Morphological response of mammalian cells to pulsed ac fields

Bioelectrochemist~ and Bioenergetics, 33 (1994) 121-133

Morphological

121

response of mammalian cells to pulsed ac fields

Paramita M. Ghosh, Charles R. Keese and Ivar Giaever School of Science, Rensselaer Polytechnic Institute, Troy, NY 12180-3590 (USA) (Received 29 September 1993; in revised form 29 November 1993)

Abstract A newly developed method of detecting morphology and morphological changes of adherent mammalian cells cultured on thin film gold electrodes (electrical cell-substrate impedance sensing) has been used to measure the response of cells to external electrical stimuli. When sufficiently high ac voltage pulses were applied, the impedance of the cell layer changed reproducibly, indicating morphological changes in the cell layer, as no such impedance changes were seen with cell-free electrodes. The response increased with increasing voltage pulses and was distinct for different cell types. The threshold voltage across the cell layer causing an observable change was found to be of the order of 0.1 V, which is higher than that reported by others using dc fields. In certain cases, it was found that the impedance of the cell layer decreased upon pulsation and then started to recover, but decreased again before finally recovering. This suggests that two different events cause a drop in impedance of the cell layer when the adherent mammalian cells are transiently exposed to an external electrical field.

1. Introduction The effect of pulsed electric fields on mammalian tissue has been studied for a long time. A reason for these studies has been the widespread application of such fields in many areas of biology and medicine [1,2]. Reports of alterations in cell metabolism [3], protein synthesis [4], proliferation [5] and morphology [6] in response to electrical stimulus may be found in the literature. Much of the work has concentrated on cell morphology and motility changes. When the voltage applied is sufficiently high, it can cause membrane electroporation [l]; this is often accompanied by large changes in the morphology of the cells, including microvilli formation [7] and blebbing [7,8]. For lower voltages, although the alterations in cell shape are not as drastic, a loss of membrane fluidity [6] and a redistribution of stress fibers [9] have been reported. For dc fields, cells were found to align themselves and even to move physically in the direction of the field [9,10]. There is an indication that the cellular movement is directed towards the cathode [lo], although this may not be true for all cell types [ill. The response of cells to fields as low as 0.2 mV across the width of the cell (about 20 V m-r) have been reported [121; however, no cell response is seen at fields below about 10 V m-l [13]. Recently several epidemological studies have re0302-4598/94/$7.00 SSDZ 0302-4598(93)01686-H

ported that very low frequency electric fields found in the average home may cause cancer and have other health effects [lo]. Cell motion and morphology changes are usually studied with time-lapse videomicroscopy [9,101 or electron microscopy [7,8]. An alternative method is described in this paper in which cell motion changes are detected by the alterations in the impedance of the cell layer. This method, known as electric cell-substrate impedance sensing (ECES), has been developed by Giaever and Keese and has been described in detail elsewhere [14,151. The cells are cultured on thin film gold electrodes (area, about 10m3 cm’) bathed in tissue culture growth medium which acts as the electrolyte (Fig. 1). A large counterelectrode (area, about 2 cn?) is situated a short distance away. A small 4000 Hz ac voltage (to be referred to as the sampling voltage), producing a current of about 1 PA in the circuit, is used to monitor the impedance of the cell layer. Movements of the cells within the layer are detected as a small fluctuation in the measured impedance, as shown in the inset of Fig. 1. No such fluctuations are seen for cell-free electrodes, thus confirming their cellular origin 1161. These impedance fluctuations can be detected by ECIS because of the special electrode geometry employed. It is well known that the total impedance of an 0 1994 - Elsevier Sequoia. All rights reserved

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P.M. Ghosh et al. /Morphological

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response of mammalian cells to pulsed ac fields

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Fig. 1. Basic electrical set-up used in the experiments. The equivalent circuit used for calculations is shown at the top. At the bottom right-hand comer is shown the nature of curves obtained with the set-up on monitoring a cells layer not disturbed by any external stimulus. It may be seen that the impedance of the cell layer is not constant but fluctuates in time. It has been shown that these fluctuations arise owing to cell motion on the electrode. Note that both curves have been plotted on the same scale to show that the fluctuations in the resistance for this cell type (VA13) are larger than the corresponding fluctuations in the reactance.

