ANIMAL CELL CULTURE

In organ cultures, whole embryonic organs or small tissue fragments are cultured in vitro in such a manner that they retain their tissue architecture. In contrast, cell cultures are obtained either by enzymatic or mechanical dispersal of tissues into individual cells or by spontaneous migration of cells from explants; they are maintained as attached monolayers or as cell suspensions.

Freshly isolated cell cultures are called primary cultures; they are usually heterogeneous and slow growing, but are more representative of the tissue of their origin both in cell type and properties. Once a primary culture is sub-cultured, it gives rise to cell lines, which may either die after several subcultures (such cell lines are known as finite cell lines) or may continue to grow indefinitely (these are called continuous cell lines).

Usually, normal tissues give rise to finite cell lines, while tumours give rise to continuous cell lines. But there are several examples of continuous cell lines, which were derived from normal tissues and are themselves non-tumorigenic, e.g., MDCK dog kidney, 3T3 fibroblasts, etc.

Bead Bed Reactors

These reactors are packed with 3-5 mm glass beads (which provide the surface for cell attachment) and the medium is pumped either up or down the bead column. Use of 5 mm beads gives better cell yields than that of 3 mm beads.

Heterogeneous Reactors

These reactors contain circular glass or stainless steel plates stacked 5-7 mm apart and fitted to a central shaft. Either an airlift pump is used for mixing or the shaft is rotated either vertically or horizontally. The chief disadvantage of the system is very low ratio of surface area to medium volume (1-2 cm2/ml).

Porous Micro carriers

Porous microcarriers are small (170 /lm-6000 /lm) beads of gelatin, collagen, glass or cellulose, which have a network of interconnecting pores. These provide a tremendous enhancement in surface area/volume ratio, permit efficient diffusion of medium and product, are suitable for scaling up, and are equally useful for suspension and monolayer cultures.

These can be arranged as fixed bed or fluidized bed reactors or used in stirred bioreactors. It is expected that future developments will make the immobilized cell systems the most dominant production systems.

Choice of Medium and other Considerations

The choice of medium depends chiefly on the purpose of culture, e.g., ITES¬ERDF medium was found to be the most suitable for proliferation of and antibody (IgM) production by human hybridoma cells. In general, it is preferable to use rich medium formulations like 199 than simpler media like MEM.

It should be remembered that nutrients become growth limiting much before they are exhausted. The nutrients that are exhausted the earliest are glutamine, cystine (human diploid cells) and glucose (it should be supplemented after 2-3 days).

For many purposes, it is desirable to lower the serum in the medium to I % or lower; in such cases, additives like insulin, transferrin, ethanolamine and selenium need to be provided. In order to reduce cell aggregation, Ca2+ and Mg2+ ions have to be omitted from the medium, or alternatively low levels (2 μg/ml) of trypsin may be added to the medium.

Ideally, pH of the medium should be near 7.4; it should not go below 7.0. Culture pH is affected by the buffer capacity and type, headspace of fermenter and glucose concentration.

The bicarbonate buffer system is weak; the buffering capacity of the medium is increased by the phosphate present in the balanced salt solution. Improved buffering and pH stability is provided by buffers like HEPES (10-20 mM).

Somatic Cell Fusion –

Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g.,

to study the control of cell division and gene expression, to investigate malignant transformations, to obtain viral replication, for gene or chromosome mapping and for production of monoclonal antibodies by producing hybridoma (hybrid cells between an

immortalised cell and an antibody producing lyphocyte), etc.

Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes are fused with mouse continuous cell lines.

When human and mouse cells (or cells of any two mammalian species or of the same species) are mixed, spontaneous cell fusion occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet inactivated Sendai (parainfluenza) virus or polyethylene glycol (PEG).

These agents adhere to the plasma membranes of cells and alter their properties in such a way that facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cell with two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus.

A generalized scheme for somatic cell hybridization may be described as follows. Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouse and 'hybrid' cells) are plated on a selective medium, e.g., HAT medium, which allows the multiplication of hybrid cells only.

Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic and appropriate biochemical analyses for the detection of enzyme/ protein/trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a human chromosome in the hybrid clones.

If there is a perfect correlation between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait is taken to be located in the concerned chromosome.

The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis of purines and pyrimidines from simple sugars and amino acids.

However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a salvage pathway, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acids.

Hypoxanthine is converted into guanine by the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase (TK); both HGPRT and TK are enzymes of the salvage pathway.

On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) can not divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium).

For using HAT medium as a selective agent, man cells used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficinet for the other enzyme of this pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) with TK deficient mouse cells (denoted as TK- HGPRT+).

Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+ (due to the mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion.

