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
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.
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.
Cleaved macroporous microcarrier
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
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.