electrochemical system includes the solution resistance and the faradaic impedance of the electrode-electrolyte interfaces [17]. The solution resistance is ohmic in nature (i.e. it is independent of the applied voltage and the ac frequency), but the faradaic impedance is not. In a system consisting of two electrodes of large surface area, the faradaic impedance is normally small compared with the solution resistance. If these electrodes are placed relatively far apart, the total impedance is dominated by the solution resistance, and any change in impedance caused by a cell layer growing on the electrodes cannot be detected. It can be shown that, for a circular electrode of radius R, the solution resistance (also referred to as the constriction resistance) varies as l/R while the faradaic impedance varies as l/R* [17]. Thus, in a system consisting of one large and one sufficiently small electrode, the faradaic impedance of the small electrode will dominate the total impedance, and small changes in the impedance of this electrode can be easily detected.

When cells are cultured on the small electrode, the impedance of the electrode will increase because the current flows around the basically insulating cells. When a 1 PA, 4000 Hz sampling signal is applied, the voltage appearing across the cell layer (the transcellular voltage) is roughly 5 mV and therefore about 2.5 mV appears across each membrane as the cytoplasm is highly conductive. This additional voltage is small compared with the normal resting potential of cell membranes (60-110 mV) and has so far had no detectable effect on cell behavior [18]; hence, it may be assumed that the method of impedance measurement described above is non-invasive. If much larger ac fields are applied, the effect on the cells becomes observable and the system is no longer non-invasive. Earlier, we have reported the observation of large changes in the measured impedance of a cell layer during the first few seconds after the application of a high voltage pulse (to be referred to as the pulsing voltage) 1191, and it was shown that this

P.M. Ghosh et al. /Morphological

response of mammalian cells to pulsed ac fields

drop reflects alterations in the membrane resistance because of pore formation. Following this initial change, a second drop in the impedance is also detected, which may be traced to morphological changes of the cells in response to pulsation. This drop lasts for minutes in contrast with pore formation which operates on a time scale of a few seconds. At intermediate pulsing voltages, when the membrane resistance is not changed, only the second drop is observed. In this paper, we extend our studies on cell layer impedance changes to understand better this second drop which represent changes in cell morphology due to electrical fields. 2. Experimental 2.1. Cells and cell culture

Different cell types including transformed human lung fibroblast WI-38/VA13 (referred to as VA131, Madin-Darby canine kidney (MDCK) epithelial cells and bovine pulmonary microvessel endothelial (B3B5) cells were used in these experiments. The VA13 and MDCK cells were purchased from American Type Culture Collection, Rockville, MD, and the B3B5 cells were provided by P.J. Del Vecchio, Albany Medical College, Albany, NY. All cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and gentamicin (0.05 mg ml-‘) (all supplied by GIBCO Laboratories, Life Technologies Inc., Grand Island, NY) at 37°C in a humidified atmosphere containing 5% CO,. 2.2. Setup The electrical circuit used is shown in Fig. 1. The cells are grown on gold electrodes evaporated on plastic tissue culture dishes as described elsewhere [14]. The natural growth medium of the cell (DMEM + 10% FBS), which has a resistivity of 54 0 cm at 37°C is used as the electrolyte. The electrodes are connected via an external resistor (1 k&l Ma> to the oscillator of a lock-in amplifier (Princeton Applied Research model 5301). The lock-in amplifier is used both to measure the impedance of the active electrode at low voltages and to generate high voltage pulses lasting 1 s across the electrode. It is interfaced with a computer that controls the lock-in amplifier and collects the data. During impedance measurements, care is taken to ensure that the transcellular voltage does not exceed 5 mV. Normally, impedance measurements are made once every second with a 4000 Hz, 1 PA sampling signal. Under certain conditions, other frequencies are used as well. However, if a lower frequency (e.g. 700 Hz) is used to measure the impedance, the current used is also made smaller (e.g. 0.1 PA). The oscillator

123

of the lock-in amplifier has an upper limit of 10 V, thus it is necessary to reduce the external resistance to reach the higher transcellular voltages. In those cases, the sampling voltage is also decreased to keep the sampling current unchanged. For example, at 4000 Hz, the following combinations of the external resistance R, and sampling potential E,,, were used: E,, = 1 V with R, = 1 MR, E,, = 0.1 V with R,, = 100 kR, and Eapp = 0.01 V with R, = 10 k0. 2.3. Procedure

