Characterizing the Luminal Microenvironment of Human Organoids for Studies of Gastric pH Regulation

The stomach is notorious for having the steepest proton gradient in the body, which pathogenic Helicobacter pylori must traverse to infect the epithelium. This gradient across the 200-300 μm-thick gastric mucus layer enables the stomach to maintain a near-neutral pH at the epithelial surface and a strongly acidic environment in the stomach lumen. In this study, we use human gastric organoids to study the maintenance and regulation of this pH gradient. Human gastric organoids are 3D cellular constructs derived from patient biopsies that mimic the microanatomy and physiology of the stomach, as they contain both mucus and gastric acid. Organoids have been used to investigate H. pylori-induced gastritis, peptic ulcers, and stomach cancer, which claims 700,000 lives each year. Gastric mucus is critical for protection against H. pylori infection, which is the leading cause of gastric cancer. It is presently unclear how the gastric mucosal pH gradient is maintained, and how closely organoid models can mimic this microenvironment. First, we detected mucus within human gastric organoids using histological staining and bead microrheology. We also used a FITC-conjugated lectin to visualize the mucosal matrix inside of the organoids. These data were supported by our observation that a clear viscous material was produced on the apical side of two-dimensional, organoid-derived cell monolayers. Additionally, we developed a novel method for the measurement of intraluminal pH of the gastric organoids. Using a micromanipulator and stereomicroscope to maneuver a glass microelectrode into the organoid lumen, we measured and manipulated luminal pH using histamine stimulation. We determined that pH remains consistent across organoid lines, and that the intraluminal space is more acidic than the surrounding environment. In ongoing studies, we will develop the spatial resolution of our organoid microprofiling technique to fully characterize the proton gradient within the mucus-filled lumens gastric organoids. Our studies are the first to utilize pH microsensors in these models, and will lead to improved molecular and biophysical understanding of gastric physiology and potential new treatment approaches for disease conditions (such as peptic ulcers and gastric cancer) that involve dysregulated gastric acid secretion.