QUANTITATIVE EXAMINATION OF THE MECHANISM OF FREEZING INJURIES OF CELLS AS A FUNCTION OF THERMAL PARAMETERS

缓慢冷冻过程中细胞的损伤主要归因于溶液效应，即浓电解质溶液的效应。然而，由于一些研究已经表明由生长的冰晶引起的机械损伤，因此溶液效应和来自冰的机械应力对冷冻损伤的贡献仍然不清楚。本研究的目的是评估这两种机制对冷冻损伤的影响，定量作为冷冻条件的函数。为此，进行了两种实验。一个是简单的冻结实验，另一个是伪冻结实验（非冻结实验）。伪冷冻实验旨在将细胞暴露于低温下的浓缩环境中，这与冷冻过程中的环境相似，但没有任何冰形成。假冷冻实验后细胞存活率表明了溶液效应的结果，并与冷冻实验的结果相比较， ...更多信息 冻结实验是冰存在的结果，表明冰的机械应力引起的破坏。在恒温条件下进行了仅改变浓度的不冻实验，结果表明，当细胞冻存到较高的冻存温度时，细胞主要由于结冰而被破坏。而在低温冷冻条件下，细胞存活率明显低于高温冷冻条件，主要原因是NaCl浓度的增加。因此，我们的结果清楚地表明，在较低的温度下，溶液效应变得显着，它被认为是在缓慢冷冻的温度，产生足够的冰在细胞悬浮液中的细胞损伤的主要原因。它也被澄清，由于在恒定温度下的渗透胁迫的伤害发生在增加和减少的NaCl浓度，后者是占主导地位。两种类型的损伤，即高渗和高渗后损伤，表现出不同的依赖性的暴露时间。因此，假设溶液效应的主要原因是由于在高渗溶液中收缩期间细胞膜的改变，从而在NaCl浓度增加至等渗条件引起的体积膨胀期间受到损伤。少

The damage of cells during slow freezing has been ascribed mainly to the solution effect that indicates the effect of concentrated electrolyte solutions. However, since several studies have suggested mechanical injuries caused by growing ice crystals, contribution of the solution effect and the mechanical stress from the ice to cryoinjury is still unclear. The objective of the present study is to assess the effect of these two mechanisms on cryoinjury quantitatively as a function of freezing conditions. To this end, two kinds of experiments were carried out. One was a simple freezing experiment and the other was a pseudo-freezing experiment (non-freezing experiment). The pseudo-freezing experiment was designed to expose cells to the concentrated environment at low temperatures, which is similar to that during freezing, but without any ice formation. The cell survival after the pseudo-freezing experiment indicates the result of the solution effect, and the difference from that of the fr … More eezing experiment is explicitly the result of the presence of ice, indicating the damage caused by the mechanical stress from the ice. The non-freezing experiment with only concentration change at constant temperatures was also performed to obtain an insight into the mechanism of the solution effect.The result showed that, when cells were frozen to a relatively higher freezing temperature, cells were destroyed mostly due to the ice formation. In contrast, in the case of lower freezing temperature, the cell survival was much lower than that of higher temperature mainly due to the increased concentration of NaCl. Therefore, our results clearly indicated that the solution effect becomes significant at lower temperatures and it is considered as the main cause of cell injuries during slow freezing to temperatures that produce enough amount of ice in cell suspensions.It was also clarified that injuries due to osmotic stress at a constant temperature occurred both during increase and decrease in the NaCl concentration, and the latter was predominant. Two types of injuries, i.e. the hypertonic and post-hypertonic injuries, showed different dependency on the exposure time. Therefore, it was hypothesized that the major cause of the solution effect was due to the alteration of the cell membrane during contraction in the hypertonic solution and thus injured during volume expansion caused by the increase in the NaCl concentration to the isotonic condition. Less