Involvement of TFF2 and Na/H Exchanger in Gastric Wound Repair In Vitro in Gastric Organoids

Background Trefoil factor 2 (TFF2) plays an important role in promoting gastric epithelial repair of damage. We previously demonstrated that the action of TFF2 requires the sodium‐hydrogen exchanger isoform 2 (NHE2) for rapid gastric epithelial repair of damage. Dissecting molecular pathways is challenging in vivo , so the mechanism of interaction between TFF2 and NHE2 remains unknown. Recently, 3D primary culture of gastric epithelial cells known as gastric organoids has been established. Gastric organoids contain differentiated gastric epithelial cell types and to some degree mimic in vivo gastric epithelium. We tested the hypothesis that gastric organoids can be a useful in vitro model to elucidate the mechanisms of gastric epithelial repair of damage, by investigating if epithelial restitution in mouse gastric organoids was TFF2‐and/or NHE‐dependent. Methods Gastric organoids were generated from isolated fundic tissue of wild‐type (WT), TFF2 knockout (TFF2 −/− ), or NHE2 knockout (NHE2 −/− ) mice. Gastric organoids were cultured for 4–5 days prior to experiments. Damage was induced in gastric organoids by 5s exposure of 3 epithelial cells to high intensity two‐photon 720 nm light (photodamage), resulting in damage to targeted cells and subsequent cell death. Progression of damage and repair was evaluated using confocal/2‐photon microscopy, imaging cell nuclei (10 μg/ml Hoechst33342) and paracellular permeability between the basolateral and luminal compartments of organoids (20 μM Lucifer yellow [LY] added to culture medium). Results Gastric organoids derived from TFF2 −/− mice (311 ± 49 μm diameter, n=13) were significantly (p< 0.01) smaller than those derived from WT mice (514 ± 43 μm diameter, n=34) 5 days after passaging. In contrast, organoids derived from NHE2 −/− (604 ± 45 μm diameter, n=35) were larger than those derived from WT. Consistent with in vivo findings, Western blot showed TFF2 protein was upregulated in NHE2 −/− organoids, while NHE2 was downregulated in TFF2 −/− organoids. In unperturbed organoids, LY did not leak into the luminal space of the gastric organoids over 90 min, confirming integrity of the epithelial barrier. Photodamage increased LY leakage into the WT organoid. Subsequently, dead cell exfoliation occurred coincident with migration of neighboring cells to restore a continuous epithelium in the damaged area within 8 ± 1 min (n=14). In preliminary experiments, photodamage in TFF2 −/− and NHE2 −/− gastric organoids resulted in a similar intensity of LY fluorescence in the organoid, however there was a 2‐and 3‐fold delay of recovery from LY leakage in the repair of damage in TFF2 −/− and NHE2 −/− , respectively, accompanied by delay of damaged cell exfoliation. Basolateral application of EIPA (pan NHE inhibitor, 100 μM) in WT organoids also caused delay in the recovery of LY leakage and damaged cell exfoliation, while Hoe 694 (NHE 1 inhibitor, 100 μM) did not affect gastric restitution. Conclusion Gastric organoids have a TFF2‐ and NHE‐dependent epithelial repair of damage, paralleling previous in vivo studies. Therefore, gastric organoids can be a useful tool to elucidate mechanisms involved in epithelial repair of damage. Support or Funding Information Supported by NIH R01 DK102551