Cholesterol Delivery to NS0 Cells : Challenges and Solutions in Disposable Linear Low-Density Polyethylene-Based Bioreactors(BIOCHEMICAL ENGINEERING)

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概要

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• We report the successful cultivation of cholesterol dependent NSO cells in linear low-density polyethylene (LLDPE) Wave Bioreactors when employing a low ratio of cyclodextrin to cholesterol additive mixture. While cultivation of NS0 cells in Wave Bioreactors was successful when using a culture medium supplemented with fetal bovine serum (FBS), cultivation with the same culture medium supplemented with cholesterol-lipid concentrate (CLC), which contains lipids and synthetic cholesterol coupled with the carrier methyl-β-cyclodextrin (mβCD), proved to be problematic. However, it was possible to cultivate NS0 cells in the medium supplemented with CLC when using conventional cultivation vessels such as disposable polycarbonate shake-flasks and glass bioreactors. A series of experiments investigating the effect of the physical conditions in Wave Bioreactors (e.g., rocking rate/angle, gas delivery mode) ruled out their likely influence, while the exposure of the cells to small squares of Wave Bioreactor film resulted in a lack of growth as in the Wave Bioreactor, suggesting an interaction between the cells, the CLC, and the LLDPE contact surface. Further experiments with both cholesterol-independent and cholesterol-dependent NS0 cells established that the concurrent presence of mβCD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types. By reducing the excess mβCD added to the culture medium, it was possible to successfully cultivate cholesterol-dependent NS0 cells in Wave Bioreactors using a cholesterol-mβCD complex as the sole source of exogenous cholesterol. We propose that the mechanism of growth inhibition involves the extraction of cholesterol from cell membranes by the excess mβCD in the medium, followed with the irreversible adsorption or entrapment of the cholesterol-mβCD complexes to the LLDPE surface of the Wave Bioreactor. Controlling and mitigating these negative interactions enabled the routine utilization of disposable bioreactors for the cultivation of cholesterol-dependent NS0 cell lines in conjunction with an animal component-free cultivation medium.

• 2007-01-25

著者

• Salmon Peter

  Merck Research Laboratories Bioprocess R&d

• CHARTRAIN MICHEL

  Merck Research Laboratories

• ZHANG JINYOU

  Merck Research Laboratories

• Zhang Jinyou

  Merck Research Laboratories Bioprocess R&d

• Robinson David

  Merck Research Laboratories Bioprocess R&d

• Okonkowski Jessica

  Merck Research Laboratories, Bioprocess R&D

• Balasubramanian Uma

  Merck Research Laboratories, Bioprocess R&D

• Seamans Craig

  Merck Research Laboratories, Bioprocess R&D

• Fries Serena

  Merck Research Laboratories, Bioprocess R&D

• Chartrain Michel

  Merck Research Laboratories Bioprocess R&d

• Balasubramanian Uma

  Merck Research Laboratories Bioprocess R&d

• Fries Serena

  Merck Research Laboratories Bioprocess R&d

• Seamans Craig

  Merck Research Laboratories Bioprocess R&d

• Okonkowski Jessica

  Merck Research Laboratories Bioprocess R&d

• Salmon Peter

  Merck Res. Laboratories Bioprocess R&d

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