Scalable Expansion of Human Induced Pluripotent Stem Cells in Xeno-Free Microcarriers

A microcarrier-based suspension culture system was developed for scaling-up the expansion of human induced pluripotent stem cells (hiPSCs) in serum-free medium and using xeno-free synthetic beads based on a peptide-acrylate surface designed for long-term support of hiPSCs self-renewal [1]. Envisaging a homogeneous cell distribution, single-cell inoculation of hiPSCs on microcarriers was optimized using a chemical inhibitor of the Rho-associated kinase pathway. Under static conditions a 5-fold expansion of the initially inoculated cells was achieved after 5 days of culture. This microcarrier-based system was then successfully scaled-up to a dynamic culture system using 15 mL-spinner flasks. After optimization of the agitation speed, feeding regimen and impeller geometry a maximum cell fold increase of 14 was obtained after 7 days of culture in relation to the number of the initially attached cells. Envisaging the improvement of the scalability of the culture, multipassage expansion on the microcarriers was attained both using single cells and confluent microcarriers as the inoculum by taking advantage of the bead-to-bead cell transfer. In the latter case, after 4 passages, a cumulative 214-fold expansion was attained over 15 days of continuous culture. In static as well as in dynamic cultures, cells maintained their typical colony morphology and pluripotency-associated marker-expression. Moreover, their potential for spontaneous differentiation into cells of the three embryonic germ layers was demonstrated through formation of embryoid bodies containing cells expressing typical markers of endoderm, ectoderm and mesoderm. Importantly, the directed differentiation of the expanded hiPSCs into neural precursors expressing Pax6 and Nestin markers was successfully achieved both onto the xeno-free microcarriers and also after cell re-plating in tissue culture plates, by means of the dual-SMAD inhibition method. Overall, we expect these technologies to facilitate the standardized and automated scaling-up of hiPSCs expansion and integrated controlled neural differentiation for further downstream applications including potential cell replacement therapies. 

[1] Melkoumian Z, Weber JL, Weber DM, Fadeev AG, Zhou Y, Dolley-Sonneville P, Yang J, Qiu L, Priest CA, Shogbon C, Martin AW, Nelson J, West P, Beltzer JP, Pal S, Brandenberger R, “Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells”, Nat Biotechnol, (2010), 28(6): 606-10