Production of Tissue-Engineered Small Intestine in Rats with Different Ages of Cell Donors

Objective: The objective of this study was to compare the impact of different ages of cell donors on the production of tissue-engineered small intestine (TESI). Materials and Methods: Four different ages of Lewis rats were chosen as donors of intestinal organoids: E18 fetuses, 5-day-old newborns, 21-day-old weanlings, and 6-week-old adults. The harvested intestine was exposed to enzymatic digestion to release intestinal stem cell (ISC)-containing organoids. Organoids were purified, concentrated, and seeded onto tubular polyglycolic acid scaffolds. Seeded scaffolds were attached to the anterior surface of the abdominal wall in recipient nude rats. After 4 weeks of in vivo incubation, specimens from each donor age were explanted for evaluation. Results: TESI produced from E18 fetal organoids demonstrated the largest quantity of neomucosa as confirmed by gross observation and volume measurement, and intermediate quality of the neomucosa as determined by periodic acid-Schiff staining. TESI produced from newborn organoids was of intermediate quantity, but had the best quality of neomucosa. TESI produced from weanling organoids had lower quantity and poorer quality neomocosa, with TESI produced from adult organoids having the worst results. Immunofluorescence staining confirmed the presence of all epithelial cell lineages in TESI produced from all donor ages, with the highest density of proliferating Ki67-positive cells seen in TESI produced from the youngest cell donors. Gene expression for Lgr5 (indicative of actively dividing ISCs) and for Bmi1 and Hopx (indicative of quiescent stem cells) was higher in younger donor intestines. Conclusions: Among the four ages of cell donors, using 5-day-old newborn rats as cell donors produced the best quality of TESI, with a single patent lumen. TESI produced from other cell donors revealed multiple neomucosal cysts mixed with varying degrees of fibrous tissue, which may be not appropriate for future clinical application.