Development of Novel Storage Method for Highly Viable and Well-Characterized Vitrified Human Islets

项目总结/摘要 拟议研究的最终目标是实现治疗剂量的长期储存 用于逆转1型糖尿病和改善2型糖尿病的人胰岛， 了解玻璃化转变温度（TG）附近的储存损伤，使新型长 长期存储有待开发。在使用前，胰岛银行应减少或防止胰岛的恶化， 促进耐受诱导，能够在肾移植后延迟胰岛移植， 几周前从同一个捐赠者那里得到的，还有很多其他的好处。我们已经证明，在 未发表的结果，这是可能的玻璃化和复温人胰岛与良好的胰岛 数量恢复和良好的活力保留（基于活体染色），葡萄糖刺激胰岛素 释放（刺激指数）、葡萄糖刺激的耗氧量和逆转诱导的 糖尿病小鼠与最小的胰岛剂量，我们正在努力扩大规模，从13,000 IEQs每 玻璃化批次至完全治疗剂量的胰岛（450，000 - 720，000）。下一步是确定 储存至少3-4周后仍保持高度活力和功能，并确定最佳方案 胰岛的储存条件。最佳储存条件被假定为是相当非常规的， 与玻璃化来自单个供体的所有胰岛相关的大体积， 被玻璃化，并希望快速转移到和出长期储存，没有任何风险， 断裂特别是，我们认为胰岛应该储存在TG附近，甚至可能高于TG，但 在此温度范围内的储存以前没有以任何适当的方式进行过研究。因此，委员会认为， 目的1致力于确定温度对胰岛活力和功能的影响，目的2是 针对确定温度对小岛及其周围冰核的影响， 玻璃化介质和冰核和胰岛完整性之间的关系，目的3是为了 以确定储存时间超过1个月的影响。一个月的储存应该足够 大多数胰岛银行的需要，但两个月将是非常宝贵的提供一个安全边际， 为临床医生和患者提供更大的灵活性。同时，比较1个月和2个月的储存 将打开目前完全缺乏的关于TG附近成核速率随时间变化的信息， 并将开始回答有关TG附近生物稳定性的问题， 晦涩难懂。

Project Summary/Abstract The ultimate objective of the proposed research is to enable the long-term banking of curative doses of human pancreatic islets for the reversal of type 1 diabetes and the amelioration of type 2 diabetes and to understand storage injury near the glass transition temperature (TG) to enable a new type of long term storage to be developed. Banking should reduce or prevent the deterioration of islets prior to use, facilitate tolerance induction, enable delayed islet transplantation following transplantation of a kidney from the same donor weeks earlier, and have many other benefits. We have already demonstrated, in unpublished results, that it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viability (based on vital staining), glucose-stimulated insulin release (stimulation index), glucose-stimulated oxygen consumption, and ability to reverse induced diabetes in mice with minimal islet doses, and we are working on scaling up from 13,000 IEQs per vitrified batch to full curative doses of islets (450,000-720,000). The next step is to establish that islets remain highly viable and functional following at least 3-4 weeks of storage and to determine the best storage conditions for islets. The best storage conditions are postulated to be quite unconventional due to the large volumes associated with vitrifying all islets from a single donor, the fact that the islets will be vitrified, and the desirability of rapid transfer into and out of long term storage without any risk of fracturing. In particular, we believe that islets should be stored near TG, and possibly even above TG, but storage in this temperature range has not been previously studied in any adequate way. Accordingly, Aim 1 is devoted to determining the effect of temperature on islet viability and functionality, Aim 2 is directed toward determining the effect of temperature on ice nucleation in both the islets and their vitrified medium and the relationship between ice nucleation and islet integrity, and Aim 3 is intended to determine the effect of storage time beyond 1 month. One month of storage should be sufficient for most islet banking needs, but two months would be very valuable for providing a safety margin and more flexibility to the clinician and the patient. At the same time, comparing 1 and 2 months of storage will open up information about rates of nucleation over time near TG that is presently entirely lacking, and will begin to answer questions about biological stability near TG that presently remain entirely obscure.