Isochoric Pressure Based Preservation of Cells, Tissues and Organs

项目总结/摘要 在移植界和更广泛的生物医学界以及政府中得到了广泛的认可 资助机构，扩大和改善生物材料的保存是需要一个巨大的范围， 在生物医学、医学研究和药物发现、器官和组织移植、基于细胞的 治疗，生育和再生医学，应急准备和创伤护理。现行标准 器官和血管复合同种异体移植物（VCAs）的保存包括几个小时的低温静态 在冰上UW溶液中储存。这导致器官/VCA短缺和丢弃率增加，加剧了 缺血性损伤和移植物排斥反应，由于次优的供体-受体匹配，并降低生活质量 移植受者。为了应对这些挑战，我们开创了一种新的热力学 基于稳定平衡状态和等容（恒定体积）的生物保存方法 在零度以下（-5 ° C至-20 °C）的高温条件下，可有效保存器官 和VCAs，与当前临床实践相比，储存时间增加了20 - 28倍（<6- 12小时的低温 储存），同时避免细胞损伤和由在深低温下储存产生的其它挑战。 重要的是，我们将建立在第一阶段的成功可行性论证和Sylvatica 和加州大学伯克利分校合作证明了高零度等容保存：（1） 用于保存混合物的生物相容性热力学压力分布，其支持在等容环境中的人细胞存活 存活率高于75%的系统，（2）成功地将等容保存初步扩大到整个大鼠心脏和皮肤 在前所未有的温度下（-8 ° C，-10 ° C），以及（3）鱼类肌肉和整个生物体的等容保存（ 研究模型C. elegans）。因此，本阶段II提案的目的是使用动物和 VCA代表性组织和肢体保存的人类模型，延长VCA的冷冻保存时间， 5-7数天和数周或更长时间，在储存和恢复后具有良好的功能结果。在五个具体目标中， 我们将首先采用皮肤和血管模型的VCAs的冷冻鸡尾酒和协议优化，与中央 目标是在积极抑制新陈代谢和增强血液循环的同时， 抗逆性一个等容保存平台（室，压力/温度传感器），基于 在第一阶段使用的成功系统将被设计和建造，以支持冷冻方案验证， 临床尺寸的人体皮肤和血管，然后通过大鼠前肢模型的保存和评估 假移植，然后通过原位同种异体移植， 肢体功能和行为恢复。动物保存/移植模型的结果将 用于人类手指和手的高度零下等容保存的概念验证， 进行全面的失血性代谢支持质量评估。

PROJECT SUMMARY/ ABSTRACT There is wide recognition within the transplant and broader biomedical communities, as well as government funding agencies, that extended and improved preservation of biological materials is needed for a huge range of endeavors in biomedicine, medical research and drug discovery, organ and tissue transplantation, cell-based therapies, fertility and regenerative medicine, emergency preparedness, and trauma care. The current standard of preservation for organs and vascular composite allografts (VCAs) consists of few hours of hypothermic static storage in UW solution on ice. This contributes to organ/VCA shortage and increased discard rates, exacerbates ischemic injury and graft rejection due to suboptimal donor-recipient matching, and diminishes the quality of life for transplant recipients. To address these challenges, we have pioneered a novel thermodynamic approach to biopreservation, based on a stable equilibrium state and isochoric (constant volume) condition, at high subzero (-5°C to -20 °C) temperatures, that will allow effective preservation of organs and VCAs, with a 20x-28x increase in storage duration over the current clinical practice (<6-12h of hypothermic storage) while avoiding cellular injury and other challenges created by storage at deep cryogenic temperatures. Importantly, we will build on the successful demonstration of feasibility in Phase I and the results that Sylvatica and UC Berkeley have collaboratively demonstrated with regard to high subzero isochoric preservation: (1) biocompatible thermodynamic pressure profiles for preservation cocktails that support human cell survival in isochoric systems with viabilities above 75%, (2) successful initial scale-up of isochoric preservation to whole rat hearts and skin at unprecedented temperatures (-8°C, -10°C), and (3) isochoric preservation of fish muscle and an entire organism (the research model C. elegans). Therefore, the objective of this Phase II proposal is to demonstrate, using animal and human models of VCA representative tissues and limb preservation, prolonged cryopreservation of VCAs for 5-7 days and weeks, or longer, with good functional outcome post storage and recovery. Across five specific aims, we will first employ skin and vascular models of VCAs for cryostasis cocktail and protocol optimization, with the central goal of enabling high subzero isochoric preservation while actively suppressing metabolism and enhancing stress tolerance. An isochoric preservation platform (chamber, pressure/temperature sensors) based on the successful system used in Phase I will be designed and built to support cryostasis protocols validation using clinical size human skin and blood vessels, then a model of rat forelimb preservation and assessment through pseudo transplantation, and then by orthotopic allotransplantation with comprehensive characterization of upper extremity functional and behavioral recovery. The results from the animal preservation/transplantation model will be used for the proof-of-concept for high subzero isochoric preservation of human fingers and hands, with full exsanguinous metabolic support quality assessment.