Urothelial cells can be harvested for cell expansion outside the body with a minimally invasive technique. The cells can be stored for later transplantation back to the patient. In this procedure the urinary bladder is catheterized and washed with sterile saline for isolation of cells. The method can be used for adult and pediatric patients. In a clinical study, we used in vitro cultured autologous cells for urethral reconstruction in patients with severe hypospadias. Follow-up studies demonstrate good results. Our laboratory work includes quality controls of urothelial cells after long-term expansion in vitro and an optimized cell culture protocol. We also demonstrate that three different cell types can be co-cultured to achieve a complex tissue engineered transplant that mimics the wall of the urinary pathway.