Shier W T
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Minnesota, Minneapolis 55455.
Cryobiology. 1988 Apr;25(2):110-20. doi: 10.1016/0011-2240(88)90004-1.
Normally a freeze-thaw cycle is a very efficient method of killing mammalian cells. However, this report describes conditions that prevent killing of cultured mammalian cells by nucleated freezing at -24 degrees C. Optimal protection from cell killing at -24 degrees C was obtained in isotonic solutions containing an organic cryoprotectant such as dimethyl sulfoxide (DMSO; 10%, v/v), a saccharide such as sucrose over a broad concentration range from 50 to 150 mM, and glucose. Glycerol was also an effective cryoprotectant but other organic solvents were ineffective, although in some cases they appeared to protect cell membranes, while not protecting other vital components. A wide variety of saccharide structures were effective at protecting cells from freeze-thaw killing, with trehalose being particularly effective. The degree of resistance to killing by a freeze-thaw cycle under these conditions varied widely among different cell lines. If toxicity of DMSO was responsible for this variability of cryoprotection, it must have been due to short-term, not longer term, toxicity of DMSO. Studies on the mechanism by which cells are protected from killing under these conditions indicated that neither vitrification of the medium nor the concentrating of components during freezing were involved. One model not eliminated by the mechanistic studies proposes that the organic solvent cryoprotectant component acts by fluidizing membranes under the thawing conditions, so that any holes produced by ice crystals propagating through membranes can reseal during the thawing process. In this model one of the mechanisms by which the saccharide component could act is by entering the cells and stabilizing vital intracellular components. Consistent with this, a freeze-thaw cycle promoted the uptake of labeled sucrose into cultured cells.
通常，冻融循环是杀死哺乳动物细胞的一种非常有效的方法。然而，本报告描述了在-24℃下有核冷冻时防止培养的哺乳动物细胞被杀死的条件。在含有有机冷冻保护剂（如二甲基亚砜（DMSO；10%，v/v））、在50至150 mM的宽浓度范围内的糖类（如蔗糖）以及葡萄糖的等渗溶液中，可获得对-24℃下细胞杀伤的最佳保护。甘油也是一种有效的冷冻保护剂，但其他有机溶剂无效，尽管在某些情况下它们似乎能保护细胞膜，而不能保护其他重要成分。多种糖类结构在保护细胞免受冻融杀伤方面有效，海藻糖尤其有效。在这些条件下，不同细胞系对冻融循环杀伤的抗性程度差异很大。如果DMSO的毒性是这种冷冻保护变异性的原因，那一定是由于DMSO的短期而非长期毒性。对在这些条件下细胞免受杀伤的机制的研究表明，既不涉及培养基的玻璃化，也不涉及冷冻过程中成分的浓缩。机理研究未排除的一个模型提出，有机溶剂冷冻保护剂成分的作用是在解冻条件下使膜流化，以便冰晶穿过膜产生的任何孔洞在解冻过程中能够重新封闭。在这个模型中，糖类成分起作用的机制之一可能是进入细胞并稳定重要的细胞内成分。与此一致的是，冻融循环促进了标记蔗糖进入培养细胞。
