O2168: Transplantation of Intestinal Epithelial Organoids for Regeneration of Gut Injury

Background: Intestinal epithelial organoid (IEO) transplantation has been shown to repopulate and regenerate the injured intestine in experimental inflammatory bowel disease. Further, a clinical investigation on IEO transplantation in adults with inflammatory bowel disease is currently ongoing. However, IEO transplantation has not been investigated in small bowel diseases such as necrotizing enterocolitis. The aim of this study is to evaluate the regenerative capability of transplanted IEOs in a novel ex vivo perfusion system (EVPS) (Fig. A - arrow) that simulates small intestinal epithelial injury. The EVPS allows for the assessment and observation of tissue explants in a controlled environment.

Methods: Distal ileum from C57BL/6 mice (AUP 58119), from ages postnatal day (P) 19-21, were resected (2-3 cm) and studied. Four groups were evaluated: (i) control; (ii) control maintained in the EVPS for 6h; (iii) injured intestine maintained in the EVPS for 6h; (iv) injured intestine with transplanted IEOs maintained in the EVPS for 6h. Intestinal injury was induced via mechanical mucosal scratching. IEOs (Fig. B) were derived from P9 red fluorescent protein expressing mice and dissociated into fragmented crypts before transplantation. IEOs were transplanted intraluminally into the intestine in the EVPS, mimicking enema. Tissue viability and IEO engraftment were compared between groups by histology, immunofluorescence, and two-photon laser scanning microscopy.

Results: Peristaltic movements were observed after 6h in the EVPS in all groups, including the injured intestine, confirming that the injury was confined to the mucosa. Intestinal tissue viability was maintained in the EVPS as seen by preserved morphology (Fig. C), maintained barrier function (E-Cadherin - red) (Fig. D) and low cell death (Cleaved-caspase 3; CC3 - red) (Fig. E) in the intestinal crypts and villi. Intestinal epithelial injury was also maintained in the EVPS (Fig. F - arrow). Importantly, the IEOs transplanted (red) successfully engrafted onto the injured intestinal epithelium as indicated by two-photon laser scanning microscopy (Fig. G - arrow), and immunofluorescence (Fig. H - arrow).

Conclusion: This study demonstrates the successful transplantation and engraftment of IEOs in small bowel injury. Through preservation of intestinal viability, our study indicates that EVPS can be a viable model for evaluation of IEO regenerative capacity in the ex vivo environment. This study provides the scientific basis for the use of the EVPS as an important and effective regenerative medicine model for neonatal small bowel diseases.