The impedance of the cell-free electrode is first measured at several frequencies. Cells are then seeded, usually at a concentration of lo5 cells cm-‘, and grown to confluency. The impedance of the cell-covered electrode is then measured again at the same frequencies. The cell-covered electrode is pulsed when the ratio of the resistance of the cell-covered electrode to that of the cell-free electrode at 4000 Hz exceeds 5. For pulsation, the current in the circuit is raised to the desired level for a predetermined length of time (about 1 s) and then returned to the original level. No measurements are made during the pulse. Before and after the pulse, the impedance of the cell-covered electrode is monitored with a small ac signal as described above. At the end of the experiment, the cell layer is removed by trypsinization, and the cell-free electrode pulsed again in the same manner. 2.4. Calculation of the solution resistance Figure 2(a) shows the measured impedance as a function of frequency for a typical cell-free electrode. The open circles represent the measured resistance and the open squares represent the measured reactance. Following Warburg [20], we have represented the electrode as a series RC circuit where the electrode impedance 2 can be written in complex notation as Z=R+

1 -=R-ix iwC

(1)

where R is the resistance and X = l/wC is the reactance of the electrode (C is the capacitance), w = 2rf (f is the ac frequency), and i = (- 1)1/2. From the equivalent circuit in Fig. 1, we see that in the cell-free case the measured impedance Z,, is Z,, = Z” + Rsol+ Zl (2a) where Z, and Z, are the impedances of the small electrode and the large electrode respectively and Rso, is the solution resistance. Because the impedance of the large electrode is small in comparison with the other impedances, it can be ignored and we have Z,, = Z, + R,,

(2b)

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P.M. Ghosh et al. /h4orphological response of mammalian celis to pulsed ac j?ekb

cludes the solution resistance, does not. However, when the asymptotic value of the resistance is subtracted from the measured resistance, the plot of resistance vs. frequency also becomes linear, as shown by the curve with full circles. Hence, the asymptotic value of the measured resistance is the solution resistance. This resistance is independent of frequency and is represented by the broken line in Fig. 2(a). 2.5. Calculation of voltage across cell layer and electrode The resistance and reactance of the same electrode when covered with VA13 cells are shown as functions of frequency in Figs, 2(b) and 2(c) respectively. The broken curves represent the corresponding impedances of the cell-free electrode. It is possible to obtain a reasonable estimate of the average transcellular voltage appearing across the cell layer by a simple calculation. The measured impedance of a cell-covered electrode is

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(3)

where Z, is the impedance of the cell-covered electrode and includes the impedances of both the cell layer and the gold electrode. The impedance of the cell layer alone is therefore Z, - Z, = Z,, - Z,,. If EaPP is the applied potential, from Fig. 1, the total current in the circuit is

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(5b’) where E,* is the complex conjugate of E,, and E,* is the complex conjugate of E,. Similarly, for the cell-free case, the voltage appearing across the faradaic resistance of the gold electrode is E aPP

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response of mammalian cells to pulsed ac fields

2.6. Cell model

and Keese [15] where cells are treated as disks suspended a distance h above the electrode surface. The model shows that Z, depends on Z,, the cell membrane impedance Z,, the resistance R, between the

A more accurate but also more complex analysis of the change in impedance of a cell-covered electrode can be done based on a model developed by Giaever

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t/n-&in Fig. 3. Resistance R and reactance X of VA13 cells pulsed at 4000 Hz for 1 s durations at 17 min intervals with a 100 k0 external resistor and measured with a 0.1 V sampling voltage at 4000 Hz. The pulsing voltages used in each case’are (a), (b) 1 V, (c), (d) 3 V, (e), (f) 4 V and (g), (h) 5 V. The corresponding transcellular voltages may be found in Table 1. The solution resistance (840 fl) has not been subtracted from the total measured resistance in this figure. The positions of the pulses are indicated by arrows.

