Avikpe, F. R.; Alibhai, F. J.; Romero, D. A.; Mostofinejad, A.; Bauer, J. E. S.; Montague, C.; Laflamme, M.; Amon, C. H.

Human pluripotent stem cells (hPSCs) hold significant promise for regenerative medicine, yet optimizing their expansion in three-dimensional bioreactor systems remains challenging due to complex interactions between mechanical forces, metabolic constraints, and aggregate formation dynamics. This study developed and validated a mechanistic mathematical model to predict hPSC proliferation dynamics in vertical-wheel bioreactor (VWBR) systems, incorporating the effects of shear stress and energy dissipation rate (EDR) on cell growth and aggregate dynamics. Seven model variants employing different kinetic formulations for shear stress and energy dissipation rate effects were systematically evaluated through model selection, identifiability analyses, and experimental validation. Experimental data from six bioreactor conditions varying in initial cell density (2.0E4 - 15.0E4 cells/mL), agitation rate (30 - 60 RPM), and working volume (100 - 500 mL) were used for model calibration and selection. Bayesian Information Criterion analysis identified a model combining Michaelis-Menten kinetics for shear stress inhibition with a EDR-mediated aggregate detachment formulation as the best-performing variant, achieving a Mean Relative Prediction Error of 14.0%, comparable to the experimental variability of 16.3%. Independent validation experiments using leave-out data gathered under different media exchange schedules confirmed model accuracy with prediction errors below 14%, consistent with observed experimental variability around 12.0%. The validated model was used to optimize the media exchange protocol, leading to a 37.5% reduction in media consumption with only a 13.5% reduction in final cell yield, demonstrating its utility for prospective, quantitative bioprocess design in VWBR systems.