INTRODUCTION AND OBJECTIVES: For the surgical treatment of long and complicated urethral strictures, bioartificial matrixstabilized urothelium offers an innovative therapeutic option for urethral reconstruction. Collagen matrices increase the stability of cell-based implants and promote cell viability and proliferation of urothelial cells. In this study a Collagen Cell Carrier (CCC) seeded with autologous urothelial cells was implanted in minipigs after induction of a urethral stricture. METHODS: Six minipigs underwent surgical procedures for urethral stricture induction by circular thermocoagulation and vesicostomy to guarantee urinary diversion together with bladder tissue harvest to isolate autologous porcine urothelial cells(PUC). PUC were expanded, labelled by PKH26, and seeded onto the CCC in high density. Stratification was induced in confluent PUC cultures. Constructs were applied after seven days. Three weeks after stricture induction, minipigs were treated with autologous urothelium-matrixtransplants for urethroplasty at stricture side. After one, two, and four weeks, animals were sacrificed and urethral tissues were histologically examined for integration and survival of the seeded CCC. In vivo phenotype of multilayered urothelium-matrix-constructs were characterized via immunofluorescence staining of AE1/AE3, CK20, p63, Ecadherin, and ZO-1. RESULTS: All animals tolerated the required interventions and recovered quickly. The seeded CCC displayed excellent stability characteristics and suturability when manipulated and applied in the minipigs. Transplanted cells could be detected via positive PKH26 fluorescence in all minipigs, although cells were partly shifted from the CCC. Urothelium-matrix transplants integrated very well into the host tissue without any sign of visible inflammation. Throughout the complete experimental period, none of the animals showed symptoms of rejection or impairments. Blood counts did not reveal major aberrations. Immunofluorescence analysis confirmed epithelial phenotype, junction formation and ongoing differentiation two weeks post-op. CONCLUSIONS: Urethroplasty using autologous urothelial cells seeded on CCC was feasible in minipigs after induction of urethral stricture. This study demonstrates excellent suitability of the cellseeded CCC for urologic reconstruction purposes and recommends its use in clinical application.