P.M. Ghosh et al. /Morphological

126

response of mammalian cells to pulsed ac fields

cells and a parameter (Y= r,(p/Zz)‘/* where p is the resistivity of the electrolyte and rc is the cell radius: 1

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According to this model, cell motion and morphological changes influence only cy and R,. Changes in R, usually result from horizontal motion of the cells while changes in (Y result mainly from vertical motion. Changes in Z, are seen only when the voltage applied across the cell layer is sufficiently high to cause electrical breakdown of the cell membrane. It has been found that generally the cell membrane resistance is of the order of lo3 0 cm*, while the membrane capacitance is of the order of 10m6 F cm-*. The values of R, and cr for VA13 cells may be found from the curves shown in Figs. 2(b) and 2(c) by fitting it with impedance values obtained using eqn. (7). These parameters change as the cells become more confluent over a period of days. For example, for confluent VA13 cells 1 day after plating, R, = 2.2 R cm* and (Y= 7 L?l/* cm, while for the VA13 cells shown in Figs. 2(b) and 2(c), which are 2 days old, these values are 4.3 0 cm* and 4.5 fii/* cm respectively. For the B3B5 endothelial cells, these numbers are approximately the same, but they are much larger for the MDCK epithelial cells. In this case, 1 day after plating, R, = 60 0 cm* and (Y= 35 fli/* cm. When the cells move under normal conditions on the electrode, the parameters R, and (Y undergo small changes, and these changes are reflected in the fluctuations in the cell layer impedance seen in Fig. 1. Large changes in cell morphology caused by external stimuli, however, cause large changes in these parameters and indicate how cells react to the stimulus. 3. Results 3.1. Effect of increasing voltages

Figure 2(a) represents a cell-free electrode with an area of 6.5 cm X 10e4 cm. As can be seen from the figure, at 4000 Hz, the total measured resistance of the

electrode is about 1200 0 and the total measured reactance is about 4500 R. The solution resistance is 840 0, i.e. the faradaic resistance at 4000 Hz is about 360 0. Figures 2(b) and 2(c) show that, 2 days after the VA13 cells are seeded, the measured resistance increased to about 11000 R (a ratio of about 9.2) while the measured reactance increased to about 6400 0. The result of pulsing this electrode with different voltages at 4000 Hz for a duration of 1 s once every 17 min is shown in Fig. 3. For all curves in this figure, the external resistor was 100 k0, and the impedance was monitored with a 0.1 V sampling voltage at a frequency of 4000 Hz. The measured impedance shown here includes the cell layer impedance, the faradaic impedance of the small gold electrode and the solution resistance. When the pulse voltage was 1 V, no effect is seen in the electrode impedance. Upon raising the pulse voltage to 3 V, small dips in the reactance begin to appear, but no effect is seen in the resistance. At 4 V, dips are also observed in the resistance, and those in the reactance become more prominent. At this point the recovery is fast and the observed dips are basically superimposed on a rise in impedance due to cell changes unrelated to the pulses. At 5 V the drops are much deeper and the recovery slower. The pulse voltage is the total voltage applied by the amplifier. and not the transcellular voltage appearing across the cell layer. The corresponding transcellular voltages have been calculated and are given in Table 1, together with

TABLE 1. Potentials across cell layer and across the gold electrode calculated from the pulsing voltage when pulsation is carried out at 4000 Hz Pulsing voltage (9

1 3 4 5

Cell-covered electrode Potential across cells/V

Potential across electrode /V

Cell-free electrode, potential across electrode /V

0.084 0.245 0.348 0.467

0.044 0.133 0.177 0.220

0.043 0.130 0.175 0.220

The potentials were calculated using eqns. (l)-(6) from the data presented in Figs. 3 and 4. Each number shown is the average of four voltages estimated to appear across the cell layer or electrode during the four pulses shown in each part of these figures. In all cases, the solution (or constriction) resistance is assumed to be 840 n. For the cell-covered electrodes, the faradaic resistance of the gold electrode under the cells has been taken to be 360 J2, and the faradaic reactance to be 4500 0. It has been assumed that the impedance of the gold electrode remained constant while the impedance of the cell layer alone changed. However, in case of the cell-free electrode, the impedances considered for voltage calculations were the actual values measured just before pulsing.

P.M. Ghosh et al. /Morphological

response of mammalian celLs to pulsed ac fields

estimates of the approximate voltage appearing across the gold electrode. To show that the observed effect is due to the changes in the cell morphology, the electrode is

ttypsinized to remove the cells, and the cell-free electrode is pulsed in the same manner for comparison. The results of this pulsation is shown in Fig. 4. The solution resistance has been subtracted from the mea-

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Fig. 4. Resistance R and reactance X of cell-free electrodes treated the same way as in Fig. 3. As before, the pulsing voltages used are (a), (b) 1 V, (c), Cd) 3 V, (e), (0 4 V and (B), (h) 5 V. The external resistor was 100 k0, the frequency 4000 Hz and the duration of the pulse 1 s. The slight drift in the impedance seen may be due to a slight warming of the medium or pH changes. The constriction resistance has been subtracted from the measured resistance to show the effect of the applied voltages on the faradaic impedance alone. Again, the positions of the pulses are indicated by arrows.

