PhD Student Hannover Medical School Hannover, Niedersachsen, Germany
Abstract: Human pluripotent stem cells (hPSCs) have great potential for revolutionizing regenerative medicine. Despite recent progress in early clinical applications, differentiation and production of hPSC-progenies has been mainly studied in laboratory scale adherence-dependent (2D) cultures. This approach has substantial limitations regarding bioprocess efficiency, control and scalability for cell production in clinically relevant quality and quantities. For overcoming these, we have established different 3D suspension culture platforms, focusing on fully controlled stirred tank bioreactors (STBRs). By leveraging the application of STBRs, the mass production of 5.25^9 pluripotent hPS cells in a 150 mL working scale has been achieved within a 7-day process, resulting into a 70-fold expansion of the inoculated cell density. Nevertheless, pre-expansion of cryopreserved hPSC stocks via poorly controlled 2D culture was previously required before STBRs inoculation for 3D cultivation, creating challenges for good manufacturing practice (GMP)-compliant bioprocess development. Here we present a novel approach enabling the “high-density seeds” (HDS) for 3D bioprocess inoculation directly from cryopreserved hPSC stocks, thereby overcoming the need of any 2D culture. However, our novel strategy critically depends on the efficient recovery of viable hPSCs from cryopreservation followed by the impact of stirring shear stress in suspension. We have thus established screening assays for testing the efficiency of different “cell recovery reagents” with the HDS approach and will present solutions enabling efficient hPSC recovery and aggregation particularly during the critical phase post-inoculation. The resulting cultivation strategy is notably compatible with subsequent serial passaging of hPSCs combined with process upscaling, while all relevant hPSC characteristics such as exponential cell growth, pluripotency-associated marker expression, genomic stability and differentiation potential are maintained. Moreover, when combined with a directed differentiation protocol, the successful mass production of bona fide hPSC-cardiomyocytes in suspension culture has been demonstrated, fulfilling future clinical applications and high throughput screening platforms for drug testing as well.
