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

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.