Factors affecting ice formation in cells and their relevance to cryopreservation

描述(申请人提供)：冷冻保存的关键是不发生致死性细胞内冷冻(IIF)。它的发生取决于两个主要因素。其一是冷却速度。它必须足够低，以便细胞在冷却到可以进行IIF的温度之前，在渗透作用下几乎失去所有的水。其次是IIF发生的温度。温度越高，通过缓慢冷却避免IIF的难度就越大。我们目前的赠款和这项续签建议主要涉及后者。在小鼠、非洲爪哇卵母细胞和拟南芥原生质体中发现，IIF需要细胞外的冰与细胞膜密切接触。小鼠卵母细胞中的一个强有力的证据是，间歇性受精发生在大约95%的外部培养液已经冻结的温度下。该温度从-14°C到-40°C不等，这取决于介质中低温保护化合物的浓度。另一个影响间接受精温度的因素可能是细胞大小，因为间接受精发生在1毫米非洲爪哇卵中的温度比在0.1毫米小鼠卵母细胞中发生的温度高得多。上面使用的两个工具是一个冷冻阶段，它允许我们在冷却和升温期间观察细胞，并将IIF显示为“闪光”，以及差示扫描量热仪(DSC)，它检测IIF是热爆发。在这次续签申请中，我们建议引入两个新的工具。其中一个是与橡树岭国家实验室合作的扫描和透射电子显微镜(STEM)，它配备了一个新设计的样本室，可以对水溶液中的水化细胞进行高分辨率成像。另一种仪器是定向冻结台。它允许将冷却速度分解为热梯度(G)和晶体生长速度(V)。G和V的不同影响冰晶的形态，进而影响IIF的温度。我们将在这项研究中增加三种新的细胞类型：酵母和两种类型的仓鼠组织培养细胞。这些细胞比小鼠卵母细胞小10倍，从而使我们能够进一步探索细胞大小的作用。其次，细胞必须这么小才能放入STEM液体样品室。第三，在这些细胞中从未直接观察到IIF，我们打算使用DSC来实现这一目的。这项更新建议的其他主要目的是(1)确定观察到的小鼠卵母细胞IIF温度与冷冻分数之间的高度相关性是否适用于其他类型的细胞。(2)确定质膜上的气孔是否与IIF温度有关。为此，我们将研究小鼠桑椹胚的冷冻，其中8到12个细胞具有缝隙连接和水通道蛋白孔。其次，我们将在小鼠卵母细胞和植物原生质体中引入毛孔，方法是将它们暴露在来自葡萄球菌的毒素中。最终目标将是利用IIF上的机械数据来设计更好的方法来避免它，从而提高目前不能很好地冷冻保存的细胞的冷冻保存成功率。

DESCRIPTION (provided by applicant): Critical to cryopreservation is that lethal intracellular freezing (IIF) not occur. Its occurrence depends on two major factors. One is the cooling rate. It has to be low enough so that the cells lose nearly all their water osmotically before cooling to the temperature at which IIF becomes possible. Second is the temperature at which IIF occurs. The higher that temperature, the greater the difficulty in avoiding IIF by slow cooling. Our current grant and this renewal proposal deal primarily with the latter. IIF in mouse and Xenopus oocytes and in Arabidopsis protoplasts was found to require the presence of extracellular ice in close contact with the cell membrane. One strong piece of evidence in the mouse oocyte is that IIF occurs at temperatures where about 95 percent of the external medium has frozen. That temperature varies from -14¿C to -40¿C depending on the concentration of cryoprotective compounds in the medium. Another factor affecting the IIF temperature may be cell size, for IIF occurs at much higher temperatures in 1 mm Xenopus eggs than it does in <0.1 mm mouse oocytes. The two tools used above were a cryostage that permits us to observe cells during cooling and warming and manifests IIF as "flashing", and a differential scanning calorimeter (DSC) that detects IIF as an outburst of heat. In this renewal application we propose to introduce two new instruments. One, in collaboration with the Oak Ridge National Laboratory, is a Scanning and Transmission Electron Microscope (STEM) equipped with a newly designed sample chamber that will permit high resolution images of hydrated cells in aqueous solutions. The other instrument is a directional freezing stage. It permits a cooling rate to be resolved into the thermal gradient (G) and the crystal growth velocity (V). Differences in G and V affect the ice crystal morphology which in turn may affect the temperature of IIF. We will add three new cell types to the study: Yeast and two types of hamster tissue culture cells. These cells are 10-times smaller than mouse oocytes thus permitting us to further explore the role of cell size. Second, cells have to be that small to fit in the STEM liquid sample chamber. Third, IIF has never been observed directly in these cells and we intend to use DSC for this purpose. Other major aims in this renewal proposal are (1) to determine whether the high correlation between observed IIF temperature in mouse oocytes and the frozen fraction holds for other cell types. (2) To determine whether there is a relation between pores in the plasma membrane and the IIF temperature. For this, we will study the freezing of mouse morulae, the 8 to 12 cells of which possess gap junctions and aquaporin pores. Second, we will introduce pores in mouse oocytes and plant protoplasts by exposing them to a toxin from Staphylococcus bacteria. The final goal will be to use the mechanistic data on IIF to devise better methods of avoiding it and thus improve success in the cryopreservation of cells that currently can not be well cryopreserved.