Modification of cellular membranes conveys cryoprotection to cells during rapid, non-equilibrium cryopreservation

Rapid cooling and re-warming has been shown promising to cryopreserve living cells, which cannot be preserved by conventional slow freezing methods. However, success is limited by the cytotoxicity of highly concentrated cryoprotective agents. Recent results have shown that cryoprotective agents do not need to suppress intracellular ice crystals completely to allow for survival after cryopreservation. Cryoprotective agents like DMSO or ethylene glycol can also lead to a tolerance of cells towards intracellular ice. It is however unclear by which mechanism this tolerance is achieved. These substances are also known to modulate properties of cellular membranes. It is shown here that cryoprotective DMSO and ethylene glycol have a clear influence on the mobility of lipids in the plasma membrane of HeLa cells. To isolate changes of the properties of plasma membranes from effects on ice formation, the membrane properties were modulated in absence of cryoprotective agents. This was achieved by changing their sterol content. In cells with elevated sterol content, an immobile lipid fraction was present, similar to cells treated with DMSO and ethylene glycol. These cells showed also significantly increased plasma membrane integrity after rapid freezing and thawing in the absence of classical cryoprotective agents. However, their intracellular lysosomes, which cannot be enriched with sterols, still got ruptured. These results clearly indicate that a modulation of membrane properties can convey cryoprotection. Upon slow cooling, elevated sterol content had actually an adverse effect on the plasma membranes, which shows that this effect is specific for rapid, non-equilibrium cooling processes. Unraveling this alternative mode of action of cryoprotection should help in the directed design of novel cryoprotective agents, which might be less cytotoxic than classical, empirically-found cryoprotective agents.