| Autor: |
Brockbank KGM; Tissue Testing Technologies LLC, 2231 Technical Parkway, Suite A, North Charleston, SC 29406, USA.; Department of Bioengineering, Clemson University, Charleston, SC 29425, USA.; Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA., Bischof JC; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA., Chen Z; Tissue Testing Technologies LLC, 2231 Technical Parkway, Suite A, North Charleston, SC 29406, USA., Greene ED; Tissue Testing Technologies LLC, 2231 Technical Parkway, Suite A, North Charleston, SC 29406, USA., Gao Z; Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA., Campbell LH; Tissue Testing Technologies LLC, 2231 Technical Parkway, Suite A, North Charleston, SC 29406, USA. |
| Abstrakt: |
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. |