Hybridoma Technology -

A hybridoma is a hybrid cell obtained by fusing a B-lymphocyte with usually a tumour cell of the antibody forming system or of B-lymphocytes (these are called myelomas). The hybrid cells thus produced possess the ability to produce antibodies due to the B-lymphocyte genome and the capacity for indefinite growth in vitro due to the tumour (myeloma) cell involved in the fusion.

Therefore, hybridomas are either cultured in vitro or passaged through mouse peritoneal cavity to obtain monoclonal antibodies; this is called hybridoma technology.

This technology was developed by G. Kohler and C. Milstein in 1975 for which they (alongwith N. Jerne) were awarded the Nobel Prize for Physiology and Medicine in 1984. Antibodies are produced by B-lymphocytes, each B-lymphocyte cell being preprogrammed to respond to a single antigenic determinant. Antigenic determinant denotes that region of an antigen molecule, which reacts with an antibody that is specific to it.

When an antigen reacts to the cell surface receptor of a B-lymphocyte, it proliferates rapidly to yield a population (clone) of B cells all of which produce antibodies of the same specificity; this is called clonal selection.

Thus a B-lymphocyte cell produces antibodies of only one specificity, i.e., specific to only one antigenic determinant. In addition, an antibody producing B-Iymphocyte cell, called plasma cell, is fully differentiated and does not divide when cultured in vitro; these features are critical to hybridoma technology.

B-Iymphocytes are isolated from the spleen of an animal, e.g., mouse, which had been immunized with the antigen against which monoclonal antibodies are to be raised. Immunization is achieved by injecting the antigen along with a suitable adjuvant (a nonantigenic preparation known to stimulate the immune response) either subcutaneously or in peritoneal cavity followed by booster doses of the antigen.

Immunization enhances the population of B-¬lymphocytes producing antibodies specific to the antigen used (clonal selection), which greatly increases the chances of obtaining the desired hybridoma clone. A large number of these B-Iymphocytes are mixed with the cells of selected myeloma and induced to fuse to form hybrid cells.

The myeloma cells are selected for mainly two features:

these cells must not produce antibodies themselves, and they must contain a genetic marker, e.g., HGPRT- trait, which permits an easy selection of the

resulting hybrid cells.

When HGPRT cells are fused with B-lymphocytes, the resulting cell population will consist of

hybrid cells (hybridomes), myeloma cells, and B-Iymphocytes.

This cell population is now cultured in HAT medium containing the drug aminopterin. The HGPRT myeloma cells are unable to divide in the HAT medium due to aminopterin.

Similarly, the B-Iymphocytes do not grow for long periods of time in tissue culture and eventually die. In contrast, only the hybridoma cells proliferate on the HAT medium since the B-lymphocyte genome makes them HGPR'I* and they have the capability for indefinite growth from the myeloma cell. Thus hybridomes (myeloma + B-Iymphocyte hybrid cells) are selected by using a suitable selective medium like HAT, which allows only the hybridomes to proliferate. The next step consists of identification and

isolation of the hybridoma cells producing antibodies specific to the antigen used for immunization of the animals.

The supernatant from each micro well is sampled anq assayed for the presence of antibodies specific to the antigen in question using one of the methods based on either precipitation or agglutination caused by the antibodies specific to the given antigen. ELISA is the most sensitive and rapid of these assays (see, Appendix¬2.VIII).

Wells containing the antibodies specific to the antigen are identified and the hybridoma cells from them isolated and cloned (Appendix-3.I) to ensure that a hybridoma clone produces antibodies of a single specificity.Once the desired hybridoma clone has been obtained, it is multiplied either in vitro or in vivo to obtain monoclonal antibodies.

The in vivo production system involves injection of the hybridoma cells into the peritoneal cavity of isogenic animals, collection of the ascitic fluid and separation of the antibodies from it. Alternatively, hybridoma cells are grown in vitro in a suitable large scale culture system and the monoclonal antibodies are purified from these cultures.

Cloning -

Cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning. (Asexual progeny of a single individual make up a clone).

In simple terms, cloning consists of trypsinisation of a monolayer culture to prepare a cell suspension, 3-4 dilution steps to achieve a suitable cell density (10-200 cells/ml), and seeding in Petri dishes or flasks or multi well dishes.

directly from multi well dishes by trypsinisation of usually such wells, which contained only one cell at the start, or which have a single distinct colony originating from a single cell lying away from other nondividing cell or cells. This is confirmed by microscopic observation.

Isolation of clones from petriplates is done by using cloning rings, which are placed around the desired colonies after the medium is poured. The cell colony within each porcelain, teflon or stainless steel ring is trypisnized, cells are suspended in medium and seeded in a well of multiwell plate or in a flask. Alternatively,

the desired colony may be shielded and the remaining colonies are irradiated by a lethal dose (3,000 rads) of X-rays or gamma-rays. The protected colony is trypsinised, and the cells are cloned in the same plate; the irradiated cells serve as a feeder layer

Plating efficiency (per cent of cells forming colonies) of continuous cell lines is generally 10% or higher, while that of finite cell lines may be quite low, say, 0.5-5% or even zero.

