Isochoric Pressure Assisted Vitrification of Testicular Tissue and Whole Testes

该项目旨在开发一种新的人类睾丸组织和整个睾丸的冷冻保存方法，该方法也可以应用于卵巢组织和性腺的保存，最终适用于任何细胞、组织和整个器官的保存。这种多管齐下的方法建立在机器灌流、自然启发的细胞保护策略和无毒冷冻保护剂解决方案的最新进展基础上，将它们与我们用于恒容、压力辅助冷却的新方法相结合。我们的方法有望实现活组织的玻璃化，而不需要有毒浓度的冷冻保护剂的帮助，在热力学平衡的系统中，可能在高达-80°C的温度下(能够运输干冰)。它改善或完全避免了大组织传统冷冻保存方法的许多局限性，包括破坏冰结晶、有害的溶质浓度变化和体积变化。这种方法有可能使睾丸组织、整个睾丸和其他生物材料无限期储存，同时极大地限制目前与体外储存相关的组织损伤。这一第一阶段提案的技术目标是开发一种优化的鸡尾酒和方案，用于保存和无限期储存睾丸组织。我们将在三个具体目标上发展我们的方法。在证明等时保存可行性的初步研究的基础上，在目标1中，我们将从扩展的可行性研究开始，重点研究在平衡无冰等时保存后温度、压力和CPA对睾丸细胞存活率的联合影响。具体地说，我们将使用半高通量筛选来测量目标压力高达160兆帕(红细胞显示低温保存得到改善的压力)下人类支持细胞、间质细胞和内皮细胞的活性。在目标2的平行研究中，我们将使用我们更广泛的团队开发的成熟方法，在等容(恒容)系统中为新颖的、受自然启发的低温保护剂溶液定义新的热力学曲线；这将使我们能够选择允许在低至中压和远高于传统无冰低温保存所需的存储温度下进行压力辅助玻璃化的活体材料的解决方案。在目标3中，我们选择了在目标1-2中建立的最有效的冷冻保护剂溶液，以评估我们在人类睾丸组织条中的高玻璃化程度，并将结果与传统的最先进的等压保存进行比较。组织将通过测量组织活力以及组织学、免疫组织化学和异种移植到不育的裸鼠进行评估。我们还将通过亚常温机器灌流来测试这些方法的增强，从而进一步减少缺血组织的损伤。这些新方法单独或组合的成功可能会使肿瘤受精和生物保存研究和临床实践取得突破。

This program aims to develop a novel method for cryopreserving human testicular tissue and whole testes, which can also be applied to ovarian tissue and gonadal preservation and ultimately any cells, tissues, and whole organs. This multi-pronged approach builds on recent advances in machine perfusion, nature-inspired cytoprotection strategies, and non-toxic cryoprotectant solutions, combining them with our novel method for constant-volume, pressure-assisted cooling. Our method promises to achieve vitrification of living tissues without the aid of toxic concentrations of cryoprotectants, in a system at thermodynamic equilibrium and potentially at temperatures as high as -80°C (capable of dry-ice shipment). It ameliorates or entirely circumvents many of the limitations of conventional cryopreservation methods for large tissues, including damaging ice crystallization, deleterious changes in solute concentrations, and volumetric changes. This approach has the potential to enable indefinite banking of testicular tissues, whole testes, and other living materials while dramatically limiting tissue injury currently associated with ex-vivo storage. The technical objective of this Phase 1 proposal is to develop an optimized cocktail and protocol for preservation and indefinite banking of testicular tissue. We will develop our method across three specific aims. Building upon preliminary studies that demonstrate the feasibility of isochoric preservation, in Aim 1 we will begin with extended feasibility studies focused on investigating the combined effects of temperature, pressure and CPA on testicular-cell viability following equilibrium ice-free isochoric preservation. Specifically, we will use semi- high-throughput screening to measure the viability of human Sertoli cells, Leydig cells and endothelial cells with targeted pressures of up to 160 MPa (pressure at which red-blood cells show improved cryopreservation). In parallel studies in Aim 2, we will use well established methods developed by our broader group to define new thermodynamic profiles for novel, nature-inspired cryoprotectant solutions in an isochoric (constant- volume) system; this will allow us to select solutions that allow for pressure-assisted vitrification of living materials at low to intermediate pressures and much higher storage temperatures than traditionally needed for ice-free cryopreservation. In Aim 3, we select the most effective cryoprotectant solutions established in Aim 1-2 to evaluate our “high sub-zero vitrification” in human testicular tissue strips and compare outcome with conventional state-of-the-art isobaric preservation. Tissue will be evaluated by measuring tissue-viability, as well as histology, immunohistochemistry and xenotransplantation into infertile nude mice. We will additionally test the augmentation of these methods with subnormothermic machine perfusion, enabling further reduction of ischemic tissue injury. Success of these novel approaches individually or in combination will likely enable breakthroughs in oncofertility and biopreservation research and clinical practice.