A parametric study of freezing injury in BPH1^CAFTD-2 human prostate tumor cells

Freeze injury in BPH1^CAFTD-2 cells frozen/thawed in suspension was studied through a two-level four-parameter (2⁴) experimental design and analysis. The four parameters considered were end temperature, hold time, TNFα concentration, and thawing rate. Thermal parameter values chosen were based on the approximate thermal history cells would experience in the peripheral region of a cryosurgical iceball. Cell suspensions were frozen at a constant 10°C/min on a directional solidification stage and viability was assessed within 1 hr post-thaw using a dye exclusion assay. The parameters affecting cell survival were determined through calculation of the individual parameter effects (E) and interactions (I) according to factorial design guidelines; data set curvature (C) was also determined. Cell viability ranged from a maximum of 87.55% to a minimum of 17.59% indicating trends in cell survival were sensitive to the parameters chosen. Survival was affected by the following parameters in order: lowering the end temperature, increasing the hold time, adding TNFα, and reducing the thawing rate. In addition, all 2-way parameter interactions except TNFα-hold time were statistically significant. Curvature analysis showed that cell viability at the midpoint of the data was nearly 20% lower than predicted based on linear interpolation. These results were verified and extended using analysis of variance (ANOVA). We conclude that cryoinjury in this tumor line can be influenced by multiple interacting thermal parameters, most importantly end-temperature and hold time, as well as the presence of the cytokine TNFα. Finally, although the cell type is tumorigenic results suggest the possibility of using freezing to control benign prostatic hyperplasia (BPH) in addition to cancer within the prostate.