## 3 Results
### Determination of Optimal Density and FBS Concentrations for Caco-2 Cells Growth
What is more, the significant drop in CI (\(\sim\)40%) was observed after the media changing procedure at 190 h. The cell death phase started at 240 h of incubation and finished around 350 h, 60 h later than in the previous assay (Figure 3). Figure 3: The CI curves of Caco-2 seeded with cell density 5000/well (blue lines) **(A)** and 3000/well (red lines) (**B**) and cultured for 360 h (15 days) in full media (20% of FBS). The baselines include a cell-free medium well (violet) and PBS (green). Both experiments indicate that the proposed cell densities of 3000 and 5000/well and FBS concentration of 20% are inappropriate for investigation of fully differentiated Caco-2 cells; thus, we decided to decrease the cell density again to 2000/well together with incubation in media containing a low concentration of 1% FBS. As was shown in Figure 4, the cell adhesion phase was recorded between 0 and 160 h when the cells were incubated with 20% of FBS. The addition of fresh media with 20% of FBS at 165 h of incubation resulted in acceleration of proliferation and an increase in CI from around 1.0 to 3.0. Thus, in the middle of this fast proliferation state (190 h of post-seeding incubation), we decided to add 1% of FBS. It resulted in the decrease in CI back to 1.0, probably due to too dynamic change in media and FBS concentration recorded by sensitive microelectrodes, and next the cells entered the long proliferation phase. The cells reached a maximum CI of 4.5 after approximately 310 h of incubation. The plateau horizontal line was observed from 310 h up to 360 h of incubation when the experiment was terminated. The experiment was repeated next under the same conditions, but the device was set up for a longer real-time monitoring of the cell growth, i.e., 660 h (27.5 days). As was shown in Figure 5A, the decreasing of cell density to 2000/well and replacement of 20% FBS with 1% in the middle of the proliferation phase seemed to resolve the problem of too short a plateau period and too early cell death. The media changing from 20% FBS to 1% FBS was performed more carefully again in the middle of the proliferation state at 190 h of incubation, when the cells reached CI = 6.0. This time the observed before CI fluctuations during media changing from 20 to 1% of FBS were not recorded. The fresh media with 1% FBS were next added every 3-4 days. The real-time CI indicated a formation of a monolayer of differentiated Caco-2 cells ready to perform the investigations of epithelial barrier functions after the desired 432 h (18 days) of incubation (Figure 5A). The viability of cells was also monitored and confirmed with bright-field microscopy (Figure 5B,C). The long plateau horizontal line was observed from 360 h of incubation up to 520 h. Next, the quick cell death phase was recorded, which ended at around 620-640 h of incubation. To confirm the formation of TJs in the XCELLigence device, the Caco-2 cells were stained in the next experiment directly on the E-16 plate after 425 h incubation post-seeding. The expression of a tight junction protein, namely occludin, is shown in Figure 5D. Figure 4: The CI curve of Caco-2 with cell density 2000/well cultured for 360 h in full media (20% of FBS), which were replaced next by 1% of FBS at 190 h of incubation (red lines). The baselines include a cell-free medium well (violet) and PBS (green). Figure 5: The CI curve of Caco-2 with cell density 2000/well cultured for 660 h (**A**). The cells were incubated in full media (20% of FBS) which were replaced next by 1% of FBS at 190 h (red lines). The  
baselines include a cell-free medium well (violet) and PBS (green). The Caco-2 cells on E-plate 16 after 120 h (5 days) (**B**) and 336 h (14 days) (**C**) of incubation. The formation of a monolayer was observed by Leica DMi8 microscopy (10\(\times\)). The cells were seeded in the concentration of 2000 cells/well and incubated in 20% FBS-containing media for the first 6 days and then for 8 days in 1% FBS.