Radiol Oncol 2001; 35(3): 193-202. 

Cell electropermeabilization to small molecules in vitro: 
control by pulse parameters 

Alenka Ma.ek Lebar and Damijan Miklav.i. 

University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia 

A systematic study concerning the role of the different electric field parameters (pulse number, duration and 
amplitude) on electropermeabilization of DC3F cells to small molecules (propidium iodide) and on cell viability 
is presented. Cell permeabilization and viability dependence on the pulse amplitude was determined 
by twenty different sets of electrical parameters. The number of pulses varied between 1 and 64 and pulse 
duration between 20 ms and 1 ms. The most important parameter was the pulse amplitude because it triggered 
the electropermeabilization process and the process of cell death. Either in the case of electropermeabilization 
as well as in the case of cell viability experiments, the parameter U50 (the pulse amplitude leading 
to permeabilization or to the death of 50% of cell population) was not changed if the set of electrical 
parameters consisted of more than 16 pulses. This was independent of the pulse duration. The efficiency of 
permeabilization was enhanced by using of longer pulses. Such a systematic study of the influence of different 
electric field parameters on electropermeabilization and cell viability may serve as a base for optimization 
of the electropermeabilization conditions for different applications. 

Key words: electroporation; electromagnetic fields; cell survival; propidium iodide 

Introduction 

The phenomenon of cell membrane electropermeabilization 
can be described as a dramatic 
increase in the transmembrane permeability 
induced by an externally applied electric 
field. This transient state has important 

Received 17 July 2001 
Accepted 10 August 2001 

Correspondence to: Damijan Miklav.i., Faculty of 
Electrical Engineering, University of Ljubljana, 
Triaäka 25, 1000 Ljubljana, Slovenia. Phone: +386 1 
4768 456; Fax: +386 1 4264 658; E-mail: damijan@ 
svarun.fe.uni-lj.si 

practical applications like the fusion of cells 1 
and the introduction of the biologically active 
substances like drugs 2 and genetic material 3 
into cells. Electropermeabilization is nowadays 
widely used to manipulate biological 
cells, organelles, cell aggregates and tissue. 
The clinical applications gain increasing importance, 
particularly in oncology.4,5 Electropermeabilization 
can be achieved with different 
sets of pulse parameters, i.e. the strength 
of applied field (voltage of applied pulses), 
the number and the shape of pulses, their duration 
and repetition frequency. An identical 
set of electrical parameters is not necessarily 
efficient for all applications. While eight 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 

square-wave electric pulses of 100 ms delivered 
at 1 Hz is the most common used set of 
electrical parameters in electrochemotherapy, 
the pulses must be much longer (eight 
square-wave electric pulses of 20 ms delivered 
at 1 Hz or 2 Hz) for DNA electrotransfer 
in vivo.5 Likewise, it has been shown in vitro 
that only pulse duration equal to or longer 
than 1 ms was associated with the detection 
of macromolecules in pulsed and viable cells, 
while the permeabilization of cells to small 
molecules was already detected for the microsecond 
range.6 Therefore it is important to 
know how the pulse parameters affect the 
electropermeabilization process. 

The role of pulse parameters in obtaining a 
higher efficiency of electropermeabilization 
in vitro was investigated in a number of studies.
6-9 The permeabilization as a function of 
the parameters of applied electric field was 
quantified in two ways. Either the fraction of 
electropermeabilized cells in suspension was 
measured using fluorescence optical microscopy 
10, or the permeability of each cell 
was integrated over the whole cell population 
by measuring radioactive incorporation 11 or 
ATP leakage.6 Both types of information were 
gathered in some studies using flow cytometry.
12 Independently of the method, it was 
shown that the electric field intensity is the 
crucial parameter for inducing membrane 
permeabilization. The permeabilization occurs 
only if the electric field intensity is higher 
than a certain threshold value. This threshold 
value is a function of the pulse number 
and the pulse duration. It decreases by increasing 
either the pulse duration or the number 
of pulses until it reaches a "real" threshold 
value below which no permeabilization occurs 
even if using longer pulses or higher 
number of them. The "real" threshold value 
was obtained using 10 pulses or more with 
the duration longer than 100 ms.13 The permeability 
threshold depends on the molecular 
size of the probe used for its measurement: 
the larger the test molecule, the higher 

the apparent threshold.14 It also depends on 
the cell line because of their differences in 
size 15 and membrane properties.16,17 

