MNC-RED A Chemically-Defined Method to Produce Enucleated Red Blood Cells from Adult Peripheral Blood Mononuclear Cells

Many methods have been developed to produce red blood cellsin vitrobut translational applications have been hampered by the high cost of production. We have developed R6, a chemically-defined, albumin-free, low-transferrin culture medium, and MNC-RED, a protocol to differentiate peripheral blood mononuclear cells into enucleated erythroid cells that does not require any albumin or any animal components. Erythropoiesis requires large amounts of iron for hemoglobin synthesis. In all existing protocols, these large iron needs are met by increasing the concentration of holo-transferrin. This is necessary because transferrin recycling does not take place in existing erythroid culture conditions. In the R6 medium, iron is provided to the differentiating erythroblasts by small amounts of recombinant transferrin supplemented with FeIII-EDTA, an iron chelator that allows transferrin recycling to take place in cell culture. As a result of the absence of albumin and the use of low amounts of transferrin, the production of cultured red blood cells using the MNC-RED protocol is much less expensive than with existing protocols. The MNC-RED protocol should therefore help make the many translational applications of cultured RBCs economically more feasible.HighlightsWe have developed R6, a chemically-defined, albumin-free low-transferrin culture medium, and MNC-RED, a protocol to differentiate peripheral blood mononuclear cells into enucleated erythroid ER6 is suitable for red blood cell culture despite the low transferrin amounts because of the presence of FeIII-EDTA, an iron chelator that allows transferrin recycling to take place in cell culture.The MNC-RED protocol should help make the many translational applications of cultured RBCs more economically feasible.