Distinguishing heat-treated dead cells from viable cells using frequency dependence of electrical impedance

Abstract

BACKGROUND:

There is currently no methodology for evaluating the accuracy of ablation in ablation therapy, and thus normal cells in the surrounding area can be damaged, possibly leading to complications.

OBJECTIVE:

The aim of this study was to distinguish heat-treated dead cells from viable cells using the electrical impedance-to-frequency ratio as an evaluation index.

METHODS:

Rat heart striated myocytes were cultured in a monolayer on collagen-coated microelectrodes placed in the center of an electrode-loaded chamber. The cells in the chamber were killed by heat treatment for 5 minutes at 50 °C, and the frequency response of the cell impedance was measured before and after heat treatment. The frequency of the input current was varied from 10 to 100 kHz. The measured electrical impedance at each frequency was divided by the value at 100 kHz, and we refer to the resulting values as the impedance ratio.

RESULTS:

The impedance ratio was high at low frequencies and low at high frequencies. Furthermore, the impedance ratio was lower at lower frequencies after heat treatment than before heat treatment.

CONCLUSIONS:

The electrical impedance ratio can be used to distinguish viable and dead cells after heat treatment.

Keywords

Distinction of cell survival frequency dependence of electrical impedance heat-treated cell

1. Introduction

Catheter ablation has recently become a common treatment for arrhythmias, leading to research on and development of 3D Computed tomographic (CT) imaging [1] and 3D mapping equipment [2] to assess cardiac function during catheter ablation. 3D mapping equipment allows the visualization of cardiac electrical activity in real time [3], helping to improve the postoperative cure rate in non-paroxysmal atrial fibrillation [4]. However, currently it is impossible to evaluate whether ablation has been performed correctly, and normal cells in the surrounding area can be damaged during ablation, possibly leading to complications [5].

The electrical properties of biological tissue can exhibit anisotropy, nonlinearity, and a frequency dependence [6–8]. For example, the frequency response is different in three different frequency bands due to its complex configuration [9], with β-dispersion frequencies from several kHz to 10 MHz or higher being widely used for biomedical measurements and quantitative evaluation of cells and tissues [10,11]. In this frequency band, the tissue can be considered as an electrical equivalent circuit, with the extracellular and intracellular fluids providing the electrical resistance components ( $R_e, R_i$ ) and the cell membrane providing the electrical capacitance component ( $C_m$ ). A simplified equivalent circuit for tissue is shown in Fig. 1. The cell membrane and intracellular fluid are arranged in series, with the extracellular fluid being connected in parallel [12]. When the synthetic electrical impedance of the tissue is obtained using this circuit, the complex impedance is affected by $R_e, R_i,$ and $C_m$ with respect to the real and imaginary parts of the impedance, respectively.

Fig. 1.

Simplified equivalent circuit model for biological tissue.

The cell membrane can be regarded as an electrical insulator and it acts as a capacitor with a large electrical capacitance due to its extremely small thickness. Generally, when a current is passed through a biological cell, it flows mainly through the extracellular fluid because the capacitive component of the electrical impedance is high at low frequencies. At higher frequencies, the capacitive component of the cell membrane decreases, allowing current to flow into the cell. When a viable cell dies, membrane integrity is lost, resulting in a decrease in electrical impedance, especially at low frequencies, due to the change in the electrical capacitance of the cell membrane. This suggests that cell viability can be determined using the frequency characteristics of the electrical impedance. Such measurements have been attempted in the past [12–14], but the electrical impedance has different values depending on the measurement environment, such as temperature, composition of the fluid inside and outside the cell, cell type, cell arrangement, cell density, and electrode arrangement. It is therefore relatively easy to determine the correspondence between the measured electrical impedance and cell viability, but it is difficult to determine cell viability in different environments from electrical impedance values. In addition, the electrical impedance can vary depending on the cause of cell death, but it is unclear how death due to cell denaturation caused by heating is reflected in the electrical impedance value. Electrical impedance measurements can be monitored in real time and have been applied to electrical impedance tomography. This technique applies a micro current from a pair of electrodes in the area to be measured. The potential difference or voltage generated in the area allows the rate of change of the electrical impedance or the resistivity distribution in the area to be measured, generating an image [15]. If this technology could be incorporated into electrode catheters in ablation therapy, one could visually determine the extent of dead cells based on electrical impedance data, allowing the appropriateness of localized ablation to be assessed in real time.

In this study, we measured the frequency characteristics of monolayer cultured rat heart striated myocytes before and after heat treatment, and examined whether the electrical impedance ratio (defined as the impedance at a certain frequency divided by that at 100 kHz), can be used as an evaluation index to distinguish between viable and dead cells.

