There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft-versus-host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC-based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In the present study, we developed and optimized serum-free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum-free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum-containing adherent MSC cultures. Our results suggest that serum-free media for MSC spheroids may pave the way for scale-up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors. (C) 2014 American Institute of Chemical Engineers