An assessment of tumor cell viability after in vitro freezing

The identification of the minimum lethal temperature for tumor cells in vivo is difficult because of the secondary factors that are associated with the cryoinjury. This study attempts to identify this temperature by a combination of in vitro and in vivo techniques. Suspensions of Walker carcinoma cells were frozen at a rate of 1 degree C/min without cryoprotection, to either -10, -15, -20, -25, -30, -35 or -40 degrees C and held at that temperature for either 0, 10, 20, or 30 min. After spontaneous rewarming viability was assessed by a combination of vital dye studies and the growth of tumor cells inoculated into the liver and subcutaneous tissue of male, Sprague-Dawley rats. Trypan blue studies indicated that less than 1% of the cells frozen to -35 degrees C were considered viable, yet significant tumor take rates were noted, suggesting that for some cells the cryoinjury is reversible. As expected tumor take rates were reduced by lowering the temperature but were independent of the holding time. The volume doubling time and final tumor volume of the subcutaneous tumors was similar to that of controls, indicating that the growth potential of the cells which survive freezing is normal. The minimum lethal temperature was dependent upon the site of inoculation, subcutaneous tumors developing from cells frozen to -35 degrees C, whereas liver tumors did not develop from cells frozen beyond -25 degrees C, this may have important clinical implications.