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：
8336889
负责人：
PETER MAZUR
金额：
$ 25.56万
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/8336889
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.

描述（由申请人提供）：细胞内冰形成（IIF）通常是致命的。这笔赠款前四年的主要关注点是确定小鼠和武士卵母细胞以及酵母中IIF的细节。细节包括IIF发生的温度以及冷冻保护剂（CPA）类型的影响以及对这些温度的浓度。从这些研究中，我们发现，在1至1.5 m或甘油中，成核发生在-400C附近，即水的均质成核温度。但是在较低的浓度下，它是通过异质成核发生的，外部冰是成核的可能性很高。（请参阅《进度报告出版物列表》中的论文139-140、142-143、145-147、149-150、153论文。在过去的三年中，在当前的续签授予下，我们将重点转移到确定内部冰的玻璃化和重结晶期间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，细胞内冰的形成不是致命的。致命是通过重结晶在变暖过程中这些晶体的生长。这意味着，变暖速率在确定“玻璃化”小鼠卵母细胞的存活中起着主要作用，而不是冷却速率。（论文157、154和160）。当前的续订应用程序直接建立在上面段落中的项目上。到目前为止，我们已经基于“生存”基于解冻细胞以及细胞膜和细胞质的形态正态性的渗透特性的保留。该论点是，基于这些标准产生低“存活”的一组条件几乎可以肯定会表现出非常低或零功能的生存。但是，我们发现，如果变暖率很高，许多条件会产生80％至90％的渗透/形态生存。冷却速率较小或没有影响。现在，我们打算以这种方式确定卵母细胞的功能生存。另一个发现是，如果卵母细胞温暖> 100,0000c/min，则在形态上，即使将玻璃化培养基稀释为一半，约有80％的卵母细胞在渗透渗透，形态上也可以生存至2细胞胚胎。可能，即使更快的变暖也可以允许使用更稀释的玻璃化解决方案。我们建议通过使用特殊激光来实现高100倍的变暖率来检验这一假设。大多数人认为，玻璃化溶液的功效取决于它们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将冷却启动至-1960C之前将细胞渗透脱水的程度，我们建议以定量的方式检验该假设。