Several approaches have been used to enhance the plating efficiency, e.g.,

use of a rich medium, use of serum, especially foetal calf serum, in the medium,

using conditioned medium (a medium in which homologous cells were grown for a period of time),

use of feeder layers, addition of hormones like insulin, dexamethasone, etc., providing intermediate metabolites like keto acids, nucleosides, etc.

A feeder layer is a layer of cells, which have been treated to prevent their growth and ultimately cause their death (cells irradiated with X-rays or gamma-rays at 3,000 rads die in 3 weeks); these cells, however, provide the necessary metabolites to enhance the plating efficiency of cells being cloned.

A feeder layer may be established by trypsinising embryo fibroblasts from a primary culture and reseeding the cells at 105 cells/ml density. When the monolayer reaches 50% confluence (50% of the substrate surface is covered by the monolayer) the cells are either treated with mitomycin-C (0.25 μg/ml solution at the rate of 2 μg/106 cells) overnight, or are irradiated with 3,000 rad X-'rays.

After treatment, the cells are incubated for 24 hours in fresh medium, tryspinised and reseeded at 5 x 104cells/ml, and incubated for 24-48 hours. This yields the feeder layer on which cells to be cloned are seeded, incubated and desired colonies isolate.

Feeder layer may be established using homologous cells (cells from the same species), but it is perferable to use heterologous cells for easy detection of accidental cross contamination in the isolated clones.

The culture vessels are incubated for 1-3 weeks with a medium change after 1 week; by this time colonies will develop.

Colonies may be isolated Cloning is used to

obtain homogeneous cell lines from heterogeneous cell cultures, (ii) to isolate biochemical mutants and (iii) cell strains with marker chromosomes, and (iv) to develop hybridoma clones.

Cloning is generally applied to continuous cell lines, but often their clones become considerably heterogeneous by the time they are sufficiently multiplied for use. The problem with finite cell lines is that of life span; by the time the clone is sufficiently multiplied, the cells may be approaching sensescene.

Limitation on Organ Culture

Results from organ cultures are often not comparable to those from whole animal studies, e.g., in studies on drug action, since the drugs are metabolized in vivo but not in vitro.

Organ cultures can be maintained only for a few months. But it may be desirable to study the effects of certain factors for several months. In such cases, the organs treated in vitro may be transplanted into suitable host animals, e.g., nude mice.

Applications of Organ Culture -

Studies on the patterns of growth, differentiation and development of organ rudiments, i.e., foetal organs, and the influences of various factors like hormones, vitamins, etc., on these parameters.

The action of drugs, carcinogenic agents, etc. on the animal organs is studied in vitro to at least serve as a guide for the events in whole animals.

Perhaps the most dramatic application of organ cultures is to produce tissues for implantation in patients; this is often called tissue engineering.

Human skin has been successfully produced in vitro and used for transplantation in more than 500 cases of serious burns, ulcers, etc. The ultimate objectives of tissue engineering are to reconstitute body parts in vitro

for use as grafts or transplants and to be used as models for studies on drug delivery and action.

It is expected that cartilage tissue developed in vitro (artificial cartilage) will be available for human implantation in cases of injuries, arthritis, etc.; results with rabbits have been encouraging. It is hoped that studies will permit the culturing and constitution of bones, liver, pancreas, etc.

Artificial Skin - The skin produced in vitro is in fact only the epidermis portion of skin; when the epidermis is applied to the burnt area, it leads to the regeneration of dermis (the remaining parts of skin) underneath.

But improvements in the techniques have permitted the reconstitution of virtually complete skin (both epidermis and dermis), called living skin equivalent (LSE); this technology employs a collagen matrix as a support for growth of the tissue.

The skin explants used for obtaining artificial skin may be either obtained from the patient concerned or from the foreskin (loose skin from the tip of penis) of newborn babies. Skin cells of new-borns grow more vigorously than adult skin; the use of a synthetic polymer called PGA allows the newborn skin to grow without scars.

Artificial skin from newborn skin explants is used to cover the wound till the patient's skin is cultured and artificial skin obtained for grafting. The production of artificial skin, in simple terms, is as follows and is essentially cell and not organ culture.

The bulk (Ca 90%) of epidermis is constituted by cells called keratinocytes, which produce the dead cells (corneocytes) making up the outermost cornified layers of skin.

The keratinocytes are dissociated by treating the skin explant with trypsin. These cells are cultured in vessels the bottom of which is covered with irradiated 3T3 fibroblast cell line; this is because certain products from fibroblast cells facilitate the proliferation of keratinocytes.

