Preservation of Ehrlich ascites tumor cells with dimethyl sulfoxide.

For a number of years our investigations of Ehrlich ascites tumor (EAT) in Mus musculus as a model for tumor immunity studies have yielded variable LD50 values. To explain these results, the hypothesis that the cell challenge is modified during transfer of the stock cells from animal to animal is being tested. Our first attempts at freezing a stock cell suspension of EAT in the ascitic fluid or in glycerol (F. Miya et al., Am. J. Clin. Pathol. 33:231, 1960) resulted in cells without malignant potential after thawing. The results of experiments with dimethyl sulfoxide (DMSO) are presented here. Ascitic fluid containing tumor cells was removed from donor mice and pooled. The supernatant fluid was saved and two volumes were added to three volumes of Hank's balanced salt solution (BSS). DMSO (70%, Merck, Sharp and Dohme) was added to this solution to final concentrations of 2.8 and 5.6%. The cells were counted in a standard bloodcounting chamber. At least 400 cells were counted to estimate the population, which was then adjusted to 107 per milliliter with Hank's BSS; 10-ml portions of cell suspension were distributed to test tubes and centrifuged. The cell residues were resuspended in 10 ml of the DMSO-ascitic fluidHank's solution. The cell suspensions were allowed to freeze in glass test tubes (16 by 150 mm) with screw caps by placing the tubes into a Revco TABLE 1. Mortality ratios of animals challenged with dimethyl sulfoxide-treated Ehrlich ascites tumor cells