Impedance Measurement for the Monitoring of In Vitro Cells Cultured in the Presence of Electromagnetic Waves

Abstract

This paper explores the possibility of using the impedance measurement method used to monitor morphological changes in culture cells for use in cultures in the presence of an electromagnetic field generated by a mobile phone. For this purpose, we used Electric Cell–Substrate Impedance Sensing (ECIS), which is a real-time, label-free, impedance-based method to study cell behaviors in tissue culture. As part of the work, a device enabling the connection in a climatic chamber was prepared without the need to interfere with environmental conditions, and a test culture of mouse fibroblasts was performed. The device based on the Arduino UNO programmable platform worked like a mobile phone. During cell proliferation, it was connected to the device three times and a change in electrical parameters in the measuring system was observed. During the phone call, there was a clear change in the values of the measured parameters. However, analysis of the obtained results indicated that there was little or no effect of the presence of the electromagnetic field on the cell culture, while the observed changes in the values of impedance, resistance, and capacitance are most likely due to the separation of positive and negative medium ions in the electromagnetic field. The application of the presented method seems possible; however, in order to eliminate the separation of ions, a different type of antenna should be designed to emit a homogeneous field to the entire well.

1. Introduction

Electromagnetic waves of different wavelengths are found at virtually every point on Earth. They are of natural or industrial origin. Their impact on plants and animals still raises much controversy, which results in various studies being conducted to try to determine changes in the structure and behavior of organisms under the influence of radiation.

With the development of civilization, many sources of electromagnetic fields of different frequencies and intensities have been created. One of the greatest inventions of civilization in the field of access to information are mobile phones that use radio waves for transmission. In addition to making and receiving calls and sending text messages, they also allow access to information on the Internet. Today’s telephones, despite many advantages associated with the facilitation of daily activities, generate electromagnetic radiation that can be harmful to the human body. It can induce thermal and non-thermal effects in living organisms [1]. Radio frequency electromagnetic fields have been classified by the International Agency for Research on Cancer (IARC) as group 2B (possibly carcinogenic to humans) [2]. This means that there may be a risk of carcinogenicity, so additional research is required on the long-term, heavy use of wireless devices.

A mobile phone, through an antenna, generates electromagnetic waves used in the transmission from the radio frequency spectrum in the GSM 900/1800 MHz band (Global System for Mobile Communications) [3]. It is not ionizing radiation, which means there is not enough energy to separate electrons from atoms or molecules, to ionize them, or break chemical bonds. One of the main effects of long-term exposure is the thermal effect [4]. However, unlike infrared waves, which are absorbed mainly at the surface of objects and cause heating, radio waves can penetrate the surface and give off their energy inside biological materials and tissues. There are many results from developed research [1], from one of which it was found that a telephone conversation lasting several dozen minutes heats the head area. The temperature increase in the cranial region is temporary due to the circulatory system that acts as a thermoregulatory mechanism.

Because the effect of heating is essentially no different from other sources of heat, most research on the possible health risks associated with exposure to radio waves has focused on non-thermal effects, specifically whether radio waves have other effects on tissues besides those caused by heating. So far, no negative impact of the non-thermal effect on the human body has been confirmed [1]. The topics of current research are very broad and include, for example, carcinogenicity, cellular changes (chromosomes, genes), sleep disorders, electromagnetic hypersensitivity, effects on the reproductive system, etc. [5]. The obtained results for non-thermal effects are often related to the response of cells to temperature increases; moreover, they are usually controversial and poorly reproducible. An example may be the simultaneous research on the positive impact of the electromagnetic field on the human body. The results are inconclusive, as some have determined that several years of mobile phone use slow the development of Alzheimer’s disease by 40%, while others have determined that high-frequency electromagnetic fields increase the risk factor for the disease by increasing beta amyloid levels in the brain [6].

