Organoid structure of normal rat bladder in unilaminar and bilaminar histophysiologic gradient culture : methods and observations

Abstract

In lining epithelium of mammals certain recurrent architectural patterns are recognized that may be critical for epithelial organization in culture. Among these structural imperatives are three dimensional growth, restricted migration of cells, histophysiologic gradients, and continuity of epithelial membranes. Histophysiologic gradient culture procedures have been developed to comply with requirements suggested by normal tissue architecture. In a small chamber, 5 mm diam, epithelium grows attached to a thin permeable transparent collagen membrane or sandwiched between two apposed collagen membranes. The chamber is held in a plastic capsule so that metabolic exchange is limited to substances that diffuse across the collagen membranes to the adherent basal layer of epithelium. On a single membrane after 2 wk of growth, normal urothelium appears as a diffusely hypercellular urothelium, 6 to 10 cells thick. As the culture period is extended by 2 or more wk, multiple nodules of urothelium extend from the basal surface into the subepithelial space between the epithelium and the collagen membrane. Normal bladder, cultured between two apposed collagen membranes, gives rise in a few days to confluent epithelium that contains many extracellular cysts. Through an apparent merging of cysts, after 2 wk the urothelium appears as a highly organoid structure, a flattened cyst lined by completely stratified polarized urothelium. Such microbladders consist of a stratified epithelium without interruption of continuity. With histophysiologic gradient culture, processes in carcinoma and precursor lesions are accessible to study at the level of tissue organization.