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

Abstract

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.