Factors affecting ice formation in cells and their relevance to cryopreservation

描述（由申请人提供）：细胞内结冰（IIF）通常是致命的。这项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，成核发生在接近-400 ℃，即水的均匀成核温度。但在较低的浓度下，它是通过非均质成核发生的，外部冰是成核剂的可能性很高。(See论文139-140，142-143，145-147，149-150，153，Mazur，Seki和Kleinhans在进展报告出版物列表中）。 在过去的三年中，根据目前的更新补助金，我们已经将重点转移到确定内部冰的玻璃化和重结晶过程中的物理事件，以及它们对生存的影响。最重要的发现是，在某些条件下，细胞内冰的形成是不致命的;致命的是那些晶体在加热过程中通过重结晶的生长。这意味着升温速率在决定玻璃化小鼠卵母细胞存活率方面起着主导作用，而不是冷却速率。（第157、154、160页） 目前的续期申请直接建立在上段所列项目的基础上。到目前为止，我们已经基于“生存”的保留渗透性能的解冻细胞和细胞膜和细胞质的形态正常。他们的论点是，根据这些标准，那些产生低“存活率”的条件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/形态存活率为80%至90%;冷却速率的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。另一个发现是，如果卵母细胞以> 100，0000 C/min的速度升温，即使玻璃化冷冻培养基被稀释一半，约80%的卵母细胞仍能存活，形态学上，以及发育成2-细胞胚胎的能力。可能，更快的升温将允许使用更稀的玻璃化溶液。我们建议通过使用一种特殊的激光来测试这一假设，以实现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到-1960 ℃之前对细胞的免疫抑制程度。我们建议以定量的方式来检验这一假设。 公共卫生相关性：在非常低的温度下保存细胞的能力在临床医学如辅助生殖、组织移植和工程化组织构建体的这些方面具有重要的意义和应用;在提高农业生产力方面;以及在遗传上重要的实验室动物的种质的成本有效的维持方面。

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.