Physical, chemical, and structural properties of human gastric organoid-derived mucus

The gastric mucus layer protects the epithelium from gastric acid and ingested pathogens. However, studies of human gastric mucus have been limited due to poor accessibility of native human mucus and the abundance of contaminants in these samples. Here, we explored the potential of human gastric organoids as models for mucus production. Immunofluorescence staining confirmed that the organoids produced mucus containing MUC5AC and MUC6. The luminal mucus had viscoelastic properties similar to those of native human gastric mucus, as determined by particle tracking microrheology. To collect organoid-produced gastric mucus, termed bioengineered gastric mucus (BGM), organoids were cultured as monolayers at the air-liquid interface (ALI), and apically-secreted mucus was harvested and analyzed by MUC5AC ELISA, proteomics, CryoFE-SEM, and bulk rheometry. BGM contained high-molecular weight molecules also found in native gastric mucus, including MUC5AC. Proteomic analysis confirmed that BGM contained MUC5AC, MUC6, MUC1, and other stomach-specific molecules such as gastricsin, olfactomedin 4, and gastrokine. CryoFE-SEM showed that both BGM and native mucus had a porous structure and a characteristic honeycomb scaffold. Bulk rheometry confirmed that BGM exhibited shear thinning and predominantly elastic behavior, consistent with native mucus. Collectively, these findings indicate that BGM is an accessible alternative to native gastric mucus that can be produced on-demand for in vitro studies. New and NoteworthyWe demonstrate the structural and functional similarities of organoid-derived gastric mucus and native mucus collected from human patients. The bioengineered gastric mucus mimics its native counterpart in its proteomic profile, physical architecture, and viscoelasticity. This work highlights the translational potential of organoid-derived mucus for functional investigations of the human gastric mucus layer.