cell components

8/4/2019 Cell Components

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Cells are the fundamental units of all organisms. Some organisms made up of only one cell,

but many more organisms are made of billions of cells. A cell is a packaged power plant that

maintains all necessary functions in order to stay alive. All cells have certain components that

enable them to carry out vital life processes. There are several different types of specialized

cells, but emphasize to students the basic structure of the cell.

A cell has several components that perform different functions. The vital parts of a cell are

called "organelles." Among the most important are the nucleus, vacuoles, and mitochondria,

all of which are enclosed within the cell membrane and immersed in cytoplasm.

Each organelle performs a specific task that helps keep the cell alive. In both an animal and

plant cell, the nucleus, vacuoles, mitochondrion, cell membranes, and cytoplasm can be

found.

The nucleus is the control center of cell activity and contains the genetic material that is

important for cell division. The structures that resemble air bubbles are called vacuoles. Some

vacuoles store food for future use while other store wastes until they are removed. Since thecell is a living entity, it needs energy. The energy that a cell uses is produced in the

mitochondrion which releases sugar and starches that is used as fuel by the cell. Most of the

parts of the cell are surrounded by a cell membrane. The function of the membrane is to

allow only certain fluids and chemicals into and out of the cell. Cells contain a thick substance

called cytoplasm which is capable of maintaining the life processes. The name of the

protoplasm within the nucleus is called nucleoplasm.

A plant cell also contains chloroplasts and a cell wall. Chloroplasts are the photosynthesis

center of a plant cell. It converts light into energy. It contains chlorophyll which gives it the

green color we associate with plants. The cell wall gives plant cell a rigid support and protects

the cell.

CHEMISTRY OF THE CELL

The living cell is a symphony of thousands ofchemical reactions all miraculously timed

and coordinated to perform all the functions necessary for life.

Amazingly, this symphony has only a few major players; only six elements carbon,

hydrogen, oxygen, nitrogen, phosphorus, and sulfur (sometimes called CHNOPS;>) makeup about 98% of the mass of all living organisms.

Carbon is a unique element with the remarkable ability to form strong, stable chemical

bonds with other atoms (keeps you from falling apart.) Each carbon atom can form four

bonds with other atoms. (Sometimes, two atoms will form more than one bond between

themselves making a double bond or even a triple bond.)

This bonding ability allows carbon atoms to form chains of almost unlimited length. These

chains can be closed on themselves to form rings or may branch wildly. This gives great

variety to the kinds of molecules that carbon can form. Below are just a few examples of

the many ways carbon chains can be arranged to form the skeleton for different molecules.


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Atoms of hydrogen and oxygen and less frequently nitrogen, sulfur, or phosphorous are

bonded to the carbon skeleton to form giant molecules called macromolecules. A few other

elements may occur in trace amounts.

The four major types of macromolecules found in living cellscarbohydrates, lipids,

proteins, and nucleic acids--are made of smaller, repeating subunits called monomers. Themonomers are not always identical but they always have similar chemical structures. They

are joined together by a series of chemical reactions in a process called polymerization to

form large, complex molecules called polymers.

The Four Major Types of Macromolecules Found in Living Cells

Macromolecule Elements Monomer Polymer example

Carbohydrate C, H, O Simple sugars Polysaccharide Starch

Lipids C, H, O Fatty acids &

glycerol

Lipid Fats, oils, waxes

Proteins C, H, O, N,

S

Amino acids Polypeptides Insulin

Nucleic acids C, H, O, P Nucleotides Nucleic acids DNA

The chemical diversity that polymerization allows living things is similar to the diversity

that our alphabet allows our language. Although there are only 26 letters in our English

alphabet, our ability to join them together to form words gives us an almost infinite variety

of possible words. Similarly, the monomer units of macromolecules can be arranged with

an almost endless potential for variety.

The functions of macromolecules are directly related to their shapes and to the chemical

properties of their monomers. The way the monomers are arranged in the macromoleculedetermines its shape and its function in the same way that the arrangement of the letters in

a word determine its sound and meaning.

Much of a cell's activities involve the arranging and rearranging and bonding of

macromolecules. It is the job of DNA both directly and indirectly to coordinate and direct

these activities.

