A Pumpless, High-Throughput Microphysiological System Confirms Enteric Innervation of Duodenal Epithelium Strengthens the Barrier Function

Abstract

Enteric neurons, diverse in function and great in number, are heavily involved in homeostasis within the small intestine, and their dysregulation has been implicated in gastrointestinal disorders and neurodegenerative diseases. Innovations in biofabrication have resulted in advances for in vitro models of the gut, however, the majority lack enteric innervation, limiting therapeutic screening and discovery. Here, we present a high-throughput co-cultured microphysiological system (MPS), or organ chip, that supports a primary epithelial monolayer that directly interfaces with a three-dimensional hydrogel containing a primary enteric neuron culture, mimicking the close proximity present in vivo. The acrylic MPS device was fabricated with our established and cost-effective laser cut and assemble method. We have expanded this technology to include up to twelve 3D MPSs per device within the footprint of a traditional well-plate, supporting high-throughput experimentation. The inclusion of this 3D microtissue does not hinder physiologically relevant flow, standard measures of barrier function, and microscopy techniques. The device features gravity-driven flow to induce physiological shear stress on the epithelium culture and provide continuous nutrient presentation. Results show the intestinal and neural tissue maintained expected morphologies over an experimental timeline of ten days. Proximal enteric neurons extend neurites through the 3D hydrogel towards the epithelial monolayer. Barrier function was confirmed with both Transepithelial Electrical Resistance (TEER) and Lucifer Yellow diffusion on-chip. TEER confirmed a significantly more substantial barrier integrity in co-cultures compared to baseline values (1.25-fold) in epithelial cell-only. Lucifer yellow permeability assays performed in parallel supported the TEER results, with an 11.8% lower permeability of the co-cultured group than the epithelium only. The presence of the ENS on chip results in a significant (1.4 fold) reduction in epidermal growth factor (EGF). This is the first high-throughput, innervated gut on a chip device that demonstrates the importance of the autonomic nervous system on EGF expression and possibly epithelial renewal in vitro. Innervation is essential to create more biomimetic and physiologically relevant in vitro models for biological and pharmacological assays.