Objective According to the regulatory guidelines, one of the critical steps in using in‐vitro permeability methods for permeability classification is to demonstrate the suitability of the method. Here, suitability of the permeability method by using a monolayer of cultured epithelial cells was verified with different criteria. Methods Imaging with a transmission electron microscope was used for characterisation of the cells. Monolayer integrity was confirmed by transepithelial electrical resistance measurements and permeability of zero permeability marker compounds. Real‐time polymerase chain reaction was employed to evaluate expression levels of 84 known transporters. Samples for bidirectional permeability determination were quantified by ultra‐performance liquid chromatography. Key findings The Caco‐2 cells grow in an intact monolayer and morphologically resemble enterocytes. Genes of 84 known transporters were expressed at different levels; furthermore, expression was time depended. Functional expression of efflux transporter P‐glycoprotein was confirmed. We established a correlation between permeability coefficients of 21 tested drug substances ranging from low, moderate and high absorption with human fraction absorbed literature data (R2 = 0.84). Conclusions Assay standardisation assures the consistency of experimental data. Only such fully characterised model has the ability to accurately predict drug's intestinal permeability at the early stage of research or for the BCS‐based biowaiver application.

Fluorescence microscopy has become one of the most important tools for biologists to visualize and study organelles and molecules in a cell. Fluorescent markers are used to visualize specific molecules. One of the most used markers is green fluorescent protein (GFP), which can be expressed along with a protein of interest. However, it is known that the intensity of fluorescence decreases with observation time. To combat this problem, researchers and companies have developed protocols and additives to mitigate photobleaching. In this study, we tested the effects of the three most used culture media on photobleaching and developed a new approach of short-wavelength fluorescence recovery after photobleaching (FRAP). Photobleaching was analyzed by comparing pixel brightness on images taken with a fluorescence microscope. We determined photobleaching of GFP-expressing cells from images taken with a fluorescence microscope by comparing pixel brightness. Statistical analysis was performed to determine the average bleaching for specific culture media. The culture media analyzed had no significant effect on the photobleaching of GFP. However, a brief UV burst (15 sec) restores 50% of the original fluorescence and neither increases ROS nor decreases cell viability. To avoid artifacts in image analysis and interpretation, our study suggests using this simple method of GFP fluorescence recovery with UV light induced FRAP to extend the fluorescence lifetime and imaging of GFP molecules.