Induction of Tolerance to Freeze-Thaw (FT) Damage in Mammalian Cells by Pre-FT Hypothermia Treatment

Abstract Attached asynchronous exponential phase V79 Chinese hamster cells were pretreated by hypothermic cycling in Hepes growth medium by Method 3 (48 h at 25°C + trypsin) or Method 4 (48 h at 25°C + 3 h at 37°C + trypsin) prior to a freeze-thaw (FT) cycle in Hepes growth medium. Pretreatment by Method 3 or 4 increased the FT survival by a factor of 3.4. This implies that the 3 h at 37°C, after the 25°C exposure, is not necessary to confer resistance to the subsequent FT cycle in the case of V79 cells. However, with RIF-1 mouse cells, the 3 h at 37°C confers increased resistance. The increase in FT survival of V79 cells after the above pretreatments cannot be accounted for by changes in cell cycle age distribution. No heat shock proteins are produced by this pretreatment. Since pretreatment by Method 3 or 4 also makes the cells resistant to hyperthermia, three other pretreatments, making the cells thermotolerant, were tried. None of these pretreatments resulted in a change in FT survival of the cells. Interaction analysis of FT data, when pretreatment by Method 4 is combined with the presence of DMSO during the FT cycle, indicates that the pretreatment and DMSO act synergistically whether exponential or stationary phase cells are used. Furthermore, the pretreatments and l -glutamine also act synergistically. These pretreatments also increase the FT survival of the RIF-I mouse cell line; again, pretreatment and DMSO act synergistically. Hence, the method is not limited to cells of hibernating mammals.