(680c) Propagation of Embryonic Stem Cells without Loss of Their Pluripotency in a Stirred-Tank Bioreactor

Embryonic stem cells (ESCs) with their ability to self-renew and their capacity to differentiate towards multiple cell types, are an ideal renewable source of material intended for cellular therapies and for engineering of tissues. However, implementation of ESC-based therapies in the clinical setting will require the production of undifferentiated stem cells in sufficiently large quantities. Therefore, the development of culture systems which allow the generation of large numbers of uncommitted ESCs in a reproducible fashion is highly desirable. In this work, suspension bioreactors which are amenable to scale-up, accommodate high-density cell cultures and provide easier process control, were explored for the expansion of undifferentiated mouse ESCs (mESCs). Mouse ESCs were expanded as pluripotent aggregates in a stirred-tank bioreactor. An average of 40-fold expansion was achieved in a 4-day period while cell viability was maintained above 90%. In comparison with mESCs cultured in static cultures, mESCs expanded in suspension expressed similar mRNA levels of Oct-3/4, SSEA-1, Nanog and Rex-1. The number of Oct-3/4⁺ and SSEA-1⁺ mESCs did not diminish, even after multiple passages in a suspension bioreactor, indicating that cultured cells maintained their pluripotency. This is also supported by differentiation assays in which mESCs propagated in bioreactors, gave raise to cell types of the three germ layers. Furthermore, control of the average size of the pluripotent aggregates was achieved by modulating the agitation rate. Scale-up studies were also conducted aiming at increasing the total number of undifferentiated mESCs. The amount of mESCs was substantially boosted over the same culture period as above by increasing the bioreactor volume. As with smaller bioreactor cultures, there was no significant loss of cell viability while maintenance of mESC pluripotency was confirmed by the presence of corresponding markers and differentiation assays. The results support the use of suspension bioreactors for expansion of uncommitted stem cells. Although mESCs were used in this study as a model, the findings may contribute to the development of culture systems for the expansion of human stem cells in a clinically relevant scale for their utilization as therapeutics.