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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

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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.

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Authors and Affiliations

  1. Institute of Biotechnology, ETH Zürich, CH-8093, Zürich, Switzerland

    Martin Fussenegger, James A. Zanghi & Michael Graf

  2. Technische Universität Hamburg-Harburg, Biotechnologie 1, D-21071, Hamburg, Germany

    Dieter Fassnacht, Regine Schwartz & Ralf Pörtner

  3. Institute of Biotechnology, ETH Zürich, CH-8093, Zürich, Switzerland

    James E. Bailey

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  1. Martin Fussenegger
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  2. Dieter Fassnacht
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  3. Regine Schwartz
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  4. James A. Zanghi
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  5. Michael Graf
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Fussenegger, M., Fassnacht, D., Schwartz, R. et al. 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. Cytotechnology 32, 45–61 (2000). https://doi.org/10.1023/A:1008168522385

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