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Cytotechnology

, Volume 32, Issue 1, pp 45–61 | Cite as

Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation

  • Martin Fussenegger
  • Dieter Fassnacht
  • Regine Schwartz
  • James A. Zanghi
  • Michael Graf
  • James E. Bailey
  • Ralf Pörtner
Article

Abstract

Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.

apoptosis Bcl-2 fixed-bed reactor regulated gene expression 

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Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Martin Fussenegger
    • 1
  • Dieter Fassnacht
    • 2
  • Regine Schwartz
    • 2
  • James A. Zanghi
    • 1
  • Michael Graf
    • 1
  • James E. Bailey
    • 3
  • Ralf Pörtner
    • 2
  1. 1.Institute of BiotechnologyETH ZürichZürichSwitzerland
  2. 2.Technische Universität Hamburg-Harburg, Biotechnologie 1HamburgGermany
  3. 3.Institute of BiotechnologyETH ZürichZürichSwitzerland

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