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, cryo-field emission scanning electron microscopy (CryoFE-SE), and rheometry. BGM contained high-molecular weight molecules also found in native gastric mucus. Proteomic analysis confirmed that BGM contained MUC5AC, MUC6, MUC1, and other stomach-specific molecules such as pepsin C, trefoil factor 2, and gastrokine. CryoFE-SE showed that both BGM and native mucus had a porous structure and a characteristic honeycomb scaffold. However, the viscosity of the BGM was generally lower than that of native human gastric mucus, and BGM failed to exhibit gelation at low pH. Collectively, these findings demonstrate the potential as well as some limitations of BGM as an accessible model system for human gastric mucus.NEW & NOTEWORTHY We 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 and physical architecture. This work highlights the translational potential of organoid-derived mucus for functional investigations of the human gastric mucus layer.