Scale-Up of Single CellInoculated Suspension Cultures of Human Embryonic Stem Cells

We have developed a simple yet highly reproducible technique of upscaling single cell–inoculated human embryonic stem cells (hESCs) in suspension cultures, using defined, nonconditioned media. Mass expansion of hESC was readily achieved by serially upscaling 2-ml static cultures to 50-ml spinner flasks. Use of the Rho kinase (ROCK) inhibitor Y-27632 (Ri) was found to enable cell survival in suspension by promoting hESC reaggregation. This treatment resulted in 44% (vs. 5–10% in untreated controls) of inoculated cells being rescued after 24 h. We further optimized a heat shock treatment in combination with Ri, which significantly increased the percentage of surviving cells from 44% to 60%. Interestingly, our data suggest that E-cadherin plays a role in hESC aggregation. The dissociation and reaggregation upon passaging may function as a “purification” step toward an E-cadherin and pluripotency marker-enriched population. In addition to the use of Ri and heat shock treatment, a media comparison revealed that mTeSR was superior to the knockout medium in supporting cell proliferation and inhibition of cell differentiation in our expansion protocol. Our upscaling strategy proved to be highly robust and practical, with significant potential to provide pluripotent cells on a clinically relevant scale. Nevertheless, our data highlight a significant line-to-line variability with respect to culture conditions and the need for a critical assessment of novel methods with numerous relevant cell lines.