An understanding of the structure and functions of carbohydrates and lipids is not

particularly key to the understanding of molecular genealogy. However, it may be helpful

to take a quick look at the structure and function of proteins before moving on to the nitty

gritty of the nucleic acids (of which DNA is one.)


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ANIMAL CELL STRUCTURE AND ORGANELLES

CELL MEMBRANE

The cell or plasma membrane was once thought to be a simple barrier that kept the contents of

the cell, the cytoplasm, contained. It serves as a gateway which helps to control materials

going in and out of the cell. With more research, it turns out the cell membrane is very

important in a wide range of cell activities including functions related to cancer and AIDS.

Structurally, the membrane is a lipid bilayer. What this means is that, under the electron

microscope two separate layers can be seen. The layers are composed of a two part molecule

called a phospholipid. The lipids (fatty acids) are "water fearing" (hydrophobic) molecules.

Just try to mix oil and water to see what that means. The phosphate end is water loving(hydrophilic). The membrane forms when the phosphate ends point out, attracted to the


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watery environment of the cell and the lipid ends point in, trying to avoid the water.

Embedded in this lipid bilayer are several different kinds of protein molecules.

The membrane does not form a solid sheet like a piece of cellophane. Its structure is called a

"fluid mosaic" because pieces of the membrane can rip off without creating a hole or merge in

with other membranes. The membranes surrounding the internal organelles, such as the

endoplasmic reticulum, Golgi bodies, lysosomes and vacuoles use this action to help carry out

their functions.

Jobs of the Cell Membrane

I. Transport

The cell membrane is the border surrounding the entire cell. Obviously if food, oxygen, water

and wastes are going to move in and out of the cell there has to be some way to do that.

2. The Immune System

If cells are going to fight off germ invaders they have to have a way to recognize which cells

belong to you and which cells don't. Cells identify themselves by marker molecules on the

cell membrane.

Cytoplasm

Cells are filled with organelles that each do something to keep the cell alive. The jelly-like

insides of a cell is called cytoplasm. Before electron microscopes, scientists thought there was

a living fluid called "protoplasm" inside the cell. They knew of only a few large organelles

such as the nucleus.. Special staining techniques also have shown us a cell skeleton.

Throughout the cytoplasm are three kinds of fiber like proteins -- tiny actin fibers,

intermediate fibers and thicker microtubules. These fibers help the cell keep its shape, anchor

organelles and allow for movement inside the cell.


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Vacuoles

Vacuoles are surrounded by membranes. They are sort of like a storage bubble in the

cytoplasm. Vacuoles in animal cells are considerably smaller than those in plant cells. Inanimal cells vacuoles may store food that needs to be digested. Food cannot pass through

membranes until it is broken into smaller particles. The lysosome can fuse with the vacuole

membrane and squirt digestive enzymes into the food vacuole to break down what is in there.

Your white blood cells do this when they eat invading bacteria. Vacuoles can also store the

undigestible wastes until they can fuse with the cell membrane and squirt the wastes outside.

Vacuoles in animal cells can form when the cell membrane surrounds a material and pinches

off to bring the substance inside the cell. This process is called endocytosis.

The Nucleus and Nucleolus

The nucleus is one of the most important organelles in the cell. It is sometimes referred to as

the brain or control center because of its role in controling development and life activities.The nuclear membrane surrounds material made up of thin, thread-like chromosomes. The


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individual chromosomes twist up and become visible only when the cell divides.

Chromosomes are made of DNA and protein. One or more dense areas can often be seen

inside the nucleus. This area is called the nucleolus and functions in RNA and ribosome

synthesis

It is DNA in the chromosomes in the nucleus that controls life. DNA carries coded

instructions to make RNA molecules which, with the help of ribosomes, build proteins.

Proteins are made of smaller molecules called amino acids. There are only around 20 different

amino acids in all living things. Simply arranging these amino acids in different ways creates

all the variety of life on earth. It is proteins that make you different from any other human

being and every organism different from any other organism. Your DNA code tells your body

how to make your unique proteins. How can proteins be so important? Some of those proteinmolecules are the structural building blocks that make cells grow. Other proteins serve as

enzyme molecules - organic catalysts that control all of the complex chemical reactions that

makes life happen. The nucleus controls life because it controls the production of these, and

other, important protein molecules.

