Quantitative cryomicroscopic analysis of intracellular freezing of granulocytes without cryoadditive

Purified human granulocytes were frozen in isotonic saline at different constant cooling rates down to -60 degrees C and subsequently thawed on the thermally defined cryostage of a cryomicroscope. Cells monitored on videotape were examined with respect to cooling rate threshold, type, and temperature of intracellular ice formation during cooling and recrystallization during warming. Two apparently different mechanisms of intracellular ice formation (iif) were distinguished during cooling, i.e., "twitching" (no visible ice front) and "darkening" (diffuse ice front). Both types of iif are related to cooling rate and hence also to dehydration. Cooling rate thresholds and temperatures of intracellular recrystallization were determined. It was found that twitching iif occurs just about 6.3 to 7.4 degrees C above the homogeneous nucleation temperature, suggesting that it might be catalyzed by nucleators present within the cells. Darkening iif, on the other hand, was observed at much higher temperatures, i.e., 23.4 to 28.3 degrees C above the homogeneous nucleation temperature, which could possibly indicate a nucleation induced by extracellular ice crystals (at a cooling rate of 30 degrees K/min, however, darkening iif was observed to occur at a temperature lower than that required for twitching iif). The proposed mechanisms of cryoinjury are related to membrane integrity measurements presented in M. W. Scheiwe, Ch. Körber, and S. Englich, Cryo-Letters, 5, 300-306, 1984.