Cholesterol Delivery to NSO Cells: Challenges and Solutions in Disposable Linear Low-Density Polyethylene-Based Bioreactors

摘要

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 NSO 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-beta-cyclodextrin(m beta CD),proved to be problematic.However,it was possible to cultivate NSO 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 NSO cells established that the concurrent presence of m beta CD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types.By reducing the excess m beta CD added to the culture medium,it was possible to successfully cultivate cholesterol-dependent NSO cells in Wave Bioreactors using a cholesterol-m beta CD 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 m beta CD in the medium,followed with the irreversible adsorption or entrapment of the cholesterol-m beta CD 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 NSO cell lines in conjunction with an animal component-free cultivation medium.