Preface Cell culture is an essential and indispensable tool in many branches of the life sciences and the applications of cell culture is getting increased exponentially in  various fields of biological and medical research arena.  It  provides the basics for studying the regulation of cell proliferation, differentiation and product formation. Viruses replicate only within the living cells and with the advent of cell culture technique, it has  become easier to grow viruses  under in vitro condition. Besides due to agitation shown by the animal activist groups, religious people and animal ethics committee members, testing of different  metals (arsenic, cadmium, mercury etc), toxic substances, toxins, drugs, medicines, viruses etc in animals have become a nightmare. The use of cell culture  made it easier  for  testing of above mentioned substances. This book entitled “Animal Cell Culture and Virology” is intended to explain why and how the basic techniques are used  and how to set up a cell culture laboratory and detailed procedures of different techniques  of cell culture starting from media filtration, sterilization of glasswares, plasticwares etc, counting of cells, cryopreservation of cells and revival, primary cell culture, secondary cell culture, contamination and curing,  applications, advantages and disadvantages of cell culture, isolation and identification of viruses in cell culture, SNT, titration of viruses, scaling up of cell culture, collection, preservation and dispatch of samples for diagnosis of viral diseases, cell line authentication and characterization etc have been described in a simple and easily understandable manner. Lastly it will be useful not only for undergraduates, graduates, technicians,  researchers but also teachers and scientists directly and indirectly associated with cell culture in day to day experiments. In spite of my best efforts at perfection, element of human error is  still likely to creep, the author will appreciate receiving any comments on the  improvement of the book.

Cell culture has become one of the major tools used in the life sciences today. Viruses replicate only within the living cells. With the advent of cell culture technique, it has become easier to grow the viruses under in vitro condition (cell culture). Viruses are usually propagated in cell culture, embryonated hen’s egg, laboratory animals or natural hosts. In 1949, Enders, Weller and Robbins first reported that polio virus could be grown in cultured non-neural cells with the production of recognizable cytopathic changes. Although the cell culture technique has been evolved long back, it is only since the advent of antibiotics that cell culture became a matter of simple routine technique. Aseptic precautions are still essential, but the problems of contamination with bacteria, mycoplasma, fungi and yeasts can be checked by antibiotics and anti-fungal agents. The term in vitro literally means ‘in glass’ although today most cell culture is performed in or on plastic.

The cell is the unit of structure of all plants and animals. With increasing complexity of organization, masses of different cell types tend to become localized and to form recognizable patterns. An aggregation of cells forming a definite pattern is called tissue. A still higher level of organization, tissues aggregate to form organs. The animal cell is a highly organized structure. Plant cell is different from animal cell  by having a cellulose cell wall.  There are a number of well defined structures viz., nucleus, mitochondria, lysosomes, Golgi apparatus etc in the cell. Animal cells also have centrioles whereas plant cells contain plastids such as chloroplasts. The remainder of the cell is the cytoplasm.

The best environment for growing cells is similar to  the conditions what they experience in vivo. A number of various environmental factors affecting the tissues are :       (1)    Temperature : In living matter, complicated chemical reactions are happened at comparatively low temperature.  In most of the mammalian and avian tissues the temperature  is 37ºC to 38.5ºC. If the temperature is enhanced to 45ºC, cells are destroyed within  an hour but survive 12-24 hours at 42ºC. It is applicable to most of the fibroblasts and epithelial  types of cells arising  from  mammalian tissues. Some of the mammalian cells ( human skin epithelial cells)  and amphibian cells  prefer lower temperature but fish tissue will not survive beyond 20ºC.

Types of cells : Animal cells are usually defined by the tissue of their origin. On the basis of morphology, they can be divided into 5 types.     1.    Epithelial cells : Epithelial tissues consist of a layer of cells which cover organs and line cavity. Example skin and the lining of alimentary canal. They grow well as single cell monolayer in culture.     2.    Fibroblast cells : Connective tissue form the major structural component of animals. The tissue contains fibroblasts and most widely used cell in the laboratory. Fibroblasts are bound to the fibrous protein collagen in the connective tissue. They have excellent growth characteristics.

