Development of a simplified ice-free cryopreservation method for heart valves employing VS83, an 83% cryoprotectant formulation.

We have previously demonstrated storage of ice-free cryopreserved heart valves at -80°C without the need for liquid nitrogen, with the aims of decreasing manufacturing costs and reducing employee safety hazards. The objectives of the present study were a further simplification of the ice-free cryopreservation method and characterization of tissue viability. Porcine pulmonary heart valves were permeated with an 83% cryoprotectant solution (VS83) followed by rapid cooling and storage at -80°C. The cryoprotectants were added and removed in either single or multiple steps. Fresh untreated frozen controls employing 10% dimethylsulfoxide and controlled rate freezing to -80°C, and storage in vapor phase nitrogen were also performed. After rewarming and washing, cryopreserved leaflets were compared with fresh controls using the resazurin reduction metabolism assay. Comparison of valve tissues in which the cryoprotectants were added and removed in either single or multiple steps demonstrated similar viability results for the muscle, conduit, and leaflet components. The ice-free cryopreserved conduit and leaflet components were significantly less viable than either fresh or frozen tissues. The muscle component, although less viable, was not significantly different. The changes in tissue viability were a function of cryoprotectant exposure, and resulting cytotoxicity, not temperature reduction during storage. TUNEL staining showed that ice-free cryopreservation did not induce significant amounts of apoptosis, suggesting that necrosis is the predominant cell death pathway in ice-free cryopreserved heart valves. There was very little difference in cell viability when the cryoprotectants were added and removed in a single step versus multiple steps. Ice-free cryopreserved valve tissues demonstrated very low viability compared with controls. These results support further simplification of the ice-free cryopreservation method.