the cells survival extracted from connective tissue and blood in serum as

well as in plasma.

1903- Jolly reported the in vitro salamander leucocytes cell division.

1907- Frog nerve cells were cultivated by Harrison, father of cell culture and reported the in

vitro the growth of nerve fibres for several weeks.

1910- The lengthy period growth of embryo cell was reported by Burrows in plasma clots

and he made availability of explained the description of mitosis for us.

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1911- The primary liqud medium with a composition of serum, embryo extract, sea water, peptones

and salts was prepared by Lewis and Lewis.

1913- Carrel proposed and established the aseptic techniques so the cell culture could be

maintained for long periods.

1916- Proteolytic enzyme trypsin was introduced by Rous and Jones for adherent cells subculture.

1923- Carrel and Baker reported the development of first specially designed T-flask for cell culture

container and also working microscopic examination of cells.

1927- The first viral vaccine -vaccinia was produced by the Carrel and Rivera.

1933- The roller tube technique was developed by the Gey.

1940s- Antibiotics such as penicillin as well as streptomycin is used during cell culture in

medium reduce the contamination trouble in cell culture.

1948- From single cell clone Mouse L fibroblasts were isolated by Earle. Fischer developed

at the same time a chemically defined CMRL 1066 medium.

1949- Polio virus has been reported by the Enders and grown on human embryonic cells in

culture.

1952- Continuous HeLa cell lines from human cervical carcinoma was established

established by Gey.

1954- Contract inhibition motility of diploid cells was observed by Abercrombie in

monolayer culture stops when adjacent cells get in touch with them.

1955- The nutrient needs of cell cultures was studied and establishment of chemically defined

medium was done by Eagle.

1975- The first hybridoma cells secreting a monoclonal antibody was produced by Kohler

and Milstein.

1990: Recombinant products such as HBsAG, HIVgp120, factor VIII, GM-CSF, CD4, EGF,

IL-2, mAbs etc. comes in clinical trial.

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3. Types of Animal Cell Culture used in animal cell biotechnology

3.1 Primary Culture- Surgically and aseptically removed cells from an individual and kept

into a appropriate culture medium with maintained atmosphere conditions; they will adhere,

split and grow into cell culture vessel. This process is known a primary culture. Generally

two basic techniques are used for achieving this. In first treated in a plastic culture flask and

dipped in culture medium. After incubation at suitable environment condition for a specific

time period, cells will grow and move from the tissue explants onto the substrate surface and

start to split and multiply over the facade of vessel. The other way speeds up the procedure by

adding up digestion/proteolytic enzymes, for example trypsin and collagense to disturb the

interaction holding the cells and substrate. This method makes suspension of individual cell

which are than contained into culture flask having culture medium and stayed to divide and

grow. This process is described as enzymatic disaggregation. Figure 1 explains the basic

steps of primary animal cell culture.

Figure 1: Basic steps in primary animal cell culture.

3.2 Sub-culturing of the cells

Once the cells are present in primary culture flask are fully grown-up and overflowing up the

space in presented culture surface, at that time sub-culturing of these cells must be needed to

provide space for continued growth and division. This is always done by removal of these

cells as lightly as possible from the surface of flask by enzymes. These are same as enzymes

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used in prime culture obtaining and in the breakdown of the protein bonds holding the cells in

the flask surface. However, a few cell lines may be cultivated by softly removing the cells

from the underside of culture container. Some time ago the cells released the cell in the

suspension must be further divided and transferred into fresh culture flask.

When sufficient/surplus amount of cells are present, these must be given treatment with

suitable cryoprotective agents e.g. dimethyl sulfoxide or glycerol, cautiously frozen and

preserved at very low temperature (lees than -130°C) till they are further used.

4. Cell culture based systems

There are two main culture systems are in use for growth and division of cells. These are

depend mainly on the capability of cells to either they grow as adhered to a glass, known as

monolayer culture systems or floating freely in the culture medium described as suspension

culture systems.