Studies using real exposures from devices available on the market (mobile phones or other telecommunication devices) and studies using simulated exposures from generators or “test” phones with similar but unchanging parameters, such as intensity, frequency, etc., are extremely important [7]. Exposure in real life to a similarly wide variety of animals and biological samples exposed in vivo [8] and in vitro [9] has already been found to produce a wide variety of biological and clinical effects [10]. In such research, the intensity or power density of radiation and the duration of exposure varied widely. Many research institutions have conducted studies on the effects of the electromagnetic field on DNA damage, cell cycle arrest, reactive oxygen species formation, cellular morphology, proliferation and growth profile, immunological responses, cell death mechanisms, heat-shock protein signaling, and viability, weighing the genotoxic effect of such radiation. The assessment of genotoxicity was tested using different frequencies and doses under different irradiation conditions, which included continuous or pulsed exposures as well as amplitude- or frequency-modulated waves. Such effects have often been focused on for risk assessment. The results showed that the effects were dependent on the type of cells and the choice of endpoint. However, conflicting results have been observed more than once in the same cell types in the same assay [11,12].

New research techniques are constantly being developed that would enable a better understanding of the influence of the electromagnetic field on the behavior of cells. This paper describes an experiment carried out by monitoring the electrical parameters of a cell culture that was subjected to electromagnetic radiation generated by a mobile phone. The Electric Cell–Substrate Impedance Sensing (ECIS) method was used to measure impedance, resistance, and capacitance using a small, non-invasive alternating current (AC) that is applied across the electrode pattern at the bottom of the ECIS array. This results in potential across the electrodes which is measured by the ECIS instrument. The impedance is determined by Ohm’s law. The conducted analysis aimed to answer the question of whether it is possible to use this method to study the influence of electromagnetic radiation on living cells.

2. Materials and Methods

2.1. 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 µm 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. Continuously recorded results provide precise determination of changes occurring at the cellular level in real time. The process of proliferation causes changes in electrode impedance values due to morphological modifications. In this case, the morphological changes are closely related to the life cycle of the cells and cause synchronous growth until a confluent culture is obtained. Under normal conditions, cells do not proliferate at the same rapid rate. Acceleration of this process is associated with the use of a substance that accelerates their growth. The ECIS^® system has other functions in addition to its ability to detect changes in cell impedance values. One of them is the elevated electric field mode, by means of which foreign genetic material is introduced into the culture. The advantage of performing this type of testing is that the electric field only affects the cells on the electrode. Thus, it is possible to compare their condition with cells deposited outside the electrode area. The non-invasive way of conducting experiments using impedance measurement has dramatically reduced the number of toxicity studies and animal tests for new cosmetics and drugs. With the modern and relatively inexpensive method of observing cellular functions which has been made possible by the ECIS^® apparatus, substances can be evaluated for cytotoxicity and its effects on living cells [18,19,20].

2.2. Electromagnetic Field Measurement

In the present experiment, a typical cell culture was performed, in which fibroblast cells were exposed to radio frequency radiation emitted by a cell phone for approx. 1–2 h. The results of the obtained electrical measurements will allow for the estimation of the cell culture response to the appearance of the electromagnetic field and to it switching off. The main problem was that a regular mobile phone could not be used, as inserting it into the incubator by opening it during culture would affect the environmental conditions. Additionally, leaving the phone in the chamber from the beginning of the experiment would cause cells to grow in the constant presence of an electromagnetic field resulting from the phone connecting to the base station. Therefore, a device based on an open-source electronics platform (Arduino UNO) with a SIM800L GSM/GPRS module attached was prepared, which had a switch mounted outside the climate chamber. The selected GSM module supports frequencies in the 900 MHz range, and the microcontroller was programmed in such a way as to automatically answer incoming calls. This made it possible to turn on the power at any time and connect to the device after a short while. A GSM antenna with a U.FL connector was attached to the telephone module, which was attached at the edge of the test substrate at approx. 1 cm from wells 4 and 8, and for the remaining pairs (3 and 7, 2 and 6, 1 and 5) by another about one centimeter away.

The intensity of the electromagnetic field generated by a mobile phone usually depends on the location of base stations in the vicinity, traffic in the mobile network, and the distance of the mobile phone from the base stations [21]. Therefore, before starting the cell culture, measurements of the electromagnetic field strength of the manufactured device were carried out. Measurements were made using the EMI Test Receiver R&S ESCI3 and a dedicated RS E02 near-field probe, which was placed at the following distances: 0 cm, 2.5 cm, and 5 cm from the antenna. The probe measures frequencies from 30 MHz to 3 GHz. It is a passive element connected to the 50 Ω input of the receiver via a 20 dB amplifier. An R&S^® HZ-16 amplifier was used to obtain the most accurate measurement results. It was placed between the spectrum analyzer and the measurement probe when studying weak high-frequency fields. Due to the specifications of the preamplifier, the use of which results in results being increased by 20 dB, the user is forced to subtract this value when analyzing measurements.