A finite growth capacity is a characteristic of all cells derived form the normal animal tissue. The cells pass through a series of age related changes before being incapable to divide further. The finite number of generations of growth is a characteristic of the cell type, age and species of origin and referred to as the “Hayflick limit”. The cells derived from embryonic tissue have a greater growth capacity than those derived from adult tissue.

A cell culture laboratory should allow the sterile handling of cultured cells with no or minimum level of contamination. The growth of bacteria and fungi is very high and no level of contamination can be allowed in animal cell culture. However, it is impractical to design a laboratory free from potentially contaminated microorganism. Further, it is possible to establish a laboratory with minimal risk of contamination. Several points should be taken into consideration while designing a new cell culture laboratory.

Once cells are obtained from a culture collection or isolated from animal tissue,, cells are put in the flask with growth medium. The cells are inoculated at a density of 104-105 cells/ml to get a concentration of 106 cells/ml or 105 cells/cm2 in a solid surface in 2-3 days because of nutrient limitation, accumulation of toxic metabolite or lacking of growth surface.

Cell culture medium : To grow cells in in vitro, environment should be as close as possible to in vivo condition. But a number of factors are important to grow cells in vitro namely substrate, temperature and medium. There is a number of ready made medium available commercially and made the cell culture work easier. Any successful medium consists of isotonic buffer solution containing inorganic salts, amino acids, vitamins, a energy source and various supplements. Usually serum or serum replacement is used as supplement.

Primary cells are often preferred to established cell lines as they better represent the cells in vivo in terms of activity and functions. On the other hand, there is significant difference between original culture and present culture and even between cultures of the same cells in different laboratories. The rate of growth increases and there is change in chromosome numbers of lines over the time. The differences in phenotypes can cause problems in comparison and interpretation of results. The advantage of cell lines are ease handling, grow continuously, large yield and readily available. Primary cell cultures compared to established cell line are more fastidious in their growth requirements because many cell lines are derived from tumour tissue and demonstrate reduced growth factor requirements.

Cell growth and cell passage : Different types of cells have different growth requirements but in all cases the surrounding medium must supply all the essential nutrients. The temperature, pH, osmolarity and humidity must be kept within limits. Again, toxic or inhibitory substances must not be allowed to accumulate. Serum, a source of macromolecular growth factors, is essential to the growth of many cell types. Anchorage dependent cells require nontoxic or biologically inert glass or plastic surface for attachment.

In may cases, it becomes necessary to send the cells from one laboratory to another laboratory within or outside the country. The ideal method is to fill the flask containing cells at log/exponential phase with growth medium, tightly close the cap, seal it with parafilm and tape and pack it properly with polythene containing tissue paper and cotton. The tissue paper and cotton is used to soak the medium in case of accidental breakage. The polythene containing tissue culture flask having the adherent cell sheet should again be packed with thick paper. Finally, it is to be kept in a wooden/metal box or container.

Vero (monkey, African green, kidney)  Morphology: Fibroblast Species:, African green monkey  Tissue: Kidney Properties: Virology, virus titration, virus replication, plaque assays, bacterial cytotoxicity

If the concentration of cells in suspension cell culture has reached to 7X105 cells/ml, it is to be subcultured. Suspension cells are diluted to concentration of approximately 1X105 cells/ml although it varies from cell line to cell line. So, to subculture into a 3 litre glass flask using 500 ml of suspension of cells, there is a need of 5X107 cells. As the concentration of cells is 7X105 cells/ml, there is a need of 71.4 ml of cells. Adherent cells are put in the culture flasks at a concentration of 5X104 cells in a 25 cm2 flask. For example, after trypsinization, the cells have been resuspended in 5 ml of medium and cell concentration is 1X105 cells/ml or total number is 5X105 cells. If one wants to put cells into two 25 cm2 flasks each containing 5X104 cells in 5 ml volume, there is a need of 1X105 cells, which can be obtained from one ml of cell suspension (1X105 cells/ml).

