Probing the Luminal Compartment of 3D Organoids via Particle Tracking Microrheology

The mucus layer lining the human stomach is a critical barrier that protects the underlying epithelium from gastric acid and harmful pathogens such as Helicobacter pylori. The efficacy of this barrier relies on the structural integrity of the mucus, which is determined by various biochemical and biophysical features. Human gastric organoids-3D cellular models that resemble the stomach-contain mucus and have been used to investigate gastric disease. The luminal compartment of three-dimensional epithelial organoids represents a physiologically relevant but experimentally inaccessible microenvironment. In gastric organoids, luminal accumulation of mucus creates a confined viscoelastic hydrogel that mimics native gastric mucus. However, the small volume and topological confinement of organoids preclude conventional bulk rheometry. Here, we describe a particle tracking microrheology (PTM) protocol to measure the viscoelastic properties of the mucus within intact organoid lumina following microinjection of fluorescent microspheres. High-speed fluorescence imaging and particle trajectory analysis enable the quantification of viscous and elastic properties of the mucus through calculation of mean squared displacement (MSD), diffusive scaling exponent (alpha), and frequency-dependent storage (G') and loss (G'') moduli. This method enables rheological measurements in nanoliter-scale compartments without disrupting organoid architecture. We further discuss the impact of mucus heterogeneity and microstructure on scale-dependent mechanical behavior. This protocol is broadly applicable to other organoid systems and can be adapted to Transwell or organ-on-chip platforms for in situ luminal measurements. Key features • Enables rheological measurements in small (nanoliter scale) volumes. • Compatible with intact, Matrigel-embedded 3D organoids. • Allows in situ measurement without mucus harvest or purification. • Resolves microscale heterogeneity inaccessible to bulk rheometry and is compatible with functional screening of mucus-modifying drugs.