Induction and Suppression of Apoptosis in the Bioreactor Environment

The features of the bioreactor environment that may result in cell death were outlined in the introduction. In the present section, studies that have considered these factors in terms of their ability to induce apoptosis are described and the implications of suppression of apoptosis are discussed.

Death during Batch Cultivation. The nutrient limitations encountered by cells in the bioreactor may be classed into two groups: cycling or terminal limitation. The former may be encountered at all stages of large-scale or intensive culture systems. For example, in large-scale stirred tank reactors, the cells may be exposed to fluctuating nutrient levels because of inhomogeneity due to poor mixing. In intensive culture systems, the high cell densities reached will result in low local-nutrient concentrations. As a result, the level of cell death in such systems is relatively high. Terminal nutrient limitations will occur at the end ofbatch cultivation and, as a result, will become more extreme with time, invariably leading to cell death. In the case of hybridoma batch cultures, the first nutrient to become limiting is glutamine, and its exhaustion coincides with the onset of the death phase of the culture. As stated above, cell death under these conditions is almost exclusively by apoptosis. Two studies have reported on the effect of bcl-2 overexpression on cell survival during the death phase of hybridoma cultures. Itoh et al. (78) found that bcl-2 overexpression significantly extended the duration of the culture by reducing the rate of cell death. Moreover, they reported a fourfold increase in the Mab (monoclonal antibody) titre in the culture medium. Simpson et al. (8) have also reported an extension in culture duration, although there was only a 40% improvement in Mab titre. Necrosis became the predominant mechanism of cell death in the bcl-2-transfected cell line, indicating near-complete suppression of apoptosis under batch culture conditions.

More recently, Suzuki et al. (79) have reported that trans-fection of COS-1 cells with bcl-2 and then with the vector pcDNA-k carrying the immunoglobulin k gene for transient expression of k protein have resulted in higher expression of the protein when compared to the control transfectant (i.e., bcl-2 negative). In the same study, the mouse plasmacytoma p3-X63-Ag.8.653, which is used as a fusion partner in the generation of hybridomas, and the hybridoma cell line 2E3 were transfected with the human bcl-2 gene. In both cases an extension of batch culture duration was reported. The bcl-2-transfected 2E3 cells survived 2 to 4 days longer in culture, producing a 1.5- to 4-fold larger amount of antibody in comparison with the control vector transfectants. A further enhancement in survival and antibody production in hybridoma 2E3 cultures was observed when cells were cotransfected with bcl-2 and bag-1.

In contrast with these promising studies, Murray et al. (83) found that bcl-2 transfection of the murine plasma-cytoma NS0 failed to provide any protection from apopto-sis. Although there was no endogenous Bcl-2 expression, they did report expression of Bcl-xL, a functional homologue of bcl-2. Thus, they suggested that Bcl-2 may be functionally redundant in this cell line.

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