The nuclear membrane is connected to the endoplasmic reticulum (ER) - a canal system used

to transport molecules throughout the cell. The ER is in turn associated with golgi bodies

which collect proteins made by the ribosomes and package them in little bags called vessiclesto be sent out of the cell.

Endoplasmic Reticulum (ER)


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The endoplasmic reticulum or ER is like a maze of membrane canals running throughout

cells. It is closely related to several other membrane bound organelles in the cell. Through an

electron microscope you can see that some ER is covered with tiny dots called ribosomes.

This is called rough endoplasmic reticulum. Ribosomes help to make proteins and the canal

system of the endoplasmic reticulum will transport these proteins around the cell. Sometimes

other molecules are combined with the proteins. The transported protein may end up in the

Golgi complex to be packaged for shipment out of the cell. Or the protein may end up as part

of a cell organelle or the cell membrane.

Especially in cells that make or use lipid molecules, there is also smooth ER with no

ribosomes. Lipids include steroid hormones like testosterone. The liver also has a lot of

smooth ER which may help it to take harmful material such as certain drugs out of the blood.

Golgi Complex


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The Golgi complex, endoplasmic reticulum, lysosomes and cell membrane can all work

together. In case you haven't read about the cell membrane yet, it is important to know that

membranes are not solid like a piece of plastic wrap. The membrane structure is called a

"fluid mosaic" because it can break apart or join together without leaving a hole. It might help

to picture a solid layer of ping pong balls floating in a bucket of water. You can push your

hand right through them and when you pull it out, they fill in the hole again. Of course

bonding in the membrane lets it hold the cell together better than floating ping pong balls

would, but being able to break apart and fuse together is important to the function of the Golgi

complex.

Structure and Function

The Golgi complex is like the cell's packaging and shipping department. It is made up of a

stack of flattened membrane sacks. Some of the protein being transported through the canals

of the endoplasmic reticulum ends up in the Golgi complex. Here it may be joined with other

molecules before being "packaged". The packages are little pieces of the Golgi complex

which break off and form "vesicles". The vesicles move to the cell membrane and fuse with it.It may the squirt its contents outside of the cell as a secretion. Or the product assembled in the


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Golgi complex may be a new piece of the cell membrane itself. In that case the vesicle fuses

with the membrane and becomes a part of it. In plant cells the Golgi complex can assemble

material needed for the cell wall

Lysosomes

Lysosomes are sometimes called "suicide bags". Can a cell commit suicide? Yes, whether

intentionally or not. A lysosome is a membrane bag containing digestive enzymes. When a

cell needs to digest food, the lysosome membrane fuses with the membrane of a food vacuole

and squirts the enzymes inside. The digested food can then diffuse through the vacuole

membrane and enter the cell to be used for energy or growth. The only thing that keeps the

cell itself from being digested is the membrane surrounding the lysosome. (We don't really

know what keeps the membrane from being digested!)

Lysosomes are formed when the Golgi complex packages up an especially large vessicle of

digestive enzyme proteins

Centrioles

Centrioles generally appear in animal cells as two cylinders at right angles to one another,

close to the nucleus. When viewed with an electron microscope, the cylinders show up as nine

bundles of tiny microtubules arranged in a circle. The centrioles help to form the spindle

fibers. Spindle fibers are microtubules that move chromosomes aroud when the cell is

dividing.

Mitochondria


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Mitochondria can be called the "powerhouses" of the cell. This means that they help produce

the energy needed to live. We know that we get energy from food, but what most people don't

know is that we cannot use food's energy directly. There is only one usable form of energy

throughout the entire living world -- a molecule called ATP. Organisms differ in how they get

their supply of ATP. Most living organisms use food and oxygen to make ATP in a process

called cellular respiration. Most of cellular respiration takes place inside the mitochondria.

This equation shows what happens to the main molecules involved in cellular respiration:

glucose sugar (food) + 6 oxygen molecules --> 6 carbon dioxide molecules + 6 water

molecules + 38 ATP molecules

The Structure of a Mitochondrion

Mitochondria are a lot like chloroplasts in their structure. Their function is also similar,

though in many ways, the reverse of each other. (Check out the chloroplast page to see for

yourself.) In both cases there are two layers of membranes. The membranes form little spaces.

