Biomedical Engineering Reference
In-Depth Information
As cells are placed in a suitable culture environment, they will divide and grow.
This is called the “primary culture.” When the cells in the primary cell culture
have grown, they are subcultured to allow continued growth with a fresh medium.
For mammalian cells, cell subcultures are normally used to inoculate intermediate
bioreactors to generate enough cell mass or cell density ( > 1.5 × 10 6 cells/ml) so
that they can be used to inoculate the large production bioreactors.
Process Overview The schematic in Figure 12.5 represents a typical fermenta-
tion/cell culture process. In brief, the fermentation/cell culture process can be
described as follows: the first steps involve the removal and thawing of a vial
from the WCB or MCB into an appropriate medium in the presence or absence
of selective agents; the next steps are the production of an active, pure culture in
sufficient quantity to inoculate the production vessel, followed by growth of the
organism in the production fermenter/bioreactor under optimum conditions for
product formation. The final steps involve the collection of the product containing
harvest fluid and the disposal of the effluent waste.
12.3.3.2 Quality Risk Management for Fermentation/Cell Cultures The major
risks associated with the fermentation/cell culture process are discussed in the
following section.
Contamination One of the primary risks in cell culturing and fermentation is
contamination. There are two main types of contamination: chemical and bio-
logical. Chemical contamination is usually harder to detect and can be caused
by agents such as endotoxins and extractables/leachables from plastic storage
vessels and tubing, metal ions, or minute traces of chemical disinfectants. Bio-
logical contamination is usually easier to detect and is caused by fast-growing
yeast, bacteria, and fungi that usually have a visible effect on the cell culture
(e.g., phage in a fermenter can destroy a culture in an hour with mass cell lysis;
the first hints are the production of large amounts of foam in the culture ves-
sel or increased oxygen consumption). However, two other sources of biological
contamination, mycoplasmas and viruses, are not easily detectable and usually
require special detection methods, such as sensitive PCR-based methods and
immunocytochemical procedures.
Therefore, in order to minimize risks of both chemical and biological con-
tamination, it is essential that personnel engaged in cell culture are appropriately
trained in aseptic techniques; and that properly designed, maintained, and steril-
ized equipment is used during the process.
Suitable Environment There is a significant risk to production if microorgan-
isms/cells are not provided quality materials, selection agents, and a suitable
environment in which to grow, proliferate, and carry out their desired physiolog-
ical and biochemical functions. Therefore, appropriate control and monitoring of
equipment, culture media, environment, etc. is required to ensure provision of
a good suspension/substrate for attachment (i.e., for anchorage-dependent cells),
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