H. Liebe1, C. Schlegel1, X. Cai1, A. Golubkova1, T. Leiva1, C. J. Hunter1 1University Of Oklahoma College Of Medicine, Pediatric Surgery, Oklahoma City, OK, USA
Introduction: Necrotizing enterocolitis (NEC) is a severe disease that predominantly affects premature neonates. We have previously shown that three dimensional organoids harvested from neonatal intestinal tissue are an innovative model for NEC. However, standard enteroid models have a basolateral-out configuration (BO). This does not provide easy access to the “luminal” surface. Access to the lumen (apical surface) is necessary to allow physiologically comparable evaluation of the effect of bacteria and endotoxins on the intestine. We hypothesize that an apical surface-out (AO) enteroid will provide a more biologically relevant model of NEC than BO enteroids.
Methods: Following institutional review board approval (#11610-11611), discarded intestinal tissue was collected from premature human neonates undergoing surgical resection. Intestinal stem cells were collected via crypt isolation to grow small organoids known as enteroids. Enteroids were grown to maturity in the BO conformation embedded in basement membrane matrix. After reaching maturity, enteroids were re-suspended in media alone for 72 hours prior to experimentation to induce an AO conformation. This was confirmed via immunofluorescent staining for the apical protein zonula occludens-1 and basolateral protein beta-catenin (figure 1). Enteroids (AO and BO) were then either untreated (control) or treated for 24 hours with 100ug/mL lipopolysaccharide (LPS) +/- 6-hour hypoxic conditions. The inflammatory cytokine TNF-α and Toll-like receptor 4 (TLR-4) were analyzed via enzyme-linked immunosorbent assay (ELISA). Gene expression for TNF-α and the tight junction protein occludin were analyzed via qPCR.
Results: AO conformation was confirmed via immunofluorescence of the apical protein zonula occludens-1 and basolateral protein beta-catenin. AO and BO enteroids treated with LPS and hypoxia showed significantly increased gene expression (p=0.001 and p=0.04) and protein levels (p<0.0001) of the pro-inflammatory cytokine TNF-α compared to controls. TLR-4 protein expression was significantly increased in BO but not in AO enteroids (p=0.0026 and p=0.54). The tight junction protein occludin had significantly decreased gene expression in both BO and AO enteroids compared to control (p=0.02 and p=0.003).
Conclusion: Apical-out enteroids present a novel model for necrotizing enterocolitis, allowing easier access to the surface of the epithelium. Enteroids in an apical out conformation show increased inflammatory markers and decreased tight junction proteins similar to NEC. By exhibiting an apical-out conformation, this model more closely mimics in vivo NEC and may be used to elucidate potential therapeutic strategies in the management of NEC.
