Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask

In previous publications, we reported the benefits of a high-aspect rotatin g-wall vessel (HARV) over conventional bioreactors for insect-cell cultivat ion in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the p resent study characterizes the mode and kinetics of Spodoptera frugiperda c ell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumu lation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth an d bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic co nstants reveal that total cell death is reduced in the HARV and the accumul ation of apoptotic cells in this vessel results from reduced depletion by l ysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apo ptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cell-cycle analysis sho ws the percent of tetraploid cells gradually decreases during cultivation i n both vessels. For example, between 90% and 70% viability, tetraploid cell s in the HARV drop from 43 +/- 1% to 24 +/- 4%. The data suggests the tetra ploid phase as the likely origin for apoptosis in our cultures. Possible me chanisms for these changes in bimodal cell death are discussed, including h ydrodynamic forces, cell-cell interactions, waste accumulation, and mass tr ansport. These studies may benefit insect-cell cultivation by increasing ou r understanding of cell death in culture and providing a means for further enhancing culture longevity. (C) 1999 John Wiley & Sons, Inc.