(28ai) A Microphysiological System for Modeling Enteric Neuron and Epithelium Interactions in the Gut

Microphysiological systems (MPS), or organ-chip devices, provide a physiologically relevant in vitro model with broad applications including developmental biology, disease modeling, and drug testing. The relationship between the enteric nervous system and epithelium is an understudied area with several unknown mechanisms regarding epithelium barrier dysfunction, a symptom of inflammatory bowel disease and irritable bowel syndrome. Together, these disorders affect over 15% of the global population. In this work, a MPS incorporating epithelial cells (EC) and enteric neurons (EN) was optimized for further studies of the gut. MPSs were assembled by layer using laser cut acrylic, semi-permeable membranes (1 um pore), and 3M 966 pressure sensitive adhesive. The MPS features a top channel for an EC monolayer on a membrane base. The permeable membrane allows for passage of medium components, metabolites, and neurite extensions. EC were obtained from rat duodenal organoids and seeded into the chips as cell monolayers. ENs were also obtained from neonatal rat intestines and encapsulated into a Matrigel-collagen gel in three dimensions below the epithelium layer.

Epithelial barrier integrity was assessed in co-cultured and monocultured devices using transepithelial electrical resistance (TEER) and lucifer yellow permeability assays. Results showed higher permeability for epithelial only cultures compared to co-cultured conditions with epithelial cells and enteric neurons present. These results highlight a supportive role of enteric neurons in gut health and barrier formation. Co-cultured MPSs also show a higher percent of epithelial confluency, suggesting ENs may support increased epithelial stem cell growth. In addition, early ELISA results measured lower levels of calcitonin gene related peptide (CGRP), a protein involved in neuroinflammation and pain, in co-cultures of EN and ECs compared to neuron only. These findings suggest that the neuron-epithelial interactions are critical for maintaining homeostasis. Work in progress includes incorporating the proinflammatory compound lipopolysaccharide (LPS) into mono-culture and co-culture MPSs. CGRP and cytokine levels will be compared between the experimental groups to further assess the role of neuron-epithelium interactions in mitigating inflammation, a suspected key component of the etiology for many gastrointestinal disorders and chronic abdominal pain. Gene expression of the epithelium is also being quantified to show how enteric neurons influence epithelial stem cell proliferation and enterocyte differentiation as epithelial subtype ratios may be contributing to the changes in barrier function seen.

This MPS supports applications beyond those discussed here, including the ability to research the effects of the microbiota and its metabolites on EN and EC function (microbiota-gut-brain-axis). Several different disease models can also be created using this culture system and additional cells of interest, such as immune cell types, can be incorporated by adjusting the MPS geometry.