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: 7725346
• 负责人: PETER MAZUR
• 金额: $ 12.45万
• 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 2011-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7725346
• 关键词: Affect; Agriculture; Animals; Arabidopsis; Area; Award; Belief; Biological Preservation; Cell membrane; Cells; Chinese Hamster Ovary Cell; Companions; Cryopreservation; Detection; Development; Differential Scanning Calorimetry; Educational workshop; Electron Microscopy; Elements; Embryo; Equilibrium; Freezing; Funding; Gap Junctions; Glass; Grant; Growth; Hamsters; Hemolysin; Human; Ice; In Situ; Institution; Investigation; Laboratory Animals; Laboratory Research; Life; Maintenance; Medicine; Methods; Morula; Mus; National Center for Research Resources; Neuroblastoma; Oocytes; Permeability; Prevention; Procedures; Process; Productivity; Protoplasts; Research; Research Personnel; Role; Route; Solutions; Staging; System; Temperature; Tissue Transplantation; Transfection; Water; Work; Yeasts; Zebrafish; assisted reproduction; authority; cell type; cell water; cold temperature; college; cost; cryobiology; embryo cell; improved; public health relevance; simulation; solute; sperm cell; tissue/cell culture; transmission process; water channel

DESCRIPTION (provided by applicant): Cryopreservation demands that lethal intracellular freezing (IIF) not occur. There are two routes to its prevention. One is referred to as equilibrium slow cooling. In this procedure, cells are cooled slowly enough so that they lose nearly all their water osmotically before reaching the nucleation temperature at which IIF becomes possible. Many cell types can be easily and successfully cryopreserved by this method, but many others can not, one example being mouse and human oocytes. The second route to avoiding IIF is to subject cells to vitrification procedures, which convert cell water into a glass. To achieve vitrification, the belief is that cells have to be suspended in high concentrations of a permeating CPA and cooled and warmed at high rates, with a reciprocal relation between CPA concentration and rate. This has led us into an investigation in mouse oocytes of the effects of cooling and warming rates and holding temperatures on the growth of intracellular ice by recrystallization during warming. We have found that this lethal process occurs over a range of at least -80¿C to -50¿C with a large increase in rapidity with increasing temperature. Companion studies with "vitrified" oocytes indicate that their survival depends almost entirely on the warming rate with cooling rate having relatively little effect. A new specific aim will pursue further studies on this matter. An important question is how does external ice cross the cell membrane to initiate ice nucleation within the cell. A corollary in multi-cellular systems is how does internal ice in one cell propagate to its neighbors? To investigate these questions, we are proposing in this supplemental application to intensify our study of whether two types of pores in two types of cell systems serve as routes for ice transmission. The two types of pores are those in aquaporins and those in gap junctions. The first cell type is the mouse 8-cell/morula embryo. Early 8-cell embryos possess neither aquaporins or gap junctions; late 8-cell (= early morula) embryos possess both. The second cell type are tissue-culture cells; namely, hamster V79 cells and human neuroblastoma cells. By appropriate transfection, both can be obtained with or without functional aquaporins or gap junctions. An important element of the latter work will be the involvement of Dr. David Spray (Einstein College of Medicine) as a collaborator. Dr. Spray is a world authority on gap junctions. In April, 2007, NCRR held a workshop to review the status of the cryopreservation of sperm, oocytes, and embryos of important laboratory research animals, including the zebrafish; however, to date, all attempts to cryopreserve their embryos have failed. For several reasons, immature oocytes may be more amenable. One reason is that their permeability to water and CPA is considerably higher than that of mature oocytes or embryos. Consequently, we propose in a new specific aim to investigate IIF in the immature oocytes. This study will be strongly enhanced by the addition of Dr. Mary Hagedorn (Smithsonian Institution) as a co-investigator. Her expertise is zebrafish cryobiology, an area in which she has made major contributions. PUBLIC HEALTH RELEVANCE (provided by applicant): The ability to preserve cells by freezing to low temperatures has important implications and applications to assisted reproduction and tissue transplantation in medicine, to improving agricultural productivity, to the maintenance of the gemplasm of genetically important laboratory animals far more cost effectively that its maintenance by living colonies, and to the preservation of the germplasm of endangered species.

描述（由应用程序提供）：冷冻保存要求未发生致命的细胞内冷冻（IIF）。预防有两条路线。一种被称为等效的慢冷却。在此过程中，细胞的冷却得足够缓慢，因此在达到IIF的成核温度之前，它们几乎渗透失去所有水。许多细胞类型可以通过这种方法轻松而成功地冷冻保存，但是许多细胞类型不能，一个例子是小鼠和人类卵母细胞。 避免IIF的第二个途径是对细胞进行玻璃化程序，然后将细胞水转化为玻璃。为了实现玻璃化，人们认为必须以高浓度的渗透CPA悬浮细胞，并以高速冷却和温暖，并在CPA浓度和速率之间存在相互关系。这使我们对小鼠卵母细胞进行了投资，以使冷却和变暖速率的影响以及保持温度对细胞内冰的生长在变暖过程中。我们发现，这种致命过程至少在-80€至-50 c的范围内发生，并且随着温度的升高，速度迅速增加。使用“玻璃化”卵母细胞的伴侣研究表明，它们的存活几乎完全取决于变暖速率，而冷却速率的作用相对较小。一个新的特定目标将对此事进行进一步的研究。 一个重要的问题是，外部冰如何跨越细胞膜以启动细胞内的冰成核。多细胞系统中的推论是一个细胞中的内部冰如何向其邻居传播？为了调查这些问题，我们在此补充应用中提出，以加强我们对两种类型的细胞系统中两种类型的孔的研究，作为冰的传播途径。两种类型的孔是水通道蛋白和间隙连接处的孔。第一种细胞类型是鼠标8细胞/莫拉拉 胚胎。早期的8细胞胚胎既没有水通道蛋白也没有间隙连接；晚期8细胞（=早期毛拉）胚胎都具有两者。第二个细胞类型是组织培养细胞。即，仓鼠V79细胞和人类神经母细胞瘤细胞。通过适当的转换，可以在有或没有功能性水通道或间隙连接的情况下获得两者。后一项工作的重要组成部分是戴维·斯雷斯（David Spray）博士（爱因斯坦医学院）作为合作者的参与。 Spray博士是差距连接的世界权威。 2007年4月，NCRR举行了一个研讨会，以回顾重要实验室研究动物（包括斑马鱼）的精子，卵母细胞和胚胎的冷冻保存状况；但是，迄今为止，所有冷冻固定胚胎的尝试都失败了。由于几个原因，未成熟的卵母细胞可能更适合。原因之一是它们对水和CPA的渗透性高于成熟的卵母细胞或胚胎。因此，我们提出了一个新的特定目的，以研究未成熟卵母细胞中的IIF。通过添加玛丽·哈迪恩（Mary Hagedorn）博士（史密森尼机构）作为共同投资者，这项研究将大大增强。她的专家是斑马鱼冷冻生物学，这是她做出了重大贡献的领域。 公共卫生相关性（通过应用程序提供）：通过冷冻到低温来保护细胞的能力具有重要的含义和应用，在医学中的辅助繁殖和组织移植，提高农业生产力，维护一般重要的实验室动物的gemplasm gemplasm gemplass，从而有效地维持生物的成本，以使生物的维持以及生存的生物和生物的维持，以维持生命的生产物，以及生存的物种。