Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures

Cryopreservation of biological materials is predominantly done using liquid nitrogen, and its application involves high maintenance costs and the need for periodical refilling of liquid nitrogen. Stable storage in mechanical freezers at -80°C would eliminate these issues and allow for shipment of frozen specimens using dry ice. In this work, the possibility of increasing the storage temperature of cryopreserved samples to -80°C by using combinations of DMSO and sucrose has been studied. Preservation efficacy was studied by measuring stability of liposomes encapsulated with carboxyfluorescein during storage at -150, -80 and -25°C for up to three months. Thermal and molecular mobility properties of the different DMSO-sucrose formulations were measured using differential scanning calorimetry, whereas hydrogen bonding interactions of the formulations were probed by Fourier transform infrared spectroscopy. It was found that addition of sucrose to DMSO solutions increases the Tg, and decreases molecular mobility in the glassy state at a particular temperature. Although it was expected that storage above or close to Tg at -80°C would affect liposome stability, stability was found to be similar compared to that of samples stored at -150°C. Higher molecular mobility in the glassy state could not be associated with faster CF-leakage rates. Distinct differences in storage stability at -25°C, far above Tg, were found among the sucrose/DMSO formulations, which were explained by the differences in permeability of sucrose and DMSO resulting in different levels of osmotic stress in the formulations.