ixing is required in animal cell bioreactors to provide a homogeneous environment throughout the bioreactor and to increase the mass transfer rates of oxygen and other nutrients to the cells. It is well known that intense agitation can be detrimental to mammalian cells in culture.Thecurrentunderstanding of the damage mechanisms for both attached and suspended cells in bioreactorshas been reviewed recently (Croughan &L ~ a n g 1991; , ~ a p o u t s a ~ i1991a). s,
There are several assays available to quantitate cell death in bioreactors. The simplest assay uses Trypan blue dye exclusion from intact cells to determine live and dead cell concentrations. Cells that have died and lysed, however, cannot be accounte~for using this assay (see Chapter 2, section 2.2). Cell lysis, as determined by the concentration of intracellular proteins present in the culture supernatant, is a valuable indicator of cell damage due to excessive agitation.The cytoplasmic enzyme lactatedehydrogenase ( used as a measure of cell damage for both suspension and attached cells in a variety of culture systems (see Chapter 2, section 2.5). Lactate dehydrogenase activity as a measure of cell injury should be used with care, however, because blems may complicate interpretation of the results. The concentration the culture supernatant can very likely be correlated with cell damage becauseintracellular H levels areconstantduringtheexponential growth er, intracellular has been seen to decline towards the ay also affect the level of intracellular Culture conditi For example, the activity of LDH has been shown t se in oxygen-limited was found to be relacultures (Geaugey et al., 1990). In one culture system, tively stable in the culture supernatant, with the loss i ity not exceeding 5% ner et al., 1992). The generality of this finding has not yet been estabCell damage under different agitation conditionscan be assessed by monitoring the increase in LD release. To equate the LDH content in culture supernatant to the number of cells that have lysed, a standard curve must be established for each cell type and (where applicable) for different environmental conditions to which the cells were exposed. The procedures for the Trypan blue dye exclusion H assay are described in Chapter 2, sections 2.2 and 2.5.
Cell and Tissue Culture: Laboratory Procedures in Biotechnolo~y,edited by A. Doyle and J.B. Criffiths. Wiley & Sons Ltd.
0 1998 John