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）：生殖（GP）的冷冻保存（精子，卵母细胞，胚胎，干细胞，卵巢组织）对于保留遗传动物的遗传变异，人类的生殖健康，动物繁殖产业和野生动植物保护至关重要。尽管存在许多用于缓慢冷冻和快速冷却（玻璃化）的方法，设备和设备，但实际上每种方法，细胞类型和物种都需要自己的最佳保存方案。玻璃化（VF）在为许多类型的GP（包括精子和干细胞）开发的成功方案中广受欢迎。但是，所有现有的VF方法都需要复杂且仔细的时间安排，可能容易出现技术错误，通常不可扩展，并且仅限于很小的样品量（0.5-5？l）。因此，诸如精液，绳索干细胞以及足够量的多能干细胞和卵巢组织等样品的冷冻保存非常困难。另一个方面是，虽然大量可能有毒的冷冻保护剂（CPA）大大降低，但对于大多数GP类型而言，浓度仍然相对较高，除了毒性外，CPA的浓度仍然相对较高。加法时间和洗脱时间必须得到精确控制。玻璃化的主要因素之一玻璃化的临界冷却速率（BCR）是否强烈依赖于CPA浓度，例如，需要数十万c/min来玻璃化水甘油溶液，该溶液可容忍所有CPA物种浓度。所有现有的方法旨在实现此类高速，但实际上许多方法并没有这样做，这主要是由于Leidenfrost效应（LFE） - 在样品周围形成了沸腾的氮气蒸气涂层。这种蒸气涂层会通过数量级损害热导率，甚至使液滴均为a的一小部分？ m l无法杀死。速度约为500,000？k/min，我们假设我们可以使用统一的方法，设备和用品实际上可以验证所有种质。我们的CellTronix团队开发了一种用于HyperFast冷却的全新系统，称为“ Krioblast（R）”，该系统完全消除了LFE，并且可以比目前以数十万C/min的速度使用的样品冷却得多。我们已经建立了系统的试点模型（第一代），即手动操作的krioblast-1，与之一起我们可以用稀释的CPA溶液来验证大型样品量，并为人类和牛精子的两个试验获得了一些有希望的结果。获得更高的冷却速率后，我们将接近设计“通用冷冻保存方案”。在这个项目中，我们将建造一个半自动系统磷酸群-2，该系统将产生2-3倍的更快冷却速率，目标为200,000？c/min，并使细胞体积高达4,000 mL（比当前可能高1-2个数量级）。我们认为，这种速率足以使用实际统一的协议来验证GP的所有TYE。在第二阶段，我们将建立一个封闭的模块化茎，用于超快速冷却，低温储存和运输以及细胞的超快速解冻，并在真实的种质细胞上测试磷布拉斯特-3。公共卫生相关性：种质的冷冻保存（精子，卵母细胞，胚胎，干细胞，卵巢组织）对于保留遗传动物的遗传种类，协助人类生育能力，动物育种产业和野生动植物保护至关重要。目前存在各种各样的冷冻保存方法，设备和设备，但是每种方法，细胞类型和物种都需要其自己的最佳协议。该项目的目的是开发一种新型的可扩展设备，用于使用统一的设备和方案允许玻璃化多种种质细胞和物种的效率，这不仅会显着有益于种质冷冻保存，而且最终可能会改变冷冻保存范式。