Keratinocytes grow to form colonies, which are again dissociated into single cells and cultured in the same manner. The process is repeated till a confluent sheet of pure epithelium is formed; this sheet is detached from the culture vessels, cleaned and used for grafting.

The explant for preparing artificial skin for the graft must come from the patient himself to avoid rejection. A 3 cm2 skin explant can yield about 7 m2 artificial skin in 3-4 weeks representing a 5000-fold increase.

In about 5 years after grafting of the artificial skin, all the essential components of skin are regenerated. Artificial skin grafts have been used to successfully repair several types of skin defects, including chronic skin ulcers.

Cell Cultures -

Cell cultures may contain the following three types of cells:

stem cells, precursor cells and differentiated cells.

Stem cells are undifferentiated cells, which have unlimited capacity for poliferation, and they can differentiate under correct inducing conditions into one of several kinds of cells; different kinds of stem cells differ markedly in terms of the kinds of cells they will differentiate into.

Precusor cells are derived from stem cells, are committed to differentiation, but are not yet differentiated; these cells retain the capacity for proliferation. In contrast, differentiated cells, usually, do not have the capacity to divide. Some cell cultures, e.g., epidermal keratinocyte cultures, contain all the three types of cells.

In such cell cultures, stern cells constantly provide new cells which develop into precursors; the precursor cells proliferate and mature into the differentiated cell types. Thus stern cells are necessary for the maintenance of such cultures, which by their nature are heterogenous.

On the other hand, other cell cultures, e.g., fibroblast cultures, contain a more or less uniform population of dividing cells at low cell densities ( 104 cells/cm2), but at high cell densities 005 cells/cm2) are uniformly composed of nonproliferating differentiated cells. The cells begin to proliferate once the cell density is appropriately reduced.

Differentiation and cell proliferation are affected by, in addition to cell density, factors like serum, Ca2+ ions, hormones, cell to cell and cell to matrix interactions, etc. Generally, cell proliferation is promoted by low cell density, low Ca2+ (100-600 µM), and high growth factor levels, while differentiation is promoted by the exact opposite conditions and by the presence of differentiation inducing factors, e.g., cortisone, nerve growth factor, etc.

The proportions of stern, precursor and differentiated cells are markedly affected by the source tissue used for obtaining the cultures. For example, cultures derived from embryos and those derived from even adult tissues where continuous cell renewal occurs naturally, e.g., intestinal epithelium, haematopoietic cells (cells producing blood cells), etc., stern cells are likely to be more frequent than in other cell cultures.

In contrast, cell cultures from tissues where renewal occurs only under stress, e.g., fibroblasts, muscle, etc., may contain only precursor cells. Cell cultures can be grown as

monolayers or as suspension cultures.

Propagation in suspension cultures is limited to haematopoietic cell lines, ascites tumours and transformed cells (those cells that have become phenotypically modified during in vitro culture to become anchorage independent and are able to grow in layers of several cells thick, as against monolayer growth of nontransformed cells).

Therefore, cells in culture need a surface or substrate to adhere to so that they are able to proliferate.Cells that are unable to adhere to a substrate are unable to divide, i.e., their growth is anchorage-dependent.

Substrate

The surface available for attachment of cultured cells is called substrate. The various kinds of substrates used in cell cultures may be grouped into the following three categories:

glass, plastics, and metals.

Glass. It is the most commonly used substrate in the form of slides, test tubes, flasks, etc. It can be easily washed, readily sterilized and is optically clear to allow microscopic examination of cultures. Alum-borosilicate glass, e.g., Pyrex, is preferred to soda lime glass since the latter releases alkali into the medium.

Soda lime glass can be detoxified by boiling in weak acid before use. Repeated use tends to reduce cell attachment; this can be regained by a treatment with 1 µM magnesium acetate for several hours. Glass ware must be washed with a nontoxic detergent.

Plastics. Plasticware are nonautoclavable, are supplied sterile and are for a single use. Polystyrene is the most commonly used, but polyethylene, poly carbonate, perspex, PVC, teflon, cellophane and cellulose acetate are all suitable substrates.

All the organic substrates must be suitably treated to make them wettable and negatively charged. Thin teflon films are available as Petri dishes or as membranes to be used as rafts; these films are permeable to O2 and CO2 and can be sectioned for microscopy.

Modification of Substrate Surface - The most important factor in cell attachment to a substrate is the net negative charge of its surface and the high electrostatic energies present. Since animal cell surface also has a net negative charge, cross linking with glycoproteins and/or divalent cations (Ca2+, Mg2+) is required for cell adhesion.

A glycoprotein involved in cell attachment is fibronectin which is present in the serum used in many culture media, and is synthesized by many cells. In view of this, the substrate surface may need modifications, some of which are summarised below.

