Dairy Biotechnology

Module 4. Cell culture and fusion technology

Lesson 16
ANIMAL CELL CULTURE

16.1 Definition

Animal cell culture is the process of culturing animal cells extracted from tissues or organs under in vitro aseptically controlled laboratory environment (temperature, gases and pressure) simulating that of in vivo system. Under the controlled environment, the animal cells are able to survive and proliferate as under in vivo conditions.

16.2 History

The development of animal cell culture can be traced back to 1880 when Arnold showed that leucocytes can divide outside the body. Later, in 1903, Jolly studied the behavior of animal tissue explants immersed in serum and lymph, or ascites fluid. Ross Harison was able to culture frog tadpole spinal chord in a lymph drop hanging from a cover slip of a cavity slide in 1907. In 1912, Carrel initiated a culture of chick embryo heart cells which were passaged for a period of 34 years. The use of trypsin (a proteolytic enzyme) by Rous and Jones in 1916 was another significant break through required for the subculture of adherent cells and in the 1950s, the technique of trypsinization was exploited to produce continuously growing cell lines (HeLa cells). The practice of incorporating antibiotics such as penicillin and streptomycin, to the cell culture medium from 1940s onwards alleviated the problem of microbial contamination. Later, development of chemically defined media (Eagle and Eagle, 1950) led to the advantages of consistencies in various batches, easy sterilization and reduced the chances of contamination. The first product produced for mass vaccination was the polio vaccine which became the first major commercial product of cultured animal cells in 1950s. Animal cell culture has now become an alternative for animal experiments for drug discovery, evaluation of efficacy of several nutrients, herbals, probiotics, absorption and bioavailability studies etc.

16.3 Types of Animal Cell Culture

Animal cells may grow either as adherent monolayers or suspension cells.

16.3.1 Adherent cells

Adherent cells are said to be anchorage-dependent and the attachment to a substrate is a prerequisite for their proliferation. 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. 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, CaCO₂, 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.

16.3.2 Suspension cells

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. 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.

16.3.3 Primary cultures / cell lines

Primary culture involves culturing of cells removed surgically from an animal tissue. The whole process of primary cell culture has been presented in Fig. 16.1. 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. upto 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.

Fig. 16.1 Flow diagram of Primary cell culture development

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.

16.3.4 Continuous cultures / cell line

Continuous cell lines are developed from the cells that can be passaged indefinitely and express a reasonably stable phenotype. These cell lines have arisen spontaneously in normal cells being passaged in culture, but majority of them have been obtained by culturing tumor cells. In addition to being immortal (infinite life span), they share several additional properties that distinguish them from 'normal' cells in culture. Once a continuous cell line has been established, it is customary to clone the cells in order to obtain a genetically homogeneous population.

16.4 Cell Culture Conditions

16.4.1 Media

Basal Medium

Since cell culture medium affects the growth and proliferation of cell lines, it is extremely important to select a suitable medium. Moreover, 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.

16.4.2 Supplements

A number of supplements are added to the basal media to enable them to be used for culturing the cells. The optimum pH for most of the cell cultures lies between 7.4 – 7.7. Hence, the type of buffering that is used for the media depends on the growth conditions. Bicarbonate plusCO₂ 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.

16.4.3 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.

16.4.4 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.

16.4.5 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.

16.4.6 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.

16.4.7 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.
.
16.5 Equipment and Facilities Required in Animal Cell Culture

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

16.5.1 Biosafety cabinet class II

Biosafety cabinet class II is a pre requisite for safe handling of human carcinoma cell lines. A class II Bisoafety cabinet from Labconco has been illustrated in Fig. 16.2.

Fig. 16.2 Class II, type A2 Biological Safety Cabinets from Labconco

16.5.2 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 (Fig. 16.3). 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).

Fig 16.3 Carbon dioxide incubator from Sanyo

16.5.3 Microscope

An inverted microscope is essential to examine cell culture in dishes and flasks for their morphology and differentiation. An inverted microscope from Leica has been shown in Fig. 16.4. 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 good healthy status.

Fig. 16.4 Inverted Microscope from Leica

16.5.4 Cell culture ware/vessel

A variety of cell culture polystyrene plastic ware on which adherent cells can proliferate well, have been shown in Fig. 16.5. Cells can generally be maintained in petri dishes or flasks (25 cm² or 75 cm²) and multi well dishes etc.

Fig. 16.5 Cell culture plastic ware

16.6 Applications

Cell culture techniques are widely used in cellular and molecular biology research. Some of the areas where cell culture finds applications are listed below:

16.6.1 Model systems

Cell cultures provide a good model system for studying basic eukaryotic cell biology, biochemistry, effect of drugs/nutrients on cells etc.

16.6.2 Drug testing and efficacy

Cell culture plays an important role in pharmaceutical industry since these can be used to test the toxicity, efficacy and efficiency of a new drug. The cell lines can be used for high throughput screening of several compounds that may hold promise as drugs.

16.6.3 Production of genetically engineered therapeutic proteins

Animal cell cultures are being extensively used in the production of genetically engineered therapeutic proteins like insulin, hormones, monoclonal antibodies etc. from eukaryotes since they have the ability to introduce post transcriptional and post translational modifications in the expressed proteins to make them biologically active.

16.6.4 Gene therapy

Cells can be removed from a patient lacking a functional gene and then replacing the damaged gene and grown in culture before placing into the patient again.

16.6.5 Cancer research

Cell culture can be used to study normal versus cancer cells and also to look for drugs which can destroy cancer cells selectively.

16.6.6 Vaccine production

Several vaccines have been produced using cell lines like polio, rabies, chickenpox and measles etc.

Books

Culture of Animal Cells: A Manual of Basic Technique, 4^th Edition R. Ian Freshney (Editor), Wiley-Liss Publishers, ISBN: 0471348899

Animal Cell Culture and Technology: The Basics Michael J. Butler (Editor), Irl Pr Publishers, ISBN: 0199634165

Animal cell culture : A practical approach, John R.W. Masters (Ed.) Oxford University Press

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, R Ian Freshney, John Wiley and Sons

Internet Resources

http://en.wikipedia.org/wiki/Cell_culture

http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/intro_animal_cell_culture.pdf

http://www.ncbi.nlm.nih.gov/books/NBK21682/

http://www.uta.edu/biology/wilk/classnotes/tissue_culture/Primary%20culture.pdf

http://www.microbelibrary.org/component/resource/laboratory-test/3111-animal-cells-in-culture-protocols