Han Bumsoo, Bischof John C
Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Cryobiology. 2004 Feb;48(1):8-21. doi: 10.1016/j.cryobiol.2003.11.002.
Freezing induced direct cell injury has been explained by a two-factor hypothesis-intracellular ice formation (IIF) at rapid cooling rates, and solution effects at slow cooling rates. Even though IIF is generally believed to be a major injury mechanism at rapid cooling rates, injury by solution effects is not fully understood and several injury mechanisms have been suggested. Solution effects have generally been considered the result of the elevated electrolyte concentration within the intracellular and extracellular space during freezing. In addition to the injury by this elevated electrolyte concentration, freezing injury associated with eutectic crystallization was investigated. To examine the injury associated with eutectic crystallization, two different experiments were designed and performed. In the first experiment, two groups of AT-1 rat prostate tumor cell suspensions were frozen and thawed on a cryomicroscope in the same way except that eutectic crystallization was initiated in only one group. During the second experiment, AT-1 cells were suspended in several different media, which have different eutectic crystallization temperatures, and exposed to a single cooling-warming cycle with varying end temperature of the protocol on a directional solidification stage. After both experiments, post-thaw viability was evaluated and compared. The post-thaw viability drops significantly upon the occurrence of the eutectic crystallization regardless of suspending media, which suggests direct cell injury associated with eutectic crystallization. Based on these observations, two possible injury mechanisms are anticipated: (i) mechanical damage to the cell membrane due to eutectic crystallization, and (ii) intracellular eutectic formation (IEF). The proposed mechanisms provide a more comprehensive physical explanation of freezing induced cell injury and extend the understanding on solution effects.
快速冷却速率下的细胞内冰形成（IIF）以及慢速冷却速率下的溶液效应。尽管一般认为IIF是快速冷却速率下的主要损伤机制，但溶液效应导致的损伤尚未完全理解，并且已经提出了几种损伤机制。溶液效应通常被认为是冷冻过程中细胞内和细胞外空间电解质浓度升高的结果。除了这种电解质浓度升高导致的损伤外，还研究了与共晶结晶相关的冷冻损伤。为了研究与共晶结晶相关的损伤，设计并进行了两个不同的实验。在第一个实验中，两组AT-1大鼠前列腺肿瘤细胞悬液以相同方式在低温显微镜下冷冻和解冻，只是其中一组引发了共晶结晶。在第二个实验中，将AT-1细胞悬浮在几种具有不同共晶结晶温度的不同培养基中，并在定向凝固阶段进行单次冷却-升温循环，该循环具有不同的最终温度。两个实验后，评估并比较解冻后的活力。无论悬浮培养基如何，共晶结晶发生后解冻后的活力都会显著下降，这表明与共晶结晶相关的直接细胞损伤。基于这些观察结果，预计有两种可能的损伤机制：（i）共晶结晶对细胞膜的机械损伤，以及（ii）细胞内共晶形成（IEF）。所提出的机制为冷冻诱导的细胞损伤提供了更全面的物理解释，并扩展了对溶液效应的理解。
