A parametric study of freezing injury in ELT-3 uterine leiomyoma tumour cells

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 (2⁴) 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°C/min; ET, -20 to -30°C; HT, 0-5 min; TR 20-200°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°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°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.