From Water to Ice: Investigation of the Effect of Ice Crystal Reduction on the Stability of Frozen Large Unilamellar Vesicles

The solid phase of water, ice, is generally detrimental to cells because it causes freeze injury of cells during cryopreservation. Nonpermeable cryoprotective agents (CPAs) such as sucrose, trehalose, and glucose and/or permeable CPAs such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG) are often added to freezing solutions to reduce such effects. The effect of ice formation on the stability of phospholipid bilayers was investigated using a model membrane: 1,2 - dipalmitoyl - rac - glycero - 3 -phosphocholine (DPPC) large unilamellar vesicles (LUVs) encapsulated with carboxyfl uorescein (CF) solution. The LUV dispersion was added with a CPA, cooled to 40 ° C, and heated to 20 ° C at 10 ° C/min. LUV stability was described by the degree of CF leakage. In the presence of sugars, DMSO, or EG, ice formation was reduced. Simultaneous to the ice reduction, an increase of unfrozen fraction prevented LUV aggregation and leakage. The leakage was observed to decrease above a defi ned sugar concentration and as the concentration of DMSO or EG increased. Below the defi ned sugar concentration, LUV leakage increased with increasing sugar concentrations. Nonpermeable CPAs were more effective for the frozen LUVs compared to permeable CPAs. The current result suggests that LUVs were spaced out from one another in the presence of nonpermeable CPAs. On the other hand, LUVs in 10% (wt/wt) of permeable CPAs could probably undergo structural destabilization.