Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and Embryoid Body–Based Differentiation

Authors

  • Elaine Y.L. Fok,

    1. Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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  • Peter W. Zandstra Ph.D.

    Corresponding author
    1. Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
    • Institute of Biomaterials and Biomedical Engineering, Room 407, Roseburgh Building, 4 Taddle Creek Road, Toronto, Ontario, Canada M5S 3G9. Telephone: 416-978-8888; Fax: 416-978-4317
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Abstract

To facilitate the exploitation of embryonic stem cells (ESCs) 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 ESCs—microcarrier and aggregate cultures—and compared them with tissue-culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (∼14–17 hours) comparable 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 100 rpm impeller speed. Upon removal of leukemia inhibitory factor, 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-cadherinprotein expression, reverse transcription–polymerase chain reaction for Flk-1, HNF3-β, MHC, and Sox-1 gene expression, and EB differentiation analysis, demonstrated that the suspension-cultured ESCs 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 ESCs and ESC-derived cells and for investigations into adhesion, survival, and differentiation phenomena during ESC propagation and differentiation.

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