| Microbiology @ Leicester: Microbiology Video Library: Cell culture | Search |
Eukaryotic cells are much more difficult to culture than most prokaryotes. They demand complex media and are very susceptible to contamination and overgrowth by microbes such as bacteria, yeasts and fungi. In simple terms, there are three types of eukaryotic cell culture:
Primary cells are explanted directly from a donor organism, e.g. white blood cells or nasal brushings. They may be capable of one or two divisions in culture, and given the right conditions can survive for some time, but they do not continue to grow and eventually senesce and die. So why do we bother growing these demanding cells? Because they are thought to represent the best experimental models for in vivo situations, and because they may express characteristics which are not seen in cultured cells, e.g. epithelial cells with beating cilia:
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Lymphoid (white blood) cells can be cultivated and made to undergo a limited number of divisions before sensecence by adding cytokines and lectins to the growth media. Lectins are proteins which react with specific sugar residues on cell surfaces, cross-linking them and often causing intracellular signalling. Different lectins stimulate the outgrowth of different types of lymphoid cells, e.g. phytohaemagglutinin (PHA) stimulates T-cells only, while conconavalin-A has mitogenic activity (inducing cell division) for both T-cells and B-cells. The following video shows primary human peripheral blood lymphocytes stimulated with conconavalin-A after one week in culture. Activation of cells causes the expression of many cell surface receptors and the formation of large clumps of cells.
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Secondary cells were originally explanted from a donor organism, and given the correct culture conditions, divide and grow for some time in vitro, e.g. 50-100 generations. However, they do not continue to divide indefinitely and eventually, their physical characteristics may change, after which the cells will eventually senesce and die. The factors which control the replication of such cells in vitro are related to the degree of differentiation of the cell - in general, terminally differentiated cells are harder to maintain than less specialized cells.
MRC5 cells are secondary human lung fibroblasts which undergo between 60-70 doublings before senescence. These cells are widely used to study viruses in vitro and for vaccine production:
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BEAS-2B cells are human bronchial epithelial cells, transformed by SV40 T-antigen:
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Unlike primary and secondary cells, immortalized cells continue to grow and divide indefinitely in vitro for as long as the correct culture conditions are maintained. Immortalized cell lines are also known as transformed cells - i.e. cells whose growth properties have been altered. This does not necessarily mean that these are "cancer" or "tumour" cells, i.e. able to form a tumour if introduced into an experimental animal, although in some cases they may do. Transformation is a complex process and can occur by many different routes, e.g. infection by transforming tumour viruses or chromosomal changes.
HeLa cells are the classic example of an immortalized cell line. These are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18). (Henrietta Lacks, 1951) HeLa cells are adherent cells (they stick to surfaces) which maintain contact inhibition in vitro, i.e. as they spread out across the culture flask, when two adjacent cells touch, this signals them to stop growing. Loss of contact inhibition is a classic sign of oncogenic cells, i.e. cells which form tumours in experimental animals. Such cells not only form a monolayer in culture but also pile up on top of one another in foci. HeLa cells are not oncogenic in animals, but they may become so if further transformed by a virus oncogene:
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Virus
Culture: A Practical Approach
A.J. Cann (Ed).
Viruses have properties that are distinct from other living organisms and
so require different methods to culture them. This volume provides a broad treatment
of the principles and practice of virus culture and will be of interest to all
those involved in virus culture including academic, industrial, and clinical
research groups. Contains over 90 tried and tested protocols for virus culture.
(Amazon.co.UK)