Caco-2 Cell Permeability Assay
Also known as: Caco-2 assay, intestinal permeability, ADME screening
The Caco-2 assay is an in vitro model system using human colon carcinoma cell monolayers to screen drug intestinal permeability. Developed by Hidalgo and colleagues in 1989, Caco-2 cells differentiate into an epithelial barrier resembling intestinal mucosa, enabling rapid assessment of drug absorption potential and identification of transporter-mediated transport.
Key highlights
- Excellent correlation with human oral bioavailability, especially for passive drugs
- Identifies transporter-mediated transport (P-gp efflux, OATP uptake) affecting intestinal absorption
- Relatively rapid and cost-effective compared to animal models; results available in weeks
- Standardized assay with established acceptance criteria; reproducible across labs
Intuition
This section is available to Pro members. Upgrade to Pro
How it works
This section is available to Pro members. Upgrade to Pro
When to use it
Use Caco-2 early in drug discovery to rank compounds by intestinal permeability and predict oral bioavailability. Useful for identifying transporter interactions that may affect co-administered drugs.
Strengths & limitations
- Excellent correlation with human oral bioavailability, especially for passive drugs
- Identifies transporter-mediated transport (P-gp efflux, OATP uptake) affecting intestinal absorption
- Relatively rapid and cost-effective compared to animal models; results available in weeks
- Standardized assay with established acceptance criteria; reproducible across labs
- Monolayer transport may not fully capture in vivo intestinal environment (pH, food effects, mucus layer)
- Tight monolayer may overestimate permeability for compounds that use paracellular transport
- Transport mechanism in Caco-2 may differ from other regions of GI tract (duodenum vs. colon)
- Does not account for intestinal metabolism (e.g., first-pass CYP3A metabolism); Papp does not equal systemic bioavailability
Common pitfalls
This section is available to Pro members. Upgrade to Pro
Applications
This section is available to Pro members. Upgrade to Pro
Frequently asked
What is TEER and why is it important?
TEER (transepithelial electrical resistance) measures monolayer integrity. High TEER indicates tight junctions are closed and drug transport is primarily transcellular (through cells). Low TEER suggests paracellular leakage; results are unreliable. Typical acceptance criterion is TEER > 200 ohm·cm.
What is the difference between Papp and bioavailability?
Papp is the transport rate in Caco-2; it correlates with intestinal permeability but does not equal oral bioavailability. Bioavailability also depends on absorption rate, first-pass metabolism, and drug solubility. A high-Papp drug can still have poor bioavailability if it undergoes extensive metabolism.
How do I interpret A→B and B→A flux ratio?
If A→B flux >> B→A flux, the drug likely permeates passively. If B→A >> A→B, an active efflux transporter (e.g., P-gp) is present. Ratio near 1 suggests no active transport. Test with transporter inhibitors to confirm mechanism.
Can I use Caco-2 for drugs with poor solubility?
Yes, but use solubilizing vehicles (DMSO, ethanol) at low concentrations to avoid monolayer toxicity. Alternatively, use dissolution media that mimic intestinal fluid. Document vehicle effects on permeability; some vehicles enhance apparent Papp by opening tight junctions.
Sources
- 1.Hidalgo, I. J., Raub, T. J., & Borchardt, R. T. (1989). Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology, 96(3), 736-749.
- 2.Artursson, P. (1990). Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. Journal of Pharmaceutical Sciences, 79(6), 476-482.
You have read it. What now?
Cite this page
ScholarGate. (2026, June 3). Caco-2 Permeability. ScholarGate. https://scholargate.app/pharmacology/caco-2-permeability