In almost all studies, the effect of pulse duration 
was studied at a given field intensity 
and pulse number. Similarly, the effect of 
pulse number was studied at a given field intensity 
and pulse duration, and vice-versa the 
effect of field intensity was studied at a given 
pulse duration and pulse number.6-8 However, 
the fraction of electropermeabilized 
cells as a function of pulse duration at a given 
number of pulses is strongly dependent on 
selected field intensity (Fig. 1). Namely, a 
plateau is reached at shorter pulses if higher 
field intensity is used. 

In this paper, we present a systematic 
study concerning the role of different electric 
field parameters (field intensity, pulse number, 
and duration) on electropermeabilization 
of DC3F cells to small molecules and on cell 
viability. DC3F cells have been chosen because 
considerable information about the 
electropermeabilization of that strain is avail


able.2,18,19 

The fraction of electropermeabilized 
cells was quantified by the penetration 
of propidium iodide and the viability of the 
cells by their cloning efficiency. The cells 
were pulsed with twenty different sets of 
electrical parameters (Table 1). The number 
of pulses varied between 1 and 64, the pulse 
duration between 20 ms and 1 ms and the 
pulse amplitude from 40 to 600 V. 

Materials and methods 

Chemicals 

Eagle's minimal essential medium (EMEM), 
trypsin and propidium iodide (PI) were purchased 
from Sigma Chemical Co. (St. Louis, 
MO). Fetal calf serum (FCS) and L-glutamine 
were obtained from Gibco BRL (Galthersburg, 
MD), penicillin, streptomycin, gentamicin 
from Lek (Ljubljana, Slovenia), and 
Crystal violet from Kemika (Zagreb, Croatia). 

Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 


Figure 1. Fraction of electropermeabilized cells as a function of pulse duration. Cells were pulsed eight times at 
different pulse amplitudes. The error bars in the fraction of electropermeabilized cells represent standard deviations 
of the data. 

PI was dissolved in sterile H2O at a concentration 
of 100 mM. 

Cell culture 

DC3F cells, a line of spontaneously transformed 
Chinese hamster lung fibroblasts, 
were grown in monolayers in the culture 
medium consisting of EMEM supplemented 
with 10% heat-inactivated FCS, 10mM L-glutamine, 
100 units/ml penicillin, 100 mg/ml 
streptomycin, and 11 mg/ml gentamicin. The 
cells were incubated at 37°C in a humidified 
atmosphere with 5% CO2, and were routinely 
subcultured every 4 days. 

Cell exposure to electric field 

Cells from the exponential growth phase 
were trypsinized and centrifuged for 5 min at 

4°C and 1500 rpm in the culture medium. 
They were then resuspended in the serum-
free medium supplemented with 0.5 mM 
CaCl2 at a concentration of 2.2 x 107 cells/ml. 
90 ml cell suspension was mixed with 10 ml PI 
for the determination of electropermeabilization, 
or with medium supplemented with 0.5 
mM CaCl2 for determination of electropulsed 
cell viability. A 50 ml droplet of the cell suspension 
was placed between two flat, parallel, 
stainless steel electrodes (length = 6 mm, 
width = 6mm, interelectrode distance = 2 
mm). The electrodes were connected to a 
voltage generator (Jouan GHT 1287 B, France) 
generating monophasic square-wave electric 
pulses with independently adjustable electric 
parameters (voltage, number of pulses and 
duration). The cells were pulsed at 1 Hz frequency. 
The pulse parameters were monitored 
by an oscilloscope (Hameg HM 205-3, 

Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 

Table 1. Sets of electrical parameters. Repeated pulses 
were delivered at 1 Hz frequency 

Set of electric Number Pulse duration 
field parameters of pulses [ms] 
1.20 1 20 
1.100 1 100 
1.500 1 500 
1.1000 1 1000 
4.20 4 20 
4.100 4 100 
4.500 4 500 
4.1000 4 1000 
8.20 8 20 
8.100 8 100 
8.500 8 500 
8.1000 8 1000 
16.20 16 20 
16.100 16 100 
16.500 16 500 
16.1000 16 1000 
64.20 64 20 
64.100 64 100 
64.500 64 500 
64.1000 64 1000 

Germany). All experiments were performed 
under sterile conditions in a laminar flow 
hood at room temperature. 

