Animal Cell Culture: Introduction, Types, Methods and Applications

• Animal Cell culture refers to the process by which cells are grown in a controlled artificial environment.
• Cells can be maintained in vitro outside of their original body by this process which is quite simple compared to organ and tissue culture. In a cell culture technique, cells are removed from an animal or a plant, and grown subsequently in a favorable environment.
• For animal cell culture the cells are taken from the organ of an experimental animal. The cells may be removed directly or by mechanical or enzymatic action.
• The cells can also be obtained by previously made cell line or cell strain. Examples of cells used to culture are fibroblast, lymphocytes, cells from cardiac and skeletal tissues, cells from liver, breast, skin, and kidney and different types of tumor cells.
• Usually, mammalian cells are found to be more delicate and more susceptible to mechanical damage. They have lesser growth rates and need more complex culture media along with special substrates.

Cell Culture Conditions

Media

Basal Medium

• It is extremely important to select a suitable medium. Different cell lines have different requirements for their growth. The most common basal media include Eagle Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle medium (DMEM), RPMI 1640, and Ham F10. All of them contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either in powder or in liquid form various commercial suppliers like Sigma, Invitrogen etc.

Supplements

• Bicarbonate plus CO2 and N-2-hydroxyethylpiperazinee -N’-ethane sulphonic acid (HEPES) are most common buffers.
• Each type of media has a recommended bicarbonate concentration and CO2 tension to achieve the correct pH and osmolarity.
• In addition to buffering the medium, essential amino acids such as cysteine and tyrosine as well as glutamine may be needed to meet certain growth requirements.
• L-Glutamine is also required by most cell lines since cultured cells use glutamine as an energy and carbon source in preference to glucose, although glucose is present in most defined media. L-glutamine is an unstable amino acid that converts to a form that cannot be used by cells, hence should be added to medium just before use.

Serum

• Serum is partially undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cell lines.
• Most cell lines require calf serum for adequate growth but often fetal calf serum provides the best growth conditions. Fetal calf serum (FCS) is often most commonly used, but for some applications less expensive sera such as horse or calf may also be used. Different serum batches should be tested to find the best one for each cell types since the quality varies a lot.

Antibiotics and fungicides

• Antibiotics and fungicides are used to prevent microbial contamination including bacteria, yeasts and molds. These include penicillin, streptomycin, kanamycin, nystatin and amphotericin B etc.

Additional supplements

• Primary cell culture requires some additional supplements such as collagen and fibronectin, hormones such as estrogen, and growth factors such as epidermal growth factor and nerve growth factor to attach to the cell culture vessel and proliferate.
• Media, serum and supplements should always be tested for sterility prior to their use by incubating
• a small aliquot at 37 °C for 24-48 hours. If microbial growth occurs, it should be discarded.

The following is the list of commercially used media for animal cell culture:

Incubation

• Cell lines should be incubated in a CO₂ incubator with a tightly regulated temperature and CO₂ concentration. Most cell lines grow at 37 °C in presence of 5% CO₂ with saturating humidity.

Preservation of cell lines

• The cell cultures are required to be stored for long term usage. The general procedure of preservation of all cell cultures is freezing.
• The cells should be frozen in exponential phase of growth with a suitable preservative like dimethylsulfoxide (DMSO). The cells are frozen slowly at 1 °C/min to -50 °C and then kept either at -196 °C immersed in liquid N2 or -70°C. Deterioration of frozen cells has been observed at -70°C, therefore, -196°C is better for storage and preservation.

Equipment and Facilities Required in Animal Cell Culture

• Animal cell culture laboratory requires some specific equipments and techniques which include the following.

1. Biosafety cabinet class II is a pre requisite for safe handling of human carcinoma cell lines.
2. Carbon dioxide incubators: Many cell lines can be maintained in an atmosphere of 5% CO₂:95% air at 99% relative humidity at around 30-40 °C using carbon dioxide incubator. The concentration of CO₂ has to be kept in equilibrium with sodium bicarbonate in the growth medium. The incubators are designed to allow CO₂ to be supplied from a gas cylinder which regulates supply of gas (2-5% as required by different cell lines).
3. An inverted microscope is essential to examine cell culture in dishes and flasks for their morphology and differentiation. Additional features of microscope include fluorescence, luminescence, CCD camera and monitor etc. to keep a check on the purity and viability of the cells in healthy status.
4. Cell culture ware/vessel: A variety of cell culture polystyrene plastic ware on which adherent cells can proliferate well. Cells can generally be maintained in petri dishes or flasks (25 cm2 or 75 cm2) and multi well dishes etc.

