PROJECT SUMMARY Intestinal failure results from conditions such as trauma-related small bowel injury, necrotizing enterocolitis, and inflammatory bowel disease. Treatment options are limited; however, an important emerging therapeutic approach is intestinal transplantation (IT). When successful, IT is associated with marked improvements in quality of life and overall well-being. However, its current success rate is <60% 5 years post-transplant, and there are multiple factors that limit its clinical use. The major impedance to more successful transplants is donor tissues' limited tolerance to current storage methods. The current standard, static cold storage (CS), has been shown to cause epithelial damage after 6 hours (hr) as well as inflammation and loss of epithelial tight junctions. Indeed, clinical studies show that intestinal allograft preservation of >6 hr of cold ischemic time correlates with a progressively higher rate of recipient post-reperfusion syndrome, translocation of enteric bacteria that cause systemic infections, induction of inflammation, and increased transplant failure. Thus, methods that could improve graft viability and organ preservation, especially reducing the incidence and severity of ischemia- reperfusion injury could have a major impact on enhancing the IT success rate, making it the preferred therapy for intestinal failure patients. Normothermic machine perfusion (NMP) has shown promise in clinical heart, lung, and liver transplant scenarios, but this potentially groundbreaking advancement has not been developed for intestinal failure. We have performed highly promising preliminary studies using NMP in porcine and human intestine and have shown compared to CS: 1) Successful allogeneic transplantation with fewer histologic signs of epithelial reperfusion injury, enhanced intestinal stem cell activation, and histologic evidence of active epithelial restitution and regeneration in 6 hr NMP-stored porcine small intestine. 2) Histologically intact epithelial barrier in 6 hr NMP-stored human donor small intestine. We hypothesize that intestinal NMP will reduce ischemia-reperfusion injury by enhancing epithelial barrier integrity and function and decreasing inflammation, thereby minimizing factors that prime the graft for failure. We will use an ex vivo pig intestinal tissue and a porcine model of IT to address the hypothesis with the following specific aims: Aim 1. To determine the extent to which NMP protects epithelial cell integrity and regenerative potential to enhance barrier function. Aim 2. To determine if NMP minimizes allograft inflammatory activation and the cellular mechanisms mediating pro- and anti- inflammatory processes during allograft storage. Aim 3. To determine if NMP enhances the suitability of injured intestine for transplantation. Successful completion of these aims will develop a mechanistic basis to enhance intestinal allograft function by mitigating preservation injury and will address the practica...