These spaces can be used to bring about the energy conversions of both processes. Click here

to see another diagram of a mitochondrion.

The Chemistry of Cellular Respiration


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Cellular respiration takes place in several stages. Basically the secret of releasing energy

stored in chemical bonds is breaking the bonds. The more bonds you break, the more energy

you can get out. The first stage of cellular respiration just sort of breaks the glucose in half.

This doesn't really release much energy. The two halves go into the mitochondria where the

enzymes stored there, piece by piece, rip apart the rest of the glucose molecule, breaking lots

of bonds and releasing a lot more energy. Finally all that is left is some carbon dioxide and

water. Oh yes, and all of the energy, which is now stored in molecules of ATP. Click here to

find out some more about how this chemical process works.

Mitochondria are one of the best examples of how organelles form compartments that can

isolate from the rest of the cytoplasm all of the enzymes necessary to carry out a specific

process. In fact scientists think that mitochondria were themselves once independentorganisms capable of reproducing on their own. They contain tiny bits of DNA - hereditary

material that controls life. An interesting fact is that the DNA code in all of the mitochondria

in your cells came only from your mother. This has been used to help identify the true parents

of children separated from their families by war.

Ribosomes

Through an electron microscope, ribosomes look like tiny dark dots. Sometimes they are

floating free in the cytoplasm and sometimes they are attached to membranes like the

endoplasmic reticulum and nuclear membrane. The job of the rtibosomes is to help make

proteins. They are needed to translate the genetic code brought from the nucleus to the

cytoplasm by messenger RNA (mRNA) molecules. This genetic code is nothing more than

the instructions for linging up amino acids in the right order to make proteins or polypeptide

chains (pieces or proteins). Proteins then help the cell to grow or are used to control chemicalreactions that make everything happen in the cell.


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THE SIMILARITIES BETWEEN ANIMAL AND PLANT CELLS

The similarity that Plant and Animal Cell have is they both have Nucleus, Cell Membrane,

Vacuole, Cytoplasm, and Mitochondrion.

Plants and Animal Cell perform respiration that is the similar function they perform. Only

plants cells perform photosynthesis which is the different function it performs.

The difference between an Animal cell and a Plant cell

The difference between an Animal cell and a Plant cell is that Plant cell has more features

than the animal cell. Plant cell contains all the special features of an animal cell but animal

cell is lacking a few of plant cell features. For example animal cell does not have chloroplast.

Another difference is the way they are shape. Since animal cell don't have a cell wall to keep

it in place like a plant cell.

Plant cells have cell walls, which make them appear rectangular-shaped. Plant cells have

chlorophyll, the light-absorbing pigment required for photosynthesis. This pigment is

contained in structures called chloroplasts, which makes plants appear green. Respiration

happens in the mitochondrion of plants and animals. Photosynthesis only happens in plants

which is chloroplast.

CELL CULTURE

Cell culture is the process by which cells are grown under controlled conditions. In practice

the term "cell culture" has come to refer to the culturing of cells derived from multicellular

eukaryotes, especially animal cells. The historical development and methods of cell culture

are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a common laboratory technique in the mid-1900s,[1] but the

concept of maintaining live cell lines separated from their original tissue source was

discovered in the 19th century.[2]

History

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the

chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating

of an isolated animal heart outside of the body.[1] In 1885 Wilhelm Roux removed a portion

of the medullary plate of an embryonic chicken and maintained it in a warm saline solution

for several days, establishing the principle of tissue culture.[3] Ross Granville Harrison,


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working at Johns Hopkins Medical School and then at Yale University, published results of

his experiments from 1907-1910, establishing the methodology of tissue culture.[4]

Cell culture techniques were advanced significantly in the 1940s and 1950s to supportresearch in virology. Growing viruses in cell cultures allowed preparation of purified viruses

for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-

produced using cell culture techniques. This vaccine was made possible by the cell culture

research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins,

who were awarded a Nobel Prize for their discovery of a method of growing the virus in

monkey kidney cell cultures.

