Caco-2 Permeability Assay Protocol

Culturing Caco-2 cells in Transwell plates is considered the reference in vitro technique to evaluate intestinal permeability in novel orally administered compounds, according to leading regulatory agencies (FDAEMA and PMDA).

The primary outcome of such technique is the transport rate of the experimental compound across the Caco-2 monolayer membrane, which positively predicts in vivo absorption across the intestinal system into the bloodstream thanks to the well-established correlation between the in vitro system and the physiologic intestinal absorption.

Which are the key steps to carry on a successful in vitro permeability test with CacoReady?

  • Caco-2 cells culture stage

The Caco-2 cells are seeded at very specific concentrations on a permeable surface, usually a polyethylene membrane, and placed in a Transwell plate to form donor-receiver compartments, artificially simulating intestinal absorption to the bloodstream. The cells are cultured at 37ºC for 15 days, applying culture medium changes every second day to allow the formation of a confluent monolayer.

  • Specialized shipping procedure

After 15 days, the cell culture medium is replaced by a Shipping Medium, a gelatinous culture medium which ensures the viability of the Caco-2 cells for up to 7 shipping days. The intestinal cells continue the differentiation process during the shipping process at room temperature

  • Assessment of cell monolayer integrity

In order to verify that cell integrity meet the standards, Transepithelial Electrical Resistance (TEER), apparent permeability coefficient (Papp) and paracellular flux index (LY) can be performed. Specifications after 21 days of differentiation are:

Measurement Value
TEER > 500 Ω·cm2
Papp < 7.5×107 cm/s
LY < 0.3 %
  • Test compound permeability assay

Studied molecule(s) is diluted to a recommended concentration of 10 µM and applied to the apical and/or basolateral kits’ compartment for 2 hours, evaluating unidirectional or bidirectional transport between compartments. Test permeability and efflux ratio should be determined in triplicate.

Compound(s) concentration is quantified by peak area analysis on LC-MS/MS.

  • Reference Compounds for Permeability Studies

Several compounds can be used as control tests; the main recommendation is to validate the assay using a high permeable molecule, such as Propanolol, and a low permeability compound, such as Atenolol, in triplicates for each group.

  • Evaluation of the Compound Permeability

The experimental molecule permeability coefficient (Papp) is calculated from the permeation rate and compound concentration at t=0h and t=2h.


dQ/dt: amount of product present in the basal (A-B) or apical (B-A) compartment as a function of time (nmol/s).

A: area of transwell (cm2).

C0: initial concentration of product applied in the apical (A-B) or basal (B-A) compartment (nmol/ml).

  • In vivo Absorption extrapolation

Based on in vitro/in vivo correlation studies, Papp values obtained from Caco-2 barrier studies are used to define and predict the intestinal permeability of the tested compounds.

In vitro Papp values Range of predicted in vivo absorption
Papp ≤ 10-6 cm/s Low (0-20%)
10-6cm/s < Papp ≤ 10 x 10-6 cm/s Medium (20-70%)
Papp > 10 x 10-6 cm/s High (70-100%)

In case it is required more specific information related to Caco-2 permeability studies and CacoReady kits, please contact

Related posts

Caco-2 based in vitro models for the screening of anti-inflammatory drugs

Caco-2 based in vitro models for the screening of anti-inflammatory drugs

Inflammatory reactions of the intestinal tract are the result of a combination of hereditary, genetic and/or environmental factors that have been clustered in the inflammatory bowel disease (IBD). ReadyCell has available CacoReady and CacoGoblet to assay compounds inflammatory properties.

Cell-based in vitro models to characterize Food-Drug Interactions

Cell-based in vitro models to characterize Food-Drug Interactions

It is well known that food intake changes luminal conditions (e.g. pH, motility, microbiota,…) in the stomach and the small intestine, modifying drugs bioavailability. Food-drug interactions are one of the major challenges for oral-administered drugs, even more so if considering the growing use of food supplements and functional foods.