Epithelial Regeneration Ability of Crohn’s Disease Assessed Using Patient-Derived Intestinal Organoids

Abstract

AbstractBackgroundThe intrinsic limitation of cell lines and animal models limits our understanding of epithelial regeneration capability in Crohn’s disease (CD). Therefore, we aimed to study epithelial regeneration ability in CD using an intestinal organoid model. Further, since tumor necrosis factor alpha (TNFα) is a major proinflammatory effector during CD pathogenesis, we also investigated TNFα-induced alteration of regeneration ability in CD patient-derived intestinal organoids.MethodsHuman intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from control subjects and patients with CD. The epithelial regeneration ability of intestinal organoids was assessed using organoid reconstitution, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2′-deoxyuridine (EdU), and wound healing assays.ResultsEx vivo cultures of ileal crypt cells revealed that organoid formation rate of CD patients were reduced compared with that of control subjects (p <.001). CD patient-derived organoids sub-cultured for more than 6 passages showed stable organoid reconstitution and identical morphological features. The organoid constitution and MTT assay revealed that the viability of TNFα-treated CD patient-derived organoids were significantly lower than that of TNFα-treated control organoids (p <.05 for each). The number of EdU+ proliferative cells was significantly lower in TNFα-treated CD patient-derived organoids than in TNFα-treated control organoids (p <.05). The wound-healing ability of TNFα-treated CD patient-derived organoids was significantly lower than that of TNFα-treated control organoids (p <.001).ConclusionsThe clinical trials are disabled to settle this issue, our results indicated that the epithelial regenerative ability is impaired in patients with CD, especially in TNFα-enriched condition.