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 definitive conclusions regarding the nature and consequences of this lipid deficiency. 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 (S/D)-extracted pooled plasma) as lipid sources. Our results reveal that S/D-extracted plasma leads to cholesterol-deficient reticulocytes with impaired characteristics, including reduced filtration 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 majority of defects observed with culturing erythroid cells with plasma alone. Importantly, this study provides the first 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.