In Vitro Permeability Assays

The Caco-2 permeability assay provides a measure of the permeability of a test compound across the intestinal barrier and its potential for interactions with drug transporters. We provide permeability assays for small molecule formulations such as pharmaceuticals, industrial chemicals and consumer products.

Our permeability assays utilize the C2BBe1 cell line, a subclone of the original Caco-2 cell line.

Initial assessment of the permeability of a test compound is typically carried out in the Caco-2 cell line, derived from a human colon carcinoma. These cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarized monolayer, well-defined brush border on the apical surface and intercellular tight junctions.

Unidirectional Assessment
Here the rate of transport of a compound across the Caco-2 cell monolayer is measured in a single direction (apical to basolateral, A-B).

Bidirectional Assessment
Here the rate of transport in both directions (A-B and B-A) across the cell monolayer is measured, which enables an efflux ratio (B-A/A-B) to be determined. The efflux ratio provides an indicator as to whether a compound undergoes active efflux (efflux ratio ≥2).  If the test compound undergoes active efflux, further assays to determine the identity of the transporter(s) involved are required (Please see Transporter Assays).
 

 Permeability Assay Protocol

 
Test System Caco-2 cell line, wild type
Test Compound Concentration 10 µM (or custom)
Passage Number 40-60
Cell Culture 21 days in 24-well Transwell® plates
Number of Replicates 3
Incubation Time 2 hours at 37°C
Compound Requirements 100 µL of 10 mM DMSO solution
Integrity Marker Lucifer yellow
Control Compounds Atenolol (low passive permeability)
Metoprolol (high passive permeability)
Analysis Method LC-MS/MS quantification of test substance
Data Delivery Assay methods
Apparent permeability rates (Papp)
Experimental recovery

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