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Objective: The objective of this study was to compare the impact of different in vivo incubation sites on the production of tissue-engineered small intestine (TESI)., Materials and Methods: Green fluorescent protein transgenic rat pups (3-5 days) were used as donors of intestinal organoids. Harvested intestine was exposed to enzymatic digestion to release intestinal stem cell-containing organoids. Organoids were purified, concentrated, and seeded onto tubular polyglycolic acid scaffolds. Seeded scaffolds were implanted in each of five locations in recipient female nude rats: wrapped with omentum, wrapped with intestinal mesentery, wrapped with uterine horn membrane, attached to the abdominal wall, and inserted into the subcutaneous space. After 4 weeks of in vivo incubation, specimens from each site were explanted for evaluation., Results: Wrapping seeded scaffolds with vascularized membranes produced TESI with variable lengths of vascularized pedicles, with the longest pedicle length from uterine horn membrane, the shortest pedicle length from intestinal mesentery, and intermediate length from omentum. The quantity of TESI, as expressed by volume and neomucosal length, was identical in TESI produced by wrapping with any of the three membranes. The smallest quantity of TESI was found in TESI produced from insertion into the subcutaneous space, with an intermediate quantity of TESI produced from attachment to the abdominal wall. Periodic acid-Schiff and immunofluorescence (IF) staining confirmed the presence of all intestinal epithelial cell lineages in TESI produced at all incubation sites. Additional IF staining demonstrated the presence of enteric nervous system components and blood vessels. Wrapping of seeded scaffolds with vascularized membranes significantly increased the density of blood vessels in the TESI produced., Conclusion: Wrapping of seeded scaffolds in vascularized membranes produced the largest quantity and highest quality of TESI. Attaching seeded scaffolds to the abdominal wall produced an intermediate quantity of TESI, but the quality was still comparable to TESI produced in vascularized membranes. Insertion of seeded scaffolds into the subcutaneous space produced the smallest quantity and lowest quality of TESI. In summary, wrapping seeded scaffolds with vascularized membranes is favorable for the production of TESI, and wrapping with omentum may produce TESI that is most easily anastomosed with host intestine.