Permeability assay (Caco-2 cells)

Aim: Assessment of in vitro bidirectional transport in Caco-2 cells to screen test compound for permeability and involvement of efflux transporters.

Background information: Caco-2 cells are a human colon adenocarcinoma cell line used as an in vitro model of human intestinal absorption of drugs. When cultured as a monolayer, they differentiate and polarize to form intercelular tight junctions and a brush border with efflux transporters, mimicking the human intestinal epithelium.

Transport assays are performed in both directions across the monolayer: absorption (apical to basolateral) and efflux (basolateral to apical). Test compound is added to either the apical or basolateral sides of the monolayer. Concentrations are quantified by UPLC-MS/MS using a calibration curve.

Compound requirement: 50 µl of 10 mM DMSO solution

Turnaround time: 4 weeks

Experimental design:

  • Test compound concentration: 10 µM
  • 24-well plate
  • Number of replicates: 2
  • DMSO concentration: 1%
  • Incubation time: 120 min
  • Control: positive controls of efflux transporters or permeability
  • Monolayer integrity marker: lucifer yellow
  • Analytical method: UPLC-MS/MS with calibration curves

Delivered results:

  • Absorption and efflux permeability
  • Absorption and efflux recovery
  • Efflux ratio