Glass and metal surfaces are naturally negatively charged, and no treatments are necessary. Repeatedly washed glassware can be treated with magnesium acetate to regain cell adhesion properties.

Organic surfaces, e.g., plastics, must be made wettable and negatively charged. This is readily achieved by certain chemical (e.g., oxidising agents, strong acids) or physical (e.g., UV irradiation, high voltage discharge, etc.) treatments. Plasticware are supplied after one such treatment.

In some special cases, e.g., for culture of neurons, muscle cells, etc., the substrate surface is made positively charged by coating it with gelatin, collagen, or polylysine. A tissue culture grade collagen is commercially available; alternatively, it is prepared from rat tails.

Suspension Cultures

- Cells of certain type can be grown in liquid media as suspension culture. Cells harvested from late log phase cultures are inoculated into fresh liquid medium maintained at 37°C; the initial cell density is usually between 5 x 104 to 2 x 105 cells/ml. The cultures are stirred at 200-350 rpm.

Several factors affect cell yield, e.g., pH O2 limitation, accumulation of toxic products, (e.g., NH4), nutrient limitation (e.g., glutamine), spatial restrictions and mechanical/shear stress; suitable strategies for overcoming these limitations have been devised.

Growth curve follows the typical sigmoidal pattern: a lag phase of 2-24 hours (no growth), followed by a logarithmic phase (rapid exponential growth) and culminating in a stationary phase (no growth); finally, the cells begin to die.

Generally, various growth factors/hormones are added to the basal medium for good cell growth; commonly used factors are insulin, transferrin, ethanolamine and selenium. Cell aggregation is often a problem; media lacking Ca2+ and Mg2+ have been devised as these cations are involved in cell attachment.

The optimum pH is 7.4, but it should not fall below 7.0; to ensure these suitable buffers are added to the medium. To ensure adequate O2 supply, the cultures are aerated by anyone or a combination of the following:

Surface aeration, sparging (bubbling of gas through medium), membrane diffusion (through thin silicone tubing, which is permeable to gases), medium perfusion (medium regularly removed from culture vessel and passed through oxygenation chamber and then returned to the culture), and environmental supply (O2 concentration in the culture vessel increased).

Suspension cultures are of the following types:

Batch cultures have a fixed medium volume; as the cell grow, medium becomes gradually depleted, and eventually cells cease to divide.

In fed batch cultures, there is gradual addition of fresh medium leading to an increase in culture volume.

In semi continuous batch cultures a constant fraction of the culture, including cells, is withdrawn at regular intervals and an equal volume of fresh medium is added to the culture.

In perfusion cultures, at regular intervals, a constant volume of spent medium, without cells, is withdrawn, aerated and added back to the culture; alternatively, an equal volume of fresh medium may be added).

In continuous flow cultures, continuous withdrawal of culture along with cells and addition of equal volume of fresh medium is done so that the culture is maintained in a steady state of growth.

Animal Cell Culture Gas Phase - Animal cell cultures require a gas phase of which O2 and CO2 are the critical gases. Since culture media are usually buffered with bicarbonate, the bicarbonate concentration in the medium and CO2 tension in the gas phase must be at equilibrium. In general, sealed flasks have low buffering capacity medium using about 4 mM HCO3-, and are generally provided with air as the gas phase.

Open or sealed culture vessels have moderately buffered media using 23 mM HC03-, and require 5% CO2 in their gas phase. When culture media require high buffering usually 8 mM HC03- is supplemented with 20 mM HEPES buffer; in .such cases, 2% CO2 is provided in the gas phase.

The O2 tension is usually maintained at the atmospheric level, but some cultures may require higher or lower O2 tensions. In case of organ cultures, particularly adult mammalian cultures, high O2 concentration (even upto 95%) is needed to avoid central necrosis.

In case of suspension cultures, particularly in larger vessels like fermenters, special arrangements have to be made for proper aeration. The elevated CO2 concentration is especially important in cultures having low cell density, while those with high cell density may generate enough or even excess CO2, which may sometimes necessitate ventilation.

Initiation of Cell Cultures - The initiation of cell cultures may be conveniently dealt with under the following heads:

preparation and sterilization of the substrate (culture vessels), preparation and sterilization of the medium, isolation of explant, disaggregation of the explant, and subculture and cloning.

Preparation and Sterilization of Substrate

Plasticware are supplied sterilized and ready for use; treatments are usually not necessary. Since they are disposable and not reused, they are costlier than glassware which is adequate for most purposes. However, glassware must be carefully washed with nontoxic detergent following, preferably, an overnight soak.

They should be thoroughly rinsed in tap water and finally in deionized or distilled water. Glassware are kept in containers or wrapped in aluminium foil and usually sterilized in dry heat (160°C for I hour), while screw caps are autoclaved separately.

