There is a constant worldwide need for blood products, traditionally obtained from donations. In vitro red blood cell (RBC) production could supplement this demand and offer benefits such as thorough screening for improved safety, the possibility of genetic manipulation to restore genetic deficiencies, and therapeutic loading. Induced pluripotent stem cells (iPSCs) are a promising cell source for transfusable RBCs due to their immortality and independence from donors. However, current iPSC differentiation protocols—including both monolayer and embryoid body-based systems—have failed to produce sufficient erythroid cells (1011-12 per unit) for therapeutic application, primarily due to developmental immaturity, inefficient enucleation (5–25%), and suboptimal, static culture conditions lacking physiological relevance. This study describes the optimization of an iPSC to RBC differentiation platform and its step-by-step translation process to dynamic culture conditions, allowing scalability and eventual bioreactor application. The optimized dynamic culture yields ≈4.6 × 103 RBC/iPSC, requiring an estimated ≈4.9 × 107 iPSCs to produce a minitransfusion unit, achieving a consistent 40–70% enucleation rate and bona fide function, demonstrated by both in vitro and in vivo assays. Our feeder-free, GMP-compatible system accomplishes an enucleated RBC production rate sufficient for large-scale application and serves as a bridge to large-scale bioreactor RBC production, facilitating clinical application.

Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def₃·Fe³⁺, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def₃·Fe³⁺ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def₃·Fe³⁺ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.

Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 10¹² RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55–2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O₂/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O₂ mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.