Types of Animal Cell Culture

A. Primary cultures

• Primary culture involves culturing of cells removed surgically from an animal tissue. The whole process of primary cell culture has been presented. There are two major steps involved in preparation of primary cultures viz. explant culture and enzymatic dissociation.
• Explant culture involves cutting tissues into small pieces and growing them into culture medium. Cells then move from explant and proliferate. The process however can be speeded up by using trypsin or collagenase. Once the cells in primary culture grow, they are subcultured for continuous growth.
• They are generally harvested by scrapping or trypsinization treatment. They are capable of only a limited number of cell divisions i.e. up to confluency state after which they enter a non-proliferative state called senescence and finally die out. At lower cell densities, however, the normal phenotype can be maintained.
• The advantages of primary cultures are that they are morphologically similar to the parent tissue and hence express tissue specific functions. Primary cells are extensively used by many researchers since they are physiologically more similar to in vivo cells. Moreover, cell lines when cultured for longer / extended periods can undergo phenotypic and genotypic changes that can lead to discrepancies when results from different laboratories are compared using the same cell line.
• Furthermore, many of the cell lines are not available as continuous cell lines. However, the
  disadvantage is that every time cells are required to be isolated afresh for each experiment.
  Secondly, proteolytic enzymes required for disruption can result into damage of membrane
  receptors; disrupt the integrity of the membrane, and loss of cellular products etc.
  

Process to obtain Primary culture

1. Primary cell cultures are prepared from fresh tissues.
2. Pieces of tissues from the organ are removed aseptically; which are usually minced with a
   sharp sterile razor and dissociated by proteolytic enzymes (such as trypsin) that break
   apart the intercellular cement.
3. The obtained cell suspension is then washed with a physiological buffer (to remove the
   proteolytic enzymes used).
4. The cell suspension is spread out on the bottom of a flat surface, such as a bottle or a Petri
   dish.
5. This thin layer of cells adhering to the glass or plastic dish is overlaid with a suitable culture
   medium and is incubated at a suitable temperature.

Depending on their origin, primary cells grow either as an adherent monolayer or in a Suspension:

(i) Adherent cells

• Adherent cells are said to be anchorage-dependent cells and propagate as a monolayer.
• The attachment to a substrate is a prerequisite for their proliferation.
• These adhere to the culture vessel with the use of an extracellular matrix which is generally
  derived from tissues of organs that are immobile and embedded in a network of connective
  tissue.
• They stop dividing when they reach confluency i.e they cover the whole surface and reach at
  such a density that they come in contact with each other.
• As being single layers, such cells can be transferred directly to a cover slip to examine under
  microscope.
• However, if they are left in confluent state for long, they lose their viability and die. Most of
  the cell lines grow in this manner e.g. HeLa cells, HT-29, INS etc.
• Adherent cells need to be separated from the culture dish by breaking the bond between
  cells and the surface using trypsin.
• The process is called trypsinization.
• The other proteolytic enzymes can also be used such as collagenase, pronase and papain etc.

(ii) Suspension cell

• Suspension cells do not adhere to the surface.
• They are generally in suspension or only loosely adherent.
• Cells from blood, spleen or bone marrow as well as some transformed cell lines and cells derived from malignant tumors can be grown in suspension.
• These cells grow much faster which do not require the frequent replacement of the medium and can be easily maintained.
• These are of homogeneous types and enzyme treatment is not required for the dissociation of cells; similarly these cultures have short lag period.
• However, the methods used to propagate these cells are very different from those for adherent cells. These methods are easy to perform since they do not need any trypsinization.