The obtained results were compared with the measurements of the mobile phone: Samsung, operating in the GSM900 system, measured with the same instruments, under identical conditions. A SIM card from the same mobile network operator on which the phones operated was inserted into the GSM module. From the graphs shown (Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6), it can be observed that the repeatability of the measurements was slightly better for the mobile phone, while it was worse for the developed device. However, the field strength in the prepared device had a value slightly higher than that of a mobile phone, but the measurements were relatively repeatable. Moreover, the distance of the measuring probe from the tested elements had little influence on the obtained values. It can be concluded that the intensity of the electromagnetic field at a distance of ≤5 cm is practically the same.

2.3. Cell Culture

Cells from mouse fibroblast line L-929 were derived from American Type Culture Collection (ATCC^® CCL-1TM) and cultured in Eagle-Mem medium produced by Sigma-Aldrich (Saint-Louis, MO, USA), and then used for test culture. This is one of the earliest continuous culture strains obtained, and it is used to study vesicular stomatitis and encephalomyocarditis virus, among others. The experiment was carried out in a Galaxy 170R incubator under controlled growth conditions, constant humidity, and constant air saturation of 5% CO₂. After cell proliferation and stabilization (10 days), when the culture reached at least 75% confluence, the next stage of research was started. It consisted of placing the ECIS arrays in the holders of the measuring stations (Figure 7) and then placing them in an incubator at 37 °C. Test plates containing electrodes were incubated for 24 h with medium prior to cell inoculation. After stabilization of the conditions, a mixture of 540 µL of fresh medium and 60 µL of cell suspension was introduced into the measurement matrix. The seeding of the arrays was performed with 600 µL of the suspension in each of the 8 wells (~1.2 × 10⁵ cells/mL). The composition of the medium included:

-

NaHCO₃,

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L-glutamine,

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Earle’s salts (5% CO₂),

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phenol red,

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bovine serum 5%,

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antibiotic mixture (100 IU/mL penicillin, 10 mg/mL streptomycin, 25 µg/mL amphotericin B, Pan-Biotech, Aidenbach, Germany).

2.4. Setting up of the ECIS System

In total, 8 identical wells with a bottom area of approximately 0.8 cm² and a maximum volume of 600 μL were placed into a medium, and after approximately 1 h, cells were added to 7 of them (except well #1). After inoculation with cells, they drifted to the bottom, where they attached and then spread to the surface of the electrode substrate. In well #1, the medium was left alone without cells for baseline measurements and verification of the changes that would occur without cells. Then, the culture was monitored for 93 h (Figure 8), during which resistance, capacitance, and impedance measurements were made for the signal frequency in the range of 62.5 Hz–64 kHz. The ECIS^® Z-Theta device from Applied BioPhisycs (Troy, NY, USA) was used for the measurements. The meter is controlled by a dedicated software that generates a control signal for 11 frequencies: 62.5 Hz, 125 Hz, 250 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, 8 kHz, 16 kHz, 32 kHz, and 64 kHz.

2.5. Electromagnetic Wave Exposure

Three connections were planned during the cell culture. One lasted at least one hour, the other over two hours, while the third connection was discontinued due to the observed cell apoptosis. Device activation and phone calls were made at the following times during the culture:

(1)

24 h 10 min–25 h 34 min (duration 84 min),

(2)

43 h 42 min–46 h 06 min (duration 144 min),

(3)

91 h 03 min–92 h 10 min (duration 67 min).

At that time, a phone call was made to the Arduino UNO device, which it automatically answers. After the phone call ended, the Arduino UNO was disconnected from the power supply. During the entire cell culture, the environmental conditions in the incubator were kept constant so that the only disturbance affecting the cells was the electromagnetic field generated by the phone call.

