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

作为热参数函数的细胞冻伤机制的定量研究

基本信息

批准号：
15360115
负责人：
TAKAMATSU Hiroshi
金额：
$ 9.79万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15360115/
关键词：
Slow freezing Cell Freezing injury Osmotic stress Mechanical stress Ice formation Cell membrane Cryopreservation 凍結細胞骨格細胞膜透過率

项目摘要

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

细胞在慢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引起的机械损伤，溶液效应和冰的机械应力对低温损伤的贡献尚不清楚。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。一个是简单冻结实验，另一个是伪冻结实验（非冻结实验）。拟冷冻实验是将细胞暴露在低温的浓缩环境中，类似于冷冻过程，但不形成冰。伪冻结实验后的细胞存活率是溶液效应的结果，而与原冻结实验的差异明显是冰存在的结果，表明冰的机械应力造成的损伤。为了深入了解溶液效应的机理，还进行了恒温条件下仅变化浓度的非冻结实验。结果表明，当细胞被冷冻到较高的冷冻温度时，细胞的破坏主要是由于冰的形成。而在较低的冷冻温度下，细胞存活率明显低于较高温度下，主要原因是NaCl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显著，并且被认为是在缓慢冷冻到细胞悬浮液中产生足够冰的温度时细胞损伤的主要原因。结果表明，在恒定温度下，NaCl浓度升高和降低时均发生渗透胁迫损伤，且以后者为主。高渗损伤和后高渗损伤对暴露时间的依赖性不同。因此，我们假设溶液效应的主要原因是由于NaCl浓度升高至等渗状态导致细胞膜在高渗溶液中收缩时发生改变，从而在体积膨胀时受到损伤。少