Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.

Abstract

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324±71μm diameter in 7days in 125ml spinner flasks (100ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000ml bioreactors (1000ml volume), finally yielding hiPSC aggregates of 198±58μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000ml scale, an additional 10-fold increase was achieved. Taken together, 16×106 hiPSCs were expanded into 2×109 hiPSCs in 14days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.