Osmotic Injury of Cells by the Solution Effect

点击翻译按钮获取中文摘要

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.