The quality of glassware washing and sterilization should be checked periodically as follows:

examining the glassware with eye after washing, followed by 2) cloning monolayer cells in a low serum or serum free medium in the vessels after they have

been sterilized.

Isolation of Explants

Explants should be dissected out following an appropriate protocol developed to avoid contamination and retain cell survival. In general, embryos and other contents of uterus are free from contamination, while in adult tissues bacterial contamination may be common.

In simple terms, the site of opening is sterilized with 70% alcohol, the tissues are removed aspectically, and usually placed in a balanced salt solution, e.g., EBSS or HBSS, supplemented with antibiotics.

All subsequent handling should be under strictly aseptic conditions, preferably under laminar flow cabinets or hoods or in sterile rooms. Care should be taken not to vio .

Preparation and Sterilization of Medium

The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration. Heat stable constituents like water, salts, supplements like peptone or tryptose, etc. are autoclaved at 121°C for 20 min.

But heat labile constituents like serum, trypsin, proteins, growth factors, etc. must be sterilized by filtration through 0.2mm porosity membrane filter.

Each filtrate should be tested for sterility to avoid failure due to contamination. In case of soda glass, caps should be left slack to avoid breaking during autoclaving.

Autoclaving is preferred to filtration since it is cheaper, needs less labour and is uniformly effective. Therefore wherever possible, autoclaving should be resorted to and autoclavable versions of the media should be used. Most of the media, however, now available commercially are usually presterilized.

Disaggreagation of Explants -

Cell cultures are generally started from disaggregated explants. Occasionally, cells do migrate out of the cultured whole tissues; such cells are then separated by trypsin or other enzymatic treatment to establish cell cultures.

But in general, the tissues are first disaggregated in one of the following ways:

mechanical disaggregation, enzymatic disaggregation, and EDTA treatment.

Mechanical Disaggregation. The tissue is chopped into fairly small pieces, say, of 1 mm3 in size. These are then cultured into a suitable vessel; the pieces attach to the substrate either due to their own adhesiveness, the dish may be scratched to facilitate attachment, or clotted plasma may be used.

Cells grow out from the tissue pieces, which are trypsinized and subcultured. The explants themselves may be retained in the same dish or transferred to a new vessel to obtain further cell growth.

Enzymatic Disaggregation. Tryspin (0.25% crude or 0.01-0.05% pure) and collagenase (200-2000 units/ml, crude) are the most commonly used enzymes, but other enzymes like elastase, mucase, pappain, etc. have also been used.

Trypsin is preferred because it is effective for many tissues, is tolerated by most cells, and its residual activity is neutralized by serum (or by a tryspin inhibitor in case of serum-free media). The dissociated cells are collected, washed free of the enzyme and cultured.

In some cases, trypsin may be either damaging, e.g., epithelial cells, or may be ineffective, e.g., fibrous tissues. Therefore, disaggregation of several normal and malignant tissues is better achieved by collagenase, which readily digests away matrices containing collagen. The trypsin and collagenase solutions are generally prepared in a balanced salt solution.

EDTA Treatment. -Some tissues like epithelium require Ca2+ and Mg2+ ions for their integrity. Disaggregation of such tissues is readily achieved by a treatment with EDT A (ethylene-diamine-tetra-acetic acid) prepared in a balanced salt solution. This treatment is generally used for separation of cells from established cultures of epithelial tissues.

Enzymatic disaggregation generally gives higher cell yields than the mechanical method, but is more selective since only certain cell types will survive dissociation. Therefore, in practice, often the tissues

are only reduced to small dusters of cells by an enzyme like collagenase, and these clusters are used to initiate cell cultures.

Subculture

When mechanically or enzymatically disaggregated tissue pieces or cells are cultured, they attach to the substrate, and a monolayer of cells develops from them. These cultures are called primary cultures.

Primary cultures do not contain many types of cells present in the tissue because they are unable to attach to the substrate and survive; the dead cells are easily eliminated when the medium is poured out during subculture.

Further selection will occur during growth of primary cultures and of subsequent cell lines derived from them as faster growing cell types will gradually increase and the slower growing types will go on declining with time.

After a period of time, the available substrate becomes occupied by the monolayer. Cells are then harvested from the primary cultures and inoculated or seeded into a fresh vessel; the cultures so obtained are called cell lines. The process of inoculating cultured cells into fresh culture vessels is termed as subculturing.

The subculturing of monolayers is done as follows: the monolayer is rinsed with phosphate buffered saline (PBSA; ) or PBSA containing 1 mM EDTA, treated with cold trypsin (0.25% crude or 0.01-0.05% pure) for 30 seconds, incubated for about 15 min after removal of trypsin solution, and finally the cells are suspended in culture medium, counted to prepare the desired cell density and seeded in new culture vessels.