Determination of electropermeabilization 

Electropermeabilization of cells was quantified 
by the penetration of impermeant dye 
PI. When the membrane is permeable, PI 
binds to nucleic acids and becomes highly 
fluorescent. Therefore, it is not necessary to 
wash the cells to eliminate nonincorporated 
PI as in case of other fluorescent dyes. A selected 
evaluation method avoids the negative 
consequences of pipetting and centrifuging 
the cells that have been already pulsed.20 The 
cells were pulsed and incubated 5 min at 
room temperature. Thereafter, 25 ml of cell 
suspension was resuspended in 1 ml of 0.01 
M phosphate-buffered saline (PBS, pH 7.4) 
and kept at 4°C till being analyzed by flow cy


tometry (FACSort, Becton Dickinson, CA). 
The flow cytometer was used to measure the 
number of fluorescent and therefore permeabilized 
cells. Excitation was set at the wavelength 
488 nm and emission was detected at 
640 nm. Fluorescence was recorded for 5000 
particles. Only particles large enough to qualify 
as cells were taken into consideration. The 
number of stained cells was determined and 
normalized to the number of all cells to get 
the percentage of permeabilized cells. 

Determination of electropulsed cell viability 

Cell viability was determined by means of 
colony-forming assay. After the exposure to 
electric pulses, the cells were incubated for 5 
min at room temperature. They were then diluted 
in the culture medium and seeded in 
triplicate (300 cells per 60 mm diameter petri 
dish). After five days, the colonies were fixed 
with 96% ethanol, stained with Crystal violet 
and counted. The survival of the cells treated 
with electric pulses was calculated as the percentage 
of the colonies obtained from the untreated 
control cells. 

Statistical analysis 

All experiments were repeated at least three 
times on different days. For each experimental 
point, mean and standard deviation were 
calculated. Using nonlinear regression, a two-
parameter sigmoid curve was fitted to the 
data 


where U is the pulse amplitude, -o
is the percentage 
of permeabilized or alive cells, and 

and b are the two parameters of the sig


U50 
moid curve. Parameter U50 is the pulse amplitude 
leading to permeabilization of 50% of 
cell population in the case of electropermeabilization 
and the pulse amplitude leading to 
the death of 50% of cell population in the case 

Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 197 
Radiol Oncol 2001; 35(3): 193-202. 
Figure 2. Effect of pulse amplitude on electropermeabilization of cells and cell viability. DC3F cells were pulsed at 
1 Hz one time (A - electropermeabilization, F - cell viability), four times (B - electropermeabilization, G - cell viability), 
eight times (C - electropermeabilization, H - cell viability), sixteen times (D - electropermeabilization, I - cell 
viability) and sixtyfour times (E - electropermeabilization, J - cell viability). Pulse duration was equal to (n) 20 ms, 
(t) 100 ms, (o) 500 ms and (l) 1000 ms. The symbols denote the means and the error bars are the standard deviations.

Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 

of cell viability experiments. Parameter b governs 
the inclination of the sigmoid curve. 
Smaller absolute value of parameter b means 
that the process reaches its plateau at smaller 
interval of pulse amplitudes, while its larger 
value means that the process reaches its 
plateau at larger interval of pulse amplitudes. 
In the literature, parameter b was paralleled 
with the efficiency of permeabilization.13 

Results 

In this study we focused on the effect of pulse 
amplitude, number of pulses and pulse duration 
on the cell permeabilization and viability. 
Repeated pulses were delivered at 1 Hz 
frequency. The cell permeabilization and viability 
dependence on the pulse amplitude was 
determined using twenty different sets of 
electrical parameters (Table 1). Figure 2 
shows the results of these measurements. 
The symbols denote the means and the error 
bars are standard deviations. A two-parameter 
sigmoid curve is fitted to the data of each 
set of electrical parameters. For easier comparison 
of different sets of electrical parameters, 
the parameter U50 and b of all electropermeabilization 
curves and curves presenting 
cell viability are collected in Table 2 and presented 
in Figures 3 and 4. 

