Pegg David E
Department of Biology, University of York, UK.
Methods Mol Biol. 2007;368:39-57. doi: 10.1007/978-1-59745-362-2_3.
Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a tradeoff between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification--the production of a glassy state that is defined by the viscosity reaching a sufficiently high value (approximatly 10(13) poises) to behave like a solid, but without any crystallization. Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system. However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in transient, and sometimes equilibrium, changes in compartment volumes and these can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread applications in biology and medicine.
低温保存是利用极低温度来保存结构完整的活细胞和组织。无保护的冷冻通常是致命的，本章旨在分析其中一些涉及的机制，并展示如何利用冷却来创造稳定条件以保存生命。冷却的生物学效应主要由水的冻结主导，这会导致溶解在剩余液相中的溶质浓度增加。关于冷冻损伤的竞争理论认为，要么是冰晶刺穿或撕开细胞，通过直接机械作用破坏它们，要么是损伤来自液相组成变化的次级效应。冷冻保护剂简单地通过增加系统中所有溶质的总浓度，减少在任何给定温度下形成的冰量；但要在生物学上可接受，它们必须能够渗透到细胞中且毒性低。许多化合物具有这样的特性，包括甘油、二甲基亚砜、乙二醇和丙二醇。事实上，两种损伤机制都很重要，它们的相对贡献取决于细胞类型、冷却速率和升温速率。已形成一种共识，即细胞内结冰是危险的，而细胞外结冰是无害的。如果细胞膜的水渗透性已知，就有可能预测冷却速率对细胞存活的影响，最佳速率将是细胞内结冰风险与浓缩溶质效应之间的权衡。然而，细胞外冰并不总是无害的：紧密堆积的细胞更有可能因它们被困的通道内的机械应力而受损，对于复杂的多细胞系统，不仅要确保细胞存活，还必须避免对细胞外结构的损伤。通过玻璃化可以避免结冰——形成一种玻璃态，其定义为粘度达到足够高的值（约10¹³泊）以表现得像固体，但没有任何结晶。毒性是使用玻璃化方法的主要问题。无论允许冷冻（传统低温保存）还是防止冷冻（玻璃化），冷冻保护剂都必须进入系统的所有部分。然而，溶质自由扩散存在许多障碍（膜），这可能导致隔室体积的瞬时变化，有时是平衡变化，而这些变化可能是有害的。因此，扩散和渗透过程在冷冻保护剂的引入、冷冻保护剂的去除、冷冻过程和解冻过程中都有重要影响。这些现象适合进行实验和分析，这使得开发有效的方法来保存非常广泛的细胞和一些组织成为可能；这些方法已在生物学和医学中得到广泛应用。
