Work

Measuring Intestinal Barrier Permeability by Detecting Flux of Fluorescent Tracers Across Human Colonoid Monolayers

Year: 2025

Type: article

Abstract: The intestinal barrier is a critical site of regulation between luminal antigens and the host immune system, and its dysregulation is implicated in multiple gastrointestinal diseases. Intestinal organoids can be a useful tool to understand and model differences in intestinal barrier integrity. Here, we describe a protocol that uses human colonoids to quantify functional intestinal barrier differences by measuring the flux of fluorescent tracers across organoid monolayers. After growing monolayers to confluence, we add a fluorescent tracer, such as FITC-dextran, to the apical chamber and detect its translocation into the basolateral chamber over time. Increased translocation of the fluorescent tracer is reflective of a more permeable, or weaker, epithelial barrier. This protocol is an adaptable platform that allows evaluation of functional intestinal barrier differences across multiple conditions in parallel. Using variations of this protocol, one can assess transepithelial permeability to molecules of various size, shape, and chemical structure, as well as permeability differences in the context of isolated treatments or microenvironmental changes on the apical or basolateral sides of epithelial cells. This allows for a deeper understanding of the mechanism and treatment of barrier disruption in disease states.

Source: Journal of Visualized Experiments

DOI: 10.3791/68857

Authors: Geetha Bhagavatula, Ciara Schaepe, Sean Colgan

Institutions: Children's Hospital Colorado, University of Colorado Anschutz, University of Colorado Denver

Language: English

Topic: Barrier Structure and Function Studies

Subfield: Neurology

Field: Neuroscience

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green

Funders: U.S. Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases