Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability

Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1-30 mL cryoprotectant formulations +/- Fe nanoparticles +/- 0.6 M disaccharides, cooled to -100 degrees C, and stored at -135 degrees C. Nanowarming was performed in a single similar to 100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability (p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with alpha-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.