3. Results and Discussion

The obtained results were analyzed for standard frequencies for each parameter. The results of the impedance (Figure 9) measured at f = 16 kHz, the resistance (Figure 10) at f = 4 kHz, and the capacitance (Figure 11) at f = 64 kHz were repeatable for all 7 wells containing cells. The obtained results indicate that the cell culture proceeded correctly and typical changes in parameters for the development of the cell monolayer could be observed. After about 92 h, there is a rapid change in the values of the measured parameters, which indicates the death of cells, causing their weaker adhesion to the substrate. Changes in the measured parameters during the phone call were smaller than typical fluctuations during the culture. However, slight reactions could be observed in the plot of well #1, with the cell-free medium.

An analysis of the results obtained for different frequencies was carried out. After normalization of the values of impedance, resistance, and capacitance for much lower signal frequencies, significant changes due to the presence of the electromagnetic field in the vicinity of the cell culture could be observed. The most noticeable changes were observed for the phone call which lasted the longest (144 min). All measured electrical parameters clearly changed their value after the phone call started, and their values returned to those before the call after the call ended (Figure 12, Figure 13 and Figure 14). For clarity, wells #4 and #7 were not included in the graphs because they contained values that distorted the other results. The results were selected for the frequencies at which changes in the measured values were the most significant. The values of impedance at f = 500 Hz and resistance at f = 62.5 Hz decreased during the phone call and started to increase after it was switched off. The capacitance values at f = 1 kHz increased when the phone call started and decreased when it ended. The response of the measuring system indicates the effect of increasing changes, as they occur gradually and last until the phone connection is terminated. All graphs show the changes in measurement values during the operation of the device generating the electromagnetic field and immediately after it was turned off.

However, identical changes could be seen in well #1, which was free of cells and contained only medium. This means that the presence of a magnetic field affects the change in electrical conditions in the medium, and this property causes changes in the measured values of electrical parameters. This probably means that the presence of the electromagnetic field caused the migration of ions contained in the medium, which directly affected the obtained results. After disconnecting the Arduino UNO, the measured values returned to previous levels. The influence of cell activity after starting a phone call does not change the measured values, or its influence is negligible.

No significant effect of the distance of the wells from the antenna was observed. The changes in parameter values for wells #2, #3, and #5 were almost identical, although each well was ~1 cm further from the antenna. This is due to the small difference in field strength at these distances, which means that the expected effect on the wells was almost the same and the resulting differences were imperceptible.

4. Conclusions

In the presented work, the use of the Electric Cell–Substrate Impedance Measurement method to monitor the vital functions of cell cultures in the presence of an electromagnetic field was presented. A testing culture of mouse fibroblasts was carried out under standard conditions in the ECIS system with a timer-activated telephone device operating in the GSM900 system.

The results obtained from monitoring electrical parameters during the cell culture clearly indicate that there is a slow increase in impedance and resistance and a decrease in capacitance during the execution of a phone call. After disconnection, the values of these parameters slowly return to their levels before the phone call. It is likely that the apparent effect of changes in the measured parameters is mainly due to the reaction of the ions contained in the medium, and it cannot be conclusively stated that any interference with cell culture is due to the presence of radio waves.

Therefore, using this method to study the effect of electromagnetic fields on cell culture would require solving additional problems related to ion migration (separation of positive and negative ions) in the medium. In the experiment, an antenna was used, which was fixed to one side of the wells; this resulted in the creation of an inhomogeneous electromagnetic field. The solution to the problem of ion separation could be to generate a constant electromagnetic field covering the entire volume of the well, which would require the use of a spherical antenna around the well.

The recorded changes in the measurements of electrical parameters indicate that conducting in vitro breeding near a mobile phone may interfere with the conduct of research. The conclusion of the experiment is the need to limit the presence of electromagnetic radiation sources in the GSM range during research on culture cells.

The potential of the method used indicates the possibility of further development. However, one-sided mounting of the antenna should be avoided, as this will affect the ion separation of the medium. One way would be to use an antenna that emits a uniform field throughout the well. This would require designing an antenna mounted around the well. In addition, for more up-to-date solutions, tests should be carried out on the frequencies of electromagnetic waves used in the 5G standard.