Takahashi T, Hammett M F, Cho M S
Cryobiology. 1985 Jun;22(3):215-36. doi: 10.1016/0011-2240(85)90143-9.
Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.
利用低温显微镜、电子显微镜和功能检测方法（吞噬作用、杀菌活性和趋化性），研究了人类多形核白细胞（PMN）在零下较高温度下的细胞外冷冻损伤。冷冻损伤至少有四个主要因素：渗透应激、冷刺激、冷休克和稀释性休克。在没有冷冻保护剂的情况下，当冷却到-2℃时，细胞外冷冻的PMN会失去功能。细胞在冷冻时体积减小，减小程度与高渗暴露时相同。PMN有一个最小体积，在该体积下收缩不会造成损伤。更大程度的脱水会对细胞功能产生不可逆损伤，细胞最终会在高渗透压下崩溃。在缓慢冷却、过冷至-5℃以下的PMN中可观察到冷敏感性；在-7℃时，1小时内功能丧失。添加二甲基亚砜（Me2SO）可预防这种损伤，但甘油不能。然而，Me2SO并不能预防冷休克（快速冷却导致的损伤），冷休克在以10℃/分钟的速度冷却至-14℃时出现，而在以0.5℃/分钟的速度缓慢冷却时不会出现。使用甘油作为冷冻保护剂的问题之一源于PMN在甘油稀释或去除过程中对稀释性休克的高敏感性。
