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Stem Cell Res. 2010 May;4(3):165-79. doi: 10.1016/j.scr.2010.03.001. Epub 2010 Mar 12.

Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

Author information

1
Institute of Medical Biology (IMB), Immunos, 138648 Singapore. harmeet.singh@imb.a-star.edu.sg

Abstract

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.

PMID:
20363202
DOI:
10.1016/j.scr.2010.03.001
[Indexed for MEDLINE]
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