Author:
Heshusius Steven,Heideveld Esther,Burger Patrick,Thiel-Valkhof Marijke,Sellink Erica,Varga Eszter,Ovchynnikova Elina,Visser Anna,Martens Joost H.A.,von Lindern Marieke,van den Akker Emile
Abstract
AbstractTransfusion of donor-derived red blood cells is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units especially for chronically transfused patients.In-vitrocultured, customizable red blood cells would negate these concerns and introduce precision medicine. Large-scale, cost effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to GMP culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+isolation, and a 3×107-fold increase in erythroblasts in 25 days. Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117−DRAQ5−red blood cells in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen binding capacity of cultured red blood cells was comparable toin-vivoreticulocytes. Daily RNA sampling during differentiation followed by RNA-seq provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1L per reactor, allowing transition towards clinical studies and small-scale applications.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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