Small volume cultures are useful for experiments such as studying cell morphology, titration of viruses, plaque assay, pock assay, SNT and to compare the effect of agents on growth and metabolism. However, there are other application where there is a need of large number of cells such as extraction of DNA/RNA from cells, production of viruses for vaccine production and a number of products namely interferons, interleukins, hormones, enzymes, antibodies, glycoproteins etc. The large quantity of cells can be obtained by using a large number of flasks/bottles but it is tedius, labour intensive, expensive and there is a risk of contiamination. Although the unit processes are more cost effective and efficient, it needs some modification to overcome limiting factors such as oxygen depletion, shear damage and metabolic toxicity. There are a number of aberrations available to scale up cell culture described below.

There are a number of different cell lines derived from human and animals. With the dramatic increase in numbers of cell lines, the risk of intraspecies and interspecies cross contamination rises proportionally. It is particularly a major problem in laboratories where many different cell lines of human and animal origin are handled at a time. The purity of the primary cell or continuous cell lines and species of its origin should be checked on a regular basis to authenticate the cell line identity. In the absence of such monitoring, inter and intra species cell line contamination are likely to occur in the laboratory resulting in loss of investigator’s time effort and resources. The advantages of working with a well defined cell line free from contaminating organisms would appear obvious.

Early passage cell lines are relatively unstable and go through a period of adaptation to culture. However, between 5th and 35th generation (human diploid fibroblasts), the culture is fairly consistent although it varies from one cell to another cell. Finite cell lines should be preserved after adaptation but before senescence (getting older). Cells should be preserved to protect cell line instability, availability in case of contamination, incubator failure or accidental loss.

Cell culture contamination continues to be a major problem and a subject of great concern. Contamination may enter the cell culture system as physical, chemical or biological component of the environment and biological contamination represent the greatest threat as living organism metabolize and replicate. Replication increases the titre of the contaminant.   Physical contamination : Physical components of cell culture systems such as radiation, irradiation (UV light and fluorescent light), temperature extremes can cause reduced cell growth and cell death. To minimize the problems, incubators should be located where ambient temperature is relatively constant and no vibration (near centrifuge). Media and media components should not be stored in glass door refrigerators, next to radioisotopes, bench tops or outside the refrigerator or in the hood for prolonged period.

Enumeration of cells is essential to determine the multiplicity of infection, efficiency of plating and to obtain the specific number of cells for an experiment. The concentration of cells of a suspension can be counted using a haemocytometer. Each chamber is divided into a grid of nine large squares. Each large square is 1mmX1mm (area 1 mm2) and the depth is o.1 mm. The volume of each square is 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equal to 1 ml, the number of cells per ml of suspension can be obtained by multiply the average count per large square by 104 and reciprocal of dilution factor. Dilution is made in such a way that one large square contain about 20-60 cells. The haemocytometer chambers should be filled by capillary action and cell clump are counted as one cell. A total count does not distinguish between living and dead cells. The number of viable cells can be determined by staining the cells with trypan blue or erythrosine B. In trypan blue staining cells appear as unstained whereas dead cells appear as blue.

Cell culture is the most convenient and widely used system for the isolation and propagation of many viruses. Either primary cell culture or established cell lines can be used to isolate the suspected viruses. One can normally get 10-100 virus particles per cell inoculated. Many details of the infection process are controllable to a high degree (including cell density, age, multiplicity of infection (M.O.I.), medium composition, cell type and length and temperature of incubation). Many cell types from different species can be susceptible to a particular virus and vice versa. For replication to occur, the cell type used must be permissive for a particular virus under study.

The most important property of a virus is its infectivity or ability to invade a cell and parasitize that cell to replicate itself. To measure infectivity, a titre is defined as the given number of infectious virus units/unit volume and an infectious unit is the smallest amount of virus that produces some recognizable effect in the host system. There are two basic types of infectivity assays (a) Quantal assays and (b) Quantitative assays. Quantal assays do not measure the number of infectious virus particles present in the inoculum but denote a value for the virus titre where measurement is based on an all or none principle, CPE + or -, dead or alive etc. In the quantitative assays, the number of virus particles present in the suspension can be quantified.