Confluent culture and the necessity of sub-culture

• After the cells are isolated from the tissue and proliferated under the appropriate conditions, they occupy all of the available substrate i.e. reach confluence.
• For a few days it can become too crowded for their container and this can be detrimental to their growth, generally leading to cell death if left for long time.
• The cells thus have to be subculture i.e. a portion of cells is transferred to a new vessel with fresh growth medium which provides more space and nutrients for continual growth of both portions of cells. Hence subculture keeps cells in healthy and in growing state.
• A passage number refers specifically to how many times a cell line has been sub-cultured.
  In contrast with the population doubling level in that the specific number of cells involved is
  not relevant. It simply gives a general indication of how old the cells may be for various
  assays.

B. Secondary cell culture and cell line

• When a primary culture is sub-cultured, it is known as secondary culture or cell line or subclone.
• The process involves removing the growth media and disassociating the adhered cells (usually enzymatically).
• Sub-culturing of primary cells to different divisions leads to the generation of cell lines.
• During the passage, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. However, as they are sub-cultured serially, they become different from the original cell.
• On the basis of the life span of culture, the cell lines are categorized into two types:

A. Finite cell lines

• The cell lines which go through a limited number of cell division having a limited life span are known as finite cell lines.
• The cells passage several times and then lose their ability to proliferate, which is a genetically determined event known as senescence. Cell lines derived from primary cultures of normal cells are finite cell lines.

B. Continuous cell lines

• When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.
• Such transformation/mutation can occur spontaneously or can be chemically or virally induced or from the establishment of cell cultures from malignant tissue.
• Cell cultures prepared in this way can be sub-cultured and grown indefinitely as permanent cell lines and are immortal.
• These cells are less adherent, fast growing, less fastidious in their nutritional requirements, able to grow up to higher cell density and different in phenotypes from the original tissue. Such cells grow more in suspension.
• They also have a tendency to grow on top of each other in multilayers on culture-vessel
  surfaces.
• In contrast to primary cell cultures, continuous cell lines, usually derived from transformed
  cells or tumors, are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in suspension) and may have lost their contact inhibition.
• As a result, continuous cell lines can grow in piles or lumps resembling small tumor
  growths.

Applications of animal cell culture technique

1. Cell cultures provide a good model system for studying basic cell biology and
   biochemistry, interaction between disease causing agents and cells, effect of drugs on cells,
   and process and trigger of ageing.
2. Toxicity Testing: Cultured cells are widely used alone or in conjunction with animal tests
   to study the effects of new drugs, cosmetics and chemicals on survival and growth in a wide
   variety of cell types. Especially important are liver- and kidney-derived cell cultures.
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3. Cancer Study: Cultured cancer cells also serve as a test system to determine suitable drugs
   and methods for selectively de-stroying types of cancer.
4. Virology: One of the earliest and major uses of cell culture is the replication of viruses in
   cell cultures (in place of animals) for use in vaccine production.
5. Cell based manufacturing: These include monoclonal antibodies, insulin, hormones, etc.
   The third is the use of cells as replacement tissues and organs. Artificial skin for use in
   treating burns and ulcers is the first commercially available product. However, testing is
   underway on artificial organs such as pancreas, liver and kidney.
6. Genetic counseling: Amniocentesis, a diagnostic technique that enables doctors to remove
   and culture fetal cells from pregnant women, has given doctors an important tool for the
   early diagnosis of fetal disorders. These cells can then be examined for abnormalities in
   their chromosomes and genes using karyotyping, chromosome painting and other
   molecular techniques.
7. Genetic engineering: The ability to transfect or reprogram cultured cells with new genetic
   material (DNA and genes) has provided a major tool to molecular biologists wishing to
   study the cellular effects of the expression of these genes (new proteins).
8. Gene therapy: The ability to genetically engineer cells has also led to their use for gene
   therapy. Cells can be removed from a patient lacking a functional gene and the missing or
   damaged gene can then be replaced. The cells can be grown for a while in culture and then
   replaced into the patient.
9. Drug screening and development: Cell-based assays have become increasingly im-portant
   for the pharmaceutical industry, not just for cytotoxicity testing but also for high
   throughput screening of compounds that may have potential use as drugs.

Plant Tissue Culture: Introduction, Types, Methods and Applications