Viable Banking of Human Tissues

项目摘要/摘要 开发一种有效的人体组织冷冻保存技术是必要的，以解决关键问题 通过解决全球可移植人体组织短缺和保质期问题来满足生物医学需求 阻碍大规模生产、储存、分配和安全/质量控制的工程化组织结构。 此外，开发一种可靠的组织保存方法将使更好的组织匹配 提高了总体成功率，减轻了免疫抑制负担。通过实现可行的银行 一种有效的保存方法还将促进生物标记物的发现和药物测试， 因此，个性化的医学朝着癌症等毁灭性疾病的新的有效疗法迈进。 目前的保存策略不适用于多细胞复杂组织。该计划的总体目标 拟议的研究是为了满足对有效的、广泛适用的组织保存技术的关键需求 开发一种新的玻璃化方法，一种冷冻保存策略，涉及液体在 玻璃态使用高浓度的低温保护剂(CPA)。玻璃化冷冻是一种很有前途的 技术，但CPA具有固有的细胞毒性。事实上，CPA的化学毒性被认为是主要的 复杂组织冷冻保存成功的障碍。通过增加冷却来降低CPA浓度 升温速度减轻了毒性，但很难实现足够快的冷却和升温速度。 并可能导致极端的热应力，导致组织破裂。要克服以下限制 目前的组织保存方法，我们建议使用一种 有针对性的干预和生物物理建模的跨学科方法。我们的初步研究显示 1，2-丙二醇(PROH)是一种首选的CPA，其初级毒性是通过线粒体Ca~(2+)发生的 超载。我们能够完全防止ProH对小鼠卵母细胞、人子宫肌瘤和小鼠的毒性 用线粒体钙单转运蛋白抑制剂建立脑组织模型。最近，我们还在数学上 优化了向内皮细胞添加高浓度CPA的程序，并将细胞毒性降至最低。基于 这些令人鼓舞的初步数据，我们的中心假设是，跨学科的方法结合在一起 通过数学建模和优化来靶向抑制CPA的毒性，可以使高 CPA浓度，导致了一种适用于不同组织的通用组织玻璃化方法。这个 建议的玻璃化方法是创新的，因为它绕过了使用高CPA的主要障碍 浓度(CPA毒性)，能够更好地抑制冰核和玻璃化；其他 创新包括开发一种新型的玻璃化介质，它可以通过 通过优化抗氧化剂的组成和使用 新的基于生物物理学的数学优化策略，以确定玻璃化冷冻的毒性最低的方案。 这项拟议的研究有望为可行的人体组织银行铺平道路。

PROJECT SUMMARY/ABSTRACT Development of an effective cryopreservation technology for human tissues is necessary to address critical biomedical needs by solving a worldwide shortage of transplantable human tissues and shelf-life problems of engineered tissue constructs that hinder mass production, storage, distribution, and safety/quality control. Moreover, the development of a reliable tissue preservation method would allow better tissue matching toward increased overall success rates and reduced burden of immunosuppression. By enabling viable banking of diseased tissues, an effective preservation method would also advance biomarker discovery and drug testing, and thus personalized medicine toward new effective therapies of devastating diseases such as cancer. Current preservation strategies are inadequate for multicellular complex tissues. The overall goal of the proposed research is to meet the critical need for effective, widely applicable tissue preservation technology by developing a novel approach to vitrification, a cryopreservation strategy involving the solidification of liquid in a glass-like state using high concentrations of cryoprotective agents (CPAs). Vitrification is a promising technique, but CPAs have inherent cytotoxicity. In fact, chemical toxicity of CPAs is considered to be the main barrier to successful cryopreservation of complex tissues. Reduced CPA concentrations with increased cooling and warming rates ameliorate toxicity, but sufficiently fast cooling and warming rates are difficult to achieve in tissues and may lead to extreme thermal stresses, resulting in tissue cracking. To overcome the limitations of current tissue preservation approaches, we propose to address chemical toxicity of CPAs using an interdisciplinary approach of targeted interventions and biophysical modeling. Our preliminary studies revealed that the primary toxicity of 1,2-propanediol (PROH), a preferred CPA, occurs through mitochondrial Ca2+ overload. We were able to completely prevent PROH’s toxicity in mouse oocyte, human fibroid and mouse brain tissue models by using inhibitors of mitochondrial Ca2+ uniporter. Recently, we have also mathematically optimized a procedure to add high CPA concentrations to endothelial cells with minimal cytotoxicity. Based on these encouraging preliminary data, our central hypothesis is that an interdisciplinary approach combining targeted inhibition of CPA toxicity with mathematical modeling and optimization can enable employment of high CPA concentrations, leading to a versatile tissue vitrification method applicable to diverse tissues. The proposed vitrification approach is innovative, because it circumvents the main barrier to the use of high CPA concentrations (CPA toxicity), enabling better suppression of ice nucleation and devitrification; other innovations include the development of a novel vitrification medium that can block ice crystal growth through synthetic polymers and mitigate free radical damage by optimized composition of antioxidants and the use of a novel biophysics-based mathematical optimization strategy to identify minimally toxic protocols for vitrification. The proposed research is expected to pave the way for viable banking of human tissues.