Home › Cell Biology › Investigating Intestinal Barrier Breakdown in Living Organoids
Cell Biology JoVE (Open Access) Citable · DOI

Investigating Intestinal Barrier Breakdown in Living Organoids

DOI: 10.3791/60546-v
What you'll learn
  • ✓ Quantify barrier integrity of small intestinal organoids in real-time
  • ✓ Apply permeability assay to test barrier-modulating substances sequentially
  • ✓ Interpret intestinal organoid barrier integrity analysis results
Protocol

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Difficulty
intermediate
Total time
~4–6 hours per experimental run (including organoid preparation and assay incubation)
Model organism
Small intestinal organoids (mouse-derived)
Biosafety
BSL-1

Steps

1
Understand intestinal barrier integrity measurement principles

Review the rationale for using living organoids to assess barrier function and the advantage of time-resolved sequential testing of substances.

▶ 00:05
2
Prepare materials for barrier integrity measurement

Set up reagents, organoid culture plates, and assay apparatus required for permeability testing.

▶ 00:57
3
Execute organoid permeability assay protocol

Perform the step-by-step permeability assay on intestinal organoids, including tracer molecule application and barrier integrity quantification.

▶ 03:04
4
Analyze representative barrier integrity results

Interpret data output from organoid barrier integrity analysis and identify patterns of barrier compromise or integrity.

▶ 05:39
5
Summarize findings and next experimental steps

Review conclusions from the assay and discuss applications for testing multiple barrier-modulating compounds.

▶ 06:32
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