Isochoric Pressure Assisted Vitrification of Testicular Tissue and Whole Testes

等容压辅助睾丸组织和整个睾丸的玻璃化

• 批准号: 9466962
• 负责人: MICHAEL John TAYLOR
• 金额: $ 28.51万
• 依托单位: SYLVATICA BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-21 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9466962
• 关键词: Animals; Autologous; Back; Biological Preservation; Biomedical Engineering; Cancer Model; Cancer Patient; Cancer Survivor; Cancer Survivorship; Cell Survival; Cell model; Cells; Characteristics; Childhood; Climacteric; Clinical; Communities; Cryopreservation; Cryopreserved Tissue; Crystallization; Cytoprotection; Disease; Dry Ice; Effectiveness; Endothelial Cells; Equilibrium; Erythrocytes; Feasibility Studies; Fertility; Fertilization; Freezing; Future; Gold; Growth; High temperature of physical object; Histology; Human; Ice; Immunohistochemistry; In Vitro; Individual; Infertility; Injury; Investigation; Life; Malignant Neoplasms; Mammalian Cell; Measures; Mechanics; Medical Research; Methods; Mus; Nature; Nude Mice; Organ; Organ Culture Techniques; Outcome; Ovarian Tissue; Patients; Perfusion; Permeability; Phase; Preparation; Primates; Process; Production; Protocols documentation; Public Health; Quality of life; Radiation therapy; Reporting; Research; Safety; Sampling; Scrotum; Seminal fluid; Ships; Skin; Stem cells; Stress Tests; Stretching; Survivors; System; Technology; Temperature; Test Result; Testicular Tissue; Testing; Testis; Thermodynamics; Time; Tissue Model; Tissue Preservation; Tissue Viability; Tissues; Toxic effect; Transplantation; Trauma; Xenograft procedure; animation; base; biobank; boys; cancer genomics; cancer therapy; chemotherapy; clinical practice; combinatorial; design; efficacy trial; experience; genomic biomarker; high throughput screening; human tissue; improved; in vivo; leydig interstitial cell; male; men; novel; novel strategies; oncofertility; preconditioning; prepuberty; pressure; prevent; programs; reproductive; risk minimization; scale up; sertoli cell; service member; solute; sperm cell; success; sugar; young adult; young man

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.

该计划旨在开发一种冷冻保存人类睾丸组织和整个睾丸的新方法，该方法也可应用于卵巢组织和性腺的保存，并最终用于任何细胞，组织和整个器官。这种多管齐下的方法建立在机器灌注，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的基础上，将它们与我们的恒定体积，压力辅助冷却的新方法相结合。我们的方法有望在热力学平衡和可能高达-80 ° C的温度下（能够干冰运输）的系统中，在没有有毒浓度的冷冻保护剂的帮助下实现活组织的玻璃化。它改善或完全避免了传统冷冻保存方法对大组织的许多限制，包括破坏性的冰结晶、溶质浓度的有害变化和体积变化。这种方法有可能实现睾丸组织、整个睾丸和其他活体材料的无限期储存，同时显著限制目前与离体储存相关的组织损伤。本1期提案的技术目标是开发一种优化的混合物和方案，用于睾丸组织的保存和无限期储存。我们将在三个具体目标上发展我们的方法。在初步研究的基础上，证明了等容保存的可行性，在目标1，我们将开始扩展的可行性研究，重点是调查温度，压力和CPA对睾丸细胞活力的综合影响后，平衡无冰等容保存。具体而言，我们将使用半高通量筛选来测量人支持细胞、间质细胞和内皮细胞的活力，目标压力高达160 MPa（红细胞显示出改善的冷冻保存的压力）。在目标2的平行研究中，我们将使用由我们更广泛的团队开发的成熟方法来定义等容环境中新型天然冷冻保护剂溶液的新热力学特征。定容系统;这将使我们能够选择允许在低到中等压力和比传统的冰所需的高得多的储存温度下对生物材料进行压力辅助玻璃化的解决方案，免费冷冻保存。在目标3中，我们选择了目标1-2中确定的最有效的冷冻保护剂溶液，以评估我们在人睾丸组织条中的“高度零下玻璃化”，并将结果与传统的最先进的等压保存进行比较。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裸鼠中来评价组织。我们还将测试这些方法的增强与亚常温机器灌注，使进一步减少缺血性组织损伤。这些新方法单独或组合的成功可能会使肿瘤生育和生物保存研究和临床实践取得突破。