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 manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose 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 cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends 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 viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) was 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 days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities.
Mastrangelo
A J
AJ
Department of Chemical Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Hardwick
J M
JM
Zou
S
S
Betenbaugh
M J
MJ
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D016466:CHO Cells; D016923:Cell Death; D002470:Cell Survival / Q000235:genetics*; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation; D019254:Genes, bcl-2