Caco-2 assay (heterogeneous human epithelial colorectal adenocarcinoma cell assay)

The Caco-2 assay is the most commonly used way of investigating compound permeability. The cell line was derived from heterogeneous human epithelial colorectal adenocarcinoma cells. Caco-2 cells are similar to cells in the human upper intestinal epithelia. They incorporate efflux transporters such as P-gp (P-glycoprotein), several active uptake transporters and even metabolizing enzymes. The permeability is optimally assessed on a relative basis using high and low permeability reference compounds.

In the Caco-2 permeation experiment the cells are cultivated on 96 well plates. The cell layer is then placed between two compartments, the donor and the acceptor compartment. The donor compartment simulates the intestinal fluid either in the fasted or fed state, hence FaSSIF or FeSSIF. The acceptor compartment contains simulated blood buffer, which is commonly a phosphate buffer with a pH 7.4. The test compound is introduced into the donor compartment. Then it permeates through the cells in a passive and/or even active way. In some cases the compound can be eliminated (by P-gp) out of the cells back into the donor compartment. The compound concentration in the acceptor compartment is measured at different time points. The time-concentration profile indicates how quickly the compound permeates through the Caco-2 cells. These results can then be translated to predict compound uptake into the body.


Stephen T. Buckley et al.: In vitro models to evaluate the permeability of poorly soluble drug entities: Challenges and perspectives (2012)

Lina Fossati et al.: Use of simulated intestinal fluid for Caco-2 permeability assay of lipophilic drugs (2008)

C. Markopoulos et al.: Biorelevant Media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness (2013)