Production of lab-grown red blood cells in hollow fiber bioreactors

Abstract Lab-grown red blood cells (lgRBCs) hold significant promise for supplementing transfusion needs and serving as vehicles for drug delivery or immune tolerization. Multiple strategies have been developed to generate lgRBCs from induced pluripotent stem cells (iPSCs), primary hematopoietic progenitor cells (HPCs), or transformed cell lines. We recently reported a method for producing self-renewing erythroblasts (SREs) from iPSCs engineered with activating mutations in KIT and JAK2 (kitJak2 SREs). These cells can proliferate in the absence of cytokines and, upon withdrawal of two small molecules, enucleate at rates exceeding 50%, yielding large quantities of lgRBCs that predominantly express fetal hemoglobin. Here, we report that this approach has now been extended to cord blood– and adult-derived SREs via CRISPR editing of HPCs. While their proliferation rate is slightly lower than that of iPSC-derived SREs, these cells are still capable of producing tens of thousands of blood units at minimal cost. Notably, these adult SREs enucleate at rates approaching 90%, and the resulting lgRBCs express predominantly adult hemoglobin. We have also developed a hollow-fiber bioreactor system that supports both the expansion of SREs and their differentiation into lgRBCs at cell densities exceeding 4×10⁸ cells/mL, in an inexpensive, cytokine-free medium. The enucleation efficiency and hemoglobin expression profiles in these high-density cultures closely match those observed in low-density settings. lgRBCs derived from adult SREs are slightly smaller than those from iPSC-derived SREs and are comparable in size and hemoglobin content to donor-derived red blood cells. The development of adult SREs and this hollow-fiber bioreactor system represents a significant advance toward cost-effective, large-scale production of lgRBCs for transfusion and other translational applications.