Gut-Ex-Vivo system as a model to study gluten response in celiac disease

Abstract

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

A Gut-Ex-Vivo System to Study Gut Inflammation Associated to Inflammatory Bowel Disease (IBD)

“…Colon from 13 days old Balb/c mice was resected and cultivated in a silicone-based Gut-Ex-Vivo System (GEVS), as previously described [7]. Briefly, the custom-fabricated fluidic chip consists of a silicone-based device divided into 6 parallel isolated chambers to allocate mouse colons (up to six).…”

“…Prior to dissection, input/output ports were flushed using a sterile culture medium and colon fragments were gently flushed in a sterile medium and fixed over the luminal input and output ports. After inserting each tissue fragment into the chamber, the device is placed on the heat-conducting adapter and sealed [7,8].…”

“…The tissue culture medium was loaded into a 5 mL syringe (for short-term experiments, 5 h) and infused into the device input ports by a syringe pump (flow rate of 99 µL/h) [7].…”

“…Tissue viability was evaluated as previously described [7], through MTS assay. Tissues were placed in a 48 well plate and cultured for 4 h with DMEM supplemented with 10% FBS), 2mM L-glutamine, 100 U/mL Penicillin, 0.1 mg/mL Streptomycin (Euroclone) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium solution following manufacturer's instructions.…”

“…To generate a colon ex vivo culturing, we used the previously described GEVS [7,8]. To this aim, colon from 13-days-old Balb/c mice was surgically resected, flushed in sterile conditions, and inserted into a silicon-based device.…”