The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.
Keywords: Human pluripotent stem cells; Neural commitment; Neural precursor cells; Stirred-tank bioreactors; Suspension aggregates.
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