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Nanowarming Of Ice-free Cryopreserved Heart Valves For Maintenance Of Cell Viability
Kelvin G. Brockbank1, Zhen Zhen Chen2, Elizabeth D. Greene2, Ulrich Alfred Stock3.
1Clemson University, North Charleston, SC, USA, 2Tissue Testing Technologies LLC, North Charleston, SC, USA, 3Royal Brompton and Harefield Hospital, Imperial College, London, United Kingdom.

OBJECTIVE: Development of an ice-free cryopreservation and rapid warming method that keeps the cells viable in banked heart valves. Vitrification, cryopreserved storage in a “glassy” rather than crystalline phase, is an important enabling approach for banking of living tissues, offering the ability to store and transport cells and tissues for biomedical uses. Application to large tissues has been limited by cryoprotectant cytotoxicity and ice nucleation during slow convection warming.

METHODS: Porcine tissues were used as a model of natural or bioengineered pulmonary heart valves. The valves were vitrified in 30-50 mLs of cryoprotectant formulations, 3 cm sample diameter, with dimethylsulfoxide, propylene glycol, formamide, 0.6M disaccharides and 7.6-10mg/mL Fe nanoparticles and stored below -135C. Rewarming was performed in a single step by inductive heating within a 35 kA/m magnetic field. Controls consisted of fresh and convection warmed vitrified heart valves without nanoparticles. After extensive washing cell viability was assessed by metabolic assay.
RESULTS: The leaflets were well preserved with viability similar to untreated fresh leaflets over days post-warming in vitro. Convection warmed leaflet viability was not significantly different from nanowarmed leaflets immediately after rewarming, however significantly higher nanowarmed leaflet viability (p<0.05) was observed over time in vitro. In contrast the pulmonary artery and fibrous cardiac muscle were at best 75% viable and viability decreased over time in vitro.
CONCLUSIONS: This work demonstrates progress towards control of ice formation and cytotoxicity hurdles for ice-free cryopreservation of a large complex tissue model by application of nanowarming and disaccharides to cryoprotectant formulations. These results also suggest that there is a greater need for nanowarming for the thicker heart valve components, the artery and fibrous cardiac muscle band where the cryoprotectant concentration achieved is likely less than in thin leaflets and ice induced-damage more likely.


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