Permeability is, besides solubility, one of the key properties that determine the oral bioavailability of substances, and its evaluation is most often based on the established Caco-2 cell system, cultured on Transwell® plates. The transfer of the use of the cell line from the Transwell® system to a microfluidic organ-on-a-chip system opens the possibility of evaluating permeability under conditions that may be closer to physiological conditions. This, however, requires a re-examination of the culture procedures for transfer to microfluidic systems, where, as a rule, CO2-independent medium is used. In our thesis, we evaluated the permeability properties of Caco-2 cells and EA.hy926 cells in two different cell media and the effect of culture time on the permeability properties of both cell lines, as preparation for the later transfer of the cell lines to microfluidics. Caco-2 and EA.hy926 cells were cultured on 24-Transwell® plates and their permeability was evaluated with the apparent permeability coefficient on a set of fifteen compounds. Additionally, we monitored the integrity of the cell monolayers with measurements of transepithelial electrical resistance. For Caco-2 cells, we first obtained reference results using standard culture conditions of 21 ± 2 days in Dulbecco's Modified Eagle Medium with reduced glucose content. The results from experiments in which the cells were grown in CO2-independent medium were then compared to this reference. The permeability of various compounds through Caco-2 cells after 21 days of culture in CO2-independent medium was much lower than in Dulbecco's Modified Eagle Medium with reduced glucose content; therefore, based on the measurements of transepithelial electrical resistance, we decided to shorten the culture time to 7 days. At the shortened culture time, the results were comparable to those of standard culture; thus, Caco-2 cells in CO2-independent medium behave similarly as in media for accelerated growth and differentiation. Shortening the time to the experiment is also key for experiments on microfluidic systems, where the capacity of the method and the possibility of parallelization are lower. EA.hy926 cells were cultured in Dulbecco's Modified Eagle Medium with reduced glucose content and in CO2-independent medium for 14 days. The values of the apparent permeability coefficient and transepithelial resistance were comparable between the two media, but the EA.hy926 cell monolayer does not distinguish well between permeability groups based on absorption from the digestive tract and verified on Caco-2 cells. Therefore, we also experimented with the extension of the culture time, which did not improve the distinction between the permeability groups. Based on this, we concluded that a longer culture time does not strengthen the barrier properties of the EA.hy926 cell monolayer and that culturing EA.hy926 cells in CO2-independent medium is comparable to culturing in Dulbecco's Modified Eagle Medium with reduced glucose content. This research continues with the investigation of permeability properties on microfluidic systems, where it will also be possible to check the influence of the presence of shear forces on the cell monolayer.
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