PF463 LARGE SCALE CULTURE AND DIFFERENTIATION OF ERYTHROBLASTS FROM PBMC
Authors: Claessen M.‐J. , Heshusius S. , Gallego Murillo J. , Sellink E. , Thiel M. , Burger P. , Kersten M.J. , Klei T. , van den Akker E. , von Lindern M. 
Published: December 2023

Background:Transfusion of donor‐derived red blood cells to alleviate anemia is the most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs or enzymes. However, the source depends on donor availability, and carries a potential risk of alloimmunization and blood borne diseases. More than 30 bloodgroup systems encode >300 bloodgroup antigens, and bloodgroup matching becomes increasingly challenging in a multiethnic society. Particularly the chronically transfused patients are at risk for alloimmunisation. In vitro cultured, customizable red blood cells (cRBC) would negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications.Aims:We aim to produce human cRBC at large‐scale and cost effective, for which we need to optimize culture conditions and reduce cost‐drivers.Methods:We adapted our protocols to GMP culture requirements, which reproducibly provided pure human erythroid cultures within 25 days with a >30x106 fold expansion from peripheral blood mononuclear cells without prior CD34+ isolation. This expansion depended on the serum free medium we produce, which is supplemented with erythropoietin (Epo, 1 U/ml), stem cell factor (SCF) and glucocorticoids. Expanded erythroblasts (CD71highCD235low/‐) were differentiated for 12 days in medium supplemented with 5% human plasma, heparin and a higher concentration of Epo (10U/ml) yielding CD71dimCD235a+CD44+CD117‐DRAQ5‐ cRBC.Results:More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability was measured by an Automated Rheoscope and Cell Analyser (ARCA), and oxygen equilibrium curves were measured with a Hemox analyzer. Both parameters were similar in cRBC and freshly isolated reticulocytes. RNA sequencing was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, and erythroid specific metabolic enzymes, concomitant with loss of expression of genes involved in the formation of organelles, and cell proliferation. The culture process is compatible with upscaling using 5L G‐Rex bioreactors. Currently we are preparing a clinical study using biotinylated cRBC. For future large scale production, we investigate how erythroblasts can be expanded and differentiated in scalable bioreactors. Impellor speed and gas sparging require optimisation to maintain oxygen supply and minimize the activation of mechanosensors such as Piezo‐1 that impair erythroblast expansion.Summary/Conclusion:In conclusion, our current protocols enable us to culture cRBC from adult peripheral blood mononuclear cells and test the crBC for their stability after transfusion. Currently we prepare for a clinical trial with cRBC and we develop novel bioreactors to upscale the production.