Unidirectional Permeability – Caco-2 Cell Monolayers
This assay is used to determine the permeability of a test article through Caco-2 cell monolayers in the apical-to-basolateral direction.
Required from Sponsor
- Either a minimum of 100 µL of test article at 10 mM in DMSO or 2 mg of powder
- Molecular mass (exact mass) of the test article and its salt form
- MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.
- Cell batch QC results
- The percent recovery of the test article from the assay wells containing cell monolayers
- The apparent permeability (Papp) of the test article
- The absorption potential of a test article classified as either low or high using the following criteria for the apical-to-basolateral (A→B) direction:
- Low: Papp <1.0 x 10-6 cm/s
- High: Papp ≥1.0 x 10-6 cm/s
- Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%
- Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21 to 28 days old
- Unidirectional permeability of the test article in Caco-2 cells
- Transport buffer: HBSSg, pH 7.4 ± 0.2
- Receiver well containing 1% BSA • Apical and basolateral side at pH 7.4
- Dose two cell monolayers (n=2)
- Dose on the apical side only
- Sample from both apical and basolateral sides at 120 minutes
- Determine the concentrations of test article using a generic LC-MS/MS method with a minimum 6-point calibration curve
- The quality of the monolayer batch is verified using control articles before the monolayers are released for use
- The quality of each monolayer used in the assay is verified by calculating the Papp for the control article lucifer yellow, dosed post-experimentally
1. The results from this assay are sent to the sponsor in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference articles.
2. This assay broadly classifies the Papp of a test article
3. For reproducible results, the solubility of the test article must be greater than 5 µM in pH 7.4 HBSSg.