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

项目摘要/摘要 拟议研究的最终目标是实现治疗剂量的长期储存 人胰岛逆转1型糖尿病和改善2型糖尿病的实验研究 了解储藏损伤附近的玻璃化转变温度(Tg)，以使新型的长 待开发的定期存储。银行应在使用前减少或防止胰岛的恶化， 促进耐受诱导，使肾移植后延迟胰岛移植成为可能 来自几周前的同一捐赠者，并有许多其他好处。我们已经演示了，在 未发表的结果表明，玻璃化冷冻和复温人胰岛具有良好的胰岛是可能的 葡萄糖刺激的胰岛素的数量恢复和良好的活性保留(基于生命染色) 释放(刺激指数)、葡萄糖刺激的氧耗和逆转诱导的能力 用最小的胰岛剂量治疗小鼠的糖尿病，我们正在努力从每IEQ13,000扩大到 玻璃化冷冻至完全治愈剂量的胰岛(450,000-72,000)。下一步是建立那个小岛 在至少3-4周的存储后保持高度存活和正常运行，并确定最佳 小岛的储存条件。最好的储存条件被认为是非常非常规的 对于与玻璃化来自单一捐赠者的所有胰岛相关的大体积，胰岛将 被玻璃化，以及快速进出长期储存的可取性，而没有任何风险 骨折。特别是，我们认为胰岛应该储存在Tg附近，甚至可能在Tg以上，但是 在此温度范围内的存储以前没有以任何适当的方式研究过。因此， 目标1致力于确定温度对胰岛活性和功能的影响，目标2是 旨在确定温度对小岛和它们的冰核的影响 玻璃化介质和冰核与胰岛完整性之间的关系，以及目标3 确定贮藏时间超过1个月的效果。一个月的储存应该足够 大多数小岛银行需要，但两个月将是非常宝贵的提供安全边际和 为临床医生和患者提供了更大的灵活性。同时，比较1个月和2个月的存储 将打开目前完全缺乏的关于Tg附近随时间的成核率的信息， 并将开始回答关于TG附近生物稳定性的问题，目前这些问题完全保持不变 默默无闻。

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 We have already demonstrated, in unpublished results, that it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viability (based on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.