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

基本信息

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
项目类别：
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

项目摘要

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.

该项目旨在开发一种冷冻保存人类睾丸组织和整个测试的新方法，该方法也可应用于卵巢组织和性腺保存，最终适用于任何细胞、组织和整个器官。这种多管齐下的方法建立在机器的最新进展的基础上。灌注、受自然启发的细胞保护策略和无毒冷冻保护剂解决方案，将它们与我们的恒定体积、压力辅助冷却的新颖方法相结合，我们的方法有望在没有有毒浓度的帮助下实现活组织的玻璃化。它改善或完全规避了大型组织传统冷冻保存方法的许多限制，包括破坏冰结晶。、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、整个测试和其他活体材料的无限期储存，同时极大地限制目前与离体储存相关的组织损伤。该第一阶段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，我们将在目标 1 中基于证明等容保存可行性的初步研究来开发我们的方法。我们将从扩展的可行性研究开始，重点研究平衡无冰等容保存后温度、压力和 CPA 对睾丸细胞活力的综合影响。我们特别将使用半高通量筛选来测量人类 Sertoli 的活力细胞,间质细胞和内皮细胞的目标压力高达 160 MPa（红细胞在该压力下冷冻保存效果得到改善）。在目标 2 的并行研究中，我们将使用我们更广泛的团队开发的成熟方法来定义新的热力学曲线。等容（恒定体积）系统中新颖的、受自然启发的冷冻保护剂解决方案，这将使我们能够选择能够在低至中压下对生物材料进行压力辅助玻璃化的解决方案；比传统无冰冷冻保存所需的储存温度高得多在目标 3 中，我们选择目标 1-2 中建立的最有效的冷冻保护剂解决方案来评估我们在人类睾丸组织条中的“高度低于零的玻璃化”，并将结果与传统方法进行比较。我们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估组织。此外，还将测试这些方法与亚常温机器灌注的增强作用，从而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在肿瘤生育和生物保存研究和临床实践中取得突破。