Biological validation of bio-engineered red blood cell productions

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies.For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34+ cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index.Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.