Abstract

Understanding How Fluidic Agitation Impacts Human Pluripotent Stem Cells in Dynamic Suspension

Daniel Nampe, Student, University of California-Riverside

Due to the unique ability to self-renew indefinitely and differentiate into any cell type, human pluripotent stem cells (hPSCs) are an ideal cell source for future cell therapy applications. However, scalable culture systems that can produce sufficient number of clinical stem cell products are currently lacking. Dynamic suspension culture is a potential platform because it is easily scalable and automated, thereby reducing cost of stem cell production. However, propagation of undifferentiated hPSCs in dynamic suspension has not been explored until recently, and microenvironmental factors that regulate fate decision of hPSCs are poorly understood. In particular, fluidic agitation is unique to dynamic suspension culture and can play an important role in survival, self-renewal, and differentiation of hPSCs. We assessed impacts of different agitation rates (0-120 rpm) on cell output using a conventional spinner flask. After 7 days of culture, moderate agitation at 60 rpm achieved the highest cell yield (55 fold increase) while maintaining high expression of pluripotent markers. This condition produced the most uniformly sized cell aggregates with 200-300 µm in diameter, whereas the other agitation rates resulted in broader size distribution. To decouple impacts of fluidic agitation and size on the observed cell yield, we investigated hPSC aggregates of prescribed sizes under static condition for 7 days. Aggregate size at 300 µm had the highest cell yield (40 fold increase) and viability with high expression of pluripotency markers. Sizes below or above 300 µm displayed a decrease in both cell yield and viability. Particularly, larger sizes (>400 µm) resulted in early germ layer differentiation. Collectively, this set of studies shows that cell aggregate size is a critical parameter through which fluidic agitation, by modulating aggregation kinetics, can affect fate decisions of hPSCs in dynamic suspension.