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

细胞在缓慢冷冻过程中的损伤主要归因于溶液效应，即浓电解质溶液的作用。然而，由于一些研究表明，冰晶生长会造成机械损伤，因此溶液效应和冰层产生的机械应力对低温损伤的作用尚不清楚。本研究的目的是定量评估这两种机制对冷冻损伤的影响，作为冷冻条件的函数。为此，进行了两种实验。一种是简单冻结实验，另一种是假冻结实验(非冻结实验)。拟冷冻实验旨在将细胞暴露在低温的浓缩环境中，这类似于冷冻过程中的情况，但不会形成任何冰。假冷冻实验后的细胞存活率表明了溶液效应的结果，并与fr…的结果不同。更多的实验是明确的冰的存在的结果，表明了来自冰的机械应力造成的破坏。此外，还进行了恒温下只改变浓度的非冷冻实验，以探讨溶液效应的机理。结果表明，当细胞冷冻到相对较高的冷冻温度时，细胞主要是由于结冰而被破坏。相反，在较低的冷冻温度下，细胞存活率远低于较高的温度，这主要是由于氯化钠浓度的增加。因此，我们的结果清楚地表明，溶液效应在较低的温度下变得明显，这被认为是在缓慢冷冻到细胞悬浮液中产生足够多的冰的温度时细胞损伤的主要原因，并阐明了恒温渗透胁迫对细胞的伤害既发生在盐浓度的增加期间，也发生在盐浓度的降低期间，而后者是主要的。高渗性损伤和高渗后性损伤对暴露时间的依赖程度不同。因此，推测溶液效应的主要原因是在高渗溶液中收缩时细胞膜发生变化，从而在体积膨胀过程中受到损伤，这是由于氯化钠浓度增加到等渗状态。较少

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