Large-scale in-vitro production of red blood cells from human peripheral blood mononuclear cells

Transfusion of donor-derived red blood cells is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units especially for chronically transfused patients. In-vitro cultured, customizable red blood cells would negate these concerns and introduce precision medicine. Large-scale, cost effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to GMP culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+ isolation, and a 3x107-fold increase in erythroblasts in 25 days. Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117-DRAQ5- red blood cells in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen binding capacity of cultured red blood cells was comparable to in-vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-seq provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1L per reactor, allowing transition towards clinical studies and small-scale applications.