Cells of a urinary bladder tumor (transitional cell carcinoma) induced in a Wistar strain rat with N-butyl-N-(4-hydroxybutyl)nitrosamine have been maintained in tissue culture for 71 generations (about 1 ½ years) in Eagle's minimal essential medium with 20% calf serum. Under a phase-contrast microscope, this cell line, Nara Bladder Tumor II, appeared in culture as mosaic sheets of epithelium. Reinoculation of the cultured cells into Wistar rats resulted in development of exactly the same type of tumor as the original, and the cells kept the morphologic characteristics of the original urinary bladder carcinoma, sometimes developing a squamous cell carcinoma with pearls. On subcutaneous inoculation, the tumor at the site of inoculation showed cystic changes, whereas on intraperitoneal inoculation, disseminated tumors occurred in the peritoneal cavity with hemorrhagic ascites. There was a correlation between the number of cells inoculated and the survival time of the recipients. The chromosome number of the cultured cells was nearly tetraploid. The in vitro growth rates of these cells in the 7th, 27th, and 71st generations were essentially the same.
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Toyoshima et al. (1971) studied this question.