Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

We are investigating the pathophysiology of eosinophilic esophagitis and trying to unravel the inflammatory cascade inducing and promoting the chronic inflammatory process of EoE. The identification of new immune mediators that contribute to the disease pathogenesis centered around the impairment of the epithelial barrier and the approval of therapeutic antibodies against said immune mediators. We have identified the IL-20 subfamily cytokines as inflammatory mediators that impair epithelial differentiation and keratinization, resulting in epithelial barrier impairment via the MAP kinase pathway.

To begin, take esophagus biopsy samples immersed in keratinocyte serum-free medium, or KSFM. Replace the KSFM with one milliliter of Dispase 1 and incubate the biopsies for 10 minutes at room temperature. After that, using a 1, 000-microliter pipette, aspirate the Dispase without touching the biopsies or the cell debris pellet.

Then rinse the biopsies with DPBS. After centrifuging the biopsies, use a 1, 000-microliter pipette to aspirate the supernatant. Next, add 500 microliters of trypsin-EDTA to the biopsies and incubate at 37 degrees Celsius for 10 minutes while shaking at 800 RPM.

Then, perform repeated pipetting until a single cell suspension is obtained. Using the rubber plunger head of a tuberculin syringe, filter the cells through a 70-micrometer cell strainer. Afterward, wash the strainer with two to four milliliters of soybean trypsin inhibitor.

Next, filter the cells through a 35-micrometer cell strainer with a snap-on cap on a five-milliliter round-bottomed polystyrene tube. After centrifuging the cell suspension, use a 1, 000-microliter pipette to aspirate the supernatant. Resuspend the cell pellet in the basement membrane extract hydrogel matrix and form 40-microliter droplets in a prewarmed suspension cell culture plate.

Then, incubate the plate without medium for 20 to 30 minutes at 37 degrees Celsius to ensure solidification of the basement membrane extract droplets. Add prewarmed KSFM supplemented with 10 micromolars of Y-27632. On the ninth day, the onion-like multilayered structure and a growing keratinized core in the center of the organoid were observed.

To begin, take cells isolated from esophagus biopsies and resuspend them in four milliliters of KSFM with 10-micromolar Y-27632. Transfer the cells into a T25 cell culture flask. Once keratinocytes form a monolayer with 60%to 80%confluency, passage P1 cells.

Then seed P2 primary keratinocytes onto transwell inserts for the air-liquid interface culture. To perform airlift on day seven, replace the medium in the lower chamber with high-calcium KSFM and aspirate the medium from the upper chamber.