Serial Cultivation of Normal Rat Bladder Epithelial Cells in Vitro

Abstract

Recent advances in culture techniques for human urothelial cells have led to the development of an improved method for growing primary rat bladder epithelial cells. We report here the conditions developed for large-scale in vitro growth and serial cultivation of normal diploid rat bladder epithelial cells. Primary cultures were initiated by attachment of bladder mucosal explants to type I collagen gels. A rapid outgrowth of epithelial cells from the explants occurred when cultured in a hormone-supplemented medium with epidermal growth factor. These primary outgrowths were passaged by nonenzymatic dispersion with 0.1 per cent ethylenediaminetetracetic acid and replating onto new gels. The capacity for routine serial passaging and maintenance of rat bladder epithelial cells required the presence of epidermal growth factor, a requirement not observed with human urothelial cells. The characteristics of the cultured rat bladder epithelial cells were similar to human urothelial cells in: ultrastructural and phase-contrast morphologic properties, showing junctional complexes, desmosomes, stratification and an apical glycocalyx; the absence of stromal cell contamination; and the ability to be serially passaged. Spontaneous cell-line formation was observed with the rat bladder epithelial cells, but has not been found with the human urothelial cells. With the method that we have developed, the number of rat bladder epithelial cells generated from a single bladder of a 4 to 6 week old rat was increased 100-fold from about 7 X 10(5) cells to 7 X 10(7) viable cells within 3 weeks of culture. The capability of culturing normal, primary rat bladder epithelial cells on this scale has not been reported previously and will facilitate comparative studies of the biological and molecular characteristics of the mammalian urothelium. Furthermore, this culture system will be useful for carcinogenesis studies, including metabolic activation of carcinogens and cellular transformation in vitro.