Nature-inspired methods for long-term banking of endocrine cells within encapsulation devices

项目摘要 本研究的目的是探索和发展细胞的长期冷冻保存方法。 胰岛移植作为1型糖尿病治疗改善胰岛健康的背景。我们的目标是 开发一种高低温保存方法，将保护性免疫隔离设备内的细胞冷却以 将保存时间延长到几周或几个月。我们的方法是基于 自然界中的耐寒和冬眠动物通过使用 最新的科学认识和生物工程原理。重要的是，我们的方法并不寻求解决 玻璃化冷冻或经典冷冻保存所需的所有问题，而不是第一个发育组织 使用较高的零下温度(范围为-5℃)在受控、部分冻结的平衡状态下保存 至-20°C)合并代谢抑制。这些是在自然界中使用的温度和策略 能够存活几个月的物种，与整个动物一起，包括 单个器官被“储存”，而不会造成损伤。特定目标1(SA1)是选择和优化低温恒温器 一种鸡尾酒，含有冷冻后细胞存活所必需的成分。不可代谢的3-O-甲基-d- 葡萄糖(3-OMG)和海藻糖的混合物将用于防止细胞脱水和细胞内结冰 在超低温保存期间。Unisol将被用作基础溶液，用于测试多剂量 3-OMG，利用βTC3细胞，海藻糖随着温度的变化而储存。具体目标2(SA2)旨在 达到主动抑制代谢率、增强应激耐力的目的。βTC3信元将存储在 在SA1中开发的冷冻鸡尾酒以及抗氧化剂(AO)、抗细胞凋亡的多剂量测试 (AA)和代谢抑制因子(MS)。人类的胰岛将被冷冻保存并储存在标准培养中， 在DMSO中，在最佳剂量下，添加剂、温度和储存时间通过SA1和 SA2.这些胰岛的质量评估包括氧耗率(OCR)，归一化为DNA，百分比 恢复率以DNA来衡量，以活力和葡萄糖刺激胰岛素的分泌来衡量 功能。特殊目标3(SA3)是在TheraCyte设备中测试封装的胰岛，使用最有希望的 由SA1和SA2组成的冷冻鸡尾酒。胰岛将被冷冻保存，并使用标准的 SA1和SA2菌株的培养方法和最佳培养条件。一旦解冻，来自SA2的胰岛质量评估 将在解冻后立即进行，并在48小时培养恢复期后进行。具体目标4(SA4)是 在糖尿病裸鼠模型上测试冷冻保存的、被包裹的人胰岛。人间小岛 超低温保存的细胞免疫隔离装置在标准培养和新的 冷冻鸡尾酒将被移植到糖尿病裸鼠体内。将通过以下方式监测小鼠的糖尿病逆转 在30天内进行每日血糖和每周一次的腹膜糖耐量试验。移植的装置 将通过设备OCR/DNA、GSIS和组织学进行评估，以分析设备内的胰岛健康状况。

Project Summary The objective of this study is to investigate and develop long-term storage of cells using cryopreservation in the context of improving health of islets for islet transplantation as a Type1 Diabetes treatment. The goal is to develop a high subzero cryopreservation method that cools cells inside protective immunoisolation devices to extend preservation time to weeks and months. Our approach is based on the best strategies employed by freeze-tolerant and hibernating animals in nature augmented with complementary strategies developed using recent scientific understanding and bioengineering principles. Importantly, our approach does not seek to solve all the problems needed for vitrification or classical cryopreservation, but rather be the first to develop tissue preservation in a controlled, partially frozen equilibrium state using high subzero temperatures (ranging from -5 to -20 °C) combined with metabolic depression. These are temperatures and strategies used in nature by species able to survive months in a state of ‘suspended animation,’ with the whole animal, including every single organ being “banked” without injury. Specific Aim 1 (SA1) is to select and optimize the cryostasis cocktail with components that are essential for cell survival post freezing. Non-metabolizable 3-O-methyl-d- glucose (3-OMG) and trehalose mix will be used to prevent cellular dehydration and intracellular ice formation during cryopreservation. Unisol will be used as the base solution for testing the interaction of multiple doses of 3-OMG, trehalose with storage time as a function of temperature using βTC3 cells. Specific Aim 2 (SA2) is to achieve active suppression of metabolic rate and enhancement of stress tolerance. βTC3 cells will be stored in the cryostasis cocktail developed in SA1 as well with multiple dose testing of antioxidants (AO), anti-apoptotic (AA) and metabolic suppressors (MS). Human islets will be cryopreserved and stored in the standard culture, in DMSO, and in the optimal dosage, additives, temperature and storage duration established through SA1 and SA2. Quality assessments of these islets include oxygen consumption rate (OCR) normalized to DNA, percent recovery as measured by DNA to assess viability, and glucose stimulated insulin secretion to measure function. Specific Aim 3 (SA3) is to test encapsulated islets in the TheraCyte device using the most promising cryostasis cocktail established from SA1 and SA2. Islets will be cryopreserved and stored using the standard culture methods and the best conditions from SA1 and SA2. Once thawed, islet quality assessments from SA2 will be performed immediately post-thaw and after the 48 hour culture recovery period. Specific aim 4 (SA4) is to test cryopreserved, encapsulated human islets in a diabetic nude mouse model. Human islets within Theracyte immunoisolation devices that have been cryopreserved and stored in standard culture and the new cryostasis cocktail will be transplanted into diabetic nude mice. Mice will be monitored for diabetes reversal by taking daily blood glucose and weekly intraperitoneal glucose tolerance tests over 30 days. Explanted devices will be assessed by device OCR/DNA, GSIS, and histology to analyze islet health within the device.