Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: Potential applications for tissue engineering technology

Purpose: Small intestinal submucosa is a xenogenic, acellular, collagen ric h membrane with inherent growth factors that has previously been shown to p romote in vivo bladder regeneration. We evaluate in vitro use of small inte stinal submucosa to support the individual and combined growth of bladder u rothelial cells and smooth muscle cells for potential use in tissue enginee ring techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. Materials and Methods: Primary cultures of human bladder urothelial cells a nd smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 10(5) cells per cm.(2), incubated and harvest ed at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were u rothelial cells seeded alone on the mucosal surface of small intestinal sub mucosa, smooth muscle cells seeded alone on the mucosal surface, layered co culture of smooth muscle cells seeded on the mucosal surface followed by ur othelial cells 1 hour later, sandwich coculture of smooth muscle cells seed ed on the serosal surface followed by seeding of urothelial cells on the mu cosal surface 24 hours later, and mixed coculture of urothelial cells and s mooth muscle cells mixed and seeded together on the mucosal surface. Follow ing harvesting at the designated time points small intestinal submucosa cel l constructs were formalin fixed and processed for routine histology includ ing Masson trichrome staining. Specific cell growth characteristics were st udied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matri x penetrance. To aid in the identification of smooth muscle cells and uroth elial cells in the coculture groups, immunohistochemical analysis was perfo rmed with antibodies to cr-smooth muscle actin and cytokeratins AE1/AE3. Results: Progressive 3-dimensional growth of urothelial cells and smooth mu scle cells occurred in vitro on small intestinal submucosa. When seeded alo ne urothelial cells and smooth muscle cells grew in several layers with min imal to no matrix penetration. In contrast, layered, mixed and sandwich coc ulture methods demonstrated significant enhancement of smooth muscle cell p enetration of the membrane. The layered and sandwich coculture techniques r esulted in organized cell sorting, formation of a well-defined pseudostrati fied urothelium and multilayered smooth muscle cells with enhanced matrix p enetration. With the mixed coculture technique there was no evidence of cel l sorting although matrix penetrance by the smooth muscle cells was evident . Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differen tiation a-smooth muscle actin and cytokeratins AE1/AE3. Conclusions: Small intestinal submucosa supports the S-dimensional growth o f human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has import ant future clinical implications with respect to tissue engineering technol ogy. The results of our study demonstrate that there are important smooth m uscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small inte stinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various dis ease processes of the bladder.