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Gastric mucus is a complex hydrogel that protects the epithelial surface of the stomach from gastric acid and prevents invasion of pathogens such as Helicobacter pylori. The viscoelastic properties of the mucus layer are considered essential for barrier function. However, since native human gastric mucus is not readily accessible, the majority of previous studies were performed with animal mucus or with purified gastric mucin that lacks functionally important mucus components. Moreover, commonly used purification protocols may damage mucin structure and impact gel-forming capacity. Therefore, the objective of this study was to engineer sterile human gastric mucus in vitro to study interactions between mucus and H. pylori bacteria. We hypothesize that our bioengineered mucus layer is both structurally and functionally relevant and applicable to in vivo gastric mucus. Based on previous studies with organoid-derived monolayers, we developed and optimized a protocol to produce mucus in 2D air-liquid-interface (ALI) cultures derived from human gastric organoids. The structure of the mucus was analyzed using cryo-scanning electron microscopy, and viscoelastic properties were quantified using particle tracking microrheology. Live imaging and particle tracking also were used to quantify the motility of H. pylori in the bioengineered mucus. 2D monolayers of human gastric organoids cultured at an ALI on transwell inserts consistently produced milliliter quantities of sterile human gastric mucus. This bioengineered mucus could be easily harvested and did not require purification or processing. Mucus production by the ALI cultures was maintained for over one month, demonstrating the robustness of our culture system. Electron microscopy confirmed that the bioengineered mucus had a characteristic honeycomb structure with pore sizes of 10.8 ± 3.4 microns, consistent with previous studies on porcine and canine gastric mucus. Particle tracking microrheology confirmed the viscous behavior of the collected mucus. Compared to liquid media, the bioengineered mucus also caused a significant decrease in H. pylori swimming speed, with 42.8 ± 0.2 microns/second in brucella broth and 10.9 ± 0.1 microns/second in mucus (P ≤ 0.001). We here leveraged the ability of gastric organoids cultured as monolayers at an ALI to secrete sterile mucus to produce native human gastric mucus for in vitro studies. Using this system, we showed that the mucus inhibited bacterial motility, limiting bacterial invasion into the mucus layer. Our observations support previous hypotheses on gastric mucus function and demonstrate the potential of organoid-derived mucus for mechanistic studies of gastric mucus layer physiology. NIH R01GM131408-01, NIH UL1 TR002319 This is the full abstract presented at the American Physiology Summit 2023 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.