Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors

• The baseline protocol for PSC aggregate expansion in Vertical-Wheel bioreactors demonstrated robustness across five commercial media, achieving high-fold expansions over six-day cultures. • Long-term serial passage revealed significant differences in cell growth and quality among media, underscoring the need for extended process validation beyond short-term optimization. • In-vessel dissociation operations critically impacted process outcomes, highlighting the importance of integrating harvest and passaging steps into bioprocess design. • The study provides a framework for evaluating commercial media and process robustness, facilitating the translation of PSC therapies toward clinical and commercial manufacturing.