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

工程优化和扩展可实现高质量胰岛冷冻保存以用于储存和移植

基本信息

批准号：
10343955
负责人：
JOHN C BISCHOF
金额：
$ 58.35万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-22 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10343955
关键词：
Adopted Affect Biological Biology Cells Cellular Stress Cessation of life Chemicals Clinical Cryopreservation Cryoprotective Agents Crystallization Cyclic GMP Data Diabetes Mellitus Disease Dose Engineering Family suidae Freezing Goals Health Health Care Costs Heating Human Ice Impairment In Vitro Individual Infusion procedures Injury Insulin Interdisciplinary Study Islets of Langerhans Islets of Langerhans Transplantation Knowledge Measures Methods Modeling Molecular Mus Organ Pancreas Pathway interactions Personal Satisfaction Phenotype Problem Solving Procedures Process Protocols documentation Recovery of Function Reporting Resources Rewarming Risk Source Survival Rate System Technology Temperature Testing Tissues Toxic effect Translations Transplantation Validation Whole Organism Xenograft Model advanced analytics analytical method cGMP production clinical translation cost cryogenics human stem cells improved in vivo innovative technologies islet potency testing preservation prevent repaired response scale up stem cells success transplant centers

项目摘要

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.

项目摘要糖尿病对受影响个体的健康和福祉产生了巨大影响，整体社会伯恩（Burnen）相当大。胰岛移植具有治愈糖尿病的潜力，但一种限制这种处理成功的主要问题是胰岛供应不足。一个来自单个的胰岛供体通常不足以实现胰岛素独立性，并且经常需要多次输注增加风险。存在两种潜在策略来增加可用胰岛的数量：（1）来自多个捐助者并执行单个程序，高剂量移植；（2）开发此类替代来源作为干细胞衍生的胰岛。这些有限资源的可用性成为供应链问题，并且无论哪种方法，胰岛制备方法都是必不可少的。我们的长期目标是为移植之前的冷冻水平或“银行”胰岛。没有以前的策略实现高可行性，单个方法中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业务，我们建议使用替代性冷冻保存策略，即玻璃化。也就是说，处于无冰玻璃状态下的低温存储。生物测量的果实挑战是玻璃化和重新加热所需的冷却和加热速率高（> 107°C/min）。通过添加抑制冰形成的冷冻保护剂（CPA）来降低这些速率，但这些试剂是自己对胰岛有毒。那就，胰岛玻璃体中的关键挑战是实现足够快的冷却和加热以避免冰，同时避免CPA的毒性，并以临床上可扩展的方式进行冰。我们的多学科研究团队使用了热量和传播的工程原理用于解决此问题的玻璃化和恢复（VR）的方法，称为“ Cryomesh VR”，用于胰岛。我们的中心假设是Cryomesh VR获得的改进的热传递，结合了优化在使用CPA时，将在避免毒性的同时，使胰岛无冰的玻璃化和重新加热。我们的初步数据达到冷却和变暖率远远超过其他方法，我们显示了CPA的加载和在小鼠，人，猪和人类干细胞衍生（SC）胰岛中具有低毒性的卸载方案。确实，在所有测试的冷冻保存胰岛模型我们已经实现了满足或超过的生存能力，恢复和功能所有以前的报告，并以临床上可扩展的方法进行。进一步改善我们的方法并移动为了临床翻译，我们提出以下目的：目标1。完善最佳的物理条件小鼠，人类和SC小岛的Cryomesh VR；目标2。测量的生存能力，功能和体内效力 Cryomesh VR后的小鼠，人类和SC小岛；目标3。定义发生的分子和细胞变化回应冷冻保存； AIM 4。用于临床吞吐量的Cryomesh VR扩展并适应 CGMP生产的过程。如果成功的话，这种方法可以彻底改变胰岛的隔离，分配，并在移植之前存储并增加了已故供体胰腺的利用来治愈糖尿病。