Understanding how mechanical stress affects erythropoiesis is crucial to produce transfusable erythrocytes in fluid-turbulent bioreactors. We investigated the effects of shear-stress on differentiating CD49d+CD235a+ primary human erythroblasts (EBL) at molecular, cellular, and functional level. Shear-stress, at differentiation onset, enhanced EBL maturation and induced upregulation of genes regulating cholesterol/lipids biosynthesis, causing changes in cell lipid composition. Of note, the osmotic resistance, and the expression of 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthesis pathway, were higher in dynamic cultures. Inhibition of the S1P-induced proteolytic cleavage, activating SREBPs, led to abrogation of HMCGR expression, and loss of EBL in dynamic cultures, similar to lovastatin administration. This data reveals a role for the S1P-SREBP-HMGCR-axis in the regulation of shear-stress induced adaptation during erythropoiesis, shedding light into mechanisms that will assist the upscaling of erythroid differentiation into bioreactors. Moreover, as shear-stress on hematopoietic cells occurs within the bone-marrow, these results introduce a novel signalling axis in the transduction pathways controlling erythropoiesis.@en

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.

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