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Concepts in mammalian cell culture

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Isolation of cells

Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easilypurified from blood, however only the white cells are capable of growth in culture.

Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes

such as collagenase, trypsin, or pronase, which break down the extracellular matrix.

Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are

available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception

of some derived from tumours, most primary cell cultures have limited lifespan. After acertain number of population doublings cells undergo the process of senescence and stop

dividing, while generally retaining viability.

An established or immortalised cell line has acquired the ability to proliferate indefinitely

either through random mutation or deliberate modification, such as artificial expression of the

telomerase gene. There are numerous well established cell lines representative of particular

cell types.


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Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically,

37C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for

each cell type, and variation of conditions for a particular cell type can result in different

phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems

is the growth medium. Recipes for growth media can vary in pH, glucose concentration,

growth factors, and the presence of other nutrients. The growth factors used to supplement

media are often derived from animal blood, such as calf serum. One complication of these

blood-derived ingredients is the potential for contamination of the culture with viruses or

prions, particularly in biotechnology medical applications. Current practice is to minimize or

eliminate the use of these ingredients wherever possible, but this cannot always be

accomplished.

Cells can be grown in suspension or adherent cultures. Some cells naturally live in

suspension, without being attached to a surface, such as cells that exist in the bloodstream.

There are also cell lines that have been modified to be able to survive in suspension cultures

so that they can be grown to a higher density than adherent conditions would allow. Adherent

cells require a surface, such as tissue culture plastic, which may be coated with extracellular

matrix components to increase adhesion properties and provide other signals needed for

growth and differentiation. Most cells derived from solid tissues are adherent. Another type of

adherent culture is organotypic culture which involves growing cells in a three-dimensional

environment as opposed to two-dimensional culture dishes. This 3D culture system is

biochemically and physiologically more similar to in vivo tissue, but is technically

challenging to maintain because of many factors (e.g. diffusion).

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Cell line cross-contamination

Cell line cross-contamination can be a problem for scientists working with cultured cells.

Studies suggest that anywhere from 1520% of the time, cells used in experiments have been

misidentified or contaminated with another cell line.[5][6][7] Problems with cell line crosscontamination have even been detected in lines from the NCI-60 panel, which are used


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routinely for drug-screening studies. [8][9] Major cell line repositories including the

American Type Culture Collection (ATCC) and the German Collection of Microorganisms

and Cell Cultures (DSMZ) have received cell line submissions from researchers that were

misidentified by the researcher.[8][10] Such contamination poses a problem for the quality of

research produced using cell culture lines, and the major repositories are now authenticating

all cell line submissions.[11] ATCC uses short tandem repeat (STR) DNA fingerprinting to

authenticate its cell lines.[12]

To address this problem of cell line cross-contamination, researchers are encouraged to

authenticate their cell lines at an early passage to establish the identity of the cell line.

Authentication should be repeated before freezing cell line stocks, every two months during

active culturing and before any publication of research data generated using the cell lines.

There are many methods for identifying cell lines including isoenzyme analysis, human

lymphocyte antigen (HLA) typing and STR analysis.[12]

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Manipulation of cultured cells

As cells generally continue to divide in culture, they generally grow to fill the available area

or volume. This can generate several issues:

Nutrient depletion in the growth media

Accumulation of apoptotic/necrotic (dead) cells.

Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing known as

contact inhibition or senescence.

Cell-to-cell contact can stimulate cellular differentiation.

Among the common manipulations carried out on culture cells are media changes, passaging

cells, and transfecting cells. These are generally performed using tissue culture methods that

rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast,

or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow

cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and

streptomycin) and antifungals (e.g. Amphotericin B) can also be added to the growth media.


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As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH

indicator is added to the medium in order to measure nutrient depletion.

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Media changes

In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

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Passaging cells

Main article: Passaging

Passaging (also known as subculture or splitting cells) involves transferring a small number of

cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it

avoids the senescence associated with prolonged high cell density. Suspension cultures are

easily passaged with a small amount of culture containing a few cells diluted in a larger

volume of fresh media. For adherent cultures, cells first need to be detached; this is commonlydone with a mixture of trypsin-EDTA, however other enzyme mixes are now available for this

purpose. A small number of detached cells can then be used to seed a new culture.

