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

• 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
• 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

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具有巨大影响， 整体社会负担。胰岛移植有治愈糖尿病的潜力，但一个 限制这种治疗成功的主要问题是胰岛供应不足。从一个单一的胰岛 供体通常不足以实现胰岛素独立性，并且通常需要多次输注，每次输注都具有 增加风险。存在两种潜在的策略来增加可用的胰岛的数量：（1）将胰岛从 多个捐赠者和执行单一程序，高剂量移植;（2）开发替代来源， 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， 无论哪种方法，胰岛保存的方法都是必不可少的。我们的长期目标是开发一种方法， 在移植前冷冻保存或“储存”胰岛。以前的策略都没有达到高可行性， 功能，以及在单一方法中移植所需的临床可扩展性。 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，在无冰的玻璃态下低温储存。生物标本玻璃化冷冻的一个重大挑战 玻璃化和复温所需的冷却和加热速率非常高（>107 °C/min）。 通过添加抑制冰形成的低温保护剂（CPA）来降低这些速率，但是这些试剂是 本身对胰岛有毒。因此，胰岛玻璃化的关键挑战是实现足够快的冷却， 加热以避免冰，同时避免CPA的毒性，并且以临床可扩展的方式这样做。 利用传热和传质的工程原理，我们的多学科研究团队开发了 玻璃化和复温（VR）的方法来解决这个问题，称为胰岛的“冷冻网VR”。我们 中心假设是，通过cryomesh VR实现的改进的传热，结合优化 在CPA使用中，将能够实现胰岛的无冰玻璃化和复温，同时避免毒性。我们的初步 数据实现冷却和升温率远远超过其他方法，我们已经表明CPA加载和 在小鼠、人、猪和人干细胞衍生（SC）胰岛中具有低毒性的卸载方案。的确，在 我们测试的所有冷冻保存的胰岛模型都实现了生存力、恢复和功能， 所有以前的报告，并在临床上可扩展的方法。为了进一步改进我们的方法， 对于临床翻译，我们提出以下目标：目标1。优化最佳物理条件， 小鼠、人和SC胰岛的cryomesh VR;目的2.测量细胞的活力、功能和体内效力， 冷冻网VR后的小鼠、人和SC胰岛; Aim 3.定义发生的分子和细胞变化 响应于冷冻保存;和目的4。扩大cryomesh VR的临床吞吐量， cGMP生产工艺。如果成功的话，这种方法可以彻底改变胰岛的分离，分配， 并在移植前储存，并增加死亡供体胰腺用于治疗糖尿病的利用率。