## 3 Results  
### Determination of Optimal Density and FBS Concentrations for Caco-2 Cells Growth  
The optimization of Caco-2 cell cultivation on E-plate 16 started with the determination of suitable cell density for the long incubation necessary for their differentiation. To this purpose various concentrations of Caco-2 cells were cultured in a complete medium(20% of FBS) in E-plate 16 and the growth of cells was observed in real-time. Results showed that when Caco-2 cells were seeded in serial dilution, including 5000, 10,000, 20,000, and 40,000 cells per well in a complete medium, concentration-dependent curves with different CI values were recorded in real-time. The cut-off time of the experiment was 50 h. During the initial 18 h of seeding the cells in 40,000/well concentration, there was a pronounced increase in the CI with a value of 8.5 on the read-outs due to rapid exponential growth of cells, followed by a confluence of cells that indicates that the cells cannot increase further and thus form a plateau, as shown in Figure 2. The wells with Caco-2 cells in 20,000 concentrations showed a comparatively slower proliferation rate than 40,000 cells. The maximum CI value after 18 h of incubation reaches up only to 1.5. The wells with 10,000 concentrations of cells in the E-plate 16 showed a CI value of 0.5, and 5000 cells showed CI similar to the baselines after 18 h of the experiment and only slightly higher than baselines after 50 h. The overall results showed concentration-dependent adhesion, proliferation, and plateau (reached only for 40,000 cells) in real-time xCELLigence RTCA S16 (Figure 2). Due to the fact that Caco-2 cells need for differentiation and TJ formation at least 17-18 days of incubation, we decided in the next experiment to seed the lowest tested amount of 5000/well (Figure 3A) [12]. Medium was changed every 72 h. In the first 48 h of cell seeding, Caco-2 cells started to adhere at the bottom of the gold-plated electrodes in each well of E-plate 16. The shape of the cells at this stage was spherical, and the total volume of medium in each well was 150 \(\mu\)L. In the next stage, the cells start the adhesion, followed by the proliferation stage, where the cells enter the rapid growth phase at around 120 h of incubation. It was observed by the device in real-time in the form of increasing CI values. When the cells reached maximum proliferation in the complete medium, a short plateau between 160 and 180 h of experiment was recorded due to the cells' highest confluence, indicating the cells will no longer proliferate. The highest CI, around 5.0, was achieved at around 170 h of incubation. In the final stage of the experiment, the cells started to lose adhesion, which marked the cell death process, which ended after 310 h of incubation (Figure 3A). Figure 2: Optimization of initial concentration of cells. Different CI curves were recorded by xCELLigence RTCA S16 from different densities of Caco-2 cells cultivated in full media containing 20% FBS. The baselines include media only, representing no cells and PBS. Due to the unsatisfactory results of the experiment and the failure to obtain the required full cell viability after the recommended incubation time required for Caco-2 differentiation, we decided to repeat the study with seeding a smaller number of cells, i.e., 3000/well. Medium was changed again every 72 h. Similarly, various phases of cell kinetics were recorded, including adhesion, proliferation, plateau, and cell death, as shown in Figure 3B. The cell adhesion phase was observed between 0 and 120 h of incubation, comparably to the previous experiment for cells seeded at 5000/well. However, an extension of the proliferation phase was observed, and the cell culture reached a maximum CI of 4.5 after approximately 237 h of incubation. On the other hand, a similar too-short plateau period, lasting about 20 h, was noted.