At low cell densities and under frequent subcultures, normal cells are retained in culture. But at high cell densities and under prolonged subcultures, the proportion of normal cells gradually declines as they tend to be overrun by transformed cells whose growth is not sensitive to high cell densities.

Thus culture conditions may act as a selection pressure and eliminate some cell types. In any case, faster growing cells will increase in frequency, while slower growing cells will be gradually eliminated.

This is the most striking after the first subculture when many cell types may be lost due to their inability to tolerate trypsinization and reseeding in fresh culture vessels. Subcultures may be done every 7 days, but some fast growing lines require a change of medium after 3-4 days (feeding).

The culture becomes relatively more stable by the third subculture. But the cell lines may still be considerably heterogeneous. Cell lines having specific cell types may be obtained by cloning (culture of cells so as to obtain single cell colonies), selective culture (use of selective media and substrates for the maintenance of specialized cell lines) or by physical cell separation (by centrifugation or flow cytometry).

Evolution of Continous Cell Lines -

Most of the cell lines will grow for a limited number of subcultures (usually, 8- 10 subcultures; total culture period about 12 weeks), after which they will either cease to proliferate and die out or will give rise to continuous cell lines that can be grown indefinitely.

The phenomenon that generates a continuous cell lines is called in vitro transformation, which may either Occur spontaneously, or may be chemically or virally induced. The word transformation is used because continuous eel/lines have marked similarities with malignant cells, and are often tumorigenic.

Many normal cells do not give rise to continuous cell lines, e.g., human fibroblast cells, human glia, chick fibroblasts, etc. Human fibroblast cells remain diploid during their entire life span of about 50 generations, after which they cease to divide; they may, however, still remain viable for up to another 18 months.

The stem cells for a continuous cell line may be already present in the cell population, but it is more likely that they arise during in vitro culture presumably due to mutations. Complementation studies have revealed senescence to be dominant to continued cell growth and that the latter may be due to one or more deletions. The common features of continuous cell lines are summarised below.

Smaller, more rounded, less adherent cells with a higher nucleus/cytoplasm ratio. 2. Increased growth rate; cell doubling time reduced to 12-36 hour from 36-48 hours for finite

cell lines. 3. Aneuploid (between 2n and 4n) chromosome number showing considerable variation among

cells of a cell line. 4. Reduced serum dependence. 5. Increased cloning efficiency. 6. Reduced dependence on substrate adhesion (anchorage) and increased tendency to grow in

suspension. 7. Many of the cell lines become malignant, but some cell lines are normal and nonmalignant.

The changes in growth control are under distinct positive (oncogene) and negative (tumour suppessor genes) control genes.

8. Ability to grow upto higher cell densities, i.e., growth is less dependent on cell density.

The faster growth rate, lower serum requirement, ability to grow in suspension, independence from cell density are distinct advantages of continuous cell lines. But their limitations relate to greater chromosome instability, loss of tissue specific markers and differences from the phenotypes of donor tissues.

Maintenance of Cell Lines

Thousands of cell lines have been developed from human and other animal tissues; many of them are finite, while others are continuous cell lines. The continuous cell lines themselves may have originated from normal (through transformation) or tumour tissues.

American Type Culture Collection (ATCC) maintains a large number of cell lines acquired from their originators; these cell lines are characterised before preservation and/or distribution on request to research workers. A cell line is multiplied and divided into a

1. seed stock 2. working or distribution stock.

The seed stock is characterised and then maintained in frozen state, preferably in liquid nitrogen at -196°C. Seed stock ampoules are used to produce distribution stocks so that the cell line does not change with time due to genetic instability, selection, senescence or transformation.In contrast, working stock or distribution stock consists of material multiplied from the seed stock and used for further studies or for distribution.

Freeze preservation of animal cells is now routine in all cell line banks. A cryoprotective agent like DMSO or glycerol is generally added to minimise injury to cells during freezing and thawing.

Frozen ampoules are generally stored in liquid nitrogen refrigerators, which are rather convenient and quite safe. Following appropriate protocols, most animal cells can be stored for quite long periods.

Large Scale Culture of Cell Lines - Cell cultures are used for obtaining useful products like biochemicals (interferon, interleukins, hormones, enzymes, antibodies, etc.) and virus vaccines (polio, mumps, measles, rabies, foot and mouth, rinderpest, etc.). For these objectives, large scale cell cultures are essential; fermenters of upto 10,000 I are used for this purpose.

The scaling up of cell cultures may be done as

monolayer cultures, suspension cultures, or immobilized cell systems.

For obvious reasons, scaling up of monolayer systems is more difficult than of others.

