Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at −10 °C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at −10 °C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at −20 °C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.