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

项目摘要 本研究的目的是调查和开发长期储存的细胞使用冷冻保存在 在改善胰岛健康的背景下，胰岛移植作为1型糖尿病的治疗。目标是 开发一种高低温冷冻保存方法，在保护性免疫隔离装置内冷却细胞， 延长保存时间至数周和数月。我们的方法是基于最好的策略， 自然界中的抗冻和冬眠动物， 最近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 玻璃化或经典的冷冻保存所需的所有问题，而是首先开发组织， 在受控的、部分冷冻的平衡状态下使用高的零度以下温度（范围从-5 至-20 °C）合并代谢抑制。这些是自然界中 物种能够在“假死”状态下存活数月，整个动物，包括每一个 单个器官被“储存”而不受伤。具体目标1（SA 1）是选择和优化冷冻箱 含有冷冻后细胞存活所必需的成分的鸡尾酒。非代谢性3-O-甲基-d- 葡萄糖（3-OMG）和海藻糖混合物将用于防止细胞脱水和细胞内冰形成 在低温保存过程中。Unisol将用作基础溶液，用于检测多剂量的 3-OMG，海藻糖，储存时间随温度变化，使用β TC 3细胞。具体目标2（SA 2）： 达到主动抑制代谢率，增强抗逆能力。β TC 3细胞将储存在 在SA 1中开发的冷冻混合物以及抗氧化剂（AO）、抗凋亡剂（AAD） (AA)和代谢抑制剂（MS）。将人胰岛冷冻保存并储存在标准培养物中， 在DMSO中，并在通过SA 1建立的最佳剂量、添加剂、温度和储存时间下， SA 2.这些胰岛的质量评估包括标准化为DNA的耗氧率（OCR），百分比 通过DNA测量的恢复以评估活力，以及葡萄糖刺激的胰岛素分泌以测量 功能具体目标3（SA 3）是使用最有前途的 由SA 1和SA 2建立的低温恒温混合物。将使用标准品冷冻保存和储存胰岛 从SA 1和SA 2两个菌株中筛选出最佳培养条件和培养方法。一旦解冻，来自SA 2的胰岛质量评估 将在解冻后和48小时培养物恢复期后立即进行。具体目标4（SA 4）是 在糖尿病裸鼠模型中测试冷冻保存的、包封的人胰岛。人体内的胰岛 Theracyte免疫隔离装置已冷冻保存并储存在标准培养物中， 将冷冻混合物移植到糖尿病裸鼠中。将通过以下方法监测小鼠的糖尿病逆转： 在30天内进行每日血糖和每周腹膜内葡萄糖耐量试验。取出的器械 将通过器械OCR/DNA、GSIS和组织学进行评估，以分析器械内的胰岛健康状况。

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.