Ice-free vitrification and nano warming technology for banking of cardiovascular structures.

ABSTRACT This proposal focuses on translation of ice-free cryopreservation by vitrification employing a novel approach of volumetric heating by nanowarming using Fe nanoparticles in an alternating electromagnetic ?eld. Vitrification, sub-zero storage below the glass transition temperature in a “glassy” rather than a crystalline frozen phase, is a form of cryopreservation that avoids ice formation. Vitri?cation can be achieved by quickly cooling the material to cryogenic storage temperatures, where ice cannot form. Vitri?cation can be maintained at the end of the cryogenic protocol by quickly rewarming the tissue to temperatures above the temperatures where ice nucleation may occur. The magnitude of the rewarming rates necessary to maintain vitri?cation is much higher than the magnitude of the cooling rates that are required to achieve it in the ?rst place. The most common approach to achieve the required cooling and rewarming rates is by convection based boundary warming in which the the specimen's surface is exposed to a temperature controlled environment, such as a fluid bath. Due to the underlying principles of heat transfer, there is a size limit in the case of surface boundary heating beyond which crystallization cannot be prevented at the center of the specimen. Furthermore, due to the underlying principles of solid mechanics, there is also a size limit beyond which thermal expansion in the specimen can lead to structural damage and fractures. Volumetric heating by nanowarming during the rewarming phase of the cryogenic protocol can alleviate these size limitations. Vitrification is already an important enabling approach for reproductive medicine with the potential to permit storage and transport of cells, tissues and organs for a great variety of biomedical uses. Unfortunately, practical application of vitrification has been limited to smaller systems such as cells and thin tissues due to diffusive and phase change limitations that preclude use for blood vessels, larger tissues and organs. To circumvent this problem we demonstrated that nanowarming effectively rewarms blood vessels in our preliminary research. Our experiments demonstrated that this innovative rewarming technique rewarmed vitrified femoral and carotid arteries in volumes ranging from 1 to 50mL with retention of cell viability and physiologic function. However, warming of thick arteries was suboptimal. We propose using large animal blood vessel, models for further optimization and evaluation of nanowarmed vessels using a combination of in vitro and in vivo studies. In Phase 1 in a single specific aim we will optimize ice-free vitrification of thick walled arteries, aorta and pulmonary, with a go/no go objective of achieving rt 90% viability for progression to Phase 2. In Phase 2 specific aims, we propose using porcine vascular models in a combination of ex vivo and in vivo studies. The magnetic nanoparticles will be distributed around and within the internal spaces of vessels. The large vessel lumen space makes them a good choice for optimization of vitrification and nanowarming. In Aim 1 we will evaluate cryopreserved arteries after real time shipping, comparing methods and validating the transport conditions that are finally approved based upon absence of tissue cracking. In Aim 2 we will characterize the post-ice-free cryopreservation state of arteries preserved for at least 2 years. In addition, during this aim we will characterize the chemistry and biomaterial properties of ice-free cryopreserved blood vessels. Effective vitrification will be evaluated using cryomacroscopy to detect ice formation and cryoprotectant residuals by Raman spectroscopy. In Aim 3 we will perform short-term transplant studies (28 days) in two porcine vascular models (femoral and pulmonary artery into the carotid and pulmonary, respectively) in order to validate our technology for a future Phase IIb SBIR proposal using clinically relevant preclinical non-human primate models and human tissues.