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。这项拨款的头四年主要关注的是确定小鼠、非洲爪蟾和酵母卵母细胞中IIF的细节。这些细节包括IIF发生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 ~ 1.5 M EG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，它以非均相成核的方式发生，外部冰很可能是成核剂。（见Progress Report Publication List中Mazur、Seki和Kleinhans的论文139-140、142-143、145-147、149-150和153）。在过去的三年里，在当前的续期拨款下，我们将重点转移到确定内部冰的玻璃化和再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再结晶变暖过程中的生长。这就意味着，在决定“玻璃化”小鼠卵母细胞存活的过程中，升温速率起着主导作用，而不是降温速率。（论文157、154和160）。当前的续期申请直接建立在上述段落中的项目之上。到目前为止，我们将“存活”建立在解冻细胞保留渗透特性和细胞膜和细胞质形态正常的基础上。他们的论点是，那些根据这些标准产生低“存活率”的条件，几乎肯定会表现出非常低或零功能存活率。然而，我们发现，在一些条件下，如果升温速率非常高，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。现在我们打算确定用这种方法处理的卵母细胞的功能性存活。另一个发现是，如果卵母细胞被加热到100 000 000摄氏度/分钟，即使玻璃化培养基被稀释一半，大约80%的卵母细胞在渗透、形态和发育成2细胞胚胎的能力上存活下来。可能，更快的升温将允许使用更稀释的玻璃化溶液。我们建议用一种特殊的激光来测试这一假设，使升温速率提高100倍。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℃之前对细胞进行渗透脱水的程度。我们建议以定量的方式测试这一假设。

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. 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.