Wilking, James N.

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.

Abstract The velocity data presented demonstrate the complicated flow behavior of nanocrystalline cellulose (NCC) suspensions even when standard rheometry shows only subtle effects. Rheo-nuclear magnetic resonance (NMR) velocimetry with spatial and temporal resolution indicates that NCC suspensions undergo varying flow behavior, which correlates with bulk rheology and includes wall-slip, shear banding, and yielding. Large-velocity fluctuations in a chiral nematic liquid crystal-phase suspension (5% w/v) indicate particle director orientation tumbling and flow. The results provide details of the mesoscale velocity distributions in space and time, which can be used to inform the interpretation of rheology data, as well as processing flow conditions to control NCC suspension microstructure and impact properties of composite and other materials.

AbstractNucleic acid amplification testing (NAAT) remains one of the most reliable methods for pathogen identification. However, conventional bulk NAATs may not be sufficiently fast or sensitive enough for the detection of clinically‐relevant pathogens in point‐of‐care testing. Here, we have developed a digital droplet RT‐LAMP (ddRT‐LAMP) assay that rapidly and quantitatively detects the SARS‐CoV‐2 viral E gene in microfluidic drops. Droplet partitioning using ddRT‐LAMP significantly accelerates detection times across a wide range of template concentrations compared to bulk RT‐LAMP assays. We discover that a reduction in droplet diameter decreases assay times up to a certain size, upon which surface adsorption of the RT‐LAMP polymerase reduces reaction efficiency. Optimization of drop size and polymerase concentration enables rapid, sensitive, and quantitative detection of the SARS‐CoV‐2 E gene in only 8 min. These results highlight the potential of ddRT‐LAMP assays as an excellent platform for quantitative point‐of‐care testing.

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.

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.