School of Science and Technology, CSU-Riverina
A cell culture is obtained by disassociating tissue into a suspension of single cells or small clumps by mechanical mincing, followed by treatment with proteolytic enzymes. After the cells are washed and counted, they are diluted in medium and are permitted to settle onto the flat surface of a specially treated glass or plastic container. Most types of cells adhere quickly and under optimal conditions they then divide about once a day until the surface is covered with a confluent monolayer of cells. The impetus for culturing cells in vitro arose from virologists wishing to have a convenient and cheap system for studying viruses (viruses are obligate intracellular parasites and cannot replicate in any cell-free medium no matter how complex), and particularly the discovery by Enders et al. (1949) that poliovirus could be propagated in cultures of non-normal human cells with the production of recognisable cytopathic changes. This milestone led to development of a vaccine against poliomyelitis within four years.
Cell culture has been greatly aided by the development of chemically defined media containing almost all of the nutrients required for cell growth, however serum needs to be added to such media to supply additional growth factors, without which most cells will not multiply satisfactorily. In recent years several growth factors have been identified and certain cell lines can now be grown in media that are chemically defined. Such serum-free media are particularly useful for the cultivation of "hybridoma" cells used for the production of monoclonal antibodies (here there is the need to ensure that all of the immunoglubulin in the medium is antibody of a single species). Defined media also present advantages for the isolation of viruses which might be neutralised by antibody present in "normal" human or animal serums, although this problem can usually be avoided if foetal calf serum (which lacks antibodies) is employed at a concentration of approximately 10% and is employed in media used to grow cells for virus isolation. Table 1 lists some factors which affect the selection of growth medium for use with cell cultures.
Table 1. Requirements of Growth Medium for Cell Cultures
(i) Reproducibility between culture, avoidance of both lot-to-lot
(ii) variations and cytotoxicity.
(iii) Potential risk of contamination (virus, myoplasma)
(v) Absence of antibodies
(vi) Ease of purification of culture products
(vii) Prevention of fibroblast overgrowth in primary cell cultures
At Charles Sturt University we regularly conduct undergraduate classes in which students prepare cell cultures, observe the effects of virus replication in cells, assay virus suspensions and determine the antibody titres to viruses. Each of these procedures uses cell cultures which require growth medium and thus the items listed in Table 1 must be examined for their relevance to our situation.
(i) Reproducibility between cultures and avoidance of lot-to-lot variations
Commercially available serum is exhaustively checked to ensure that the researcher has serum which will provide consistent growth requirements for cells. For student practical classes, only one lot of serum is required. Further, a "hardy" cell line has been chosen which supports the growth of the range of viruses used in the classes. Since cells for the practical classes are only expected to undergo 3-4 passages, low level cytotoxicity is not a significant factor.
(ii) Potential risk of contamination
Both adventitious viral agents and mycoplasmas can be present in serum. A particular cause of concern to some researchers is Bovine Virus Diarrhoea (BVD) which is able to infect the foetus transplacentally. In Australia, BVD is widespread and approximately 60% of serum samples taken from cattle throughout the country have neutralising antibodies to BVD. The incidence of foetal infection is unknown, but because each batch of foetal calf serum represents a pool of serum taken from about 500 foetuses (average batch 170 L), contamination of a batch requires only a 0.2% incidence of foetal infection by BVD. The potential contamination by BVD with regard to human viral vaccines is carefully monitored but its importance is not known. Serum free from this virus can only be obtained from a closed donor herd. Since BVD does not interfere with any viral assays conducted in the practical classes and its presence can only be demonstrated with great difficulty, contamination of serum by this virus is not a significant factor. Serum can be obtained free from mycoplasmas by filtration through a filter with 100 nm average pore diameter. Mycoplasma infection of cell cultures can be a serious problem if the cell cultures are subcultured for some time, however the practical classes are conducted over a short period of time (3-4 weeks). Also, new cultures are purchased each year and thus any problem of continuing mycoplasma infection is avoided.
The choice of serum for cell cultures for undergraduate classes is based solely on cost/unit volume. Foetal bovine serum is commonly used as a supplement in the growth medium for cells and although free from antibodies, costs approximately $400/litre. Adult bovine serum is considerably cheaper ($100/litre) and thus would appear to be an attractive alternative to foetal calf serum, if it can be shown to support cell growth to a level comparable to foetal calf serum.
(iv) Absence of antibodies
Antibodies in the growth medium could potentially react with viruses grown in the cell culture. This problem can be avoided by using a serum/virus system which is heterologous and thus extremely unlikely to react, e.g. bovine serum in the growth medium with poliovirus infecting the cell culture (there is no reported evidence of any bovine virus being antigenically similar to poliovirus).
Items (v) and (vi) from Table 1 are not relevant to the practical class work conducted in the subjects offered at Charles Sturt University.
Based on the above considerations, a decision was made to obtain fresh bovine adult blood from the local abattoir in order to process it to produce adult bovine serum. The scheme for production is outlined in Figure 1.
(v) Treated Calf Serum
A report by Inglot et al. (1975) suggested that the removal of lipoproteins and immunoglobulins (i.e. antibodies) from calf serum by precipitation with polyethylene glycol (PEG-) 6000 produced serum suitable for use in growth medium. An advantage of using this PEG-treated serum is the near absence of antibodies in the medium which could be inhibitory to viral growth. Preliminary testing of the PEG-treated serum showed growth rates of cell cultures to be comparable with untreated adult bovine serum (and quantitatively better).