Brief History

The first suggestion that apoptosis may account for cell death during the cultivation of hybridoma cells came from an electron microscopic study conducted by Al-Rubeai et al. (73). Further studies by Franek and Dolnikova (74) demonstrated the accumulation of nucleosomal DNA fragments in culture medium during the death phase of batch hybridoma cultures. Based on this observation, they estimated that around 30% of cells had undergone apoptotic death. Further evidence of apoptosis during hybridoma cultures was provided by the studies of Mercille and Massie (80) and Singh et al. (81). Upon DNA gel electropho-resis, both studies revealed the laddering pattern that, as mentioned earlier, is characteristic of apoptosis. Morphological analysis of the nuclei of the cells indicated that apoptosis accounted for 90% of the dead cells. Both groups also found high levels of apoptosis during the cultivation of murine plasmacytoma cell lines (sometimes incorrectly referred to as myeloma cells). Furthermore, Singh et al. (82) reported an absence of apoptosis during the death phase of CHO and Sf-9 batch cultures. However, studies by Moore et al. (77) indicated significant levels of apoptosis during the death phase of serum-free batch cultures of CHO cells. Recent studies in our laboratory on a CHO 320 cell line overexpressing interferon indicate that this cell line may also be susceptible to apoptosis, although the morphology was not typical, and the frequency was much lower than that seen during hybridoma cultures under comparable conditions.

Studies are now required to give an indication of variability in susceptibility to apoptosis between clones of the same cell type, and between different unrelated cell lines. The objective will be to produce a correlation between susceptibility to apoptosis and general cell robustness. Clearly, such a correlation would provide a simple and easily identifiable predictor of robustness following exposure of cells to a range of stresses, thus simplifying the process of cell-line selection.

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