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Background: Extrahepatic bile duct injuries remain severe complications during cholecystectomies and often require reconstruction by bilioenteric anastomosis (i.e., hepaticojejunostomy), which comes with further long-term complications (e.g., recurring ascending cholangitis, secondary biliary cirrhosis). In the case of inherent extrahepatic biliary atresia or during liver transplant, artificial or engineered bile ducts could allow novel surgical strategies without the need for hepaticojejunostomy. Methods: We present data on the implantation of in vitro-generated neo-bile ducts in 5 domestic pigs. The neo-bile ducts were engineered through decellularization of allogeneic blood vessels and recellularization with autologous cholangiocytes. On postoperative days 0, 1, 7, and 14, blood samples were taken and analyzed (aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, creatinine, and leukocytes). Magnetic resonance cholangiopancreatography was performed on postoperative day 14 on 1 pig. Fourteen days after implantation, the pigs were sacrificed and the bile ducts were explanted. Results: All pigs survived the complete study period without severe complications. None of the pigs showed signs of biliary leakage or peritonitis. The neo-bile ducts were infiltrated by neutrophils, and neoangiogenesis was observed around and into the implanted tissue. Conclusion: We present a novel strategy for extrahepatic bile duct replacement by implantation of an autologous neo-bile duct generated ex vivo. Whether the presented technique allows the long-term replacement of native bile ducts must be further evaluated. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]