| Budget Amount *help |
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2004: ¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 2003: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Research Abstract |
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
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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
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