The Enteric Nervous System (ENS) is a complex network of neurons and glia, which regulates sensorimotor function throughout the gastroinestinal tract (GI). Here we investigated the role of the ENS and intestinal myofibroblasts in the maintenance of a primary intestinal epithelial barrier through regulation of monolayer permeability, cytokine production, and differentiation of intestinal stem cells. Utilizing a novel, in vitro, transwell-based coculture system, murine small intestinal stem cells were isolated and cultured with ENS neurons and glia or subepithelial myofibroblasts. Results show that the ENS contributes to regulation of intestinal stem cell fate, promoting differentiation into chemosensory enteroendocrine cells, with 0.9% of cells expressing chromogranin A when cultured with ENS versus 0.6% in cocultures with myofibroblasts and 0.3% in epithelial cultures alone. Additionally, enteric neurons and myofibroblasts differentially release cytokines Macrophage Inflammatory Protein 2 (MIP-2), Transforming Growth Factor beta 1 (TGF-β1), and Interleukin 10 (IL-10) when cultured with intestinal epithelial cells, with a 1.5 fold increase of IL-10 and a 3 fold increase in MIP-2 in ENS cocultures compared to coculture with myofibroblasts. These results indicate the importance of enteric populations in the regulation of intestinal barrier function.
Here we report benchtop fabrication of multilayer thermoplastic organs-on-chips via laser cut and assembly of double sided adhesives. Biocompatibility was evaluated with Caco-2 cells and primary human intestinal organoids. Chips with Luer fluidic interfaces were economical ($2 per chip) and were fabricated in just hours without use of specialized bonding techniques. Compared with control static Transwell™ cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells on chip differentiated ~4 times faster compared to controls and produced mucus. To demonstrate the robustness of laser cut and assembly, we fabricated a dual membrane, tri-layer gut chip integrating 2D monolayers, 3D cell culture, and a basal flow chamber. As proof of concept, we co-cultured a human, differentiated monolayer and intact organoids in a chip with multi-layered contacting compartments. The epithelium exhibited 3D tissue structure and organoids formed in close proximity to the adjacent monolayer. The favorable features of thermoplastics, such as low gas and water vapor permeability, in addition to rapid, facile, and economical fabrication of multilayered devices, make laser cut and assembly an ideal fabrication technique for developing organs-on-chips and studying multicellular tissues.
Tissue‐engineered models continue to experience challenges in delivering structural specificity, nutrient delivery, and heterogenous cellular components, especially for organ‐systems that require functional inputs/outputs and have high metabolic requirements, such as the heart. While soft lithography has provided a means to recapitulate complex architectures in the dish, it is plagued with a number of prohibitive shortcomings. Here, concepts from microfluidics, tissue engineering, and layer‐by‐layer fabrication are applied to develop reconfigurable, inexpensive microphysiological systems that facilitate discrete, 3D cell compartmentalization, and improved nutrient transport. This fabrication technique includes the use of the meniscus pinning effect, photocrosslinkable hydrogels, and a commercially available laser engraver to cut flow paths. The approach is low cost and robust in capabilities to design complex, multilayered systems with the inclusion of instrumentation for real‐time manipulation or measures of cell function. In a demonstration of the technology, the hierarchal 3D microenvironment of the cardiac sympathetic nervous system is replicated. Beat rate and neurite ingrowth are assessed on‐chip and quantification demonstrates that sympathetic‐cardiac coculture increases spontaneous beat rate, while drug‐induced increases in beating lead to greater sympathetic innervation. Importantly, these methods may be applied to other organ‐systems and have promise for future applications in drug screening, discovery, and personal medicine.
Here we report benchtop fabrication of multilayer thermoplastic organs-on-chips via laser cut and assembly of double sided adhesives. Biocompatibility was evaluated with Caco-2 cells and primary human intestinal organoids. Chips with Luer fluidic interfaces were economical ($2 per chip) and were fabricated in just hours without use of specialized bonding techniques. Compared with control static Transwell™ cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells on chip differentiated ~4 times faster compared to controls and produced mucus. To demonstrate the robustness of laser cut and assembly, we fabricated a dual membrane, tri-layer gut chip integrating 2D monolayers, 3D cell culture, and a basal flow chamber. As proof of concept, we co-cultured a human, differentiated monolayer and intact organoids in a chip with multi-layered contacting compartments. The epithelium exhibited 3D tissue structure and organoids formed in close proximity to the adjacent monolayer. The favorable features of thermoplastics, such as low gas and water vapor permeability, in addition to rapid, facile, and economical fabrication of multilayered devices, make laser cut and assembly an ideal fabrication technique for developing organs-on-chips and studying multicellular tissues.
To overcome the limitations of traditional organ chips, in article number 2000133, Ryan A. Koppes and co‐workers have implemented a “cut and assemble” manufacturing technique to exploit the meniscus pinning effect, via GelPins, to constrain 3D biomaterials. These GelPins enable the formation of discrete, yet contiguous hydrogel structures in both x‐y and z planes within custom microfluidic devices, facilitating future investigation of innervation and heterogenous tissue interactions.
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