In Vitro Gut Model to Assess Bacterial Adhesion and Internalization

Begin with a Transwell insert containing a co-culture of epithelial and goblet-like cells.

Epithelial cells form tight junctions, while goblet-like cells produce mucus, replicating the in vivo intestinal epithelial barrier.

Take a suspension of the oral bacterial community that has been treated with simulated intestinal fluid to mimic small intestinal digestion.

Replace the media in the upper chamber with the bacterial suspension.

Incubate with shaking to facilitate bacterial adhesion and internalization.

Collect the media from the upper chamber containing non-adherent bacteria for subsequent viability assays.

Add a mucus-dissolving solution and incubate to solubilize the mucus and release the adhered bacteria.

Collect the solution for further analysis of bacterial viability and adhesion.

Wash the cells with buffer and add a detergent to dislodge them.

Collect the cell suspension.

Vortex the suspension to lyse the cells and release the internalized bacteria.

Centrifuge and collect the supernatant and cellular debris separately for further analysis.

After washing and adjusting the cell density of the small intestine digestion according to the text protocol, remove the apical medium from the transwell system, and add 1.5 milliliters of the bacterial suspension.

Co-culture the system under general cell-culture conditions for 2 hours. Add 0.5 milliliters of 10 millimolar n-acetylcysteine in HBSS to solubilize the mucus produced by the HT29-MTX and to recover the adhered bacteria.

Incubate the plates at 37 degrees Celsius for 1 hour under agitation. Recover the NAC-HBSS in a microcentrifuge tube, and use 1 milliliter of DPBS to wash the cells twice. Add 0.5 milliliters of 0.5% triton X-100 on top of the monolayers, and disrupt the cells by pipetting.

Then quickly recover the liquid in vortex for 1 minute. Speed is crucial to avoid damaging the bacterial cells with the detergent. Analyze the samples according to the text protocol.