Vitrification media: toxicity, permeability, and dielectric properties.
The aim of this study was to select a cryoprotectant for use in attempts to preserve tissues and organs by vitrification. The first step was to select a cell line with which to compare the toxicity of a range of commonly used cryoprotectants. An immortal vascular endothelial cell (ECV304) was exposed to vitrifying concentrations of four cryoprotectants: dimethyl sulfoxide (Me(2)SO; 45% w/w); 2,3 butanediol (BD; 32%); 1,2-propanediol (PD; 45%); and ethanediol (ED; 45%). Three times of exposure (1, 3, and 9 min) and two temperatures (22 and 2-4 degrees C) were studied. After removal of the cryoprotectant, the ability of the cells to adhere and divide in culture over a 2-day period was measured and expressed as a Cell Survival Index (CSI). There was no measurable loss of cells after exposure to the four cryoprotectants but 3-min exposure to BD, PD, or Me(2)SO at room temperature completely destroyed the ability of the cells to adhere and divide in culture. In contrast, exposure to all four cryoprotectants at 2-4 degrees C for up to 9 min permitted the retention of significant cell function, the CSIs, as a proportion of control, being 76.3+/-7.0% for BD, 63.6+/-7.1% for PD, 37.0+/-4.1 for Me(2)SO, and 33.2+/-3.0 for ED. The permeability properties of the cells for these four cryoprotectants was also measured at each temperature. Permeability to water was high, L(p) approximately equal 10(-7) cm/s/atm at 2-4 degrees C with all the cryoprotectants, but there were substantial differences in solute permeability: BD and PD were the most permeable at 2-4 degrees C (P(s)=4.1 and 3.0 x 10(-6) cm/s, respectively). Equilibration of intracellular cryoprotectant concentration was rapid, due in part to high water permeability; the cells were approximately 80% of their physiological volume after 10 min. Treatment at 2-4 degrees C with BD was the least damaging, but PD was not significantly worse. Exposure to vitrifying concentrations of ED and Me(2)SO, even at 2-4 degrees C, was severely damaging. Segments of rabbit carotid artery were treated with vitrifying concentrations of each of the two most favorable cryoprotectants, BD and PD, for 9 min. It was shown that each cryoprotectant reduced smooth muscle maximum contractility to a similar extent and abolished the acetylcholine response. However, vital staining revealed that exposure to BD also caused substantial damage to the endothelial lining, whereas the endothelium was completely intact after PD exposure, raising the possibility that the effect of PD on NO release may be reversible. In later stages of this project it is planned to use dielectric heating to rewarm the tissues and thereby avoid devitrification. The effects of each cryoprotectant on this mode of heating was therefore studied. Gelatin spheres containing vitrifiable concentrations of each cryoprotectant were rewarmed from -60 degrees C in a radiofrequency applicator. Because the uniformity of heating is related to the dielectric properties of the material, these properties were also measured. PD was the most suitable. These physical measurements, combined with the measurements of toxicity and permeability, indicate that PD is the most favorable cryoprotectant of those tested for use in subsequent stages of this study.