buy Biotin-HPDP Using the optimized operating conditions we

Using the optimized operating conditions we demonstrate serial passaging and expansion of two integration-free hiPSC lines in completely defined xeno-free media in 100-ml bioreactors. The buy Biotin-HPDP exhibited homogeneous aggregate formation, steady expansion in their pluripotent state, and normal karyotype after ~20 passages. Note that one experiment of extended expansion of TNC1 cells in spinner flask (p30+6+21) showed abnormal karyotype (47, XXY). However, there is no evidence that it should be attributed to the suspension culture. In general, recently improved iPSC culture conditions reduce selective pressure for the growth of mutated cells that acquire growth advantages. Although karyotypically abnormal cells were found occasionally after long-term cultures under both adherent and suspension conditions, the frequency is low (5% of tested batches of hiPSCs and hESCs). The standard karyotyping or other genotyping methods like what we performed here will remain necessary to manage this inherent issue of long-term cell cultures. The development of a single-cell cryopreservation protocol concluded the establishment of our chemically defined xeno-free suspension culture system. For continuous expansion, UL-SF protocol was the preferred method for recovery with small number of hiPSCs from cryopreservation. The hiPSCs can be directly thawed in a spinner flask; cells cultured in a spinner flask can be transferred back to VNT-N-coated surface for the applications that require adhesion culture, indicating good flexibility of the system. The single-cell cryopreservation protocol and the suspension culture system can significantly reduce the workload when handling cells in the scale required for translational uses. Furthermore, this xeno-free protocol of cryopreservation and recovery was theoretically proved to be compliant to a scale-up strategy for hiPSCs in suspension and under cGMP condition, allowing a yield of ~1×109 cells within 20days after thawing of 1×106 frozen cells (Fig. 7). The scale-up strategy, which includes multiple xeno-free processes, employs 100-ml and 1-l spinner flasks, enabling the transition from lab scale to pilot scale of hiPSC expansion. The CFD simulation of the 1-l spinner flask (Supplementary Fig. S5) equipped with two glass-ball stirring pendulums (such as one from Integra Bio-sciences) indicated that it could be scalable using 1-l spinner flasks toward clinically relevant quantities of hiPSCs from a single batch. The simulation revealed a steady flow at the highest agitating speed of 75rpm and an even lower maximum shear stress (0.047N/m2) compared to the 100-ml spinner flask in steady state. On one hand, the distance between the farthest points of the pendulums and the spin axis (r=38.9mm in Supplementary Fig. S5, equivalent to impeller radius of paddle impellers) in the 1-l spinner flask is not much longer than the 100-ml spinner flask (r=28.2mm); thus, the highest velocity in 1-l spinner flasks is not much higher than that in 100-ml spinner flasks (v=0.309m/s vs. v=0.224m/s). On the other hand, there is much more space between the pendulums to the flask wall in the radial direction (l in Supplementary Fig. S5, l=29.1mm vs. l=3.5mm). This causes a more moderate drop of velocity from the maximum at the far reach of the pendulums to zero at the flask wall in 1-l spinner flask compared to the 100-ml spinner flask, leading to a significant decrease of the gradient of velocity, d/dx, which contributes to the majority of the shear stress. Before reaching steady state, stepping acceleration could be used to protect the cells from sudden exposure to high-speed flow. Also, the liquid level of 600-ml medium in the 1-l spinner flask is similar to that of 60-ml medium in the 100-ml spinner flask, which was proven in this study not to cause any reduced O2 transport (from Fig. 3g). While optimization of the agitation speed should be performed for different hiPSC lines to support appropriate size aggregate formation (for example, diameter of 50–80μm after 24h), this direct scale-up process in 1-l spinner flasks with the same well-designed system should be attainable.