Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults

A number of bioreactor configurations have been developed for the manufactu re of products from mammalian cell hosts. Even in the most efficient of the se, however, problems such as nutrient exhaustion, growth factor deprivatio n, and toxin accumulations may arise. Consequently, the current effort focu sed on the feasibility of overexpressing anti-apoptosis genes in baby hamst er kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limitin g cell death upon exposure to three such insults. Extended periods of gluco se deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells , typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cul tures, though to varying degrees. For instance, the presence of Bcl-2, whic h did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contras t, bcl-x(L) overexpression provided BHK cells with enhanced protection in t he absence of glucose, allowing cultures to remain viable throughout the en tire three week study. CHO cultures, on the other hand, displayed similar t rends in survival in response to both glucose and serum deprivation. During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and vi able numbers. Death suppression following exposure to 50 mM ammonium chlori de was observed to a limited extent in both BHK and CHO cells overexpressin g bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) w as able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five day s of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures du ring rescue at-tem pts in which the death-inducing stimulus was removed. Clearly, engineering cells to bet-ter withstand and recover from the insul ts common during the large scale cultivation of mammalian cells has a numbe r of potential applications in the biopharmaceutical industries where cell death can limit culture productivities. (C) 2000 John Wiley & Sons, Inc.