Device for Hyperfast Scalable Vitrification of Germplasm in Large Volumes

大容量种质超快速可扩展玻璃化冷冻装置

基本信息

批准号：
8393582
负责人：
Igor Katkov
金额：
$ 14万
依托单位：
CELLTRONIX
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-24 至 2013-04-23
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8393582
关键词：
Achievement Animal Model Biological Preservation Biomedical Engineering Cell Volumes Cells Collaborations Computer software Cryopreservation Cryoprotective Agents Devices Electronics Embryo Engineering Equipment Equipment and Supplies Fertility Freezing Furuncles Future Generations Genetic Glycerol Goals Human Ice Industry Laboratory Animals Methods Modeling Nitrogen Oocytes Ovarian Tissue Phase Pluripotent Stem Cells Protocols documentation Relative (related person)Reproductive Health Reproductive Techniques Sampling Screening procedure Seminal fluid Shipping Ships Solutions Speed Stem cells Suspension substance Suspensions System Techniques Testing Thermal Conductivity Time Toxic effect Umbilical Cord Blood Water animal breeding animal facility cell type cryobiology cryogenics design design and construction experience innovation model design novel programs prototype sperm cell stem vapor

项目摘要

DESCRIPTION (provided by applicant): Cryopreservation of germplasm (GP) (sperm, oocytes, embryos, stem cells, ovarian tissues) is essential for preserving the genetic variety of model animals, reproductive health in humans, the animal breeding industry and wildlife conservation. Although many methods, devices, and equipment exist both for slow freezing and fast cooling (vitrification), each method, cell type and species practically needs its own optimal preservation protocol. Vitrification (VF) is gaining in popularity with successful protocols being developed for many types of GP, including spermatozoa and stem cells. However, all existing VF methods require complicated and careful timing, may be prone to technical errors, often are not scalable, and are limited to very small sample volumes (0.5-5 ?L). As such, cryopreservation of samples such as semen, cord blood stem cells, and sufficient amounts of pluripotent stem cells and ovarian tissue is extremely difficult. The other aspect is that while th amount of potentially toxic cryoprotective agents (CPA) has been greatly reduced, the concentrations are still relatively high for the majority of GP types, and beside toxicity, the CPA addition and elution times must be precisely controlled. One of the major factors for vitrification is the critical cooling rate necessary for vitrification (Bcr), which strongly and inversely depend on the CPA concentration, For example, hundreds of thousands of ?C/min are needed to vitrify a water-glycerol solution that is tolerable for ALL CPA species concentrations. All existing methods purport to achieve such high speeds, but many have not in fact done so, mainly due to the Leidenfrost effect (LFE) - where a boiling nitrogen vapor coat forms around the sample. This vapor coat impairs thermal conductivity by orders of magnitude and makes even droplets that are a fraction of a ? m L impossible to vitrify. With a speed around 500,000 ?K/min, we hypothesize that we can vitrify practically ALL species of germplasm using a unified method, equipment and supplies. Our Celltronix team has developed a completely new system for hyperfast cooling, called "KrioBlast(r)", which completely eliminates LFE and can cool much larger samples than those currently used at rates of hundreds of thousands ?C/min. We have built a pilot model (first generation) of the system, the manually operated Krioblast-1, with which we could vitrify large sample volumes with dilute CPA solutions and also achieved some promising results for two trials on human and bull sperm. Upon obtaining a higher cooling rate, we will be close to devising a "Universal Cryopreservation Protocol". In this Project, we will buil a semi-automatic system Krioblast-2, which would produce 2-3 fold faster cooling rates with a target of 200,000 ?C/min and vitrify cell volumes of up to 4,000 mL (1-2 orders of magnitude higher than is currently possible). We believe that such rates will be sufficient to vitrify all tyes of GP using a practically unified protocol. In Phase II, we will build a closed modular stem for hyperfast cooling, cryogenic storage and shipment, and hyperfast thawing of cells and test Krioblast-3 on real germplasm cells. PUBLIC HEALTH RELEVANCE: Cryopreservation of germplasm (sperm, oocytes, embryos, stem cells, ovarian tissues) is essential for preserving the genetic variety of model animals, assisting human fertility techniques, the animal breeding industry, and wildlife conservation. A large variety of cryopreservation methods, devices and equipment currently exists, but each method, cell type and species would need its own optimal protocol. The goal of this Project is to develop a novel scalable device for hyper-fast (hundreds of thousands of ?C/min) cooling that would allow vitrification of a wide variety of germplasm cells and species using unified equipment and protocols, which will not only significantly benefit germplasm cryopreservation, but may eventually shift cryopreservation paradigms.