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Transfection and transduction

Main article: transfection

Main article: transformation (genetics)

Another common method for manipulating cells involves the introduction of foreign DNA by

transfection. This is often performed to cause cells to express a protein of interest. More

recently, the transfection of RNAi constructs have been realized as a convenient mechanism

for suppressing the expression of a particular gene/protein.


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DNA can also be inserted into cells using viruses, in methods referred to as transduction,

infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA

into cells, as this is a part of their normal course of reproduction.

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Established human cell lines

One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer

that those cells originated from, the cultured HeLa cells shown here have been stained with

Hoechst turning their nuclei blue.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they

may outlive their parent organism and later be used in the discovery of lucrative medical

treatments. In the pioneering decision in this area, the Supreme Court of California held in

Moore v. Regents of the University of California that human patients have no property rights

in cell lines derived from organs removed with their consent. [13]

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Generation of hybridomas

For more details on this topic, see Hybridoma.

It is possible to fuse normal cells with an immortalised cell line. This method is used toproduce monoclonal antibodies. In brief, lymphocytes isolated from the spleen (or possibly

blood) of an immunised animal are combined with an immortal myeloma cell line (B cell

lineage) to produce a hybridoma which has the antibody specifity of the primary lymphoctye

and the immortality of the myleoma. Selective growth medium (HA or HAT) is used to select

against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused

cells survive. These are screened for production of the required antibody, generally in pools to

start with and then after single cloning.

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Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and

many products of biotechnology. Biological products produced by recombinant DNA (rDNA)technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals

(monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many

simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins

that are glycosylated (carbohydrate-modified) currently must be made in animal cells. An

important example of such a complex protein is the hormone erythropoietin. The cost of

growing mammalian cell cultures is high, so research is underway to produce such complex

proteins in insect cells or in higher plants.

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Tissue culture and engineering

Cell culture is a fundamental component of tissue culture and tissue engineering, as it

establishes the basics of growing and maintaining cells ex vivo.

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Vaccines

Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell

cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza

vaccines is being funded by the United States government. Novel ideas in the field include

recombinant DNA-based vaccines, such as one made using human adenovirus (a common

cold virus) as a vector,[14][15] or the use of adjuvants. [16]

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Culture of non-mammalian cells

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Plant cell culture methods


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See also: Tobacco BY-2 cells

Plant cell cultures are typically grown as cell suspension cultures in liquid medium or as

callus cultures on solid medium. The culturing of undifferentiated plant cells and callirequires the proper balance of the plant growth hormones auxin and cytokinin.

[edit]

Bacterial/Yeast culture methods

Main article: microbiological culture

For bacteria and yeast, small quantities of cells are usually grown on a solid support that

contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are

grown with the cells suspended in a nutrient broth.

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Viral culture methods

The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial

origin as hosts for the growth and replication of the virus. Whole wild type viruses,

recombinant viruses or viral products may be generated in cell types other than their natural

hosts under the right conditions. Depending on the species of the virus, infection and viral

replication may result in host cell lysis and formation of a viral plaque.

CELL CULTURE LABORATORY

Maintenance of trouble-free cell cultures depends on careful attention to culture conditionsand passage procedures. It is also vital to pay strict attention to three characteristics that are

fundamental to the quality of cell culture assays: purity, authenticity and stability.

Purity: Contamination with microorganisms such as bacteria and fungi will normally kill the

cells and put other cultures in the laboratory at risk. Mycoplasma contamination can have

serious effects on a cell culture (see below) without inhibiting cell growth and, furthermore,

the presence of such contamination will rarely be apparent even under microscopic

observation. This is due to the extremely small size of mycoplasma organisms that can enablethem to pass through sub-micron filters. As with bacteria and fungi, mycoplasma can spread


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readily to other cultures but are not susceptible to many of the antibiotics effective against

bacterial contamination. While viral contamination typically produces a cytopathic effect in

cell cultures, persistent non-cytopathic infections may arise that can influence virological

investigations and may represent a hazard to laboratory workers (e.g. Epstein Barr Virus

expressed by B95-8 and B95a cells). Screening for viral contamination can be extremely

costly and time consuming. Routine checks for bacteria, fungi and mycoplasma, however, are

relatively easy to establish and will provide confidence in the quality of cell culture results.

