Today more and more important in research on the drugs bioavailability are cell cultures, which allow for the study of transepithelial transport of various substances. The most commonly used cell line applied in the study of the intestinal drug absorption, due to its spontaneous differentiation to the enterocyte-like structure and function, is a colon cancer line Caco-2. In order to obtain the reliable drug permeability results the monolayer must be integral and free from defects. Hence the cultivation method validation is necessary.

The validation of the method of monolayer cultures included the assay of monolayer consistency and integrity on the basis of cell morphology. The cell nuclei and tight junctions protein (ZO-1) were stained and localized by means of confocal microscopy. The membrane integrity was also assessed by the Lucifer Yellow permeability assay. These parameters were evaluated continuously during 30 day-long growth of the Caco-2 cells as well as the transepithelial electrical resistance (TEER) – the quality control check. Resulting monolayer was also assessed for the presence or absence of areas characterized by a multilayered cell growth. In order to assess the influence of the cell origin the cells from different passages and lots were cultivated.

It has been shown that between 18 and 21 day of growth the Caco-2 monolayer was obtained. The cells shapes were regular, the tight junctions were developed and the Lucifer Yellow permeability was low. The origin of the cells and the passage number however were the crucial factors influencing the time required for monolayer development. 

This study was supported by the National Centre for Research and Development (NCBiR), Grant No. R13009706

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