Choice of lipid supplementation for in vitro erythroid cell culture impacts reticulocyte yield and characteristics.

Lipids, particularly cholesterol, are critical components of red blood cell (RBC) membranes, influencing protein function, cell stability, and deformability. Reticulocytes (young RBC) derived from in vitro erythroid cultures have been reported to possess less cholesterol than their native counterparts, compromising their functional integrity and lifespan. However, variability in starting materials and culture protocols between studies has hindered deQnitive conclusions regarding the nature and consequences of this lipid deQciency. Here, we evaluated the influence of lipid sources on reticulocyte quality using a well-established CD34+ erythroid culture system. We compared the use of human AB-serum and Octaplas (solvent/detergent-treated pooled plasma) as lipid sources. Our results reveal that detergent-treated plasma leads to cholesterol-deQcient reticulocytes with impaired characteristics, including reduced Qltration yield, heightened osmotic fragility, and altered PIEZO1 activity. In contrast, AB-serum supported the generation of functionally stable reticulocytes, with cholesterol supplementation required to rescue the defects observed with plasma. Importantly, this study provides the Qrst integrated lipidomic, metabolomic, and proteomic characterisation of in vitro-derived reticulocytes cultured under distinct lipid conditions. These multi-omic datasets offer new insights into the consequences of reduced lipid availability during erythroid culture and offer new insights into how culture media affects the development and functionality of lab grown blood.