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

• Published in: Cells (Basel, Switzerland) Switzerland), 2022-06, Vol.11 (12), p.1856
• Main Authors: Brockbank, Kelvin G M, Bischof, John C, Chen, Zhenzhen, Greene, Elizabeth D, Gao, Zhe, Campbell, Lia H
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Cardiac muscle; Cell Survival; Cell viability; complex tissue preservation; Convection; Cooling; Cryopreservation; Cryopreservation - methods; Cryoprotective Agents - pharmacology; Cryoprotectors; Cytotoxicity; Disaccharides; Heart; heart valve; Heart Valves; Ice; Medical research; Nanoparticles; nanowarming; Sucrose; Swine; Transplants & implants; Vitrification
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11121856

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 °C, and stored at −135 °C. Nanowarming was performed in a single ~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 α-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.