Preliminary BCS Permeability Classification Using Caco-2 Cell Monolayers

This non-GLP assay is used to determine a preliminary BCS permeability classification by measuring the permeability of a test compound through Caco-2 cell monolayers in both the apical-to-basolateral and basolateral-to-apical direction

Required from Customer

  • A study design—the customer must specify the values of several assay variables listed in the Options section
  • Highest human dose strength
  • Minimum 20 mg of test compound in powder form
  • Solubility of test compound in modified Hanks’ buffer (HBSSg)
  • Molecular mass (exact mass) of test compound and its salt form
  • MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.


  • Apparent permeability (Papp) of a single concentration of test compound across Caco-2 cell monolayers in the apical-to-basolateral and basolateral-to-apical directions
  • Preliminary BCS permeability classification
  • Efflux
  • Tolerability of test compound by Caco-2 cell monolayers
  • Non-specific binding of test compound to cell-free wells
  • Recovery of test compound from wells containing Caco-2 cells


  • Test compound at a single concentration (1% of the highest human dose dissolved in 250 mL) in HBSSg with <1% DMSO

Assay System

  • Confluent monolayers of Caco-2 cells, 21 to 28 days old

Assay Conditions

  • Assay buffer is HBSSg, apical and basolateral side pH 7.4 ± 0.2
  • Three monolayers (N=3) used for each direction
  • Assess tolerability of test compound by dosing the basolateral (more sensitive) side with a concentration of test compound equal to the highest human dose dissolved in 250 mL, then measure the basolateral-to-apical Papp of Lucifer Yellow
  • Dose each monolayer used in the apical-to-basolateral experiment with a solution containing the test compound, a permeability reference compound and a membrane integrity control compound
  • Sample donor side at 45 minutes and receiver side at 15 and 45 minutes
  • Analyze dosing solution to confirm dosing concentration
  • Determine the concentration of test compound using a generic LC-MS/MS method with a minimum 4 point calibration curve

Assay QC

  • Verify the quality of the monolayer batch
  • Verify the integrity of each monolayer by measuring the pre-experiment trans-epithelial electrical resistance (TEER) and post-experiment permeability of a control compound
  • Verify the suitability of the monolayers by dosing each in apical-to-basolateral direction with a solution containing an integrity control compound and the test compound


  1. For an unambiguous Papp we need to know the solubility of the test compound. If the solubility is not known, Absorption Systems can measure it for you—refer to EA901, Preliminary BCS Solubility Classification by Shake-flask. This assay would be performed prior to running the permeability assay.
  2. If the recovery of test compound from cell-free wells is calculated to be less than 80%, then for the permeability experiment the monolayers are preincubated for 10 minutes prior to permeability assessment—there is an additional charge for this preincubation.
  3. The customer will be consulted if the tolerability of the test compound by Caco-2 cell monolayers, at a concentration equal to the highest human dose dissolved in 250 mL, is unacceptable. Such a finding could affect our ability to perform a GLP compliant version of the apparent permeability assessment for BCS classification. There may be additional charges for any work that might be required to optimize assay’s conditions for your test compound.
  4. To submit a biowaiver containing in vitro permeability assay result the data need to be collected in a GLP compliant manner. Absorption Systems can collect these data for you—refer to EA904, GLP BCS Permeability Classification.


  1. The customer must specify either the standard or custom report format
  2. The customer can request:
    • additional time points
    • the use of an alternative pH on the apical side—the pH can be anywhere between 6.5 and 7.4—on the basolateral side the pH of the buffer is fixed at 7.4 ± 0.2