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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：冷冻保存要求不发生致命的细胞内冷冻（IIF），有两种预防方法，一种称为平衡缓慢冷却，在该过程中，细胞冷却得足够慢，以致它们几乎失去作用。在达到 IIF 成为可能的成核温度之前，它们的所有水分都被渗透了。许多细胞类型可以通过这种方法轻松成功地冷冻保存，但许多其他细胞类型则不能，例如小鼠和人类卵母细胞。避免 IIF 的第二种途径是对细胞进行玻璃化程序，将细胞水转化为玻璃体。人们认为，细胞必须悬浮在高浓度的渗透性 CPA 中，并以高速率冷却和加热。 CPA 浓度和速率之间存在倒数关系，这使我们对小鼠卵母细胞进行了研究，研究冷却和升温速率以及保温温度对升温过程中再结晶导致的细胞内冰生长的影响。致命过程发生在至少-80°的范围内C至-50°随着温度升高，C 的速度大幅增加。对“玻璃化”卵母细胞的研究表明，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对此问题进行进一步研究。一个重要的问题是外部冰如何穿过细胞膜以启动细胞内的冰成核。为了研究这些问题，我们在本文中提出了多细胞系统中的内部冰如何传播到其相邻细胞的一个推论。补充应用以加强我们对两种细胞系统中的两种孔是否充当冰传输途径的研究。这两种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/桑葚胚早期 8 细胞胚胎不具有水通道蛋白或间隙连接；晚期 8 细胞（= 早期桑葚胚）胚胎同时具有两种细胞类型：即仓鼠 V79 细胞和人神经母细胞瘤细胞。，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，后者工作的一个重要因素是 David Spray 博士（爱因斯坦医学院）作为合作者的参与。 Spray 是间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，所有冷冻胚胎的尝试都因多种原因而失败。我们在一项新的具体目标中提出，未成熟卵母细胞可能更容易受到影响，原因之一是它们对水和 CPA 的渗透性比成熟卵母细胞或胚胎高得多。玛丽·哈格多恩博士（史密森学会）作为联合研究员的加入将大大加强这项研究，她的专业知识是斑马鱼冷冻生物学，她在这一领域做出了重大贡献。公共卫生相关性（由申请人提供）：通过低温冷冻保存细胞的能力对于辅助生殖和医学组织移植、提高农业生产力、维护具有重要遗传意义的实验动物的宝石质具有重要的意义和应用。比通过活体群体进行维护以及保护濒危物种的种质更具成本效益。