Monolayer Assay
Monolayer Assay Systems
Monolayer systems consist of a tight cell layer grown on a porous support to separate two fluid compartments. They are widely regarded as the most sophisticated in vitro tools for medium to high throughput modeling of important pharmacokinetic barriers, such as intestinal epithelium, blood-brain barrier, etc. (see Hamilton RD et al. 2007. for a recent application).
Two systems that are applied widely in monolayer studies are the human colon carcinoma cell line Caco-2 and transfectant MDCKII cells.
Figure 1. Schematic representation of the basic principles of the monolayer assay system.
Caco2 Monolayers
Differentiated Caco-2 cells (a human colon carcinoma cell line) express a wide range of transporter proteins on its cell membranes (Siissalo S et al. 2007.), which are similar to those of intestinal endothelium cells (Calcagno AM et al. 2006.). This makes Caco-2 cells ideal for intestinal absorption simulations. In fact, in the last decade, the utilization of Caco2 cells has become an industry standard for the investigation of intestinal absorption, permeability and drug-drug interactions (DDIs) (Oh DM et al. 2002.).
The Caco-2 monolayer efflux assay is designed to model the net transport events of an important fluid compartment barrier in the organism. This method utilizes a polarized Caco-2 cell layer grown on a supportive membrane surface that separates the two compartments. The unidirectional flux of the TA is determined by applying the TA to either the apical, or to the basolateral side of the cell layer and monitoring the time resolved redistribution of TA between the two compartments. The vectorial transport ratio is determined by applying bidirectional measurements [apical-to-basolateral (A-B) and basolateral-to-apical (B-A)]. In general, a ratio higher than 2 or lower than 0.5 indicates the contribution of an active transport process to the net flux of a compound. In the absence of such transport processes this ratio is around 1. A vectorial transport ratio of 1 does not indicate the absence of active transport. For example, in the case of highly permeable compounds the overall contribution of the active transport process to the net flux might be undetectable. Yet, these compounds might interfere with the transport of other compounds, which might result in a clinical drug-drug interaction. This can be assayed by measuring the modification of the vectorial transport ratio of a reporter compound that is known to be affected by some active transport process. A typical example of an indirect assay to estimate P-gp mediated drug-drug interactions is to measure the effect of the TA on the flux of a reporter substrate (i.e., 3H-digoxin).
Figure 2. Flux of 3H-digoxin through Caco-2 monolayer in the presence of P-gp inhibitors
MDCKII Monolayers
By now it is well known that transporters can have major effects on the pharmacokinetics of drugs. There is an increasing need to look at interactions on individual transporters. The introduction of transporter transfected cell lines capable of forming tight cell layers facilitated the investigation of single transporter interactions on monolayers. MDCKII (Madin-Darby canine kidney strain II cells) cell lines have been widely used as hosts for single and/or double transfections. The difference between efflux ratios on the transfected and parental cell lines is regarded as a sign of transporter mediated active uptake or efflux process.
Double transfectant cell lines
The introduction of double transfected cell lines, where an apical efflux transporter is located opposite a basolateral uptake transporter with overlapping substrate specificities, allowed the efficient vectorial transport of substrates and thereby experiments on low passive permeability molecules (Sasaki M et al. 2004., Mita S et al. 2005., Sasaki M et al. 2002., Lui L et al. 2006., Letschert K et al. 2005., Cui Y et al. 2001.). A schematic representation of the vectorial transport is shown in the following figure.
Figure 3. Adapted from Kim 2002 Toxicology 181-2:291
This setup is able to mimic physiologically active vectorial transport processes such as transport of digestive products across the intestinal epithelium from the luminal to the blood side, and the transport of bile salts and other steroid derivatives from the blood to the biliary side in hepatocytes. Double transfected monolayers are suitable for performing both direct, vectorial transport studies and indirect, inhibitory studies (effect of the test articles on the transport of the reporter substrate is measured).
Figure 4. Example of a direct transport measurement on double and single transfectant monolayers
Figure 5. Example of an indirect, inhibitory measurement on double transfectant monolayers.