P.M. Ghosh et al. /Morphological response of mammalian cells to pulsed ac folds

128

sured resistance to illustrate that the faradaic resistance does not change on application of the pulse. As in the cell-covered electrode, the impedance remains unchanged on application of 1 V but at 3 V the reactance is slightly affected. As the pulse voltage is increased further, the resistance still does not change but the reactance drops a small amount. The voltages appearing across the cell-free electrode in each case has also been calculated and are shown in Table 1. To estimate the threshold voltage required to elicit morphological changes, we have plotted the initial drop in resistance vs. the calculated transcellular voltage for 60 pulsations (Fig. 5). The threshold voltage is defined as the transcellular voltage at which cell response is first seen. The values given are the measured drop in resistance, and it is assumed to be due to the cell layer, as Fig. 4 shows that the faradaic resistance of the electrode does not change at these voltages. From this figure we see that, for a confluent layer of VA13 cells pulsed as described, the threshold transcellular voltage is about 0.3 V. The reactance values give the same result following subtraction of the drop in reactance due to electrochemical changes (data not shown). Figure 3 showed the response of VA13 cells to transcellular voltages of 0.5 V or less. When the tran-

scellular voltage during the pulse is increased further, a peculiar phenomenon is observed (Fig. 6). Only one pulse is shown in each part of the figure to allow the impedance to settle down after the pulse. The impedance drops upon pulsation as usual and then begins to recover. However, before full recovery is achieved, a second drop in the impedance is observed. Subsequently, the impedance again recovers and sometimes even reaches a value greater than the initial value before returning to normal. Figure 6 shows that the second dip becomes more and more prominent as the transcellular voltage increases. As with lower voltage pulses, this dip is seen in the reactance before it appears in the resistance. Not is the dip to be confused with the even larger drop in impedance seen upon electroporation, at transcellular voltages of about 2 V, which lasts for only a few seconds and is not shown explicitly here. In fact, when the voltage becomes high enough to electroporate the cell membrane, as shown in Figs. 6(g) and 6(h), this phenomenon disappears, and the impedance does not fully recover in the time shown. The change in impedance of a typical cell-free electrode pulsed with a comparable voltage has been plotted in the insets of Figs. 6(e) and 6(f). As before, the

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P.M. Ghosh et al. /Morphological

response of mammalian

solution resistance (in this case, about 700 0) has been subtracted from the measured resistance of the cell-free electrode, but not the cell-covered electrode. A comparison between the responses of the cell-free and the cell-covered electrodes shows that at these voltages the faradaic resistance of the gold electrode increases slightly with pulsation, whereas the resistance of the cell-covered electrode decreases.

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3.2. Comparison of the effect of voltage on different cells We investigated the phenomenon described above with other cells as well. Figure 7 shows the response of VA13 (fibroblastic), B3B5 (endothelial) and MDCK (epithelial) cells when pulsed as above to achieve transcellular voltages of about 0.5 V. Under these conditions, the resistance change is entirely due to the cells, but a small amount of the reactance-change may have

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Fig. 6. Resistance and reactance of VA13 cells upon pulsation with the transcellular voltages (a), (b) 0.732 V, Cc),(d) 1.055 V, (e), (f) 1.76 V and (g), (h) 2.08 V. The voltage appearing across each membrane of the cell in each case will be essentially half this value. Only one pulse is shown in each part of the figure. The external resistor used in (a)-@ was 10 kfl, whereas that in (g) and (h) was 1 kfi. The sampling signals were changed accordingly, but the frequency in all cases was 4000 Hz and the duration of the pulse was 1 s. In the inset to both (e) and (f) we show the corresponding electrochemical changes of a cell-free electrode on the same scale. The higher reactance of the cell-free electrode arses because an electrode with a smaller area was used for this experiment. The solution resistance (about 700 fl) has been subtracted from the resistance data in the cell-free case only to show the response of the faradaic resistance only.

