Cellular level freeze injury was investigated after controlled freezing of an Eker rat uterine fibroid cell line in both the presence and absence of oestradiol. The connection between thermal history and cell injury in single ELT-3 cells in suspension (without oestradiol) was studied through a two-level, four-parameter (2(4)) experiment with membrane dye exclusion as the end-point. The four parameters considered were cooling rate (CR), end temperature (ET), hold time (HT) and thawing rate (TR). A high and low value of each parameter was selected as follows: CR, 5-25 degrees C/min; ET, -20 to -30 degrees C; HT, 0-5 min; TR 20-200 degrees C/min. The greatest parameter effect on freeze injury in this range was ET followed by HT, then TR and finally CR. In addition, significant parameter interactions and curvature were found. Additional CR results outside the original parameter range showed a reduction in survival at both 1 and 50 degrees C/min suggestive of an inverted U-shaped survival curve. These results show that this tumour system is susceptible to cryoinjury, particularly at temperatures below -30 degrees C with HT of >5 min and slow thawing. In addition, the presence of oestradiol was found to increase the susceptibility of these cells to cryoinjury.

The connection between thermal history and cell injury in single AT-1 cells is studied systematically through a two-level, four-parameter (24) experiment. The four parameters considered are cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Cryosurgically relevant high and low values of each parameter are chosen (CR, 5 to 50°C/min; ET, −20 to −80°C; HT, 0 to 15 min; TR, 20 to 200°C/min) to maximize applicability of the results to cryosurgery; it is important to note that any conclusions drawn from the results are valid only for the range of parameter values studied. AT-1 cell suspensions are frozen in a controlled way on a directional solidification stage, and viability is assessed postthaw with a live/dead assay using the fluorescent dyes calcein-AM and propidium iodide to indicate live and dead cells, respectively. The parameters which most significantly affect short-term survival outcome are determined through calculation of the individual parameter effect values (E) according to the factorial experimental design guidelines. In addition, any synergy between two parameters in determining short-term survival outcome is revealed by calculation of the interaction value for those parameters (I). The results suggest that survival is most significantly affected by variation in end temperature and hold time, and the only significant parameter interaction found is between these two parameters. The analysis further suggests that survival depends nonlinearly on the thermal parameters, based on calculation of the survival curvature (C) in the parameter ranges studied. These results are discussed within the context of previously proposed mechanisms of cellular injury during freezing. Although coupling between several mechanisms is possible, single mechanisms which may explain the survival results include slow-cooling injury mechanisms such as solute effects injury, dehydration-induced membrane instabilities, and volume-catalyzed nucleation of intracellular ice.