Authenticity: Accidental switching of cell lines or cross-contamination between cultures has

been identified in numerous cases and can result in erroneous or misleading data. Obtaining

documentary evidence for the authenticity of new cell lines and identity testing are therefore

important means of avoiding wasted time and effort. All cell lines used in the polio

eradication initiative should be obtained through the Global Polio Laboratory Network. To

avoid cross-contamination only one cell line should be handled at a time in a cabinet, and

between culture sessions the work area should be stringently cleaned and disinfected.

Stability: Cell cultures serially passaged over an extended period of time will invariably show

some signs of variation in genetic or phenotypic characteristics. The susceptibility to such

variation will differ between cell lines. To minimize the effects of cell line deterioration it is

strongly recommended that all cell lines used routinely for polio isolation be replaced after a

maximum of 15 sequential passages.

4.1.1 Basic requirements for cell culture

Although the cost of laboratory space and equipment necessary for the handling of cell

cultures in a diagnostic virology laboratory can be reduced to relatively modest levels, certain

essential items are required. Due to the difficulty of cleaning and recycling glassware to cell

culture quality, many laboratories have resorted to using disposable cell culture plasticware. It

is recommended that all laboratories use cell culture plasticware for as many processes as

possible. The standard list of items for cell culture is displayed in Table 4.1.

4.1.2 Laboratory layout and operation

Cell culture should be performed in an environment that is tidy and not crowded or otherwise

busy. Environmental contamination should be kept to a minimum through good housekeeping

and cleaning regimes and some provision should be made for the isolation of untested and

contaminated cultures. The important principles and approaches that may be adopted to

ensure satisfactory operation of a cell culture laboratory include:


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Only essential personnel should have access to cell culture areas.

Cell culture areas should be dedicated for this purpose and separate laboratories or

areas established for other work.

Each cell culture work station should be organized such that all items needed are

readily to hand, avoiding the necessity to withdraw from the safety cabinet while handling

cells.

Laboratory layouts should allow for easy movement of personnel between the safety

cabinet and fridges, centrifuges, incubators, etc.

The use of sinks in the cell culture area should be avoided since these can be a source

of microbial contamination.

For safety reasons liquid nitrogen storage areas should be well ventilated.

Standard operating procedures should be established for:

- waste disinfection and disposal;

- procedures for disinfecting equipment such as centrifuges and BSCs;

- water bath cleaning/disinfection;

- cleaning of work surfaces and floors;

- periodic thorough cleaning to prevent build-up of contamination and dust (e.g. on high

flat surfaces, outside of BSCs, underneath and behind equipment, inside fridges and freezers.


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Use of equipment

For successful and reliable isolation of viruses in cell culture it is vital that equipment used for

manipulation and cultivation of cells is calibrated and monitored appropriately. Eachlaboratory should maintain a file for each piece of equipment that identifies calibration and

maintenance requirements and holds records of these procedures and routine data recording.

Small laboratory equipment (pipettes, pipettors etc.) should be dedicated for cell culture

activities and should not be shared with laboratories handling microorganisms.

Biological safety cabinets: Most cell culture handling is now carried out in Class II Biological

safety cabinets. These cabinets maintain a clean working environment for cell handling and

help to provide protection to the operator and environment. Horizontal laminar flow cabinetsare useful for media preparation but are not desirable for cell culture work due to the risk of

possible contaminants in the cell culture being blown into the face of the operator. The

effectiveness of a BSC is dependent on its position, correct use, regular testing and

maintenance. An example of good practice for all of these aspects is given in the British

Standard BS5726 (accessible for a fee at the web site http://bsonline.techindex.co.uk/).

Cabinets should be sited away from doors and through-traffic. Movement in the area of a BSC

will disturb airflow and so access to the area should be restricted to essential personnel. When

working within a BSC it is important to minimize the potential for contamination of the

working environment and cross-contamination between cell lines.