130

P.M. Ghosh et al. /Morphological

response of mammalian cells to pulsed ac fields

an electrochemical origin. To ensure uniformity of cell response, all the cells were plated out 1 day before pulsing. It was found that, at 4000 Hz, the threshold voltage for B3B5 cells was about 0.15 V, while that for the MDCK cells, like that of the VA13 cells, was about 0.3 V (data not shown). This value of the threshold transcellular voltage remained constant even when the pulsing frequency was altered, or if a more (or less) confluent cell layer was used (data not shown). Figure 7 shows that for the VA13 cells the rate of recovery is approximately constant, while for the B3B5 cells the initial rate of recovery is very fast, but soon slows down. The initial recovery of the MDCK cells is even faster, both in the resistance and in the reactance. The striking feature of the pulsed MDCK cells is that the

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resistance initially increases. Subsequently, the resistance returns to its original value, then drops to a slightly smaller value and finally returns again to its original value. On the contrary, the reactance decreases upon pulsation as in the case of the other cells, and usually there is no significant subsequent change in the value of the reactance. The upward spike in the resistance is also seen when the MDCK cells are pulsed at a different frequency but sampled at 4000 Hz. In Figs. 8(a) and 8(b) we show the response of these cells to 700 Hz pulses with a transcellular voltage of about 0.5 V. As in Figs. 7(e) and 7(f) the resistance increases while the reactance decreases immediately after pulsation. The drop in resistance following the initial spike is much more

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P.M. Ghosh et al. /Morphological

response of mammalian cells to pulsed ac fields

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Fig. 8. (a) Resistance and (b) reactance values for the pulsation of MDCK cells with a transcellular voltage of 0.5 V and a pulsing frequency of 700 Hz while measuring the cells with a 0.1 V sampling signal at 4000 Hz. In this cases, the external resistance was 100 kfl. (c) Resistance and (d) reactance values for the pulsation of the same cells with the same transcellular voltage and pulsing frequency, but now the measuring frequency is 700 Hz as well. In this case, the external resistance was 1 ML& and the sampling signal 0.5 V. In both cases, the pulse duration was 1 s.

in this case. Figures 8(c) and 8(d) show a similar layer of the same cells pulsed with the same transcellular voltage at 700 Hz, but now also measured at 700 Hz. The resistance decreases upon pulsation but immediately recovers, and there is no subsequent decrease. The change in reactance shown in Fig. 8(d) is similar to the reactance changes shown in Fig. 8(b). prominent

4. Discussion The data presented above demonstrate that cell layers can be disrupted by electric fields and that the extent of the disturbances caused depend upon the applied transcellular voltage and the type of cell used. To find the effect of the applied high voltage on the cells as opposed to the effect, on the gold electrode, we have compared data obtained with cell-covered (Fig. 3) and cell-free (Fig. 4) electrodes. One problem not taken into account in the simplified calculation used in Table 1 is that the current flow is not the same when the electrode is covered by cells and when it is not. This can best be explained using Fig. 2(c). When there are no cells on the electrode, current flow is uniform at all frequencies. When the electrode is cell covered, at low frequencies, the reactance is basically unchanged (there is a small decrease in reactance that is not completely understood). The physical reason for this is

that the current flow is still uniform under the cells. Hence it is reasonable to assume that the electrochemical response of the electrode to a high voltage pulse is the same at low frequencies with and without cells. At high frequencies, the reactance increases. The cells are now effectively blocking out some of the area, and the available area for current flow is smaller. The current flow becomes non-uniform, and electrochemical effects are no longer the same with and without cells on the electrode. However, at 4000 Hz, the capacitance with and without cells is almost the same; hence this effect is negligible and it is safe to assume that the values shown in Table 1 are reasonable. Thus the impedance changes observed with the cell-covered electrode must come mainly from changes in the morphology of the cell layer. Comparing Figs. 3 and 4, and using Table 1, we see that at a pulsing voltage of 3 V, corresponding to a transcellular voltage of about 0.25 V, the magnitudes of the changes in reactance in the cell-covered and the cell-free cases are comparable, while the resistance is not affected. Hence there are no morphological changes in the VA13 cells at a transcellular voltage of about 0.25 V, and the changes in reactance seen on pulsing a cell-covered electrode with a 3 V pulse are due to the electrochemical response of the gold electrode alone. This is in contrast with reports showing cell response at