Electropermeabilization 

The fraction of electropermeabilized cells in 
population is under control of the field intensity, 
the pulse duration and the number of 
pulses. Electropermeabilization occurs only 
for pulse amplitudes higher than a certain 
threshold value. This value is lower if longer 
pulses are used, or if the number of pulses is 
higher. Permeabilization curves are completely 
shifted to the lower pulse amplitudes (Fig. 
2A-E). If the set of electrical parameters consisting 
of 16 and 64 pulses, 0.5 ms electropermeabilization 
curve more or less coincides 

with 1 ms electropermeabilization curve (Fig. 
2D, E). It means that, in the sets of electrical 
parameters that consist of 16 pulses or more, 
the usage of the pulses longer than 0.5 ms 
does not change the fraction of electropermeabilized 
cells in population at certain pulse 
amplitude. 

Parameter U50 is not changed if the set of 
electrical parameters consists of more than 16 
pulses (Fig. 3A). This is independent of the 
pulse duration. The relation between the 
pulse duration and the number of pulses at a 
given parameter U50 is linear on logarithmic 
scale, if less than 16 pulses are used. 

In the literature, parameter b was paralleled 
with the efficiency of permeabiliza-

Table 2. Parameters U50 and b at different sets of electrical 
parameters. Parameters are determined by sigmoid 
curves which are fitted to the experimental data 
of electropermeabilization and cell viability using nonlinear 
regression. 

Set of Electropermea-Cell viability 
electric field bilization 
parameters |b| |b|

U50 U50 

[V] [V] [V] [V] 
1.20 354 53 664 143 
1.100 249 59 345 36 
1.500 200 33 328 50 
1.1000 156 29 265 28 
4.20 273 58 392 54 
4.100 206 43 309 34 
4.500 141 43 210 18 
4.1000 118 27 180 24 
8.20 252 41 326 43 
8.100 173 32 253 30 
8.500 122 20 159 32 
8.1000 99 13 148 18 
16.20 211 35 290 24 
16.100 140 23 191 23 
16.500 108 17 193 17 
16.1000 98 19 110 11 
64.20 211 27 274 23 
64.100 157 27 182 25 
64.500 77 19 77 17 
64.1000 57 25 80 6 
Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 

tion.13 Parameter b of the sigmoid curve is 
smaller, if the duration of the pulses is longer 
(Fig. 3B). Therefore, the efficiency of permeabilization 
is enhanced by the usage of longer 
pulses. The enhancement is less pronounced 
if the set of electrical parameters consists of 
higher number of pulses. 

Cell viability 

Also the fraction of viable cells in population 
is under control by the field intensity, the 
pulse duration and the number of pulses as 
well. Cell death occurs at the field intensities 
higher than a certain threshold value. This 
value is lower if the pulses are longer or their 
number is higher. In that case cell viability 
curves are completely shifted to the lower 
pulse amplitudes (Fig. 2 F-J). 

Likewise, in the case of electropermeabilization, 
the relation between the pulse duration 
and the number of pulses at a given parameter 
U50 is linear on logarithmic scale if 
less than 16 pulses are used. Parameter U50 is 
not changed if the set of electrical parameters 

consists of more than 16 pulses (Fig. 4A). This 
is evident for the pulses shorter than 500 ms. 

Absolute value of the parameter b of the 
sigmoid curve is smaller if the duration of the 
pulses is longer (Fig. 4B). The viability of cells 
is changed on smaller interval of pulse amplitudes 
if longer pulses are used. 

Discussion 

The application of electric field pulses to 
DC3F cells results in the permeabilization of 
their plasma membrane. Electrical parameters, 
i.e. pulse amplitude, pulse duration and 
the number of pulses, have an important role 
in electropermeabilization process as well as 
an effect on cell viability. The most important 
parameter is the pulse amplitude because it 
triggers the electropermeabilization process 
and the process of cell death. Both processes 
have their characteristic threshold values. 
Either pulse duration or the number of pulses 
can modulate these threshold values. 

