Cryo-responses of two types of large unilamellar vesicles in the presence of non-permeable or permeable cryoprotecting agents.

Cryo-responses of two types of large unilamellar vesicles (LUV) that were made from either egg yolk L-alpha-phosphatidylcholine (EPC) or 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), in the presence of non-permeable or permeable cryoprotective agents (CPA) was investigated. Partial ternary phase diagrams of CPA-salt-water with specific CPA to salt ratio (R), were constructed to estimate the phase volume of ice and unfrozen matrix of the LUV dispersion, which could aid in understanding the mechanistic actions of CPA. Leakage of both EPC and DPPC LUV was reduced if the sugar concentrations are above 10% (w/w) for disaccharides and 5% (w/w) for monosaccharides. Above these sugar concentrations, non-permeable CPA were more effective in preventing leakage of DPPC LUV than in EPC LUV. Below these sugar concentrations, EPC and DPPC LUV with limited mobility in the remaining unfrozen matrix were more likely to approach and interact with one and another, which were not anticipated when the LUV were completely embedded in the ice matrix. In the presence of Me(2)SO or EG, EPC LUV that had been subjected to freezing and thawing processes were protected from leakage. At room temperature, Me(2)SO and EG were detrimental to the DPPC LUV. This study suggests that the choice of CPA for cell cryopreservation depends on the type of phospholipids in plasma membranes, which vary in their acyl chain length and gel-liquid crystal phase transition temperature.

Characterizing the freezing behavior of liposomes as a tool to understand the cryopreservation procedures.

Freezing behaviors of egg yolk l-alpha-phosphatidylcholine (EPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicles (LUV) were quantitatively characterized in relation to freezing temperatures, cooling rates, holding time, presence of sodium chloride and phospholipid phase transition temperature. Cooling of the EPC LUV showed an abrupt increase in leakage of the encapsulated carboxyfluorescein (CF) between -5 degrees C and -10 degrees C, which corresponded with the temperatures of the extraliposomal ice formation at around -7 degrees C. For the DPPC LUV, CF leakage started at -10 degrees C, close to the temperature of the extraliposomal ice formation; followed by a subsequent rapid increase in leakage between -10 degrees C and -25 degrees C. Scanning electron microscopy showed that both of these LUV were freeze-concentrated and aggregated at sub-freezing temperatures. We suggest that the formation of the extraliposomal ice and the decrease of the unfrozen fraction causes freeze-injury and leakage of the CF. The degree of leakage, however, differs between EPC LUV and DPPC LUV that inherently vary in their phospholipid phase transition temperatures. With increasing holding time, the EPC LUV were observed to have higher leakage when they were held at -15 degrees C compared to at -30 degrees C whilst leakage of the DPPC LUV was higher when holding at -40 degrees C than at -15 degrees C and -50 degrees C. At slow cooling rates, osmotic pressure across the bilayers may cause an additional stress to the EPC LUV. The present work elucidates freeze-injury mechanisms of the phospholipid bilayers through the liposomal model membranes.

Effect of intra- and extra-liposomal distribution of sodium chloride on the stability of large unilamellar vesicles.

Three groups of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicle (LUV) dispersions were studied: LUV (A) dispersions with only extraliposomal sodium chloride (NaCl), LUV (B) dispersions with intra- and extraliposomal NaCl, and LUV (C) dispersions with only intraliposomal NaCl. The NaCl concentrations ranged from 0 to 150 mM. An abrupt increase in leakage was observed around -10 degree C for all the three groups of LUV, which coincided with the temperature of extraliposomal ice formation. Within the three groups, leakage of LUV (C) was significantly higher than the other groups. Extraliposomal ice formation and the resulting freeze-concentration of LUV may be the major cause of the leakage. Intraliposomal ice formation observed at -43 degree C seemed to stop leakage of LUV when LUV were frozen below -43 degree C. An exotherm of eutectic crystallization of NaCl was occasionally observed at -37 degree C, with a higher probability of formation at 150 mM extraliposomal NaCl than at 50 mM. The eutectic crystals were thought to cause additional leakage from the LUV (B).