Skip to main content
Log in

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the ‘anti-apoptotic’ human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day−1) for 4 days, increasing gradually to 2 day−1 at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Al-Rubeai M, Mills D, Emery AN. Electron-microscopy of hybridoma cells with special regard to monoclonal-antibody production. Cytotechnology 1990; 4: 13–28.

    Google Scholar 

  2. Franek F, Dolnikova J. Nucleosomes occurring in protein-free hybridoma cell-culture—evidence for programmed cell-death. FEBS Letters 1991; 284: 285–287.

    Google Scholar 

  3. Mercille S, Massie B. Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells. Biotechnology and Bioengineering 1994; 44: 1140–1154.

    Google Scholar 

  4. Singh RP, Al-Rubeai M, Gregory CD, Emery AN. Cell-death in bioreactors—a role for apoptosis. Biotechnology and Bioengineering 1994; 44: 720–726.

    Google Scholar 

  5. Moore A, Donahue CJ, Hooley J, Stocks DL, Bauer KD, Mather JP. Apoptosis in CHOcell batch cultures—examination by flow cytometry. Cytotechnology 1995; 17: 1–11.

    Google Scholar 

  6. Griffiths JB. Animal-cell culture processes—batch or continuous. Journal of Biotechnology 1992; 22: 21–30.

    Google Scholar 

  7. Al-Rubeai M, Emery AN, Chalder S, Jan DC. Specific antibody productivity and the cell cycle comparisons of batch, continuous and perfusion cultures. Cytotechnology 1992; 9: 85–97.

    Google Scholar 

  8. Avgerinos GC, Drapeau D, Socalow JS, Mao J, Hsiao K, Broeze RJ. Spin filter perfusion system for high-density cell culture production of recombinant urinary type plasminogen-activator in CHO cells. Bio-Technology 1990; 8: 54–58.

    Google Scholar 

  9. Bierau H, Perani A, Al-Rubeai M, Emery AN. A comparison of intensive cell culture bioreactors operating with hybridomas modified for inhibited apoptotic response. Journal of Biotechnology 1998; 62: 195–207.

    Google Scholar 

  10. Jan DCH, Emery AN, Al-Rubeai M. Use of a spin-filter can reduce disdruption of hybridoma cells in a bioreactor. Biotechnology Techniques 1993; 7: 351–356.

    Google Scholar 

  11. Leelavatcharamas V, Emery AN, Al-Rubeai M. Use of cell cycle analysis to characterise growth and interferon-gamma production in perfusion culture of CHO cells. Cytotechnology 1999; 30: 59–69.

    Google Scholar 

  12. Reuveny S, Velez D, Riske F, MacMillan JD, Miller L. Production of monoclonal-antibodies in culture. Developments in Biological Standardization 1985; 60: 185–197.

    Google Scholar 

  13. Yabannavar VM, Singh V, Connelly NV. Mammalian cell retention in a spin filter perfusion bioreactor. Biotechnology and Bioengineering 1992; 40: 925–933.

    Google Scholar 

  14. Mercille S, Massie B. Induction of apoptosis in oxygen-deprived cultures of hybridoma cells. Cytotechnology 1994; 15: 117–128.

    Google Scholar 

  15. Simpson NH, Milner AE, Al-Rubeai M. Prevention of hybridoma cell death by Bcl-2 during suboptimal culture conditions. Biotechnology and Bioengineering 1997; 54: 1–16.

    Google Scholar 

  16. Mercille S, Johnson M, Lemieux R, Massie B. Filtration-based perfusion of hybridoma cultures in protein free medium— reduction of membrane fouling by medium supplementation with DNAse-I. Biotechnology and Bioengineering 1994; 43: 833–846.

    Google Scholar 

  17. Mercille S, Massie B. Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions. Biotechnology and Bioengineering 1999; 63: 529–543.