132

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response of mammalian cells to pulsed ac fields

much lower dc fields [12,13]. Note that we measure morphological changes only. At lower transcellular voltages, other changes may occur, but these do not cause significant alterations in cell layer impedance. At higher voltages, however, cell response is obvious. Figure 4 shows that, on application of voltages of 4 and 5 V to a cell-free electrode, the faradaic resistance is not changed, and the faradaic reactance is affected somewhat. On the contrary, large impedance changes are seen in the cell-covered electrode on application of 4 and 5 V pulses. These large impedance changes are due to cell response. When the pulsing voltage is increased further, as shown in Fig. 6, two dips in the impedance are seen, and the impedance takes about an hour to stabilize. The cell-free electrode, on the contrary, never shows this behavior, illustrating that they occur in the cell layer. At even higher voltages (transcellular voltage, less than 2 V), the cell membrane is probably damaged, and the impedance changes will be due to changes in both membrane resistance and morphology. The changes in impedance seen in Fig. 6 are not understood at this point of time. The occurrence of two dips may be due to two different events. The return of the cell layer impedance to its original value about 1 h after the pulse shows that the changes in cell morphology causing these swings are temporary. The double dip is not seen at either very low or very high voltages but only in the range of transcellular voltage between 1 and 2 V. This phenomenon is not limited to the VA13 cells and has been seen in similar experiments with MDCK cells sampled and pulsed at 700 Hz with a higher pulsing voltage (data not shown). It may be noted that the VA13 cells shown in Fig. 3 take a much longer time to recover compared with the VA13 cells shown in Figs. 7(a) and 7(b). Also, the impedance of the VA13 cells shown in Fig. 3 is much higher than the impedance of the VA13 cells shown in Fig. 7. This is because the cells shown in Fig. 3 are more confluent. In general, the impedance of a cell layer is indicative of its degree of confluency; the more crowded a cell layer, the higher is its impedance. Comparing the results from Figs. 3(g) and 3(h) and Figs. 7(a) and 7(b), both showing the response of VA13 cells pulsed at transcellular voltages of about 0.5 V, it is clear that the less confluent cell layer recovers faster than the more confluent cell. The threshold voltage, however, does not seem to be affected, showing that it is a characteristic of the cell type. Figure 7 demonstrates that every type of cell has a different response to the same voltage. The difference in impedance of the three types of cell arises from the fact that cell layer parameters R, and (Yare different for each cell type. Although the cells were all plated

out 1 day before the start of the experiment with the same plating density, they reached confluency different times after plating. Hence the endothelial cells at this point in time have a lower impedance than the fibroblasts, although when confluent their impedance will be much higher. The MDCK epithelial cells, which form tight junctions, reach confluency at the same time as the VA13 cells but have a very high impedance compared with the latter. We see that each type of cell has its characteristic recovery shape. A puzzling difference between the MDCK cells and the other cell types is that the resistance increases upon pulsation when measured at 4000 Hz. Figure 8 shows that, when these cells are pulsed at 700 Hz and measured at 700 Hz, the resistance decreases but, when measured at 4000 Hz, the resistance increases as before. It is therefore not the pulsing frequency that has this effect on the cells but the measuring fre-

40 -

Rb=40 &3,,2

20 10 n 2

15 ,

3 log of frequency

I

I

_c

Rb= 40 &a~,2 R,,=20 ih,,2

-Rb

= 10 l&m2

Rb = 80

4

5

1

I

(b)

i-&Cm2

I

I

I

J

2

3

4

5

log of frequency Fig. 9. (a) Ratio of the calculated resistance of a cell-covered electrode to the measured resistance of the corresponding cell-free electrode when the assumed value of a is 4.5. The different curves correspond to R, = 5, 10, 20, 40 and 80. (b) Corresponding ratios of calculated reactance of the cell-covered electrode to the measured reactance of the cell-free electrode. The impedances were calculated using eqn. (7).

