Freezing-driven ionic charge imbalance leads to pore formation and osmotic injury of lipid membranes

In this work, we reveal the fundamental mechanism controlling osmotic injury of lipid membranes under low-temperature preservation using an in-vitro membrane system under freezing temperature and the molecular dynamic simulations. The freezing-driven ionic charge imbalance is the major factor affecting the membrane conformation and causing the osmotic injury. Under freezing temperature, the ionic charge imbalance, originating from the preferential incorporation of anions into the growing ice crystals, results in membrane poration with the directional penetration of ion molecules. Subsequently, the osmotic efflux of water molecules through the pore causes cell dehydration, eventually leading to the lethal osmotic injury of lipid membranes during freezing. Moreover, we find a stark difference in tolerance to freezing and the times required for pore formation in membranes with different lipid compositions. Membranes enriched with cholesterol and anionic lipids exhibit increased resistance to freezing-induced osmotic injury, as the addition of cholesterol and anionic lipids in membranes delays the pore formation under freezing temperature. These findings advance in depth the molecular-level understanding of freezing injury on lipid membranes and provide an opportunity to develop an alternative strategy to protect diverse cells during preservation at subzero temperatures by regulating the composition of lipid membranes.