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    • It has been previously demonstrated

    2018-10-29

    It has been previously demonstrated that hESCs are extremely vulnerable to apoptosis after single cell dissociation (Amit et al., 2000; Reubinoff et al., 2000; Thomson et al., 1998) and are therefore often manually passaged as mechanically fragmented colonies or as small cell clumps to increase cell survival rate and plating efficiency. Recently it was demonstrated that ROCK inhibitors significantly increase the survival of hESCs upon single cell dissociation (Harb et al., 2008; Watanabe et al., 2007), allowing the use of enzymatic passaging methods. Furthermore, in the presence of a ROCK inhibitor, Y-27632, single cell-dissociated hESCs can efficiently form cell MK2 inhibitor and grow in suspension (Watanabe et al., 2007). Utilizing ROCK inhibitors, several recent studies showed that hESCs can be maintained, serial passaged, and expanded in an undifferentiated state in suspension culture (Amit et al., 2010; Amit et al., 2011; Krawetz et al., 2010; Olmer et al., 2010; Singh et al., 2010; Steiner et al., 2010; Zweigerdt et al., 2011). These studies described various types of suspension systems, including low-attachment static plates, rotated dishes or Erlenmeyer flasks, and spinner flasks. Although the studies using plate, Petri dish, or Erlenmeyer flask systems appeared promising systems, the scalability was limited. Those utilizing spinner flasks are preferred for their potential scalability as closed bioreactor systems. However, the long-term scalable expansion of hESC lines using spinner flasks has not been demonstrated. Despite the success of hESC suspension culture with spinner flasks from previous studies, the cultures were reported to yield either low expansion rates or low overall cell yield (Krawetz et al., 2010; Singh et al., 2010). Using a spinner flask for hESC suspension culture, Singh et al. showed a 2-fold expansion over 7days when cells were seeded at 1×106cells/ml. In contrast, with a much lower cell seeding density, 1.8×104cells/ml, Krawetz et al. reported a significant expansion rate but a low cell yield, approximately 5×105cells/ml over 6days. To establish a scalable and efficient suspension culture system for large-scale hESC production, it will be necessary to further optimize culture conditions to achieve a high expansion rate with a high yield of hESCs.
    Results
    Discussion Conventionally, expansion of hESC adherent culture requires passaging the cells in small cell clumps, as cells which have been dissociated to single cells show poor viability and seeding efficiency (Amit et al., 2000; Reubinoff et al., 2000; Thomson et al., 1998). Recently, several reports have demonstrated that single cell-dissociated hESCs can survive in suspension by forming cell aggregates. (Amit et al., 2010; Krawetz et al., 2010; Olmer et al., 2010; Singh et al., 2010; Steiner et al., 2010). Unlike mES cells, which can form cell aggregates from single cells in suspension (Cormier et al., 2006; Kehoe et al., 2008; zur Nieden et al., 2007), hESCs require a ROCK inhibitor, Y-27632, to form cell aggregates and propagate. Formation of cell aggregates appears to be a critical step for hESCs to survive and expand in suspension and the ROCK inhibitor is a key factor for this step. We have found that when hESC aggregates are maintained in suspension culture, it is critical to control the size of the aggregates to ensure efficient expansion and quality of hESCs. It is a reasonable speculation that nutrients and cytokines will be difficult to penetrate into cell aggregates to maintain hESC growth as cell aggregates gain in size. We selected culture conditions which can generate a great number of cell aggregates in small sizes at the initial stage to achieve a better expandability. We found passaging cells every 3–4days provided the optimal expansion rate. In addition we found that cultures in StemPro media formed small and homogeneous aggregates and resulted in a better expansion rate than cultures in mTeSR media. At lower cell seeding density, 1.5×105cells/ml, initial sizes of cell aggregates appeared slightly smaller than those of seeding at 2.5×105cells/ml but we also noticed that aggregates appeared less at lower seeding density, resulting in a lower cell yield. Previous studies have shown great expansion rates of their hESC suspension cultures using much lower seeding density, while the cell yields were not comparable with adherent cell cultures (Krawetz et al., 2010; Olmer et al., 2010). In contrast, a higher seeding cell density results in higher cell yield but lower expansion rate (Singh et al., 2010). Therefore, it is crucial to optimize cell seeding density to ensure proper expansion rates and cell yields of hESC suspension culture. As the growth characteristics of hESC lines are varied, the passaging interval and cell seeding density may also need to be optimized for different lines.