2. Materials and methods

2.1. Experimental samples

We simulated the surface condition of an electrode for electrical impedance measurement pressed against tissue. Rat heart striated myocytes (Cell Line H9c2 (2-1) European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s modified eagle medium (Gibco 10567-014; Life Technologies, Waltham, MA, USA) + 10% v/v fetal bovine serum (Gibco 26140-079) + 1% v/v antibiotics (Gibco antibiotic/antimycotic 15240-062). The medium was also used as the measuring solution. A glass chamber (MED Probe MED-P5155; Alpha MED Scientific Inc., Osaka, Japan) with an inner diameter of 22 mm and a depth of 5 mm containing 8 × 8 microelectrodes (electrode size 20 × 20 μm², distance between electrodes 150 μm) was placed on a glass substrate (50 × 50 mm²). The bottom of the chamber was covered with collagen (Atelocollagen DME-02; Koken, Tokyo, Japan) and cells were seeded on the collagen. The samples were cultured in a monolayer for 5 days in an incubator (CO₂ Incubator MCO-19AIC; Panasonic, Osaka, Japan; 37 °C, 5% CO₂) to confluence.

2.2. Electrical impedance measurement system

Electrical impedance measurements were performed using a custom designed impedance measurement device to apply a current to the sample and measure the potential difference (Fig. 2(A)). The experimental system is shown in Fig. 2(B).

Fig. 2.

Experimental apparatus for electrical impedance measurement. (A) Impedance measuring device. (B) Experimental system.

Electrical continuity was ensured by using a probe on a custom printed circuit board at the conductive part of the glass chamber. The impedance measurement device was connected to the probes corresponding to each vertex of the square to be measured. The printed circuit board was placed on top of the chamber and connected to a personal computer for configuration and recording via an impedance measurement device.

Four electrodes in the chamber were selected as squares (Fig. 2(B)), a 10 μA alternating current (frequency: 10, 20, 35, 50, 75, 100 kHz) was applied to the two left longitudinal points, and the voltage was measured at the two right longitudinal points to obtain the electrical impedance frequency characteristics. This electrode arrangement results in increased sensitivity near the center of the square measurement area [16]. This allowed us to obtain information on the electrical impedance characteristics of the cells in the measurement area, especially near the center.

2.3. Electrical impedance ratio

The complex impedance contains both real and imaginary parts, and was thus evaluated using the combined absolute value. Furthermore, to investigate the utility of this approach for future imaging, the measured data at each frequency were divided by the measured value at 100 kHz, where the electrical impedance settles to a certain fixed value, to obtain the electrical impedance ratio, which is a relative value for the measured data. By using relative values, we could reduce the variation in the data at each frequency due to the variation in solution concentration with time and differences in the electrical impedance characteristics of individual chambers, which was a problem in preliminary experiments.

2.4. Heat treatment of samples

For heat treatment, the chamber sample was placed on a copper block maintained at 50 °C in a thermostatic bath and heated for 5 min to kill the cells (Fig. 3). A temperature of 50 °C is sufficient to kill cells without causing morphological changes or detachment from the substrate, assuming that the area around the cauterization site was not affected by heating. This was confirmed by preliminary experiments showing that morphological changes and detachment occurred at 60 °C but not at 50 °C, leading to cell death.

Fig. 3.

Heat treatment with copper blocks.

2.5. Experimental procedure

Electrical impedance measurements of the experimental samples were performed at room temperature (25 °C) before heat treatment. Next, the sample was heated, and after returning to room temperature, the measurement was performed again. The cells were then stained with trypan blue exclusion assay reagent (Trypan Blue Stain 0.4%; FUJIFILM Wako Pure Chemical, Osaka, Japan) and photographed using a phase-contrast light microscope and a digital camera. The resulting images were used to confirm the viability of the cells. Six measurements in the measurement range shown in Fig. 2(B) were performed using different chambers.

3. Results

The measured electrical impedance ratio for viable cells before heat treatment and dead cells after heat treatment are shown in Fig. 4. The error bars show the standard deviation. The electrical impedance ratios before and after heating were higher at low frequencies than at high frequencies, and the values after heating were smaller than those before heating. The electrical impedance ratios were significantly lower after heating, except at 10 kHz (Student’s t-test, *p < 0.05 for n = 6), and the difference in the ratio was larger at lower frequencies than at higher frequencies. The frequency characteristics of the measured synthetic electrical impedance before calculating the impedance ratio showed a similar trend both before and after heating, and settled to a constant value at 100 kHz (not shown in the figure).

Fig. 4.

