Transfusion of donor-derived red blood cells (RBC) 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 RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD341 isolation, and a 3 3 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a1CD441CD1172DRAQ52 RBC 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 RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing 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 1 L per reactor, allowing transition toward clinical studies and small-scale applications.

Additional Metadata
Persistent URL dx.doi.org/10.1182/bloodadvances.2019000689, hdl.handle.net/1765/121568
Journal Blood Advances
Citation
Heshusius, S. (Steven), Heideveld, E. (Esther), Burger, P. (Patrick), Thiel-Valkhof, M. (Marijke), Sellink, E. (Erica), Varga, E. (Eszter), … van den Akker, E. (2019). Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells. Blood Advances, 3(21), 3337–3350. doi:10.1182/bloodadvances.2019000689