Optimized Serial Expansion of Human Induced Pluripotent Stem Cells Using Low-Density Inoculation to Generate Clinically Relevant Quantities in Vertical-Wheel Bioreactors

This publication describes optimization of aggregate-based human induced pluripotent stem cell expansion in single-use Vertical-Wheel bioreactors. The authors evaluated inoculation density, agitation, oxygen availability, nutrient supply, media exchange, and harvest conditions to improve cell growth from a relatively small starting population. The optimized process achieved more than 30-fold expansion during a six-day culture with limited media use. Repetition across four serial passages produced a cumulative expansion of approximately 1.06 million-fold over 28 days. Cells collected after serial culture maintained pluripotency-marker expression, normal karyotype, and the ability to generate tissues representing the three germ layers in vivo. The study demonstrates that systematic optimization of process variables and serial passaging can support rapid generation of high-quality hiPSCs at quantities relevant to therapeutic manufacturing.