Preservation of myocyte structure and mitochondrial integrity in subzero cryopreservation of mammalian hearts for transplantation using antifreeze proteins - An electron microscopy study

Objective: Freeze tolerant fish and insects in nature are able to survive subzero temperatures by noncolligatively lowering the freezing temperature of their body fluids using a family of thermal hysteresis proteins (antifreeze proteins, AFPs) specific for each species. Past efforts to cryopreserve mammalian hearts using these proteins were unsuccessful. We report the first successful subzero cryopreservation of rat hearts using fish derived antifreeze proteins with preservation of myocyte structure. Methods: Heterotopic heart transplantations were performed in isoimmunic Sprague Dawley rats. Donors' hearts were arrested using University of Wisconsin (UW) solution and preserved in UW solution containing AFP I (six experiments) or AFP III (six experiments) at concentrations of 15-20 mg/cc for 2-6 h at subzero temperatures ranging from -1.1 to -1.3°C. Four control experiments were performed by preserving harvested hearts in UW solution alone at -1.3°C for 6 h. In all experiments ice was added in the solution for crystallization. Heterotopic transplantations were performed in the abdomen of the recipient rats. Viability was visually assessed and graded on a scale of 1 (poor contraction) to 6 (excellent contraction). The hearts were then fixed in vivo and processed for electron microscopy study. Results: All hearts preserved at subzero temperatures using AFP I or AFP III survived displaying viability scores of 4-6 1 h after transplantation. Three of the four control hearts that were preserved at -1.3°C without the protective effect of AFP froze and died upon reperfusion. Electron microscopy study of hearts preserved with AFP demonstrated preservation of myocyte structure and mitochondrial integrity. Conclusion: Subzero cryopreservation of mammalian hearts for transplantation using AFP I or AFP III is feasible with preservation of myocyte structure and mitochondrial integrity.