The Caco-2 cells spontaneously differentiate to express morphological (polarized columnar epithelium) and functional characteristics of mature small intestinal enterocytes. The polarized Caco-2 cell layer shows 4 times higher TEER values compared to HT29 monolayers, i.e. more similar to the in vivo situation. Caco-2 cells express most receptors, transporters and drug metabolizing enzymes like aminopeptidase, esterase and sulfatase found in normal epithelium. However, no P-450 metabolizing enzyme activity has been reported. This problem can be overcome though experience of the team in conducting Caco 2 Sceening.
In comparison with normal intestinal epithelium the Caco-2 cell model has some limitations. First of all that the normal epithelium contains more than one cell type, not only enterocytes. Secondly, when using the Caco-2 cell model, no mucus and unstirred water layer is present. Furthermore, a number of non-cellular parameters will affect the absorption of a certain compound in cells. Thus, transport of lipophilic molecules is strongly influenced by the presence of bile acids and phospholipids, and also compound solubility in the mucus layer as well as the unstirred water layer close to the epithelium, will strongly influence uptake in vivo. Although Caco-2 cells in general provide a powerful tool for studying properties of the intestinal epithelium, one has to be cautious in extrapolating data from such in vitro models to the in vivo situation.
Commonly occurring problems with Caco 2 and their solutions are:
|Problem||Possible explanation||Try the following|
|Very high Papp||Leaky cell monolayers.||Validate integrity of monolayers|
|High expression level of transporter protein for active transport process||Protein expression levels may vary with passage number and culturing conditions. Use the same batch of cells with similar passage number for consistent results.|
|Very low Papp||Test compound degradation||Check stability of test compound under experimental conditions|
|Poor solubility||Verify that the test compound is fully dissolved.|
|Membrane transporter may have become saturated||Verify the concentration of the test substance. Titrate and use lower concentration.|
|Poor mass balance||High degradation||Stability of test compound|
|High cellular uptake||Analyze amount of compound in the cell monolayer by lysing the cells and extracting the compound for quantification.|
|Adsorption of compound to the filter insert or culture vessel||Monitor compound concentration before and after transfer to the filter insert system.|
|High standard deviation||Damaged cell monolayers||It is vitally important to avoid damaging the monolayers during sampling|
GVK BIO is a global leader in Cac0 2 Studies. Please feel free to reach out to our scientific team for any scientific support in preclinical drug discovery.