Modelling human gut inflammation and therapeutics in organs-on-chip

Normal homeostasis of the gut largely relies on the intestinal barrier function. Disruption or of this barrier results in local consequences associated with direct contact of bacteria/bacterial products with the epithelial cells, translocation in the systemic circulation and activation of the inflammatory response. Local intestinal inflammation underlies the development of several gastrointestinal diseases, whereas the systemic consequences link with liver, heart and neurologic diseases. The currently used in vitro assays and animal models revealed mechanisms potentially involved in the deregulation of intestinal barrier, however, they have failed to produce targeted therapeutic approaches.
The study of intestinal barrier function based on animal studies is hampered by their differences in microbiome and the innate immune responses of intestinal epithelial cells in humans, while in vitro assays overlook the importance of blood flow, mechanical forces, cell-cell interactions and in vivo cytoarchitecture. Therefore, it is paramount that research focuses on organ-on-chip approaches, to unravel key mechanisms pertaining intestinal barrier function, its role in local and systemic inflammation, and evaluate clinically relevant drugs.
Hypothesis:
Organ-on-chip approaches will facilitate the holistic characterisation of mechanisms involved in intestinal epithelial barrier function in health and disease.
Aims:
Utilise organ-on-chip to elucidate cellular mechanisms which regulate intestinal epithelial barrier function upon inflammatory insults and use as platform to evaluate novel therapeutics.
Work plan:
Months 1-12- Development and validation of reproducible gut-on-chip: Using primary and immortalised non-tumorigenic intestinal epithelial cell lines in combination with primary intestinal endothelial cells will be employed to develop a reproducible organ-on-chip. The Emulate Organ-OnA-Chip platform allows the study of the effects of laminar flow on complex cell systems in an in vivorelevant tissue architecture using ready-to-populate chips. The immortalised intestinal epithelial cell lines will be compared to epithelial cells derived from primary organoids in their ability to reproduce barrier function under physiological mechanical forces. Comparisons will include permeability,
epithelial tight junction immunostaining, cell confluency and survival.
Months 13-24 - Evaluation of inflammatory stimuli effects on barrier function: The gut-on-chips will be used to characterise the alterations in molecular mechanisms involved in endothelial/epithelial barrier integrity and morphology upon exposure to inflammatory stimuli, by measurements of permeability, tight junction immunostaining, confluency and cell death, secretion of chemokines/cytokines, RNA and microRNA. Additional experiments will utilise sera, PBMCs and
faecal filtrates collected from patients with intestinal inflammatory diseases before and after therapy.
Months 25-42 - Pathway analysis and identification of druggable targets: RNA and microRNA profiles will be subjected to MetaCore pathway analysis to identify common gene networks deregulated by inflammatory insults in intestinal epithelial cells. Druggable targets will be evaluated using commercially available agonists or inhibitors. New experimental therapeutics, small molecules, microRNA-loaded nanoparticles, metabolites will be tested on this platform against the
inflammatory insults. Selected barrier insult experiments and therapeutics will be further evaluated using Emulate's ready-to-use (gut) biokits.
Anticipated outputs:
Graduate pioneering the gut-on-chip approaches combined with expertise in cellular, molecular biology and bioinformatics.
Two high impact publications, presentations at international conferences.
A new platform for evaluation of therapeutics for inflammatory diseases.
NC3Rs: animal replacement in research