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: 7367713
• 负责人: PETER MAZUR
• 金额: $ 44.85万
• 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/7367713
• 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（适用提供）：冷冻保存至关重要的是未发生致命的细胞内冷冻（IIF）。它发生取决于两个主要因素。一个是冷却速率。它必须足够低，以便细胞在冷却至可能的温度之前，几乎所有的水都会渗透渗透。第二是IIF发生的温度。温度越高，通过缓慢冷却来避免IIF的难度就越大。我们目前的赠款和这项续签提案与后期有关。发现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细胞外冰。小鼠卵母细胞中的一个有力的证据是，IIF发生在大约95％的外部培养基被冷冻的温度下发生。该温度范围从-14¿C到-40？c，具体取决于培养基中的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尺寸，因为IIF在1 mm爪蟾卵中的温度更高，而小鼠卵母细胞中的温度要高得多。上面使用的两个工具是一个低温量，它允许我们在冷却和变暖期间观察细胞，并将IIF显示为“闪烁”，而差异扫描量热仪（DSC）将IIF视为热量的爆发。在此续订应用程序中，我们建议介绍两种新工具。第一，与橡树岭国家实验室合作，是配备了新设计的样品室的扫描和传输电子显微镜（STEM），该室将允许在水溶液中的水合细胞的高分辨率图像。另一个仪器是定向冻结阶段。它允许将冷却速率解析到热梯度（G）和晶体生长速度（V）中。 G和V的差异会影响冰晶形态，进而影响IIF温度。我们将在研究中添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，因此使我们能够进一步探索细胞大小的作用。其次，细胞必须很小才能适合茎液样品室。第三，在这些细胞中从未直接观察到IIF，我们打算将DSC用于此目的。该续签建议中的其他主要目的是（1）确定小鼠卵母细胞中观察到的IIF温度与其他细胞类型的冷冻分数之间的高相关性。 （2）确定质膜和IIF温度中的孔之间是否存在关系。为此，我们将研究小鼠Morulae的冻结，其8至12个细胞具有间隙连接和水通道蛋白孔。其次，我们将通过将毛孔暴露于葡萄球菌细菌中的毒素中来引入小鼠卵母细胞和植物原生质体的毛孔。最终目标是使用IIF上的机械数据来设计更好的避免这种情况的方法，从而改善当前无法很好地保存的细胞冷冻保存中的成功。