Monolayer cultures are generally grown in treated tissue culture dishes, roller bottles. T-

flasks, cell stack culture chambers, the choice contains is being used is depend on the number

of cells required, the environment nature condition, cost and purpose. Suspension cultures are

generally grown either in magnetically rotated spinner vessels, where the cells remain in

suspended in the medium or in T-flasks and bottles as remain stationary phase in this system.

These cells arenot kept suspended: they are unable to attach firmly to the substrate. Many cell

lines are considered as anchorage-dependent, which derived from normal tissues and these

can grow only when they attached to a suitable surface.

4.2 Types of Cells

Cell culture is a complex process and selection of cells is very important. Cultured cells

usually described on the basis of morphology appearance and functional characteristics

(Figure 2). Most of the cultured cells have three basic morphologies:

4.2.1. Epithelial:

Epithelial like cells appear flattened and polygonal in shape with more regular dimensions.

They grow and attached to a substrate in discrete patches.

4.2.2 Lymphoblast-Like:

Lymphoblast like cells are spherical in shape and do not attached to the surface. They are

usually grown in suspension media.

4.2.3 Fibroblast-Like

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Fibroblast cells are bipolar or multipolar and have elongated in shapes. They grow

attached to a substrate and frequently forming swirls in heavy cultures. The culture conditions

play an important role in determining shape of cultured cells as many cells capable of

exhibiting multiple morphologies. Cells from fusion of two different parent/both parents, it is

possible to get hybrid cell. In 1975, this method was used to form cells capable in forming

custom tailored monoclonal antibodies. Through fusion of two different but related cells,

hybrid cells produce and called Hybridomas. The spleen-derived lymphocyte was the first

hybridomas that are capable in forming desired antibody and other is a fast dividing myeloma

cell having apparatus for producing antibodies but is not commanded to form any antibody.

The larger amount of required antibody is produced from this hybridoma. Antibodies

produced are known as monoclonal antibodies due to reason of their purity. These are having

much important diagnostic, clinical and industrial benefits with a yearly value more than a

billion dollors.

4.2.4 Functional characterisation

Cultured cells characteristics appeared from both origins (heart, liver etc) as well as culture

conditions in which they well adapt. If cells are maintained and perform specilized functions

that they done in vivo (e.g. liver cells), then the biochemical markers can be used to

determine those functions. Ultra characteristics and morphological markers can also be

studied (e.g. heart beating cells). These features frequently changed or lost as consequences

of being kept in an synthetic environment. Generally few cell lines will have progressively

show symptoms of aging and inhibit dividing.

These cell lines are known as finite. Another cell lines which divide indefinitely and become

immortal are known as continous cell lines. When a finite standard cell lines becomes

immortal, it is called transformation and it shows fundamental irreversible change. This could

be taken about intentionally using radiation, drugs/viruses or may occur spontaneously.

Transformed cells have abnormal extra chromosomes and repeatedly can be easily divided

and grown in suspension. Cells having normal chromosome numbers are known as diploid

cells, while aneuploid cells have different than normal chromosome number. If the cells form

tumours when injected into the animals, then they are well thought-out to be neo-plastically

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transformed.

Figure 2: Different animal cell lines used in animal cell biotechnology.

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5. Applications of Animal Cell biotechnology

5.1. Animal cell culture present a excellent model system for investigating: basic

biochemistry, cell biology, the effects of drugs on cells, and interactions between cells and

disease-causing agents, the process of aging and factors that triggers it and for nutritional

studies.

5.2 Toxicity Testing

Growth and Survival examination of cultured cells are widely used to study the effects related

to cosmectics, novel drugs as well as chemicals. Wide variety of cells alone or in association

with animal tests used to identify the belongings of disease causing agents. Liver and kidney

derived cultured cell are especially important.

