Factors affecting ice formation in cells and their relevance to cryopreservation

影响细胞冰形成的因素及其与冷冻保存的相关性

• 批准号: 7502733
• 负责人: PETER MAZUR
• 金额: $ 42.5万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-08 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7502733
• 关键词: Affect; Arabidopsis; Bacteria; Biological Preservation; Cell Size; Cell membrane; Cells; Characteristics; Collaborations; Condition; Cryopreservation; Data; Detection; Differential Scanning Calorimetry; Electron Microscope; Electron Microscopy; Freezing; Gap Junctions; Genus staphylococcus; Goals; Grant; Grouping; Growth; Habits; Hamsters; Heating; Hemolysin; High temperature of physical object; Ice; Image; Laboratories; Liquid substance; Methods; Morphology; Morula; Mus; Oocytes; Personal Satisfaction; Plants; Probability; Property; Protoplasts; Purpose; Rate; Research; Resolution; Role; Sampling; Scanning; Solutions; Staging; Temperature; Time; Toxin; Water; Xenopus; Xenopus oocyte; Yeasts; aqueous; cell type; design; egg; extracellular; falls; improved; instrument; physical state; success; tissue/cell culture; tool; transmission process; water channel

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.

描述（由申请人提供）：冷冻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 的温度之前通过渗透失去几乎所有的水。其次是发生 IIF 的温度。该温度越高，通过缓慢冷却来避免 IIF 的难度就越大。我们当前的拨款和本次更新提案主要涉及后者。小鼠和非洲爪蟾卵母细胞以及拟南芥原生质体中的 IIF 需要细胞外冰与细胞膜紧密接触。小鼠卵母细胞中的一项有力证据是，IIF 发生在约 95% 的外部介质已冻结的温度下。该温度在 -14°C 至 -40°C 之间变化，具体取决于介质中冷冻保护化合物的浓度。影响 IIF 温度的另一个因素可能是细胞大小，因为 1 毫米爪蟾卵中 IIF 发生的温度比 <0.1 毫米小鼠卵母细胞中发生的温度高得多。上面使用的两种工具是冷冻台，它允许我们在冷却和升温过程中观察细胞并将 IIF 表现为“闪烁”，以及差示扫描量热计 (DSC)，它检测 IIF 作为热量的爆发。在本次更新申请中，我们建议引入两种新工具。其中一个是与橡树岭国家实验室合作开发的扫描透射电子显微镜（STEM），配备了新设计的样品室，可以拍摄水溶液中水合细胞的高分辨率图像。另一个仪器是定向冷冻台。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。我们将在研究中添加三种新的细胞类型：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，因此使我们能够进一步探索细胞大小的作用。其次，细胞必须小到适合 STEM 液体样品室。第三，从未在这些细胞中直接观察到 IIF，我们打算使用 DSC 来实现此目的。该更新提案的其他主要目标是 (1) 确定观察到的小鼠卵母细胞 IIF 温度与冷冻部分之间的高度相关性是否也适用于其他细胞类型。 (2)确定质膜孔隙与IIF温度是否存在相关性。为此，我们将研究小鼠桑葚的冷冻，其中 8 至 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来自葡萄球菌的毒素来引入孔。最终目标是利用 IIF 的机制数据来设计更好的方法来避免它，从而提高目前无法很好冷冻保存的细胞的冷冻保存成功率。