A comparison of basolateral versus apical out enteroids as a model for necrotizing enterocolitis

Heather Liebe; Camille Schlegel; Alena Golubkova; Tyler Leiva; Xue Cai; Catherine Hunter

Necrotizing enterocolitis (NEC) is a devastating disease of premature babies. Although there have been many animal models of NEC, the utility of these systems is limited by species-specific differences. The development of human enteroids has allowed our group to cultivate a tissue engineered human NEC model. Access to the luminal surface in the enteroid is challenging, to circumvent this we have developed an apical out (AO) model. There are benefits unique to both models: AO enteroids are more physiologically relevant while the basolateral out (BO) enteroids allow for faster culturing.

AO and BO human enteroids will provide a relevant model of NEC with AO enteroids providing a more physiologically precise model.

Following IRB approval (#11610-11611) and parental consent, four human neonatal surgical intestinal samples were collected. Intestinal stem cells were harvested, crypt isolation performed and enteroids were generated. These were grown for 5-7 days in basement membrane matrix to induce a BO conformation. Enteroids were transformed to an AO conformation using our standard protocol. AO and BO enteroids were untreated or treated for 24 hours with 100ug/mL of LPS +/- hypoxic conditions (1% oxygen, 5% carbon dioxide, 94% nitrogen). AO vs BO architecture was confirmed via immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein beta-catenin. Quantitative PCR assessed gene expression. Western blot (WB) assayed tight junction proteins and enzyme-linked immunosorbent assay evaluated TNF-alpha. Uptake of Lucifer Yellow (LY) allowed comparison of permeability between AO and BO enteroids in the treatment groups.

LY assay showed significantly increased intestinal permeability in treated AO and BO enteroids compared to controls (p=0.01, p<0.0001). Gene expression of the tight junction protein occludin was decreased in treated AO and BO enteroids (p=0.004, p=0.02). Both models had decreased claudin-4 gene expression in treated groups (p=0.02). BO enteroids had decreased gene expression of claudin-3 (p=0.007 vs 0.05) while AO enteroids had decreased claudin-1 (p=0.006 vs 0.22). WB confirmed decreased occludin in AO and BO enteroids (p=0.02). Gene and protein expression of the inflammatory marker, TNF-alpha was significantly increased in both groups (p<0.05).

Treated AO and BO human enteroids demonstrate similar pro-inflammatory markers and increased permeability expected in NEC. AO enteroids are beneficial in that they provide direct access to the luminal surface while BO enteroids allow for shorter culturing times.