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. 1998 May;26(3):219–225. doi: 10.1023/A:1007914619219

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

D Fassnacht 1, S Rössing 1, F Franěk 2, M Al-Rubeai 3, R Pörtner 1
PMCID: PMC3449549  PMID: 22358618

Abstract

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps.

Keywords: apoptosis, Bcl-2, diluted medium, hybridoma, protein-free medium

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References

  1. Amos B, Al-Rubeai M, Emery AN. Hybridoma growth and monoclonal antibody production in a dialysis perfusion reactor. Enzyme Microbiology Technology. 1994;16:688–695. doi: 10.1016/0141-0229(94)90091-4. [DOI] [PubMed] [Google Scholar]
  2. Bohmann A, Pörtner R, Märkl H. Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line. Applied Microbiology Biotechnology. 1995;43:772–780. doi: 10.1007/BF02431907. [DOI] [PubMed] [Google Scholar]
  3. Bohmann A, Pörtner R, Schmieding J, Kasche V, Märkl H. The membrane dialysis bioreactor with integrated radial-flow fixed bed - a new approach for continuous cultivation of animal cells. Cytotechnology. 1992;9:51–57. doi: 10.1007/BF02521731. [DOI] [PubMed] [Google Scholar]
  4. Bushell ME, Bell SL, Scott MF, Spier RE, Wardell JN, Sanders PG. Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population. Biotechnology and Bioengineering. 1994;44:1099–1106. doi: 10.1002/bit.260440911. [DOI] [PubMed] [Google Scholar]
  5. Cotter TG, Al-Rubeai M. Cell death (apoptosis) in cell culture systems. Trends in Biotechnology. 1995;13:150–154. doi: 10.1016/S0167-7799(00)88926-X. [DOI] [PubMed] [Google Scholar]
  6. Duval D, Demangel C, Munier-Jolain K, Miossec S, Geahel I. Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of amino acid supply. Biotechnology and Bioengineering. 1991;38:561–570. doi: 10.1002/bit.260380602. [DOI] [PubMed] [Google Scholar]
  7. Fassnacht D, Rössing S, Ghaussy N, Pörtner R. Influence of non-essential amino acids on apoptotic and necrotic death of mouse hybridoma cells in batch cultures. Biotechnology Letters. 1997;19(1):35–38. doi: 10.1023/A:1018358818937. [DOI] [Google Scholar]
  8. Fassnacht D, Rössing S, Stange J, Pörtner R. Longterm cultivation of immortalized mouse hepatocytes in a high cell density, fixed-bed reactor. Biotechnology Techniques. 1998;12(I):25–30. doi: 10.1023/A:1008895207570. [DOI] [Google Scholar]
  9. Franěk F. Starvation-induced programmed cell death of hybridoma cells: prevention by amino acid mixtures. Biotechnology and Bioengineering. 1995;45:86–90. doi: 10.1002/bit.260450112. [DOI] [PubMed] [Google Scholar]
  10. Franěk F, Dolníková J. Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: Effect of nutrient concentration. Cytotechnology. 1991;7:33–38. doi: 10.1007/BF00135636. [DOI] [PubMed] [Google Scholar]
  11. Franěk F, Dolníková J. Nucleosomes occurring in protein-free hybridoma cell cultures. Evidence for programmed cell death. FEBS letters. 1991;248:285–287. doi: 10.1016/0014-5793(91)80705-8. [DOI] [PubMed] [Google Scholar]
  12. Franěk F, Šrámková K. Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids. Cytotechnology. 1996;21:81–89. doi: 10.1007/BF00364839. [DOI] [PubMed] [Google Scholar]
  13. Franěk F, Šrámková K (1997) Unbalanced media for hybridoma cell culture - an alternative reality. In: MJT Carrondo et al.(eds.) Animal Cell Technology. Kluwer Academic Publishers: 675-680.
  14. Franěk F, Vomanstek T, Dolníková J. Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology. 1992;9:117–123. doi: 10.1007/BF02521738. [DOI] [PubMed] [Google Scholar]
  15. Hiller GW, Clark DS, Blanch HW. Cell retention-chemostat studies of hybridoma cells - analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium. Biotechnology and Bioengineering. 1993;42:185–195. doi: 10.1002/bit.260420206. [DOI] [PubMed] [Google Scholar]
