Ice-free cryopreservation of heart valve allografts: Better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6\~mol/l cryoprotectant (4.65, 4.65, and 3.31\~mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at -80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4\~mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-fre e tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P\~<\~0.05). After 3 and 6\~months in vivo valve function was determined by two-dimensional echo-Doppler and at 7\~months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P\~=\~0.0403, P\~=\~0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P\~<\~0.05) and infiltrating CD3+ T-cells (P\~<\~0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at -80°C avoids ice formation, tissue-glass cracking