Gallego-Murillo, J. S. | Search bioRxiv

bioRxiv2022

Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors

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. However, very large number of cells are required for transfusion. Here we scale-up cRBC production from static cultures to 0.5 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 could also be differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions. After defining optimal mixing and aeration strategies, erythroblast proliferation cultures were successfully scaled up to 3 L bioreactors.

bioengineering↗

bioRxiv2021

Iron-loaded deferiprone can support full hemoglobinization of cultured red blood cells in the absence of transferrin

Iron is an essential nutrient in mammalian cell cultures, conventionally supplemented as iron-loaded transferrin (holotransferrin). The high cost of human transferrin represents a challenge for the large scale production of cell therapies, such as cultured red blood cells. 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{middle dot}Fe3+) at a concentration of 52mol/L could fully replace holotransferrin during erythroblast differentiation into reticulocytes, the erythroid differentiation stage with maximal iron requirements. Reticulocytes cultured in presence of Def3{middle dot}Fe3+ or holotransferrin (1000g/mL) were similar with respect to expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Def3{middle dot}Fe3+ also supported expansion of the erythroid compartment in vitro, except for the first stage when hematopoietic stem cells committed to erythroblasts, in which a reduced erythroblasts yield was observed. This suggests that erythroblasts acquired the potential to process Def3{middle dot}Fe3+ as iron source for biosynthesis pathways. Replacement of holotransferrin by Def3{middle dot}Fe3+ was also successful in cultures of six myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). These results suggest that iron-loaded deferiprone can partially replace holotransferrin in chemically defined medium formulations for the production of cultured reticulocytes and proliferation of selected myeloid cell lines. This would lead to a significant decrease in medium cost that would improve the economic perspectives of the large scale production of red blood cells for transfusion purposes. Key pointsO_LIHolotransferrin limitations in erythroid cultures lead to lower erythroblast yields, impaired maturation and low enucleation efficiencies. C_LIO_LIIron-loaded deferiprone can replace holotransferrin in erythroblast expansion and differentiation cultures. C_LI

cell biology↗