  16. Hiller GW, Clark DS, Blanch HW. Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations. Biotechnology and Bioengineering. 1994;44:303–321. doi: 10.1002/bit.260440308. [DOI] [PubMed] [Google Scholar]
  17. Hockenberry D, Nuñez G, Milliman C, Schreiber RD and Korsmeyer S (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature (London) 348: 334-336. [DOI] [PubMed]
  18. Itoh Y, Ueda H, Suzuki E. Overexpression of bcl-2, apoptosis suppressing gene: prolonged viable culture period of hybridoma and enhanced antibody production. Biotechnology and Bioengineering. 1995;48:118–122. doi: 10.1002/bit.260480205. [DOI] [PubMed] [Google Scholar]
  19. Kerr JFR and Harmon BV (1991) Definition and incidence of apoptosis: An historical perspective. In: Tomei LD, Cope FO (eds) Apoptosis: The molecular basis of cell death. Cold Spring Harbor Laboratory Press: 5-29.
  20. Lee KH, Guerini-Sburlati A, Renner WA and Bailey JE (1997) Deregulated expression of cloned transcription factor E2F-1 in chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium. In: M.J.T. Carrondo et al.(eds.) Animal cell technology. Kluwer Academic Publishers: 621-623. [DOI] [PubMed]
  21. Lüdemann I, Pörtner R, Märkl H. Effect of NH3 on the cell growth of a hybridoma cell line. Cytotechnology. 1994;14:11–20. doi: 10.1007/BF00772191. [DOI] [PubMed] [Google Scholar]
  22. Oltvai ZN, Millman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619. doi: 10.1016/0092-8674(93)90509-O. [DOI] [PubMed] [Google Scholar]
  23. Ong CP, Pörtner R, Märkl H, Yamazaki Y, Yasuda K, Matsumura M. High density cultivation of hybridoma in charged porous carriers. Journal of Biotechnology. 1994;34:259–268. doi: 10.1016/0168-1656(94)90061-2. [DOI] [PubMed] [Google Scholar]
  24. Pörtner R, Bohmann A, Lüdemann I, Märkl H. Estimation of specific glucose uptake rates in cultures of hybridoma cells. Journal of Biotechnology. 1994;34:237–246. doi: 10.1016/0168-1656(94)90059-0. [DOI] [PubMed] [Google Scholar]
  25. Pörtner R, Rössing S, Koop M and Lüdemann I (1997) Kinetic studies on hybridoma cells immobilized in fixed bed reactors. Biotechnology and Bioengineering (in press). [DOI] [PubMed]
  26. Pörtner R, Schilling A, Lüdemann I, Märkl H. High density fed-batch cultures for hybridoma cells performed with the aid of a kinetic model. Bioprocess Engineering. 1996;15:117–124. [Google Scholar]
  27. Pörtner R, Schäfer T. Modelling hybridoma cell growth and metabolism - a comparison of selected models and data. Journal of Biotechnology. 1996;49:119–135. doi: 10.1016/0168-1656(96)01535-0. [DOI] [PubMed] [Google Scholar]
  28. Simpson NH, Milner AE, Al-Rubeai M. Prevention of hybridoma cell death by bcl-2during sub-optimal culture conditions. Biotechnology and Bioengineering. 1997;54:1–16. doi: 10.1002/(SICI)1097-0290(19970405)54:1<1::AID-BIT1>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  29. Singh RP, Emery AN, Al-Rubeai M. Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2. Biotechnology and Bioengineering. 1997;52:166–175. doi: 10.1002/(SICI)1097-0290(19961005)52:1<166::AID-BIT17>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  30. Vaux DL, Cory S, Adams TM. Bcl-2 promotes the survival of haemopoietic cells and cooperates with c-myc to immortalize pre-B cells. Nature (London) 1988;335:440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  31. Zhou W, Rehm J, Hu W-S. High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnology and Bioengineering. 1995;46:579–587. doi: 10.1002/bit.260460611. [DOI] [PubMed] [Google Scholar]

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