? Although significant advances have been made in the field, the critical issues of mechanical strength and long-term storage methods limit progress in engineered vessels. It is well established that traditional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. In Phase I studies, we demonstrated that ice-free cryopreservation, known as vitrification, can have a salutary effects on the cryopreservation of polyglycolic acid - derived engineered vessels containing smooth muscle cells which otherwise sustain significant injury from freezing. Fresh, traditionally cryopreserved (frozen), and vitrified engineered vessels evaluated for apoptosis revealed few apoptotic or necrotic cells in fresh and vitrified vessels compared with frozen vessels. The metabolic assay results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were over 82.7% of fresh controls and in marked contrast, the results for frozen vessel samples were only 10.7% of fresh controls (p<0.001). Cryosubstitution studies of frozen and vitrified engineered arteries revealed negligible ice in the vitrified specimens, and extensive ice formation in the extracellular matrix of frozen specimens. Passive mechanical testing revealed enhanced tissue strength after cryopreservation. Vitrification is a feasible storage method for tissue engineered blood vessel constructs. In this Phase II proposal, testing of vitrification as a storage method for tissue engineered blood vessels is extended to include endothelialization and large animal implant studies. In addition, several new methods of vitrification with better ice control will be screened in vitro and the two most effective methods will be tested in a large animal model in year 2. Finally, we will optimize a technique for ice-free cryopreservation of human tissue engineered blood vessels. In Phase III, these studies will be extended to include clinical trials. The long-term goal of this proposed project is to develop methods for cell and tissue storage that will make it possible for tissue engineered devices to be available in the United States and worldwide, regardless of environmental conditions. ? ?