    Google Scholar 

  18. Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in human follicular lymphoma. Science 1985; 228: 1440–1443.

    Google Scholar 

  19. Itoh Y, Ueda H, Suzuki E. Over-expression of Bcl-2, apoptosis suppressing gene—prolonged viable culture period of hybridoma and enhanced antibody-production. Biotechnology and Bioengineering 1995; 48: 118–122.

    Google Scholar 

  20. Suzuki E, Terada S, Ueda H, et al. Establishing apoptosis resistant cell lines for improving protein productivity of cell culture. Cytotechnology 1997; 23: 55–59.

    Google Scholar 

  21. Singh RP, Emery AN, Al-Rubeai M. Enhancement of survivability of mammalian cells by over-expression of the apoptosis-suppressor gene bcl-2. Biotechnology and Bioengineering 1996; 52: 166–175.

    Google Scholar 

  22. Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnology and Bioengineering 1998; 59: 90–98.

    Google Scholar 

  23. Simpson NH, Singh RP, Emery AN, Al-Rubeai M. Bcl-2 over-expression reduces growth rate and prolongs G(1) phase in continuous chemostat cultures of hybridoma cells. Biotechnology and Bioengineering 1999; 64: 174–186.

    Google Scholar 

  24. Fassnacht D, Rossing S, Franek F, Al-Rubeai M, Portner R. Effect of Bcl-2 expression on hybridoma cell growth in serum-supplemented, protein-free and diluted media. Cytotechnology 1998; 26: 219–225.

    Google Scholar 

  25. Fassnacht D, Rossing S, Singh RP, Al-Rubeai M, Portner R. Influence of Bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma. Cytotechnology 1999; 30: 95–105.

    Google Scholar 

  26. Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M. Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures. Journal of Biotechnology 2000; 79: 147–159.

    Google Scholar 

  27. Tey BT, Singh RP, Piredda L. Piacentini M, Al-Rubeai M. Influence of Bcl-2 on cell death during cultivation of a Chinese Hamster Ovary cell line expressing a chimeric antibody. Biotechnology Bioengineering 2000; 68: 31–43.

    Google Scholar 

  28. Al-Rubeai M, Singh RP, Goldman MH, Emery AN. Death mechanisms of animal-cells in conditions of intensive agitation. Biotechnology and Bioengineering 1995; 45: 463–472.

    Google Scholar 

  29. Mercille S, Johnson M, Lemieux R, Massie B. Filtration-based perfusion of hybridoma cultures in protein free medium— reduction of membrane fouling by medium supplementation with DNAse-I. Biotechnology and Bioengineering 1994; 43: 833–846.

    Google Scholar 

  30. Kroemer G, Dallaporta B, RescheRigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annual Review of Physiology 1998; 60: 619–642.

    Google Scholar 

  31. Tey BT, Singh RP, Al-Rubeai M. Programmed cell death-An overview of apoptosis in cell culture. Asia Pacific Journal of Molecular Biology and Biotechnology 2001; 9(2): 1–28.

    Google Scholar 

  32. Ishaque A, Al-Rubeai M. Role of Ca, Mg and K ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture. Apoptosis 1999; 4/5: 335–355.

    Google Scholar 

  33. Ishaqe A, Al-Rubeai M. Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture. Apoptosis 2002; 7: 231–239.

    Google Scholar 

  34. Al-Rubeai M, Emery AN. Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures. Journal of Biotechnology 1990; 16: 67–85.

    Google Scholar 

  35. Mohan S, Chohan S, Eade J, Lyddiatt A. Molecular integrity of MABs produced by hybridoma cells in batch and continuous flow culture with integrated product recovery. Biotechnology and Bioengineering 1993; 42: 974–986.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tey, B.T., Al-Rubeai, M. Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity. Apoptosis 9, 843–852 (2004). https://doi.org/10.1023/B:APPT.0000045792.63249.5a

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:APPT.0000045792.63249.5a

Navigation