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Factors affecting ice formation in cells and their relevance to cryopreservation

影响细胞冰形成的因素及其与冷冻保存的相关性

基本信息

批准号：
8237381
负责人：
PETER MAZUR
金额：
$ 22.59万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-08 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8237381
关键词：
Affect Agriculture Animals Anopheles Genus Appearance Back Behavior Cattle Cell membrane Cells Clinical Medicine Cryopreservation Cryopreserved Cell Cryopreserved Tissue Cytoplasm Dehydration Disease Drosophila genus Drosophila melanogaster Effectiveness Embryo Event Exhibits Fertilization Fishes Food Genetic Genetic Variation Germ Cells Glycerol Grant Growth Human Ice Injury Invertebrates Laboratory Animals Lasers Maintenance Marrow Modeling Mus Normalcy Oocytes Organ Paper Permeability Plants Play Probability Procedures Productivity Progress Reports Property Publications Relative (related person)Reproducibility Research Role Solutions Staging Stem cells Temperature Testing Time Tissue Engineering Tissue Transplantation Transgenic Organisms Translating Vertebrates Water Xenopus Yeasts Zebrafish assisted reproduction base cell type cell water cold temperature cost effective cryogenics design developmental genetics embryo cell human tissue improved interest leukemia mutant research study solute sperm cell warm temperature

项目摘要

DESCRIPTION (provided by applicant): Intracellular ice formation (IIF) is usually lethal. The main concern of the first four years of this grant was to determine details of IIF in oocytes of mice and Xenopus and in yeast. The details included the temperatures at which IIF occurred and the effect of cryoprotectant (CPA) type and concentration on those temperatures. From these studies, we have found that in 1 to 1.5 M EG or glycerol, nucleation occurs near -400C, the homogeneous nucleation temperature of water. But in lower concentration, it occurs by heterogeneous nucleation, with a high probability that external ice is the nucleator. (See papers 139-140, 142-143, 145-147, 149-150, 153 by Mazur, Seki, and Kleinhans in the Progress Report Publication List). In the past three years under the current renewal grant, we have shifted the emphasis to determining physical events during vitrification and recrystallization of internal ice, and their effects on survival. The most important finding has been that under certain conditions, the formation of intracellular ice is not lethal; what is lethal is the growth of those crystals during warming by recrystallization. This translates to the fact that the warming rate plays the dominant role in determining survival of "vitrified" mouse oocytes-and not the cooling rate. (Papers 157, 154, and 160). The current renewal application builds directly on the items in the above paragraph. Up to now, we have based "survivals" on the retention of osmotic properties by thawed cells and by the morphological normality of cell membranes and cytoplasm. The argument was that those sets of conditions that yielded low "survivals" based on these criteria, would almost certainly exhibit very low or zero functional survival. We found, however, that a number of conditions yielded osmotic/morphological survivals of 80 to 90% if the warming rates were very high; the cooling rate had less or no effect. Now we intend to determine the functional survival of oocytes treated in this way. Another finding has been that if the oocytes are warmed > 100,0000C/min, some 80% survive osmotically, morphologically, and in their ability to develop to 2-cell embryos even if the vitrification medium is diluted in half. Possibly, even faster warming would permit the use of even more dilute vitrifying solutions. We propose to test this hypothesis by using a special laser to achieve warming rates that are 100- times higher. Most believe that the efficacy of vitrification solutions depends on the very high solute concentrations in them. Another possibility is that their efficacy depends more on the degree to which they osmotically dehydrate the cells prior to initiating cooling to -1960C, We propose to test that hypothesis in a quantitative manner. PUBLIC HEALTH RELEVANCE: The ability to preserve cells at very low temperatures has important implications and applications in such aspects of clinical medicine as assisted reproduction, tissue transplantation, and engineered tissue constructs; in improving agricultural productivity; and in the cost-effective maintenance of the germplasm of genetically important laboratory animals.

描述（由申请人提供）：细胞内结冰（IIF）通常是致命的。这笔资助的前四年主要关注的是确定小鼠和非洲爪蟾卵母细胞以及酵母中 IIF 的细节。详细信息包括 IIF 发生的温度以及冷冻保护剂 (CPA) 类型和浓度对这些温度的影响。从这些研究中，我们发现在 1 至 1.5 M EG 或甘油中，成核发生在 -400C（水的均质成核温度）附近。但在较低浓度下，它是通过异质成核发生的，外部冰很可能是成核剂。（参见进度报告出版物列表中 Mazur、Seki 和 Kleinhans 的论文 139-140、142-143、145-147、149-150、153）。在过去的三年里，根据当前的更新拨款，我们将重点转向确定内部冰的玻璃化和重结晶过程中的物理事件及其对生存的影响。最重要的发现是，在某些条件下，细胞内冰的形成并不致命。致命的是这些晶体在升温过程中通过重结晶而生长。这意味着升温速率在决定“玻璃化”小鼠卵母细胞的存活中起主导作用，而不是冷却速率。（论文 157、154 和 160）。当前的续订申请直接建立在上一段中的项目的基础上。到目前为止，我们的“存活”基于解冻细胞的渗透特性保留以及细胞膜和细胞质的形态正常性。争论的焦点是，根据这些标准产生低“存活率”的那些条件组几乎肯定会表现出非常低或零的功能存活率。然而，我们发现，如果升温速率非常高，许多条件下的渗透/形态存活率可达 80% 至 90%；冷却速率影响较小或没有影响。现在我们打算确定以这种方式处理的卵母细胞的功能存活率。另一个发现是，如果卵母细胞升温 > 100,0000C/min，即使玻璃化介质稀释一半，大约 80% 的卵母细胞在渗透、形态和发育成 2 细胞胚胎的能力方面仍能存活。可能，更快的升温将允许使用更稀的玻璃化溶液。我们建议通过使用特殊激光来测试这一假设，以实现高 100 倍的升温速率。大多数人认为玻璃化溶液的功效取决于其中非常高的溶质浓度。另一种可能性是它们的功效更多地取决于它们在开始冷却至-1960°C之前对细胞进行渗透脱水的程度，我们建议以定量方式检验该假设。公共健康相关性：在极低温度下保存细胞的能力在辅助生殖、组织移植和工程组织构建等临床医学方面具有重要意义和应用；提高农业生产力；以及以具有成本效益的方式维护具有重要遗传意义的实验动物的种质。