5.3 Cancer research

The fundamental differences among cancer cell and normal cell can be closely studied

through animal culture technique, since both normal cells and grown in culture. A normal

cultured cell can be transformed to cancer causing cells by the use of chemicals, viruses and

radiation. In addition, it is possible to understand the mechanisms that cause cancer. Cultured

cancer cells also use as a test system to decide appropriate methods and medicines for chosen

destructing variety of cancer.

5.4 Virology

Study of virus replication in various cell lines (in place of animals) is widely use for vaccine

production. Cell cultures are also mostly used in isolation and detection of animal viruses

along with basic research to know their how it grow and infect organisms.

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5.5 Cell based manufacturing

Many important products produced from cultured cell, three broad areas are gaining the more

interst. Primarily is in the vaccine construction by the large scale cultivation of viruses. This

includes vaccines production for hepatitis B, polio, chicken pox, rabies and measles. Other

one, the great scale formations of proteins that have medicinal or commercial value from

genetically engineered cultured cells. For example- insulin, hormones, monoclonal

antibodies, etc. Third is the replacement of organs and tissues with cultured cells. In treating

ulcers and burns, artificial skin is primary commercially accessible product. Conversely,

testing is a process on artificial organs includes kidney, liver and pancreas. Adult and

Embryonic stem cells have the potential to supply replacement of tissues and cells which may

come out of experiment and great success in animal cell culture technology. These cells

contains the capability to divided into wide varieties of cell types. It anticipates new

treatment methods for a huge variety of medical situations and understanding how to prevent

the growth of these cells may provide.

5.6 Genetic Counselling

Amniocentesis is a crucial technique for the primitive control of fetal abnormalities, it is

analytical method that enables doctors to excise and culture fetal cells of pregnant women.

The abnormalities in chromosomes and genes can be determined through cell examined by

using chromosome painting, karyotyping and other molecular methods.

5.7 Genetic Engineering

Genetic engineering provided a big method to molecular scientists wish to identify cellular

effects of genes expression. It facilitates to reprogram cultured cells (transfect) with novel

genetic material (genes and DNA). For further study these tools has been used to form large

quality of novel proteins in cultured animal cells. Insect cells after being infected with

baculoviruses, genetically engineered vector are mainly used as factories to express and

produce substantial qualities of proteins.

5.8 Drug Screening and Development

Cytotoxicity testing and high throughput screening of compounds have the potential to

determine the proper use of drugs. Cell dependent assays have become increasingly crucial to

study these pharmaceutical products. Usually same cell culture tests were performed in 96

well plates. In modern molecular biology, Gene Therapy is an experimental technique that

involves insertion of cloned/altered genes into cells using r-DNA technology (Figure 3). It is

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very helpful to replace defective genes causing genetic abnormalities or to prevent potential

disorders.

Gene Therapy involves the swapping of harmful mutant alleles with functional ones by

selective reverse mutation. It also involves the deactivating of mutated gene having improper

functioning, inserting a novel gene in body to battle a illness and interchanging non-

functional gene with normal gene through homologous recombination.

Figure 3: Principle behind gene therapy

6. Conclusion

Animal cell as culture system is crucial to every branch of biosciences; mainly for pharmacy

and agriculture development. It is a necessary technique to identify the specific functions of

cell biochemical and physiological components. It can be useful in antiviral vaccines

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production, which requires the multiplication and assay of viruses. The major benefit of

animal culture is its reproducibility and consistency of experimental outcomes obtained from

clonal cells. Production of monoclonal antibodies and stem cells is the major application of

animal cell culture technology. Cell fusion methods and genetic modification is simple to

take out in cells as well as organ cultures. In addition we can use the animal cell

biotechnology for the study cancer and genetic disorders. Keeping these things in mind we

can use the animal conclude that cell culture is highly important in biotechnology so priority

should be given for the establishment of good laboratories practices particularly in emergent

countries, where they are not till completely developed.

paper no. : animal cell biotechnology module :06 basics of

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