Human organoid modeling of gastric mucus layer physiology

Abstract

The stomach has two major secretory modes of self-defense: acid that facilitates digestion and kills harmful microorganisms, and a sticky, selectively permeable mucus layer that acts as a protective barrier. Helicobacter pylori, a pathogen linked to gastric inflammation, ulcers, and cancer, seeks to bypass these defenses to colonize and infect the stomach lining. Human gastric organoids--3D cellular constructs derived from patient tissue--have proven valuable for modeling such diseases in vitro. However, the ability of gastric organoid models to accurately replicate these gastric defenses has not been thoroughly examined. In this research, we first developed a novel technique for the measurement of pH inside of organoids using microelectrodes, demonstrating reproducible pH measurement and evidence of a gradient. The pH was alkaline however, indicating a lack of acid secretion in the model. To further investigate the luminal microenvironment, we used particle tracking microrheology and showed that the organoids contain heterogeneously distributed, viscoelastic mucus. As organoids are topologically closed, we next explored a method for culturing the cells as a monolayer at the air-liquid interface, which allowed access to the mucus. This approach enabled the harvesting of milliliter quantities of clean, sterile "bioengineered gastric mucus" (BGM) over several weeks. We found that BGM shared key protective properties with native mucus, including molecular composition, internal architecture, and rheological behavior--all of which contribute to its ability to act as a barrier, maintain structural integrity, and exhibit flow properties essential for defense and maintenance. Proteomic analysis of BGM compared to native human mucus highlighted the impurity of native mucus, underscoring its limitations for functional studies and emphasizing the need for physiologically relevant in vitro models. Overall, this research contributes to a more complete biophysical understanding of mucus and acid production by human gastric organoids. These insights enhance the utility of organoid models in gastric physiology studies and may inspire translation of such models toward personalized treatment approaches for gastric disease.