2020 JoVE - Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

This study combines CFD simulations with hPSC suspension culture experiments to investigate scale-up in Vertical-Wheel® bioreactors. The authors compare conventional Eulerian metrics, including volume-averaged shear stress and energy dissipation rate (EDR), with Lagrangian analysis of aggregate trajectories. Aggregate size, agitation rate, and bioreactor scale were shown to influence hydrodynamic exposure, while EDR correlated more strongly with aggregation efficiency, aggregate size, and cell expansion than shear stress. The findings highlight the value of tracking aggregate exposure histories to guide more representative scale-up and agitation strategies for hPSC manufacturing in Vertical-Wheel bioreactors.

This protocol provides step-by-step guidance for transitioning pluripotent stem cells (PSCs) from 2D adherent culture to scalable 3D aggregate culture using the PBS-Mini Vertical-Wheel® bioreactor. It covers seed train preparation, inoculation, aggregate formation, expansion, media exchange, sampling, morphology assessment, cell counting, viability analysis, and harvest. Workflows are provided for both PBS-Mini 0.1 and 0.5 vessels, including aggregate dissociation in conical tubes or directly within the bioreactor. The protocol offers a practical starting point for developing, optimizing, and scaling robust PSC aggregate processes on the PBS Vertical-Wheel platform.

Step-by-step protocol for transitioning PSCs from 2D adherent culture into scalable 3D aggregate culture using PBS-Mini Vertical-Wheel® bioreactors. Procedures cover seed-train expansion, inoculation, aggregate formation, media exchange, sampling, morphology assessment, dissociation, counting, viability analysis, and harvest as a starting point for process development and scale-up.