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Blood Adv. 2019 Nov 12;3(21):3337-3350. doi: 10.1182/bloodadvances.2019000689.

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

Author information

1
Sanquin Research, Department of Hematopoiesis and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
2
​Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands.
3
Laboratory for Cell Therapy, Sanquin Research, Amsterdam, The Netherlands; and.
4
Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands.

Abstract

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 CD34+ isolation, and a 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 CD71dimCD235a+CD44+CD117-DRAQ5- 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.

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