Scaling-Up Vertical-Wheel Bioreactors Based on Cell Aggregate Exposure to Shear Stress and Energy Dissipation Rate

PURPOSE: Large quantities of human pluripotent stem cells (hPSCs) are required for clinical applications. 3D suspension cultures are suitable for large-scale manufacturing of hPSCs but yield, viability, and quality are affected by the hydrodynamic environment. This paper characterizes the hydrodynamic environment inside vertical-wheel bioreactors (VWBRs) as a function of size and agitation rates, measures its effect on cell aggregation and proliferation, and proposes the use of Lagrangian-based shear stress and energy dissipation rate (EDR) exposures to support scale-up. METHODS: In silico: Transient, 3D, turbulent flow simulations are conducted for two VWBR sizes (100, 500 mL) at five agitation rates between 20 and 80 rpm. Trajectories of cell aggregates of sizes from 200 to 1,000 microns are calculated, and shear stress and EDR exposures are collected along these trajectories. In vitro: ESI-017 hPSCs were cultured in VWBRs for 6 days. Aggregation efficiency and daily fold ratios were calculated based on cell counts and initial inoculation density. RESULTS: Aggregate size, agitation rate, and bioreactor size modulate cell aggregate exposures to EDR and shear stress, which significantly depart from maximum or volume average metrics used for scale-up. Combined in vitro/in silico results show EDR affects aggregation efficiency, cell counts, and aggregate size, and has a small effect on daily fold ratios but a significant effect on total fold ratio. CONCLUSION: History of trajectory-based cell aggregate exposures to EDRs provides a better scale-up basis for VWBRs than volume-averaged EDR. Shear stress does not significantly affect hPSC aggregation, proliferation and expansion in VWBRs under the tested conditions.