 Chinese Hamster Ovary cells |
Cell culture techniques have become vital to the study of animal cell
structure, function and differentiation. Cell culture techniques are also
important for the production of many important
biological materials such as vaccines, enzymes, hormones, antibodies,
interferons and nucleic acids. The majority of animal cells are
anchorage-dependent and require attachment to a surface for their survival and
replication. For large-scale production an extensive surface is necessary for cell growth. Previously, the most popular methods for providing this surface involved multiple glass or plastic bottles. The surfaces available for growth were only those of the inside of the bottles. Such systems are labor intensive and require both a large amount of space and specific equipment handling. A further disadvantage in the bottle technique is the variation that can arise between different bottles within a batch since it is not practical to control pH, for example, in every bottle. Finally, the risk for contamination increases as the number of units to handle increases.
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 Principles of micocarrier cell culture |
In 1967 Van Wezel describes the use of small particles (0.2 mm), microcarriers, for the growth of anchorage-dependent cells. These microcarriers are suspended in the culture medium by gentle agitation and a homogeneous environment is obtained. The scale-up is done by increasing the fermentor volume. The culture environment is easily controlled. Since the cells are located on the surface they are subjected to mechanical stress. The physical characteristics of solid beads limit the number of cell doublings that can be obtained in each culture step. At the start, each bead has to be inoculated with 5-10 cells and cell growth terminates when cells make contact with each other, usually at a cell number of 200-300 cells on each bead. At this time the cells are released from the carriers and collected for inoculation in a larger fermentor. This release is usually made with an enzyme attacking cell surfaces. Cell harvesting is thus a delicate balance between release and cell death. These factors have resulted in limited success when used in large-scale systems.
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 Cleaved macroporous microcarrier |
Macroporous beads, in which the anchorage-dependent cells have the possibility to utilize the interior surface, substantially reduce the problems associated with the culture of these cells. Microcarriers have been manufactured from different synthetic materials including dextran, polyacrylamide and polystyren. Cell attachment to these charged microcarriers are mediated by ionic attractions. Cells will also attach to gelatin, but through a different mechanism: a protein, fibronektin, has a biospecific binding to gelatin and as the cells has an affinity to this protein they will attach to microcarriers of gelatin. A further advantage is the susceptibility of gelatin to proteolytic enzymes. Cells may thus be released with almost 100% viability by dissolution of the matrix with trypsin.
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