Protocol for Expansion of PSC Aggregates in the PBS-Mini Vertical-Wheel Bioreactor

This protocol provides step-by-step guidance for forming and expanding pluripotent stem cell aggregates in PBS-MINI Vertical-Wheel bioreactors. The workflow begins with thawing and adherent seed-train culture in T-flasks, followed by transfer to three-dimensional suspension culture in 0.1 L or 0.5 L vessels. Instructions cover inoculation, medium exchange, representative sampling, morphology assessment, metabolite and pH analysis, cell counting, and viability testing. The document presents starting parameters for seeding density, working volume, agitation, discrete medium exchange, and continuous perfusion. It also describes alternative harvest workflows for aggregate dissociation in an external container or directly inside the bioreactor. The methods are intended as starting points for optimization because performance may vary by cell line, medium formulation, aggregate-size target, and downstream process objective.