Incubators: There are two classes of incubators, standard and carbon dioxide (CO2). The

following steps should be taken concerning the correct use of incubators:

New incubators should be installed, calibrated and maintained according to the

manufacturer's instructions. Refer to Section 3.4 of the Polio Laboratory Manual concerning

laboratory equipment.

Refer to Section 4.3 of the Polio Laboratory Manual for a description of the types of

media recommended for growing cell cultures in each class of incubator.

Incubators designated for cell culture must not be used for incubating microorganisms

or biochemical specimens.


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The incubator temperature should be set to 36C.

An independent traceable thermometer should be set inside the incubator chamber and

monitored every day by the first person to open the chamber door. A record of the readingsneeds to be maintained and archived.

Every two to three months the incubator should be emptied and all shelves pulled out.

Everything, including the shelves, walls, top and bottom, should be cleaned thoroughly with

either a disinfectant or 10% bleach solution, then rinsed thoroughly with clean distilled water

to remove residues than can cause corrosion and toxicity to cell culture, then dried and put

back in place.

All spills must be contained and disinfected immediately.

In addition to the thorough cleaning listed above, it is recommended to wipe shelves

with 70% ethanol solution weekly.

Standard incubators (non-CO2): These incubators are simple, effective and usually veryreliable but require all cell culture vessels to be well sealed in order to maintain proper pH for

cell culture (between 7.2 and 7.4) as well as prevent evaporation of the culture medium.

All cell culture flasks, tubes or plates in a non-CO2 incubator must be well sealed. Close the

lids of flasks or tubes securely. Seal the lids of microtitre plates with non-toxic sealing tape or

place them in a sealed plastic box with moistened paper in the bottom. This helps to reduce

the risk of cross-contamination with other cultures.

CO2 incubators: These incubators provide humidity and a 5% CO2 atmosphere for cell

culture. In addition to the steps listed in the general information above, the following should

be considered:

A constant and reliable supply of clean, high quality CO2 is required.


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To allow proper exchange of the humid 5% CO2 atmosphere with the medium, cell

culture flasks, tubes and plates should not be sealed, but culture vessels with vented caps

should be used.

CO2 levels should be checked and recorded three to four times a year using a Fyrite

apparatus (or equivalent) as internal calibrations often drift over time.

A water tray placed in the incubator supplies the humidified environment.

Every week the water tray needs to be emptied, wiped with 70% ethanol and refilled

with autoclaved distilled water to prevent bacterial and fungal contamination. Alternatively,antibacterial/antifungal agents as recommended by the manufacturer may be added to the

water, but many chemicals can be toxic to cells, corrosive, and dangerous to humans.

In addition to the cleaning procedures listed above, special care must be taken in humid

chambers to check the water tray and chamber daily. Wipe all condensation from the shelves

and bottom of the incubator as well as all the gaskets to prevent fungus, mould and mildew

from becoming established.

Temperature, CO2 and relative humidity readings should be taken daily and a record of

the readings maintained and archived.

Water-purifying apparatus: The use of high purity, toxin-free, water (type I water) is essential

for successful cell culture. Glass distillation or other water purification techniques (e.g.

deionization) may be used but the water for cell culture should be autoclaved before use. One

of the best sources of water for preparing cell culture media is pharmaceutical grade water forinjection. All water for cell culture should be used as soon as possible after collection, as

storage in any container will allow degradation of the purity of the water unless both the water

and container are sterilized.

Double glass distillation: This was the first system to be used widely for cell culture and is

still effective. The still should be electric and the first still should automatically feed the

second still. Distillation has the advantage that the water is heat-sterilized and of high purity

but disadvantages include the large volume of water required for the distillation processes,slow production rate, high cost of operation and requirement for constant maintenance.


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Deionization: Deionization requires a multistage process where the feed water is purified

step-wise. The first step is usually reverse osmosis and subsequent steps can remove organic

or inorganic materials (activated carbon) with further purifying steps (deionization) which

may include membrane filtration depending on the quality of the feed water. The quality of

the final product can be monitored using a conductance meter, which ideally should be around

18 megohm/cm. Advantages of the deioniser system are the high throughput of pure water,

low wastage of water and the fact that the system can be modified according to the quality of

the feed water.

cell components

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