br Materials and methods br Results br Discussion Following

Materials and methods
Results
Discussion Following the demonstration that a ROCK inhibitor such as Y27632 permits the survival of single-cell hESCs (Watanabe et al., 2007), several groups utilized it to establish suspension culture systems for the expansion of hPSCs based on single-cell inoculation (Amit et al., 2011; Olmer et al., 2010; Zweigerdt et al., 2011; Abbasalizadeh et al., 2012; Chen et al., 2012; Fluri et al., 2012; Krawetz et al., 2010; Olmer et al., 2012; Singh et al., 2010; Steiner et al., 2010). Zweigerdt et al. and Amit et al. established detailed protocols for the hiv protease inhibitor and suspension culture of hPSCs in a variety of vessel types (Amit et al., 2011; Zweigerdt et al., 2011). Recently, Olmer et al. reported a high yield culture in a monitored spinner flask with 100-ml working volume. Up to 2×108 of hiPSCs were obtained in a single run of 7days (Olmer et al., 2012). Chen et al. showed long-term culture of hESCs in suspension for at least 20 passages with an average 4.3-fold expansion rate within 3 to 4days of interval, and demonstrated a complete strategy for hESC banking under cGMP or cGLP condition (Chen et al., 2012). Most of these approaches were based on serum-free media that contain BSA Fraction V or human serum albumin as the major protein component. The batch-to-batch variance of this animal-source product led to compromised defined medium (Chen et al., 2011). Toward this, we first transferred hPSCs into the newly developed E8 medium on human vitronectin substrate, to avoid BSA and have a completely xeno-free condition with low concentration of protein components. We then demonstrated that E8 medium (with Y27632 on the first day of seeding) is sufficient to support hiPSC aggregation and survival, and serial passaging in the undifferentiated state using static suspension culture. We next aimed to optimize the protocol for dynamic suspension culture in spinner flasks. While there are many experimental and computational studies for assessing the flow dynamics in spinner flasks equipped with various types of paddle-shaped impellers (Bilgen and Barabino, 2007; Collignon et al., 2010; Papoutsakis, 1991; Venkat et al., 1996; Youn et al., 2006), the relevant analysis for glass-ball spinner flasks has not been reported. We simulated the velocity field and shear stress of the flow in the glass-ball type spinner flask, providing reasonable prediction of the effect of flow on single cells during the first several hours after inoculation. Some rational approximations were applied to simplify the model, such as using the properties of 37°C water instead of the unknown data of the medium, omitting gravity due to its negligible effects on single cells compared to the agitating force, and ignoring the cell aggregation due to the complexity it would bring to the model. Although the model could not estimate the shear stress on the surface of cell aggregates after emergence, the method provides an efficient tool for the design and analysis of the bioreactors with complex vessel structures or non-standardized impeller designs (Bilgen and Barabino, 2007; Venkat et al., 1996). Using CFD analysis, we found a steady flow in all agitation speeds (i.e. 40rpm, 60rpm and 75rpm). Although small turbulent flow may occur at high agitating speeds according to the calculation of the local Reynolds number, the shear stress generated by the glass-ball impeller is considerably lower than by other types of impellers. Experimentally, we further found that an agitation speed of 60rpm supports the formation of homogeneous cell aggregates, showing significantly higher expansion rates and relatively better cell viability than the other agitation speeds. As a comparison, hiPSCs formed relatively large aggregates on day 1 at 40rpm, which reduced the potential of further proliferation due to the diffusion limitation. Moreover, occasions of undesired aggregate agglomeration, resulting in aggregates larger than 800μm, were observed on later days due to insufficient agitating force (from Supplementary Fig. S3b). At 75rpm, cell viability dropped significantly on day 1, leading to slower recovery and expansion. Based on these results, the optimal split interval at 60rpm was next determined. We noticed a reduction in cell expansion after day 3 or day 4, which might be due to the diffusion limitation as cell aggregates grow. It is widely accepted that the diffusion limitation of oxygen in human tissue is 100 to 200μm (Hoeben et al., 2004). Therefore, we determined to split the cells on day 3 or day 4 when most of the cell aggregate diameters reached ~150–200μm. The concentration of glucose and lactate in the culture media also suggested a reduced growth after day 3, when the pH of the media drop to ~6.75 as cell density increased, which might be harmful for the hiPSC lines that are more sensitive to changes in pH and influence the expansion. This issue may be solved by gradually increasing the culture volume or the media change frequency. The inclusion of a non-invasive O2 probe-patch made it possible to monitor pO2 levels real-time without disturbing the flow and the rotation of the impeller in the glass-ball bioreactor, which are normally difficult to equip with a standard large O2 probe (Zweigerdt et al., 2011; Niebruegge et al., 2009).