The tentative diagnosis of viral diseases of livestock can be made by clinical symptoms alone. The clinical diagnosis is substantiated by the laboratory to obtain a precise, correct and prompt diagnosis. The success however depends on the collection of suitable materials in proper transport medium at a particular stage of disease condition and its dispatch to the laboratory in a manner that little or no alterations occurs in the specimen. This would facilitate the isolation and identification of the causative viral agents easily.

Bovine   1.    PPR    Eye, mouth, rectal swab in PBS on ice, 10 ml blood  in anti-coagulant at height of temperature, Prescapular lymph node, spleen, piece of intestine on ice, lung, liver, spleen, tonsil in 10% neutral formol saline solution.

Processing On arrival in the laboratory, the specimens are processed immediately or refrigerated until processed. For inoculation into animal/cell culture/embryonated eggs (EE), swabs are shaken in fluid medium, faeces are dispersed in fluid medium and tissue specimens are homogenized in a high speed blender/pestle and mortar with the help of an abrasive like powdered glass or sand. Good quality hard sand should be properly washed in distilled water, treated with HCl, washed thoroughly, dried and sterilized before use. Generally the infective tissue is homogenized in a few ml of buffer usually having a pH of 7.2 to 7.3 to make about 10% tissue suspension. The suspension is centrifuged at about 1000g for 10-15 minutes to sediment the tissue debris and coarse materials.

Principle The infectivity of a virus may be neutralized by specific antibody when they are mixed and incubated. In some cases, the addition of homologous or guinea pig complement to the mixture enhances neutralization, particularly when early antiserum is used. Serum must be inactivated by heating at 560C for 30 minutes to inactivate complement or to remove non-specific virus inhibitors. Serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the only virus controls develop cytopathic effects. Antibody by neutralizing the infectivity of the virus, protects the cells against virus destruction.

Early and accurate diagnosis of disease is of prime importance for both the successful treatment of the patient and in limiting the spread of disease in the community. It is in this context that immunodiagnostic methods are now being used extensively since they can be extremely sensitive and highly specific. Among the most successful methods are those utilizing labelled antibodies or antigens. Examples of these are immunofluorescence in which antibodies labelled with fluorescent dyes are utilized and radioimmunoassay in which antigens or antibodies are labeled with isotopes. Both of these procedures have proved to be of great value for particular purposes.

Precautions to be taken while working in the laboratory for personal safety and to prevent any hazards.     1.    Be vaccinated against dangerous viruses viz., rabies and hepatitis B as persons can contract infection while working with these organisms.     2.    Use eye protection viz., goggles, spectacles etc to avoid splashing microbes into the eyes.      3.    Do not rub eyes with contaminated hands or objects.     4.    Do not eat or drink anything in the microbiology laboratory as it will contaminate the laboratory.

REFERENCES Barnes, D., Sirbsku, D. and Sato, G. (1994). Cell Culture : Methods for Molecular and Cell Biology, 4 volumes, Wiley-Liss, New York. Burleson, F.G., Chambers, T.M. and Wiedbrauk, D.L. (1992). Virology a Laboratory Manual. Academic Press. Butler, M. (1987). Animal Cell Technology : Principles and Products, Open University Press, New York. Butler, M.J. (1997). Animal Cell Culture and Technology, IRL Press, Oxford, U.K. Darling, D.C. and Morgan, S.J. (1994). Animal Cell Culture. Bios Scientific Publishing  Ltd. Wiley, New York. Darling, D.C. and Morgan, S.J. (1994). Animal Cells : Culture and Media. Wiley, New York. Davis, J.M. (1994). Basic Cell Culture : A Practical Approach . IRL Press, Oxford, U.K. Doyle, D., Hay, R. and Kirsop, B.E. (1991). Animal Cells : Living resources for Biotechnology. Cambridge University Press, Cambridge, U.K.