A Robust Ex Vivo culture System for Production of Mature Human Reticulocytes From a Single Donor

Abstract ²⁰⁹⁶

We have formulated a robust culture system simulating human erythropoiesis, producing mature reticulocytes from adult peripheral blood CD34+ progenitor cells in 20 days. Expansion of cell numbers over the course of the culture exceeded 10⁴-fold and enucleation efficiency was up to 90% without the use of feeder layers. Mature reticulocytes and erythrocytes were isolated from culture by passage through a standard leucocyte filter. Scanning and transmission electron microscopy of the reticulocytes revealed morphological features indistinguishable from those of mature reticulocytes isolated from the peripheral blood of normal donors. Post-filtration cells expressed adult haemoglobin and had a capacity to bind and release oxygen comparable to that of adult peripheral blood erythrocytes. Examination of blood group active- and other cell surface antigens revealed a normal glycosylation profile and normal cell surface protein expression. Most notably, the cells did not express cryptantigens T and Tn. LORCA analysis indicated that cultured cells had slightly reduced membrane stability and deformability compared to peripheral blood erythrocytes. Haematology parameters demonstrated a typical mean cell volume of 131fL and mean cell haemoglobin of 37.7pg. This, scalable approach has allowed us to culture cells from a single apheresis cone to produce 10¹⁰ reticulocytes, which represents a packed cell volume of 1ml. These results clearly demonstrate the feasibility of producing cultured red cell products suitable for transfusion therapy ex vivo from adult peripheral blood stem cells.