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 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. Cell-cycle 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. Copyright 1999 John Wiley & Sons, Inc.

[1]  K. O'Connor,et al.  Effects of simulated microgravity on DU 145 human prostate carcinoma cells , 2000, Biotechnology and bioengineering.

[2]  Kim C. O'Connor,et al.  Characterization of Autocrine Growth Factors, Their Receptors and Extracellular Matrix Present in Three-Dimensional Cultures of DU 145 Human Prostate Carcinoma Cells Grown in Simulated Microgravity , 1997 .

[3]  K. O'Connor,et al.  Cultivation of fall armyworm ovary cells in simulated microgravity , 1997, In Vitro Cellular & Developmental Biology - Animal.

[4]  Gordana Vunjak-Novakovic,et al.  Microgravity tissue engineering , 1997, In Vitro Cellular & Developmental Biology - Animal.

[5]  M Al-Rubeai,et al.  Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. , 1997, Biotechnology and bioengineering.

[6]  K. O'Connor,et al.  Influence of simulated microgravity on the longevity of insect-cell culture. , 1997, Enzyme and microbial technology.

[7]  M. Poot,et al.  Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. , 1997, Cytometry.

[8]  T. Frey,et al.  Nucleic acid dyes for detection of apoptosis in live cells. , 1995, Cytometry.

[9]  H. Ueda,et al.  Overexpression of bcl‐2, apoptosis suppressing gene: Prolonged viable culture period of hybridoma and enhanced antibody production , 1995, Biotechnology and bioengineering.

[10]  M. Al‐Rubeai,et al.  Cell cycle and cell size dependence of susceptibility to hydrodynamic forces. , 1995, Biotechnology and bioengineering.

[11]  M. Al‐Rubeai,et al.  Death mechanisms of animal cells in conditions of intensive agitation , 1995, Biotechnology and bioengineering.

[12]  M. Al‐Rubeai,et al.  Cell death in bioreactors: A role for apoptosis , 1994, Biotechnology and bioengineering.

[13]  R. J. Clem,et al.  Control of programmed cell death by the baculovirus genes p35 and iap , 1994, Molecular and cellular biology.

[14]  Z. Darżynkiewicz,et al.  The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. , 1993, Cancer research.

[15]  Thomas J. Goodwin,et al.  Three-dimensional modeling of T-24 human bladder carcinoma cell line: A new simulated microgravity culture vessel , 1993 .

[16]  D. Wolf,et al.  Reduced shear stress: A major component in the ability of mammalian tissues to form three‐dimensional assemblies in simulated microgravity , 1993, Journal of cellular biochemistry.

[17]  T. Goodwin,et al.  Advances in cellular construction , 1993, Journal of cellular biochemistry.

[18]  J. Becker,et al.  Three‐dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating‐wall vessel: Morphologic and embryologic considerations , 1993, Journal of cellular biochemistry.

[19]  M. Moyer,et al.  Rotating-Wall Vessel Coculture of Small Intestine as a Prelude to Tissue Modeling: Aspects of Simulated Microgravity , 1993, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[20]  J. Liao,et al.  Kinetic characterization of baculovirus‐induced cell death in insect cell cultures , 1993, Biotechnology and bioengineering.

[21]  E. Papoutsakis,et al.  Damaging agitation intensities increase DNA synthesis rate and alter cell‐cycle phase distributions of CHO cells , 1992, Biotechnology and bioengineering.

[22]  R. I. Scott,et al.  Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells. , 1992, Enzyme and microbial technology.

[23]  E. Alnemri,et al.  Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Kalogerakis,et al.  Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures , 1992, Biotechnology and bioengineering.

[25]  L. David Tomei,et al.  Apoptosis: The Molecular Basis of Cell Death , 1991 .

[26]  W. F. Hink,et al.  Protective Effect of Methylcellulose and Other Polymers on Insect Cells Subjected to Laminar Shear Stress , 1990, Biotechnology progress.

[27]  H. Miltenburger,et al.  Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures. , 1990, Experimental cell research.

[28]  E. Papoutsakis,et al.  Physical mechanisms of cell damage in microcarrier cell culture bioreactors , 1988, Biotechnology and bioengineering.

[29]  Larry V. McIntire,et al.  Shear sensitivity of cultured hybridoma cells (CRL-8018) depends on mode of growth, culture age and metabolite concentration , 1988 .

[30]  M. Al‐Rubeai,et al.  Apoptosis and cell culture technology. , 1998, Advances in biochemical engineering/biotechnology.

[31]  Z. Darżynkiewicz,et al.  Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). , 1997, Cytometry.

[32]  D. Green,et al.  The end of the (cell) line: methods for the study of apoptosis in vitro. , 1995, Methods in cell biology.

[33]  D. Wolf,et al.  Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity. , 1992, Journal of tissue culture methods : Tissue Culture Association manual of cell, tissue, and organ culture procedures.

[34]  Z. Darżynkiewicz,et al.  Features of apoptotic cells measured by flow cytometry. , 1992, Cytometry.

[35]  D. O'reilly,et al.  Baculovirus expression vectors: a laboratory manual. , 1992 .

[36]  J. Kerr.  Definition and incidence of apoptosis : A historical perspective , 1991 .

[37]  M. Ashburner.  A Laboratory manual , 1989 .

[38]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[39]  David F. Ollis,et al.  Biochemical Engineering Fundamentals , 1976 .