Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and EB-Based Differentiation

¹ Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

^* To whom correspondence should be addressed. E-mail: peter.zandstra{at}utoronto.ca.

	Abstract

To facilitate the exploitation of ESC and ESC-derived cells, scale-up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large-scale cell production and lack robust microenvironmental control. We developed two stirred-suspension culture systems for the propagation of undifferentiated ESC - microcarrier and aggregate cultures - and compared them with tissue culture flask and Petri dish controls. ESC cultured on glass microcarriers had comparable population doubling times (14-17 hours) to tissue culture flask controls. ESC growth could be elicited in shear-controlled stirred suspension culture, with population doubling times ranging between 24 and 39 hours at 100rpm impeller speed. Upon removal of LIF, the size-controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct-4, SSEA-1 and E-cadherin protein expression, RT-PCR for Flk-1, HNF3-, MHC and Sox-1 gene expression, and EB differentiation analysis, demonstrated that the suspension-cultured ESC retained the developmental potential of the starting cell population. Analysis of E-cadherin^-/- and E-cadherin^+/- cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell culture tools should prove useful for both the production of ESC and ESC-derived cells and for the investigations into adhesion, survival and differentiation phenomena during ESC propagation and differentiation.

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