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

• 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
• 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

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（由申请方提供）：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式动物的遗传多样性、人类生殖健康、动物育种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。玻璃化冷冻（VF）越来越受欢迎，成功的协议正在开发的许多类型的GP，包括精子和干细胞。然而，所有现有的VF方法需要复杂和仔细的时间，可能容易出现技术错误，往往是不可扩展的，并限于非常小的样品体积（0.5-5？L）。因此，冷冻保存样品如精液、脐带血干细胞和足够量的多能干细胞和卵巢组织是极其困难的。另一方面是，虽然潜在毒性的冷冻保护剂（CPA）的量已经大大减少，但是对于大多数GP类型，CPA的浓度仍然相对较高，并且除了毒性之外， 必须精确控制加入和洗脱时间。玻璃化的主要因素之一 玻璃化所需的临界冷却速率（Bcr）与CPA浓度成反比，例如，数十万？玻璃化水-甘油溶液需要C/min，该溶液可耐受所有CPA物质浓度。所有现有的方法都声称可以达到如此高的速度，但实际上许多方法都没有做到这一点，这主要是由于莱顿弗罗斯特效应（Leidenfrost effect，LFE），即在样品周围形成沸腾的氮蒸气涂层。这种蒸汽涂层损害热传导的数量级，甚至使液滴是一个？不可能玻璃化。速度在五十万左右？K/min，我们假设我们可以使用统一的方法，设备和用品玻璃化几乎所有种类的种质。我们的Celltronix团队开发了一种全新的超快冷却系统，称为“KrioBlast（r）"，它完全消除了LFE，可以以数十万的速度冷却比目前使用的更大的样品。我们已经建立了一个试点模型（第一代）的系统，手动操作Krioblast-1， 我们可以用稀释的CPA溶液玻璃化大体积的样品，并且在人类和公牛精子的两个试验中也取得了一些有希望的结果。在获得更高的冷却速度后，我们将接近于设计一个“通用冷冻保存协议”。在这个项目中，我们将建立一个半自动系统Krioblast-2，这将产生2-3倍的冷却速度快，目标是200，000？C/min，玻璃化细胞体积高达4，000 mL（比目前可能的高1-2个数量级）。我们相信，这样的速度将足以玻璃化所有类型的GP使用一个几乎统一的协议。在第二阶段，我们将建立一个封闭的模块化茎超快冷却，低温储存和运输，超快解冻的细胞和测试Krioblast-3对真实的种质细胞。 公共卫生关系：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式动物的遗传多样性、辅助人类生育技术、动物育种业和野生动物保护至关重要。目前存在各种各样的冷冻保存方法、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的可扩展设备，用于超快速（数十万？C/min）冷却，这将允许使用统一的设备和方案玻璃化多种种质细胞和物种，这将不仅显著有益于种质冷冻保存，而且可能最终改变冷冻保存范例。