OBJECTIVE Augmentation of the urinary bladder using a tissue-engineered approach with autologous cells is a very promising technique. To prevent risks of necrosis after transplantation, the graft vascularization process could be markedly enhanced by incorporation of autologous endothelial cells in the tissue-engineered organ. The purpose of this study was to develop a separation technique to extract four bladder cell types from the same biopsy, and to prepare an endothelialized reconstructed bladder. METHODS Fibroblasts, smooth muscle cells (SMC), urothelial cells (UC) and endothelial cells (EC) were extracted from a small porcine bladder biopsy. The SMC, fibroblasts and EC were seeded on the top of the sponge and cultured for 10 days. Then, the UC were seeded on top of these cells for 15 additional days to produce a three-dimensional bladder wall. RESULTS The UC and EC extracts from a single porcine biopsy were 97.2+/-0.6% keratin 8/18-positive and 97.7+/-0.3% PECAM-1-positive pure cells, respectively, as assessed by flow cytometry. The SMC could not be dissociated from fibroblasts, and were present as 37+/-0.5% desmin-positive cells. UC differentiated into a urothelium characterized by umbrella cells and a laminin-positive basal membrane. The EC reorganized in the matrix to form PECAM-1-positive capillary-like tubes. CONCLUSIONS This new model of tissue-engineered bladder has the main advantages of being at least 2mm thick, autologous, and able to promote the formation of capillary-like tubes. It could be a promising alternative to the use of gastrointestinal segments to improve bladder capacity.