Stirred Bioreactors

These are glass (smaller vessels) or stainless steel (larger volumes) vessels of 1-1000 I or even 8000 I (Namalva cells grown for interferon; but in practice, their maximum size is 20 I since larger vessels are difficult to handle, autoclave and to agitate the culture effectively).

These are closed systems with fixed volumes and are usually agitated with motor driven stirrers with considerable variation in design details, e.g., water jacket in place of heater type temperature control, curved bottom for better mixing at low speeds mirror internal finishes to reduce cell damage, etc. Many heteroploid cell lines can be grown in such vessels.

The needs for research biochemicals from cells are met from 2-50 I reactors, while large scale reactors are mainly used for growing hybridoma cells for the production of monoclonal antibodies although their

yields from cultured cells is only 1¬2% of those obtained by passaging the cells through peritoneal cavity of mice.

Continous Flow Cultures

These culture systems are either of chemostat or turbidostat type. In both the types, cultures begin as a batch culture. In a chemostat type bioreactor, inoculated cells grow to the maximum density when some nutrient, e.g., a vitamin, becomes growth limiting.

Fresh medium is added after 24-48 hours of growth, at a constant rate (usually, to support a growth rate lower than the maximum growth rate of culture) and the culture is withdrawn at an equal rate.

When the rate of growth equals the rate of cell withdrawl, the cultures are in a 'steady state', and both the cell density and medium composition remain constant. One of the constituents of the medium is used at a lower concentration to make it growth limiting. However, chemostat is the least efficient or controllable at the cell's maximum growth rate, hence the steady state growth rates in such a system is much lower than the maximum.

In contrast, cells in a turbidostat grow to achieve a predecided density (measured as turbidity using a photoelectric cell). At this point, a fixed volume of culture is withdrawn and the same volume of fresh normal (not having a growth limiting factor) medium is added; this lowers the cell density or turbidity of the culture.

Cells keep growing, and once the culture reaches the preset density the fixed volume of culture is replaced by fresh medium. This system works really well when the growth rate of the culture is close to the maximum for the cell line.

The continuous flow cultures provide a continuous source of cells, and are suitable for product generation, e.g., for the production of viruses and interferons. It is often necessary to use a two-stage system in which the first stage supports cell

Airlift Fermenters

Cultures in such vessels are both aerated and agitated by air bubbles introduced at the bottom of vessels. The vessel has an inner draft tube through which the air bubbles and the aerated medium rise since aerated medium is lighter than non aerated one; this results in mixing of the culture as well as aeration.

The air bubbles lift to the top of the medium and the air passes out through an outlet. The cells and the medium that lift out of the draft tube move down outside the tube and are recirculated. O2 supply is quite efficient but scaling up presents certain problems. Fermenters of 2-90 I are commercially available, but 2000 I fermenters are being used for the production of monoclonal antibodies.

Immobilized Cultures -Cultures based on immobilized cells offer several advantages:

higher cell densities (50-200 x 10-6 cells/ml), stability and longevity of cultures, applicability to both suspension and monolayer cultures, protection of the cells from shear forces due to medium flow (in case of many systems), and less dependence of cells at higher densities on external supply of growth factors, which saves

culture cost. There are two basic approaches to cell immbolization immurement and entrapment.

Immurement Cultures - In such cultures, cells are confined within a medium permeable barrier. Hollow fibers packed in a cartridge are one such system. The medium is circulated through the fiber, while cells in suspension are present in the cartridge outside the fiber.

This is extremely effective for scales up to 11 and gives cell densities of 1-2 x 108 cells/ml; sophisticated units can yield upto 40 g monoclonal antibodies/month. Membranes permitting medium and gas diffusion are also used to develop bioreactors of this type; both small scale and large scale versions of membrane bioreactors are available commercially.

The cells may be encapsulated in a polymeric matrix by adsorption, covalent bonding, cross linking or entrapment; the materials used as matrix are gelatin, polylysine, alginate and agarose. This approach

effectively protects cells from mechanical damage in large fermenters and allows production of hormones, antibodies, immunochemicals and enzymes over much longer

periods than is possible in suspension cultures. The medium diffuses freely into the matrix and into the cells, while cell products move out into

the medium.

For production of larger molecules like monoclonal antibodies, agarose in a suspension of paraffin oil is preferable to alginate since the latter does not allow diffusion of such products out of the alginate beads. Reactors of up to 31 are available commercially.

Entrapment Cultures -

In this approach, cells are held within an open matrix through which the medium flows freely. An example is the Opticell in which the cells are entrapped within the porous ceramic walls of the unit. Opticell units of up to 210m2 surface are available, which can yield upto 50 g monoclonal antibodies per day.

The cells can also be enmeshed in cellulose fibres, e.g., DEAE, TLC, QAE, TEAE. These fibers are autoclaved and washed as prescribed, and added in a spinner/ stirred bioreactor at a concentration of 3 g/1.