SBIR-STTR Award

Effective methods of preservation are irnportant for the remerging technology of tissue engineering. We recently demonstrated, for the first time, that vitrification can have a salutary effect on the cryopreservation of a complete tissue, rabbit jugular vein, which otherwise sustains significant injury from freezing. Restriction of the amount and size of ice crystal formation during cryopreservation can be achieved by promotion of amorphous solidification (vitrification) rather than crystallization In this Phase I SBIR proposal, the primary objective is to test the feasibility of employing vitrification for long-term storage of tissue engineered blood vessels (TEBV). The effects of vitrification and conventional cryopreservation involving ice formation will be assessed using biomechanical tests, cell viability assays and vascular physiology methods. If vitrification is shown to be feasible for storage of TEBVs, these studies will be extended to a Phase II SBIR proposal to include in vivo testing of the Duke University TEBVs and TEBVs being developed by other organizations. PROPOSED COMMERCIAL APPLICATION: Customers are tissue engineering, tissue processing and banking organizations, and companies supplying reagents and biological materials to research organizations. The tissue engineering industry addresses diseases and disorders associated with over $500 billion expended annually in the U.S. for health care costs.

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.

Thesaurus Terms:
blood vessel, cryopreservation, technology /technique development, tissue /organ preservation, tissue engineering biomechanics, blood vessel transplantation, carotid artery, extracellular matrix human tissue, swine