| A reliable source of qualified ES cells would enable the development of protocols to test their potential in regenerative medicine models and the design of robust assays to study stem cell differentiation and signaling. Unfortunately, conventional ES cell culture methods are impractical for large-scale cell production under controlled conditions. Two stirred-suspension undifferentiated ES cell culture systems were developed and compared with tissue culture flask and Petri dish controls. Fifteen-day ES cell microcarrier cultures expanded 192 +/- 11.3-fold, with a doubling time of 13.9 +/- 0.7 hrs. (versus 14.8 +/- 1.3 hrs. for tissue flask controls). Cells cultured as aggregates, with agglomeration inhibited by shear, expanded 53.4 +/- 9.6-fold and had a doubling time of 23.5 +/- 5.8 hrs. (versus 20.1 +/- 4.5 hrs. for Petri dish controls). ES cells remained undifferentiated in both systems (i.e., predominantly SSEA-1+, E-cadherin +, Oct-4+). Expected differentiation kinetics and markers were demonstrated upon EB formation. Results suggest that with optimization these systems can support large-scale ES cell production. |