描述（由申请人提供）：种质（GP）（精子、卵母细胞、胚胎、干细胞、卵巢组织）的冷冻保存对于保存模型动物的遗传多样性、人类的生殖健康、动物饲养业和野生动物保护至关重要。尽管存在许多用于缓慢冷冻和快速冷却（玻璃化）的方法、装置和设备，但每种方法、细胞类型和物种实际上都需要其自己的最佳保存方案。随着为多种类型的全科医生（包括精子和干细胞）开发成功的方案，玻璃化冷冻 (VF) 越来越受欢迎。然而，所有现有的 VF 方法都需要复杂且仔细的计时，可能容易出现技术错误，通常不可扩展，并且仅限于非常小的样本量（0.5-5 µL）。因此，精液、脐带血干细胞、足够量的多能干细胞和卵巢组织等样本的冷冻保存极其困难。另一方面是，虽然潜在有毒的冷冻保护剂 (CPA) 的量已大大减少，但大多数 GP 类型的浓度仍然相对较高，并且除了毒性之外，CPA 必须精确控制添加和洗脱时间。玻璃化的主要因素之一是玻璃化所需的临界冷却速率 (Bcr)，它与 CPA 浓度密切相关，例如，需要数十万℃/分钟来玻璃化水-甘油溶液，该溶液可耐受所有 CPA 物质浓度。所有现有方法都声称能够实现如此高的速度，但许多方法实际上并未做到这一点，这主要是由于莱顿弗罗斯特效应（LFE）——在样品周围形成沸腾的氮蒸气涂层。这种蒸汽涂层会导致导热性降低几个数量级，甚至会产生几分之一的液滴。 m L 不可能玻璃化。以大约 500,000 ?K/min 的速度，我们假设我们可以使用统一的方法、设备和用品来玻璃化几乎所有物种的种质。我们的 Celltronix 团队开发了一种全新的超快速冷却系统，称为“KrioBlast(r)”，该系统完全消除了 LFE，并且可以以数十万℃/分钟的速率冷却比目前使用的样品大得多的样品。我们已经建立了该系统的试点模型（第一代），即手动操作的 Krioblast-1，我们可以用稀释的 CPA 溶液玻璃化大量样品，并且在人类和公牛精子的两项试验中也取得了一些有希望的结果。在获得更高的冷却速率后，我们将接近设计出“通用冷冻保存协议”。在这个项目中，我们将建造一个半自动系统 Krioblast-2，它将产生 2-3 倍更快的冷却速率，目标为 200,000 °C/min，玻璃化细胞体积高达 4,000 mL（1-2 个数量级）幅度高于目前可能的水平）。我们相信，这样的速率足以使用实际上统一的协议来玻璃化所有类型的 GP。在第二阶段，我们将构建一个封闭的模块化干细胞，用于超快速冷却、低温储存和运输以及细胞超快速解冻，并在真实种质细胞上测试 Krioblast-3。公共健康相关性：种质（精子、卵母细胞、胚胎、干细胞、卵巢组织）的冷冻保存对于保存模型动物的遗传多样性、协助人类生育技术、动物饲养业和野生动物保护至关重要。目前存在多种冷冻保存方法、装置和设备，但每种方法、细胞类型和物种都需要其自己的最佳方案。该项目的目标是开发一种新型可扩展设备，用于超快速（数十万摄氏度/分钟）冷却，允许使用统一的设备和协议对多种种质细胞和物种进行玻璃化，这不仅显着有利于种质冷冻保存，但最终可能会改变冷冻保存模式。