Difference in electrical impedance ratio between viable cells and dead cells (Student’s t-test, *p < 0.05 for n = 6).

Figure 5 shows representative optical micrographs of viable cells before heat treatment (A) and dead cells after heat treatment (B) in a chamber. The nuclei of the cells before heating were not stained but the nuclei of all cells after heating were stained blue. Thus, the cell viability of the samples used for the electrical impedance measurement was 100% before heat treatment and 0% after heat treatment. In addition, no cell detachment from the substrate was observed following heat treatment.

Fig. 5.

Representative micrographs of rat heart striated myocytes. (A) Viable cells before heat-treatment. (B) Dead cells after heat-treatment.

4. Discussion

The electrical impedance ratio for viable cells before heating (Fig. 5(A)) was as high as 7.78 ± 3.75 and 5.38 ± 0.93 at the low frequencies of 10 and 20 kHz, respectively, and decreased with increasing frequency. The frequency dependence of the electrical impedance reflected the capacitance of the cell membrane, and the electrical characteristics were as shown in the equivalent circuit in Fig. 1. Therefore, the electrical characteristics of biological tissues can be simulated in experiments using monolayer cultured cells, as shown in this study. The electrical impedance ratio for dead cells after heating (Fig. 5(B)) was generally lower than that for viable cells before heating, and a gradual decrease was observed with increasing frequency. This increase was due to an increase in the electrical capacitance of the cell membrane, assuming that the response is as shown in the equivalent circuit (Fig. 1). However, if the effect is due to an increase in C _m, the frequency dependence curve would shift to lower frequency after heating. For example, in Fig. 4, the electrical impedance ratio at 75 kHz after heating would be 1, the same as that at 100 kHz. However, in reality, the frequency characteristic curves before and after heating showed a similar trend. This is consistent with a report on the electrical impedance frequency characteristics following cell death due to drug toxicity [12]. Therefore, dead cells in biological tissue after heat treatment can be best represented not by the equivalent circuit shown in Fig. 1, but by a circuit with C _m and R _ir (insulation resistance) in parallel, as shown in Fig. 6. Furthermore, this relationship may not be unique to heat-induced cell death, but may be common to dead cells in general.

Fig. 6.

Simplified equivalent circuit model for dead cells after heat-treatment of biological tissue.

Changes in electrical impedance may be due to degradation of the cell membrane or membrane damage in some cells. Denaturation of the cell membrane by heating was accompanied by a decrease in electrical impedance in the frequency range of 10 to 75 Hz due to a decrease in the insulation resistance of the cell membrane caused by loss of membrane integrity. The electrical impedance ratio at each frequency decreased, showing that current flowed more easily in the cells at frequencies lower than 100 kHz due to cell death caused by heating.

Preliminary experiments showed that the measured electrical impedance varied greatly at each frequency due to time variation of the solution concentration and the difference in the electrical impedance characteristics of each chamber. These variations were minimized by using the electrical impedance ratio. The error in the electrical impedance ratio shown in Fig. 4 decreased as the frequency increased, suggesting that the magnitude of the electrical impedance change (magnitude of the slope) in relation to the frequency change has an effect. The frequency dependence of the electrical impedance ratio was different for viable and dead cells, suggesting that this ratio is a simpler index for evaluating cell viability compared to handling raw data. The results indicate the possibility of discriminating cell survival in different environments by using an electrical impedance ratio that utilizes the frequency characteristics of cells and tissues. The absolute value of electrical impedance varies depending on the measurement environment, making it difficult to discriminate cell survival from the electrical impedance value, which is a limitation. However, by using the electrical impedance ratio introduced in this study, and by making the measured values after treatment relative to the initial measured values, it is possible to reduce the effects of differences in the measurement environment and system, and to determine the survival of cells under any conditions.

These results suggest that killing cells by heating causes a decrease in the insulation resistance of the cell membrane, as is the case for other causes of cell death, and that the insulation resistance can be represented by an equivalent circuit that is different from that for viable cells. Our findings show that viable rat heart striated myocytes can be discriminated from dead cells by setting the electrical impedance ratio and the slope of the frequency characteristic curve as threshold values.

5. Conclusion

The electrical impedance frequency characteristics of monolayer-cultured rat heart striated myocytes, which simulate living tissue, were measured for viable cells and heat-treated dead cells. A difference in the electrical impedance ratio was observed between live and dead cells, and the impedance of dead cells was represented by an equivalent circuit different from that for viable cells. Therefore, the survival of cells around a cauterized area can be discriminated by the electrical impedance ratio for the cells.

Footnotes

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (19K04222) from the Japan Society for the Promotion of Science.

Conflict of interest

None to report.

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