Novel cholesterol feeding strategy enables a high-density cultivation of cholesterol-dependent NS0 cells in linear low-density polyethylene-based disposable bioreactors

Abstract

We have developed a perfusion-based high cell density (HD) cell banking and inoculum expansion procedure for a cholesterol-dependent NS0 myeloma cell line using linear low-density polyethylene-based disposable bioreactors. Challenges associated with cholesterol–polymer interactions, which suppress cholesterol-dependent NS0 myeloma cell growth, were overcome using a novel cholesterol feeding protocol that included a combination of two cholesterol formulations: an ethanol-based formulation and an aqueous formulation. Using a cholesterol feed optimized for HD cell culture in a disposable bioreactor perfusion system, cell densities of >25 × 10⁶ viable cells/ml at ≥90 % cell viability were achieved. Vials of high density cell banks were created by filling 90–100 × 10⁶ viable cells/ml in 5 ml cryotube vials. Implementation of the HD cell banks enabled a significant reduction in the number of step operations in the inoculum expansion phase in a large-scale manufacturing setting.