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（由申请方提供）：冷冻保存要求不发生致死性细胞内冷冻（IIF）。有两种预防方法。一种称为平衡缓慢冷却。在这个过程中，细胞被冷却得足够慢，使得它们在达到IIF成为可能的成核温度之前几乎完全失去它们的水。许多细胞类型可以通过这种方法容易地和成功地冷冻保存，但许多其他细胞不能，一个例子是小鼠和人类卵母细胞。 避免IIF的第二种途径是将细胞进行玻璃化程序，将细胞水转化为玻璃。为了实现玻璃化，人们认为细胞必须悬浮在高浓度的渗透性CPA中，并以高速率冷却和升温，CPA浓度和速率之间存在倒数关系。这使我们在小鼠卵母细胞的冷却和升温速率和保温温度对细胞内冰的生长的影响，在升温过程中重结晶的调查。我们发现，这种致死过程发生在至少-80 ° C至-50 ° C的范围内，随着温度的升高，其速度大幅增加。对“玻璃化”卵母细胞的相关研究表明，它们的存活几乎完全取决于升温速率，而降温速率的影响相对较小。一个新的具体目标将进一步研究这一问题。 一个重要的问题是外部的冰如何穿过细胞膜在细胞内引发冰成核。在多细胞系统中的一个推论是，一个细胞中的内部冰如何传播到它的邻居？为了研究这些问题，我们建议在本补充申请中加强我们的研究，即两种类型的细胞系统中的两种类型的孔是否作为冰传输的途径。两种类型的孔是水通道蛋白中的孔和间隙连接中的孔。第一种细胞类型是小鼠8-细胞/桑椹胚 胚胎早期的8细胞胚胎既不具有水通道蛋白，也不具有缝隙连接;晚期的8细胞胚胎（=早期桑椹胚）两者都具有。第二种细胞类型是组织培养细胞;即仓鼠V79细胞和人神经母细胞瘤细胞。通过适当的转染，两者都可以在有或没有功能性水通道蛋白或间隙连接的情况下获得。后一项工作的一个重要组成部分是大卫斯派尔博士（爱因斯坦医学院）作为合作者的参与。Spray博士是缝隙连接方面的世界权威。 2007年4月，NCRR举办了一次研讨会，审查了包括斑马鱼在内的重要实验室研究动物的精子、卵母细胞和胚胎的冷冻保存状况;然而，迄今为止，所有冷冻保存胚胎的尝试都失败了。出于几个原因，未成熟的卵母细胞可能更容易接受。一个原因是它们对水和CPA的渗透性比成熟卵母细胞或胚胎高得多。因此，我们提出了一个新的具体目标，研究未成熟卵母细胞的IIF。增加玛丽哈格多恩博士（史密森学会）作为合作研究者将大大增强本研究。她的专业知识是斑马鱼低温生物学，她在该领域做出了重大贡献。 公共卫生相关性（由申请人提供）：通过冷冻至低温保存细胞的能力对医学中的辅助生殖和组织移植、提高农业生产力、以远比通过活菌落更经济的方式维持遗传上重要的实验室动物的种质以及濒危物种种质的保存具有重要的意义和应用。