Propagation of embryonic stem cells in stirred suspension without serum

Embryonic stem cells (ESCs) with their unlimited capacity for self-renewal and ability to differentiate along multiple cell lineages are a superb starting material for biotechnology applications and cellular therapies. However, realization of the potential of ESCs requires the development of scalable systems for their production in large quantities and in a regulated manner. Here, we describe a methodology for the expansion of mouse ESCs (mESCs) as pluripotent aggregates in a stirred suspension bioreactor and in medium without serum. Initially, the culture of feeder cell-independent mESCs in dishes was adapted to serum-free conditions. Also, we explored whether spinner flasks equipped with a triangle-shaped impeller and baffles support the culture of mESC aggregates. Serum-free culture in these vessels resulted in an almost 20-fold increase in the live mESC concentration over 4 days without significant loss of cell viability. Even after consecutive passages, mESCs retained high expression of pluripotency markers Oct3/4, Rex1 and SSEA-1. More importantly, when differentiation was induced these cells adopted fates of all three germ layers namely neuroectoderm, cardiac mesoderm and definitive endoderm. These findings demonstrate that stem cells can be propagated under serum-free conditions in a scalable stirred-suspension culture without loss of their pluripotency.