Team Leader CiRA Foundation / RIKEN Osaka, Ibaraki, Japan
Abstract: Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Although many attempts have been made to develop suspension culture technologies enabling rapid and large-scale preparation of hiPSCs, completed processes from clonal hiPSC generation to mass production of hiPSCs based on the precise control of cell status have not yet been achieved. we have investigated what hampers the stable maintenance of undifferentiated cell states in suspension conditions. hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix (ECM) components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. From screening candidate molecules to suppress the spontaneous differentiation of hiPSCs, we have identified that inhibitors of PKCĪ² and Wnt signaling pathways suppress their differentiation into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells (PBMCs) with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our methods are validated in several conventional culture media and many hiPSC lines. Thus, our findings show that suspension culture conditions with Wnt and PKCĪ² inhibitors can precisely control cell conditions and are comparable to conventional adhesion cultures regarding cellular function and proliferation. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.