P.M. Ghosh et al. /Morphological

response of mammalian cells to pulsed ac fields

quency. A qualitative explanation for this effect may be found using the cell model described earlier and has been found to be related to the high value of R, for these cells. We have seen that the impedance of the cell-covered electrode (Z, = R, - LX,> can be calculated from the impedance of the cell-free electrode (Z, = R, - ix,,) using eqn. (7). In Fig. 9 we have plotted the ratios RJR, and X,/X,, vs. the logarithm of frequency for several values of the parameter R, and using typical values for the other variables. It is clear from the figure that, if R, decreases owing to the pulse, the resistance will decrease if measured at a low frequency and will increase if measured at a high frequency. As can be seen from the figure, the value of R, determines what constitute low and high frequencies. We define the cross-over frequency for a particular R, as the frequency below which the resistance decreases, and above which the resistance increases. Since R, is large for MDCK cells and has typical values of around 60 0 cm’, the cross-over frequency falls somewhere between 700 and 4000 Hz, explaining a decrease in resistance when measured at the low frequency and an increase at the high frequency. For the other cell types used, R, is of the order of 5 0 cm’. Here, the cross-over frequency is higher than the regular measuring frequency of 4000 Hz, and therefore the measured resistance would decrease with a decreasing R, as observed. If these cells were measured at maybe 20000 Hz, the measured resistance would increase with decreasing R,. On keeping R, constant while changing (Y, the cross-over frequency changes, and the changes in resistance are not as large (data not shown). 5. Conclusions The ECIS system is a very sensitive method of measuring morphological changes of cells in tissue culture and we have shown that in this case we observe morphological changes by subjecting the cells to large electrical fields. The observed effects can be attributed to changes in the resistivity of the cell layer contained in the parameter R, or changes in the spacing between the ventral surface of the cells and the substratum expressed in the parameter CL From the measured impedance changes we can conclude that, on pulsing,

133

the cell layer resistivity decreases (Rb decreases) and/or the cells move further away from the surface ((u decreases). In principle it is possible to distinguish between these two events by using eqn. (7), and to find the contribution of each parameter separately. This will be considered in a later, more theoretical report. Acknowledgment

This work was performed in part pursuant to a contract with the National Foundation for Cancer Research. References 1 E. Neumann, A.E. Sowers and C.A. Jordan, Electroporation and Electrofusion in Cell Biology, Plenum, New York, 1989. 2 C.T. Brighton and S.R. Pollak, Electromagnetics in Medicine and Biology, San Fransisco Press, San Fransisco, CA, 1991. J. Assailly, J.D. Monet, Y. Goureau, P. Christel and A.A. Pilla, Bioelectrochem. Bioenerg., 8 (1981) 515. K.J. McLeod, R.C. Lee and H.P. Ehrlich, Science, 236 (19871 1465. Vodovnik, L., D. Miklavcic and G. Sersa, Med. Biol. Eng. Comput., 30 (1992) CE21. M. Yaoita, H. Shinohara, M. Aizawa, Y. Hayakawa, T. Yamashite and Y. Ikariyama, Bioelectrochem. Bioeng., 20 (1988) 169. 7 M.L. Escande-Geraud, M.P. Rols, M.A. DuPont, N. Gas and J. Teissie, Biophys. Biochim. Acta, 939 (1988) 247. 8 G.V. Gass and L.V. Chernomordik, Biochim. Biophys. Acta, 1023 (1990) 1. 9 P.W. Luther, H.B. Peng and J.J.-C. Lin, Nature, 303 (1983) 61. 10 R. Goodman, C.A.L. Bassett and AS. Henderson, Science, 220 (19831 1283. 11 J. Ferrier, SM. Ross, J. Kanehisa and J.E. Aubin, J. Cell. Physiol., 129 (1986) 283. 12 CA. Erickson and R. Nuccitelli. J. Cell Biol., 98 (1984) 296. 13 L. Hinkle, C.D. McCaig and K.R. Robinson, J. Physiol., 314 (1981) 121. 14 1. Giaever and C.R. Keese, IEEE Trans. Biomed. Eng., 33 (1986) 242. 15 I. Giaever and CR. Keese, Proc. Natl. Acad. Sci. USA, 88 (1991) 7896. 16 I. Giaever and C.R. Keese, Physica D, 38 (1989) 128. 17 R. Holm, Electrical Contacts Handbook, Springer, Berlin, 1985, pp. 20-25. 18 P. Mitra, CR. Keese and I. Giaever, Biotechniques, 11 (1991) 504. 19 P.M. Ghosh, C.R. Keese and I. Giaever, Biophys. J., 64 (1993) 1602. 20 E. Warburg, Ann. Phys. Chem., 67 (1899) 493.