## 2 Materials and Methods  
### Impedance Measurements  
The classic method of monitoring cell culture and single cells is to observe them under a microscope. The use of impedance measurement is an interesting alternative that can replace the time-consuming optical method, and in many applications, can also expand its capabilities. In measuring electrical parameters, including changes in impedance, resistance (the real part of impedance), and capacitance (the imaginary part of impedance), a system is excited by a frequency-dependent signal and the response of that system is measured. Typically, an AC voltage signal (e.g., sinusoidal) of relatively small amplitude is used as the excitation, and the response of the electric current is measured. One such technique for monitoring vital functions of cells is the measurement of Electric Cell-Substrate Impedance Sensing [13]. It is a non-invasive in vitro method that allows for the analysis of cell activity based on their structure, morphology, and ability to reproduce, divide, or translocate. Currently, this technique, among others, is used for determining the invasive nature of cancer cells, substance toxicity, and drug testing. The main element of the measurement system is a small gold or platinum electrode deposited on the test substrate at the bottom of the cell culture vessel. Often, there is an additional insulating polymer on the metal layer that limits the contact area between the electrode and the culture, which translates into monitoring changes in the measured parameters for fewer cells [14]. The ECIS system measures the change in impedance as cells cover a 250 \(\upmu\)m diameter gold electrode and block the current flow. The measurement is made by switching the AC current, usually less than 1 mA, that flows through the cells. At frequencies below 2 kHz, much of the current flows through the intercellular spaces, providing information about cell adhesion. The use of frequency (-40 kHz) causes current to flow directly through the cell membrane. As a result, information about the amount of electrode coverage by cells is obtained. The best response to analysis for impedance, resistance, and cell culture capacity may occur at different frequencies. By default, the optimal frequencies are resistance (\(R\)) at 4 kHz, impedance (\(Z\)) at 16 kHz, and capacity (\(C\)) at 64 kHz. The optimal frequencies can also be selected empirically by analyzing the obtained results for each frequency. The total acquisition time of the experiment depends on the user and can last from a few seconds to several days. Figure 1 shows a typical measurement of the life cycle of cells (animal fibroblasts) with marked phases of proliferation (1), stabilization (2), and death (3) [15]. After thawing, multiplication, and stabilization of the cell culture, the plate is inoculated in the ECIS system. The multiplication of the culture causes the current flow through the electrodes to be blocked, thus increasing the resistance and decreasing the capacity (Figure 1). After a certain period of time, cellular stabilization occurs, in which only movement of the culture on the surface of the capacitor electrodes takes place, causing a slight release of free spaces on the capacitor. This results in slight fluctuations of the measured values. In the last phase of the cycle, the capacitance of the electrode increases and the resistance decreases. This is due to the death of the cell culture and the loss of adhesion properties to the substrate. Cells with electrodes can be considered as electrical circuits made of resistors and capacitors, and thus considered as a simple mathematical model [17]. Monitoring cellular functions using impedance measurements brings with it a whole range of possibilities. First of all, the results obtained make it possible to estimate the changes occurring in the life cycle of cells under the influence of different frequency values. During all studies, the process of deposition of cells on the electrode surface promoted the determination of their adhesive properties.