Engineering optimization and scaling enables high quality pancreatic islet cryopreservation for banking and transplant

项目摘要 糖尿病对受影响个人的健康和福祉有巨大影响，以及 相当大的整体社会负担。胰岛移植有治愈糖尿病的潜力，但只有一个 限制这种治疗成功的主要问题之一是胰岛供应不足。来自单个 供者往往不足以实现胰岛素独立，而且经常需要多次输注，每次 增加风险。有两种可能的策略可以增加可用胰岛的数量：(1)从 并进行单一程序、大剂量移植；以及(2)开发替代来源，如 作为干细胞来源的胰岛。这些有限资源的可获得性成为供应链问题，对于 无论是哪种方法，一种保存胰岛的方法都是必不可少的。我们的长期目标是开发一种方法来 在移植前对胰岛进行冷冻保存，或“银行处理”。以前的任何策略都没有达到如此高的生存能力， 单一方法移植所需的功能和临床可伸缩性。 为了实现长期的胰岛银行，我们建议使用一种替代的冷冻保存策略，玻璃化。 也就是说，在无冰玻璃状态下的低温存储。生物有机体玻璃化冷冻面临的重大挑战 玻璃化和复温所需的冷却和加热速度非常高(&gt；107℃/分钟)。 这些速率可以通过添加抑制结冰的低温保护剂(CPA)来降低，但这些试剂 对小岛来说是有毒的。因此，胰岛玻璃化的关键挑战是实现足够快的冷却和 升温以避免结冰，同时避免CPA的毒性，并以临床可扩展的方式做到这一点。 利用热质传递的工程原理，我们的多学科研究团队开发了 一种解决这一问题的玻璃化冷冻复温(VR)方法，称为“冷冻冷冻VR”，用于胰岛。我们的 中心假设是，与优化相结合，低温网格VR实现的热传递改善 在CPA使用中，将实现无冰玻璃化和胰岛复温，同时避免毒性。我们的预赛 数据获得的冷却和升温速度远远超过其他方法，我们已经显示了CPA加载和 在小鼠、人、猪和人类干细胞来源(SC)的胰岛上低毒的卸载方案。事实上，在 所有冷冻保存的胰岛模型都经过测试，我们已经实现了活性、恢复率和功能达到或超过 所有以前的报道，并以临床可扩展的方法进行。进一步改进我们的做法和行动 对于临床翻译，我们提出了以下目标：目标1.优化临床翻译的最佳物理条件 小鼠、人和干细胞胰岛的冷冻网格VR；目的2.测量细胞的活性、功能和体内效力 冷冻网格VR后的小鼠、人类和SC胰岛；目标3.定义发生的分子和细胞变化 对冷冻保存的响应；以及目标4。扩大冷冻网格VR以满足临床吞吐量并使其适应 CGMP生产工艺。如果成功，这种方法可能会彻底改变孤岛的隔离、分配 并在移植前储存，并增加对已故供体胰腺的利用，用于治疗糖尿病。

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 cryopreservation; and Aim 4. Scale-up cryomesh VR for clinical throughput and adapt the processes for cGMP production. If successful this approach could revolutionize how islets are isolated, allocated, and stored prior to transplant and increase utilization of deceased donor pancreases for the cure of diabetes.