NS0 cells are an important industrial cell line for the production of therapeutic monoclonal antibodies. Culturing these cells is challenging because they are cholesterol auxotrophs, and providing cholesterol to the cells is hampered by the low solubility of lipids in aqueous medium. Limited loading capacity, precipitation, instability, and toxicity are associated with traditional delivery methods that involve solvents or carrier molecules. In this work, nanoparticle cholesterol mixtures (NCM) were produced by electrohydrodynamic spraying and added directly to a cholesterol auxotroph NS0 cell line. Compared to a cholesterol-cyclodextrin solution and a commercial proprietary cholesterol solution, SyntheChol (TM) NS0 supplement, NCM is significantly less cytotoxic. In the fed batch cell culture process, product titer was increased by 32% when the NCM supplement replaced SyntheChol (TM) NS0 supplement. An even greater product titer improvement, 64%, was achieved when both NCM and SyntheChol (TM) NS0 supplements were used in the fed-batch process. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 796-802, 2011