Factors affecting ice formation in cells and their relevance to cryopreservation

描述(申请人提供)：细胞内结冰(IIF)通常是致命的。在这笔赠款的头四年，主要关注的是确定小鼠和非洲爪哇卵母细胞和酵母中IIF的细节。这些细节包括发生IIF的温度以及冷冻保护剂(CPA)的类型和浓度对这些温度的影响。从这些研究中我们发现，在1~1.5MEG或甘油中，成核发生在水的均匀成核温度-400℃附近。但在较低浓度下，它以非均相成核方式发生，很有可能外部冰是成核剂。(见进度报告出版名单中Mazur、Seki和Kleinhans的139-140、142-143、145-147、149-150、153号文件)。在过去的三年里，根据目前的续期赠款，我们已经将重点转移到确定内部冰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，细胞内冰的形成不是致命的；致命的是这些晶体在升温过程中通过重结晶生长。这就意味着，升温速度是决定“玻璃化”小鼠卵母细胞存活率的主要因素，而不是降温速度。(157、154和160号文件)。目前的续期申请直接建立在上段所述项目的基础上。到目前为止，我们基于解冻细胞对渗透特性的保持以及细胞膜和细胞质的形态正常来确定存活率。他们的论点是，根据这些标准，那些产生低存活率的条件集几乎肯定会表现出非常低的功能存活率或零功能存活率。然而，我们发现，如果升温速度非常快，许多条件下的渗透/形态存活率为80%至90%；降温速度影响较小或没有影响。现在，我们打算确定用这种方法处理的卵母细胞的功能存活率。另一个发现是，如果卵母细胞以每分钟100,000℃的速度升温，即使玻璃化培养液被稀释一半，大约80%的卵母细胞在渗透压、形态和发育到2-细胞胚胎的能力方面仍能存活。也许，更快的升温速度将允许使用更稀薄的玻璃化溶液。我们建议通过使用一种特殊的激光来测试这一假设，以达到升温速度的100倍。大多数人认为，玻璃化溶液的效果取决于其中非常高的溶质浓度。另一种可能性是，它们的有效性更多地取决于它们在开始冷却到-1960摄氏度之前渗透脱水细胞的程度，我们建议以定量的方式检验这一假设。

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.