Human Gastric Organoid-Derived Mucus Shares Key Properties with Native Gastric Mucus and Inhibits Motility of Helicobacter pylori

Abstract

To prevent and treat gastric disease caused by Helicobacter pylori, it is crucial to understand the role of the stomach’s first line of defense: the gastric mucus layer. Gastric mucus is a complex hydrogel that has been studied extensively in animal models, and multiple groups have collected and purified porcine gastric mucus to yield pure solutions of mucin proteins. However, purified mucin—the functional viscoelastic component of mucus—cannot accurately recapitulate the molecular complexity of mucus in its entirety since it lacks salts, lipids, electrolytes, and other proteins. Additionally, access to human samples for functional analyses remains a critical barrier to mucus research. Therefore, the objective of this study was to engineer sterile human gastric mucus in vitro to evaluate its translational potential to native mucus (NM), for further study of interactions between mucus and H. pylori bacteria. In vivo, H. pylori use flagellar motility to traverse the mucus layer and colonize the underlying epithelium. We hypothesize that the microarchitecture and gel-forming capacity of our lab-grown mucus (LGM) significantly restrict H. pylori motility. Inspired by previous studies, we developed and optimized a protocol for the in vitro production of sterile LGM using epithelial monolayers derived from human gastric organoids and cultured at an air-liquid interface. This mucus could be easily harvested and did not require purification or processing. Additionally, mucus production was maintained for over one month, demonstrating the robustness of our culture system. Mass spectrometry and size exclusion chromatography revealed that the LGM has a molecular composition similar to that of native mucus. Further, we have confirmed with cryo-scanning electron microscopy that the LGM has a characteristic honeycomb structure with similar pore size distributions to those found in native mucus. Rheometric frequency sweeps demonstrated comparable, predominantly elastic gel behavior in both LGM and NM samples. Acid flow experiments in a 2D Hele Shaw cell have also demonstrated that LGM enables viscous fingering—a previously studied hydrodynamic phenomenon typical of mucus gels that is thought to be involved in gastric acid transport mechanisms in vivo. Finally, live imaging and particle tracking were used to quantify the motility of H. pylori in the LGM. Compared to liquid media, the LGM caused a significant decrease in H. pylori swimming speed. Overall, our human gastric lab-grown mucus recapitulates key functions of native mucus and will enable future investigations into gastric mucus pathophysiology and H. pylori infection. NIH Grants: R01 GM131408-01 & UL1 TR002319. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.