Factors affecting ice formation in cells and their relevance to cryopreservation

描述（由申请人提供）：低温保存的关键是不发生致命的细胞内冷冻（IIF）。它的发生取决于两个主要因素。一个是冷却速率。它必须足够低，以便在冷却到可以实现IIF的温度之前，细胞几乎会渗透掉所有的水分。其次是IIF发生时的温度。温度越高，通过缓慢冷却来避免IIF的难度就越大。我们目前的拨款和这项续期建议主要针对后者。小鼠和爪蟾卵母细胞以及拟南芥原生质体中的IIF需要与细胞膜紧密接触的细胞外冰的存在。在小鼠卵母细胞中，一个强有力的证据是，IIF发生在大约95%的外部培养基冻结的温度下。根据介质中低温保护化合物的浓度，温度在-14℃到-40℃之间变化。影响IIF温度的另一个因素可能是细胞大小，因为IIF在1mm的爪蟾卵中发生的温度远高于在<0.1 mm的小鼠卵母细胞中发生的温度。上面使用的两个工具是冷冻器，它允许我们在冷却和加热过程中观察细胞，并显示IIF为“闪烁”，以及差示扫描量热计（DSC），它检测IIF为热爆发。在本次续期申请中，我们建议引入两种新仪器。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品室，可以在水溶液中获得高分辨率的水合细胞图像。另一种仪器是定向冻结阶段。它允许将冷却速率分解为热梯度(G)和晶体生长速度(V)。G和V的差异会影响冰晶形态，进而影响IIF的温度。我们将在研究中加入三种新的细胞类型：酵母和两种仓鼠组织培养细胞。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，细胞必须小到可以装进STEM液体样品室。第三，IIF从未在这些细胞中直接观察到，我们打算使用DSC来实现这一目的。该更新提案的其他主要目的是：(1)确定小鼠卵母细胞中观察到的IIF温度与冷冻部分之间的高度相关性是否适用于其他细胞类型。(2)确定质膜孔隙与IIF温度之间是否存在关系。为此，我们将研究小鼠桑葚胚的冷冻，其中8 - 12个细胞具有间隙连接和水通道蛋白孔。其次，我们将通过将小鼠卵母细胞和植物原生质体暴露于葡萄球菌毒素中来引入气孔。最终目标将是利用IIF的机制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.