In our study, we quantified the fraction of 


Figure 3. Values of parameter U50 and b in the field of pulse duration and number of pulses in the case of electropermeabilization 
experiments. Each curve represents one value of the parameter U50 (A) or b (B). The value is 
written on the curve. Scales are logarithmic. 

Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 


Figure 4. Values of parameter U50 and b in the field of pulse duration and number of pulses in the case of cell viability 
experiments. Each curve represents one value of the parameter U50 (A) or b (B). The value is written on the 
curve. Scales are logarithmic. 

electropermeabilized cells by the penetration 
of PI. Because PI also leaks into healthy cells 
over time 21, we noticed some fluorescence 
cells also in the untreated control cells. 
Therefore, it was difficult to determine the 
pulse amplitude, which triggers electropermeabilization 
process, i.e. threshold value. 
That is why our conclusions were made by 
observing electropermeabilization curves, 
their parameters U50, i.e. the pulse amplitude 
leading to permeabilization of 50% of cell 
population in the case of electropermeabilization 
and the pulse amplitude leading to the 
death of 50% of cell population in the case of 
cell viability experiments, and parameters b, 

i.e. parameter which governs the inclination 
of the sigmoid curve. Our conclusions can be 
summarized as follows: (1) In the sets of electrical 
parameters consisting of 16 or more 
pulses, the usage of pulses longer than 0.5 ms 
does not change the fraction of electropermeabilized 
cells in the population at a selected 
pulse amplitude. (2) Both in the electropermeabilization 
as well as in the cell viability 
experiments, parameter U50 is not changed if 
the set of electrical parameters consists of 
more than 16 pulses, which is independent of 
the pulse duration. (3) The efficiency of permeabilization 
is enhanced by the usage of 
longer pulses. (4) The fraction of electropermabilized 
cells and viability of cells vary at a 
smaller interval of pulse amplitudes if longer 
pulses are used. 

In spite of a variety of studies investigating 
the role of pulse parameters in the electropermeabilization 
efficiency, the studies analyzing 
the control of the cell viability by pulse 
parameters are rare.9,18 This is probably due 
to the effects of pulsing media which can contain 
a variety of undesirable and even toxic 
substances. In our study, these undesirable 
substances are Ca2+ ions. We performed 
electropermeabilization experiments in 
EMEM medium, which is a culture medium 
of DC3F cells. Because of the compatibility of 
results the pulsation of the cells in case of the 
cell viability, experiments were made in 
EMEM like in electropermeabilization experiments. 
Although EMEM Ca2+ concentration 

(1.8 mM) is in the range of approximate 
harmless limits of the extracellular fluids concentrations 
for short periods (0.5 - 2.0 mM) 22, 
Radiol Oncol 2001; 35(3): 193-202. 


Ma.ek Lebar A and Miklav.i. D /Cell electropermeabilization to small molecules 

it can be toxic due to the impairment of Ca2+ 
cellular transports during cell injury. During 
and some time after the cells are exposed to 
electrical pulses, Ca2+ easily diffuses through 
the transiently permeable membrane due to 
very low cytosolic Ca2+ free concentration 
(~0.1 mM). An increase in cytosolic Ca2+ concentration 
can directly lead to cell lysis by 
causing disruption of the cytoskeleton, DNA 
fragmentation or extensive damage to other 
cell components.23 Therefore, the cell viability 
is affected at lower pulse amplitudes as in 
the experiments prepared in media without 
Ca2+. However, the effects of electrical parameters 
have the same trends as if performed 
in Ca2+ free medium (i. e. SMEM) (data 
not shown). 

Our results were obtained using DC3F cell 
line and small test molecules. In one of our 
previous studies, we showed that electropermeabilization 
curves, obtained by PI for a given 
set of electrical parameters, are comparable 
with the electropermeabilization curves 
obtained by using anticancer drug bleomycin 
as a marker of cell permeabilization.19 So, we 
can conclude that our observations are valid 
also for bleomycin, which is the drug of 
choice for electrochemotherapy.4,5 

A systematic study of the influence of different 
electric field parameters (field intensity, 
pulse number, and duration) on electropermeabilization 
and cell viability may serve as 
a base for the optimisation of the electropermeabilization 
conditions for different applications. 


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