Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications, technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype, three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified, cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research.
BACKGROUND Human induced pluripotent stem (hiPS) cells provide a fascinating tool for exploring disease mechanisms, compound screening in pharmaceutical drug development, and might also represent a renewable source of cells for regenerative medicine applications. This requires increased cell quantities, generated under Good Manufacturing Practice‐compatible conditions in a scalable system. RESULTS A microcarrier‐based suspension culture was explored for scaling‐up hiPS cell expansion in serum‐free medium using synthetic peptide‐acrylate surface microcarriers, developed for long‐term support of hiPS cell self‐renewal. After a 7 day‐culture in a spinner flask, cells maintained their typical morphology, pluripotency‐associated marker expression and their differentiation capability. Envisaging improvement of the scalability of the culture, long‐term expansion on the microcarriers was attained using confluent microcarriers as the inoculum of successive spinner flask cultures. Importantly, bead‐to‐bead cell transfer allowed four consecutive sub‐culture procedures and a cumulative 241‐fold expansion was achieved within 15 days, leading to a total viable cell number of 3.3 × 108 cells. CONCLUSION This work is expected to enable the scale‐up of hiPS cell culture under defined conditions and potentially leading to the use of pluripotent stem cell derivatives in cell replacement therapies. © 2016 Society of Chemical Industry
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