Enhancing Supercooled Red Blood Cell Storage: The Membrane-Stabilizing Effect of Ethanol

Hypothermic storage is constrained by the progressive depletion of energy reserves, curtailing the shelf-life of organs, cell therapies, and blood products. High sub-zero supercooling helps preserve energy homeostasis by slowing catabolic processes; however, the resulting injury in this setting is not primarily driven by energy depletion. Here, we investigated whether low-dose ethanol could prevent forms of injury that arise independently of disrupted energy homeostasis and remain unaddressed in supercooled storage. Human red blood cells treated with 4% (v/v) ethanol were stored 4 {degrees}C, -4 {degrees}C, or -8 {degrees}C and subject to a series of functional assessments and integrated metabolomic/lipidomic profiling after 21 and 42 days of storage. Metabolomics data showed that energy homeostasis was better preserved at lower temperatures, yet these supercooled conditions simultaneously intensified hemolysis and caused a marked depletion of lysophospholipid species that did not occur at 4 {degrees}C. Ethanol blunted these effects, cutting hemolysis by [~]50 % at -4 {degrees}C, by [~]85 % at -8 {degrees}C, and attenuating lysophospholipid depletion. These results uncover a previously unrecognized, lipid-centric injury that arises during supercooled storage and establish low-dose ethanol as a simple, readily deployable countermeasure that could help extend storage intervals of diverse biological systems. SUMMARYCellular preservation has traditionally focused on maintaining energy metabolism during hypothermic storage, but whether this is sufficient at high sub-zero temperatures remains unclear. Human red blood cells were stored for up to 42 days at 4{degrees}C, -4{degrees}C, or -8{degrees}C with or without 4% ethanol and evaluated using functional assays and integrated metabolomic and lipidomic profiling. Although supercooling preserved energy homeostasis, it increased hemolysis and caused pronounced lysophospholipid depletion. Ethanol reduced hemolysis by approximately 50% at -4{degrees}C and 85% at -8{degrees}C, attenuated lysophospholipid loss, and produced comparatively modest changes in cellular metabolism. These findings identify membrane integrity as a critical determinant of preservation outcome and support strategies that protect membrane stability alongside metabolic homeostasis.