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

Abstract

In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation 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 and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis 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. Cellcycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport. These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity.

Characterisation of Tetraploid and Diploid Clones of Spodoptera frugiperda Cell Line

“…The onset of cell death coincided with a drop in viability to 74% which is in agreement with Cowger et al (1999), who obtained flow cytometric evidence that S. frugiperda cells grown in rotatingwall vessel or shaker flask became apoptotic at 70% cell viability.…”

Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells

“…There is a decrease by a factor of 2.1 ± 0. apoptotic cells form at a rate equal to k EA XòXdt instead of k EA X. Similar results were obtained modeling bimodal cell death in fed cultures of insect cells (Cowger et al 1999;O'Connor et al 2002). The improvement in model fit suggests that early apoptosis involves the interaction between cells, represented by X, and the accumulation over time of some cellular factor(s), represented by òXdt, akin to the ''bystander effect'' in which apoptotic cells signal neighboring cells to die (Chaudhry 2006).…”

“…Cell growth and bimodal death was simulated as a balance between distinct cell populations (Cowger et al 1999;O'Connor et al 2002). Model equations describe the accumulation of each cell population in terms of its rate of formation and depletion with kinetic rate constants, k.…”

“…Computational analysis has the potential to expedite future developments in this field given the intrinsic complexity of population dynamics in animal-cell culture. Our research group has developed a novel mathematical model of cell population dynamics to facilitate the rational design of bioreactors for insect cells (Cowger et al 1999;O'Connor et al 2002). The model is unique in its ability to account for the time-dependent kinetic events that govern the rate of apoptotic and necrotic cell death.…”

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

“…While some articles have been published on the cultivation of insect cell lines in airlift reactor and its application to the production of baculovirus and recombinant proteins, there are few systematic studies for optimization of the geometrical parameters and operation in processes involving these reactors, nor models for their scaling-up (King et al, 1992;Maiorella et al, 1988;Visnovsky et al;. In addition to the classical airlift and stirred tank reactors, other reactor designs were applied successfully to the cultivation of lepidopteran insect cells, such as the rotating-wall vessel (Cowger et al, 1999). However, given the level of production scale that should be achieved for the development of economically feasible processes, only stirred tank and airlift reactors appear to have the potential to be used for the large scale production of insecticidal baculoviruses in insect cell cultures.…”