Caco-2 Permeability

In the development of small molecule drugs, the bioavailability of compounds needs to be evaluated early. In addition to using experimental animals to evaluate bioavailability, various in vitro analytical methods have been developed to evaluate the ability of compounds to penetrate the intestinal wall. Caco-2 monolayer cell permeability analysis has become one of the standard in vitro permeability analysis methods. For rapid assessment of membrane permeability, BOC Sciences offers standard and custom bi-directional permeability assays in a variety of cell lines, including human Caco-2 and MDCK cells. Our Caco-2 permeability assay is designed to help determine the absorption and bioavailability of drug candidates, facilitating the lead optimization process in drug discovery.

Caco-2 is an infinitely dividing human colon carcinoma cell line. Caco-2 undergoes epithelial-like differentiation spontaneously after forming a single cell layer during in vitro culture, with microvilli structure and tight junctions between cells. Caco-2 expresses numerous cell efflux transporters, including p-glycoprotein (p-gp), breast cancer resistance protein (BCRP), multidrug resistance protein (MPR), etc. Carrier systems involved in the transport of substances such as glucose, amino acids, and oligopeptides are also included. Therefore, Caco-2 cell model can not only predict the passive transport of compounds, but also characterize compound efflux and active absorption. That is, the determination of Caco-2 cell permeability not only helps to determine intestinal permeability, but also can identify specific transporters or efflux proteins and intestinal phase II drug metabolizing enzymes.

Fig. 1 Caco-2 cell monolayer model.

Brief Protocol

Caco-2 Cells Culture

Caco-2 cells are cultured on the semipermeable polycarbonate surface of the insert, which fits into the assay chamber, establishing apical and basolateral compartments. These two chambers are connected only by a monolayer of cells and their semipermeable matrix. Typically, Caco-2 cells are cultured for approximately 21 days to reach confluence and differentiate into enterocytes displaying transporters and tight junctions.

Compound Permeability Assay

Compounds were diluted at a concentration of 10 µM and applied either apically or basolaterally to the cell monolayer. Apparent permeability and efflux rates of test compounds were determined in duplicate. Compounds were quantified by LC-MS/MS analysis based on peak area.

Apparent Permeability Coefficient (Papp) Calculation

The transport rate for a specific concentration of a test compound is usually expressed as the apparent permeability coefficient (Papp), calculated using the following equation:

Efflux ratio = Papp [BL>AP] / Papp [AP>BL]; %Recovery = 100 x [(Vr x Cr) + (Vd x Cd)] / (Vd x C0).

where:

dQ/dt: the permeability rate.

A: the surface area of the cell monolayer.

C0: the initial concentration in the donor compartment.

Vr: the solution volume in the receiver chamber.

Vd: the volume in the donor chambers.

Cd and Cr: the final concentrations of transport compound in donor and receiver chambers, respectively

Case Study

BOC Sciences has the capability to perform parallel culture and testing of Caco-2 monolayer cells using 6-well, 12-well, 24-well or 96-well cell culture plates. These experiments were designed to assess the permeability of the epithelium and endothelium, the involvement of intestinal absorption and active transport. If you are interested in our Caco-2 permeability services, please contact us for more information.