Abstract Thu035: Growth Factor-Based Manufacturing of Human Pluripotent Stem Cell-Derived Cardiomyocytes Using the Vertical Wheel Bioreactor System

Background: Although several methods have been reported for the large-scale generation of hPSC-derived cardiomyocytes (hPSC-CMs) in bioreactors exclusively using small molecules, no comparable bioreactor-based protocol has been established using growth factors, despite evidence that differentiation with optimized concentrations of BMP4, Activin A, and bFGF offers finer control of cardiomyocyte phenotype. Here, we develop scalable hPSC expansion and growth factor based cardiac differentiation protocols using the Vertical Wheel Bioreactor (VWBR) system. Methods and Results: The expansion of undifferentiated hPSCs in VWBRs was optimized by varying parameters including cell inoculation density, agitation rate, and media exchange schedule. Next, we examined the impact that different hPSC culture conditions had on subsequent cardiac differentiation potential. Notably, hPSCs expanded at different densities and agitation rates all yielded high purity hPSC-CMs when differentiated in either 6-well plates or VWBRs. However, hPSCs initially expanded in VWBRs showed significantly lower cardiomyocyte yields than hPSCs expanded in 2D culture. Examination of differentiation kinetics revealed that 3D expanded hPSCs exhibited accelerated mesoderm commitment relative to their 2D counterparts, as well as a significant fraction of endoderm contamination by as early as day 3 of differentiation. Modification of our aggregation and mesoderm induction steps greatly improved cardiogenesis and enabled reliable production of >1x10 6 /mL cardiac troponin T + (cTnT) hPSC-CMs expressing MLC2v. Highlighting the versatility of our protocol, simple changes in the BMP4:Activin A ratio enabled second heart field-based differentiation and production of atrial-like cardiomyocytes in VWBRs. Lastly, we show that our developed expansion and differentiation protocols were reproducible and economic in 500mL VWBRs, enabling the production of 1.1x10 6 hPSC-CMs/mL at a mean purity of 93% cTnT + . Conclusions: This novel bioreactor-based protocol will enable cardiac cell therapy and tissue engineering applications by providing scalable and consistent production of hPSC-derived cardiac cell products.