We report the successful cultivation of cholesterol dependent NS0 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-beta-cyclodextrin (mbetaCD), 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 mbetaCD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types. By reducing the excess mbetaCD added to the culture medium, it was possible to successfully cultivate cholesterol-dependent NS0 cells in Wave Bioreactors using a cholesterol-mbetaCD 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 mbetaCD in the medium, followed with the irreversible adsorption or entrapment of the cholesterol-mbetaCD 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.