Osmotic Injury of Cells by the Solution Effect

为了对冷冻过程中由于高渗溶液造成的细胞损伤有一个基本的了解，我们开发了一种新的灌注技术，将暴露于高渗溶液的影响与冰晶相关的其他影响分离开来。该技术采用两个速度可变的注射泵，在新的灌注室中给细胞周围的溶液张力任意改变。实验用人前列腺腺癌细胞系PC-3进行。首先，将细胞突然暴露于高渗HEPES缓冲NaCl溶液中3 - 40分钟，然后恢复等渗状态。随着暴露时间的延长，高渗后活力逐渐降低。而在3.8 M NaCl溶液中，仅暴露3分钟后，存活率就下降到50%，并随着暴露时间的增加而进一步下降。其次，对细胞施加三种强直性变化模式：NaCl浓度随时间线性增加后突然降低，NaCl浓度突然增加后线性降低，NaCl浓度线性增加后下降。在每个实验中，细胞在最高浓度（2.0或3.8 M）下保持12分钟。在1.0 M/min速率下，突变与线性变化的存活率无统计学差异。在0.1 M/min时，NaCl浓度线性升高后的细胞存活率高于突然升高后的细胞存活率。然而，活力与浓度的降低方式无关。结果显示时间依赖性存活表明细胞可能部分受到化学破坏。另一方面，细胞损伤不取决于浓度的减少方式，而取决于浓度在较低速率下的增加方式，这一事实是有趣和重要的。这可能表明细胞在渗透收缩过程中受到了机械损伤。

To develop a fundamental understanding of cell damage due to the hypertonic solution during freezing, we have developed a new perfusion technique that isolates the effect of exposure to hypertonic solutions from other effects associated with ice crystals. This technique employs two speed-variable syringe pumps to give arbitrary change in the solution tonicity around the cells in a new perfusion chamber.Experiments were performed with a human prostatic adenocarcinoma cell line PC-3. First, the cells were exposed suddenly to a hypertonic HEPES buffered NaCl solution for variety of time between 3 and 40 minutes, and then returned to the isotonic condition. The post-hypertonic viability gradually decreased with increasing the time of exposure in 2. OM NaCl solution, while at 3.8 M viability decreased to 50 % after exposure of only 3 minutes and further decreased with increasing the time of exposure. Next, three patterns of change in tonicity were applied to the cells : the NaCl concentration was linearly increased with time and suddenly decreased, suddenly increased and linearly decreased, and linearly increased and decreased. In each experiment the cells are kept in the highest concentration, 2.0 or 3.8 M, for 12 minutes. No statistical difference was found between the viability obtained with sudden changes and linear changes with the rate of 1.0 M/min. At 0.1 M/min, however, more cells survived after the linear increase of NaCl concentration compared with the sudden increase. The viability was however independent of the decrease manner in concentration. The result that showed time-dependent survival indicates that the cell might be damaged chemically in part. On the other hand, the fact that the cell damage depends not on the decrease manner but the increase manner in concentration at lower rate is interesting and important. It may suggest that the cell is injured mechanically during osmotic shrinking.