Project Summary Diabetes has a tremendous impact on the health and well-being of affected individuals, as well as a considerable overall societal burden. Pancreatic islet transplantation has the potential to cure diabetes, but one of the main problems limiting the success of this treatment is an inadequate supply of islets. Islets from a single donor are often insufficient to achieve insulin independence, and multiple infusions are often required, each with increasing risk. Two potential strategies exist to increase the number of islets available: (1) pool islets from multiple donors and perform single procedure, high-dose transplants; and (2) develop alternative sources such as stem-cell-derived islets. The availability of these limited resources becomes a supply chain problem, and for either approach, a method for islet preservation is essential. Our long-term objective is to develop a method for cryopreserving, or “banking,” islets prior to transplant. No previous strategy has achieved the high viability, function, and clinical scalability required for transplant in a single approach. To achieve long-term islet banking, we propose to use an alternative cryopreservation strategy, vitrification. That is, cryogenic storage in an ice-free glassy state. A significant challenge in the vitrification of biospecimens is that the cooling and heating rates needed for vitrifying and rewarming are tremendously high (>107 °C/min). These rates are reduced by adding cryoprotective agents (CPA) that inhibit ice formation, but these agents are themselves toxic to islets. Thus, the critical challenge in islet vitrification is achieving fast enough cooling and warming to avoid ice, while avoiding toxicity from the CPA, and doing so in a clinically scalable manner. Using engineering principles of heat and mass transfer, our multidisciplinary research team has developed an approach for vitrification and rewarming (VR) to solve this problem, termed “cryomesh VR,” for islets. Our central hypothesis is that the improved heat transfer achieved by cryomesh VR, combined with optimizations in CPA use, will enable ice-free vitrification and rewarming of islets while avoiding toxicity. Our preliminary data achieve cooling and warming rates far exceeding other methods, and we have shown CPA loading and unloading protocols with low toxicity in mouse, human, pig, and human stem-cell-derived (SC) islets. Indeed, in all cryopreserved islet models tested we have achieved viability, recovery, and function that meets or exceeds all previous reports and does so in a clinically scalable method. To further improve our approach and move towards clinical translation, we propose the following aims: Aim 1. Refine the optimal physical conditions for the cryomesh VR of mouse, human, and SC islets; Aim 2. Measure the viability, function, and in vivo potency of mouse, human, and SC islets following cryomesh VR; Aim 3. Define the molecular and cellular changes occurring in response to cryop...