Tissue engineering (TE) is a new therapeutic approach to cope with the lack of donor organs or tissues. It aims to create organ substitutes to improve the function of the recipient’s organ or to replace it. The three basic principles of TE comprise the use of: 1) isolated cells or cell substitutes, 2) signal molecules that promote cell proliferation such as growth factors and 3) matrices i.e. natural or synthetic scaffolds. Using biomaterials as scaffolds, autologous cell-based transplants can be stabilised and consequently, a solid artificial organ can be constructed in vitro. This investigation was supposed to verify the suitability of a cell seeded collagen based scaffold (collagen cell carrier – CCC) to repair urethral lesions especially urethral strictures as an innovative therapeutic concept. Therefore, viability and proliferation of human and porcine urothelial cells as well as their adherence on the new cell carrier were examined. In vivo biocompatibility was tested by ectopic transplantation in nude rats and finally, cell seeded collagen matrices were applied in minipigs’ urethras after induction of a urethral stricture. In vitro as well as in vivo investigations proved the excellent suitability of CCC as a cell carrier to create artificial autologous urothelial transplants. Metabolic activity and proliferation of urothelial cells as well as their adherence on CCC were comparable to plastic seeding when high numbers of cells were used. The nude rat model and the minipig model proved the biocompatibility, integration, and degradation of the cell matrix constructs in vivo. Hence the results of this study are of greatest value for future therapeutic options for urethral strictures and lay the foundations for potential clinical application.