The epithelial cell barrier of the intestine controls the absorption of metabolites from the intestinal lumen. Improper function of this barrier can lead to inflammation and a ‘leaky gut', where tight junctions do not form between enterocytes and toxins or bacteria may pass between these cells and into the body. This dysregulation plays a critical role in intestinal diseases such as irritable bowel disease (IBD), which affects up to 15% of the population of the United States (1).
Current in vitro models of the epithelial cell barrier typically consist of polarized epithelial colorectal adenocarcinoma cells (Caco-2) grown on a polymer transwell insert (2). Barrier function may be studied in various conditions based on the tight-junction resistance and flux through the transwell membrane. These models fail to consider the extensive neural network that lines the gastrointestinal tract (GI), the enteric nervous system (ENS), which modulates intestinal secretion and motility (3). Enteric glia, the support cells of the enteric nervous system, have also been reported to impact epithelial survival and maturation (4). Therefore, it is the hypothesis of this work that a co-cultured model consisting of both Caco-2 and enteric neurons and/or glia will provide a more realistic in vitro model of the epithelial cell barrier and will help to elicit the role of the enteric nervous system in barrier function regulation of healthy tissue and disease states.
Materials and Methods:
A mixed culture of enteric neurons and glia was isolated from 10 week old, adult black 6 mouse small intestine, (5) and seeded onto laminin coated glass cover slips in 24 well plates. Neurons were grown under standard incubated culture conditions in Neurobasal media supplemented with NGF and GDNF to promote neural survival in vitro.
At day 8 of neuron culture, Caco-2 were seeded onto 0.4 um PET transwells at approximately 60,000 cells/cm2. The neurons remained on coverslips beneath the transwell. Control cultures were Caco-2 with no enteric cells, and enteric cells with no Caco-2. NGF/GDNF Supplemented Neurobasal media was used for both cell lines. After 5 days of coculture, TEER (transepithelial electrical resistance) measurements were taken with an EVOM from World Precision Instruments (in PBS, 37°C), and then both neurons and Caco-2 were fixed and stained. Enteric neurons were stained for Beta-III-tubulin to label neurites and nuclei (DAPI); Caco-2 were stained for ZO-1, a tight junction associated protein, and nuclei (DAPI). Cells were mounted on glass slides and imaged using an inverted Olympus fluorescent microscope.
Results and Discussion:
Enteric neurons and Caco-2 were cocultured with the Caco-2 in transwells and neurons on cover slips in the bottoms of the wells. The cells lines were kept spatially separate, but shared media and could thus interact through excreted metabolites and soluble proteins. Preliminary results show that the addition of enteric neurons to Caco-2 increases the transepithelial resistance and thus tight junction formation. After 5 days of culture, TEER measurements for the cocultured Caco-2 (Fig 1) were 270.8 +/- 3.9 ohms, while those of the Caco-2 alone were 238.6 +/- 10.0 ohms, a ~14% increase in a short duration culture. This indicates that soluble factors released from the enteric neurons impact the initial formation of tight junctions with this cell line.
Figure 1: Transepithelial Resistance (TEER) for co-cultured Caco-2 and enteric neurons is 14% higher than the control after 5 days of culture
These preliminary results imply a role of the enteric nervous system in the regulation of epithelial barrier function and flux across the epithelium. Further work will investigate the formation of tight junctions with coculture for an extended length of time (10-28 days) and determine if the addition of neurons and enteric glia to an in vitro model impacts transepithelial flux. Additionally, the epithelial cell line used in the coculture will be changed to include primary small intestinal stem cells (6) and/or mucous producing cells such as HT-29 (7) to determine if neural inclusion impacts barrier function. Neurite extension and functionality will be examined via neural tracing software and electrophysiology. This platform may provide mechanistic insight into the cross-talk between the epithelium and the enteric nervous system, which may lead to a more realistic pharmacological test bed that recapitulates the complex biological environment of the small intestinal niche.
Acknowledgements: The authors thank members of the ABNEL group and Northeastern University for their support. Authors also thank Dr. Rebecca Carrier for her insight into epithelial model systems.
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