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Innovative Food Science & Emerging Technologies 1 Ž2000.

135᎐149

Effects of pulsed electric fields on cell membranes in real


food systems

Alexander AngersbachU , Volker Heinz, Dietrich Knorr


Department of Food Biotechnology and Food Process Engineering, Berlin Uni¨ ersity of Technology, Konigin-Luise-Str.
¨ 22, D-14195
Berlin, Germany

Received 19 October 1999; accepted 15 March 2000

Abstract

High frequency current and voltage measurements were used to determine passive electrical properties, such as the
polarization effect at intact membrane interfaces and field-induced electropermeability changes in the cellular materials during
direct current pulses. The time sequence of the electropermeabilization at the levelc of the cell membrane showed a similarity to
the breakdown phenomena observed in cell systems Žpotato, apple and fish tissues, as well as plant cell suspension cultures . when
a single pulse with critical or supercritical field amplitude is applied. A slight membrane breakdown phenomenon occurred in the
first few microseconds after the initiation of the pulse at a critical electric field strength of 150᎐200 Vrcm. Significant membrane
breakdown was observed when the field strength of the electric pulses applied directly on the cell systems was in the range of
400᎐800 Vrcm. At various field intensities, the electrical potential across a cell membrane reached a critical value of
approximately 0.7᎐2.2 V. The initiation of conductive channels across the membrane occurred within nanoseconds during the
charging process of the membrane, whereas the formation of a high-conductance membrane due to pore expansion took place
within a few microseconds. The application of a single pulse, even with supercritical field amplitude, does not necessarily cause an
irreversible membrane rupture. The insulating properties of the cell membrane can be completely recovered within several
seconds after the termination of the pulse. The biological and engineering aspects of the membrane permeabilization are
discussed in this paper. These data are utilized as the basis for the design and optimization of high-intensity pulsed electric field
applications in the areas of food science and biotechnology. 䊚 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Pulsed electric fields; Cell membrane; Electropermeabilization

Industrial rele¨ ance: This paper demonstrates the complexity of the membrane permeabilization effects of high intensity electric field pulses and
the potential to convert this basic knowledge into effective processes where controlled membrane permeabilization is required. It is further
encouraging to note how quantifiable the kinetics of reversible or irreversible membrane permeabilization can be identified.

1. Introduction first field effect results in a drastic increase in perme-


ability due to the appearance of pores in the mem-
The application of an external electric field can branes. Based on this phenomenon, many practical
induce a critical electrical potential across the cell applications of high electric fields for the reversible or
membrane, which leads to rapid electrical breakdown irreversible permeabilization of various biological sys-
and local structural changes of the cell membrane. This tems have been studied in the fields of medicine and
biology ŽChang, Chassy, Saunders & Sowers, 1992;
Zimmermann & Neil, 1996; Lynch & Davey, 1996; Ho
U
Corresponding author. Tel.: q49-30-314-71250; fax: q49-30-832- & Mittal, 1996.. Most of the studies in food science
7663. have concentrated on the effects of high-intensity elec-
E-mail address: foodtech@mailszrz.zrz.tu-berlin.de ŽA. Angersbach.. tric field pulse ŽHELP. treatments on the inactivation

1466-8564r00r$ - see front matter 䊚 2000 Elsevier Science Ltd. All rights reserved.
PII: S 1 4 6 6 - 8 5 6 4 Ž 0 0 . 0 0 0 1 0 - 2
136 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

Nomenclature data are commonly based on studies of secondary,


␶: Polarization time constant post-field effects such as the inactivation rate for mi-
t: Time crobial populations, stress reactions on biological mate-
t pore : Time of beginning pore formation after pulse
rise
rials, or diffusions from cells. The secondary effects can
Am: Membrane area of an average cell in the field also depend on other process parameters, such as pulse
direction duration, energy input and the number of pulses. Very
d: Average cell size little information is available regarding membrane
lm: Membrane thickness permeabilization kinetics, and on reversible᎐irreversi-
␴: Effective electrical conductivity of the sample
␴c : Conductivity of the cell electrolyte
ble structure changes of cells in real food systems
␴a : Conductivity of the extracellular electrolyte during and after the application of HELP. A funda-
␴ma x : Conductivity of the cells system at t ª 0 mental understanding of these phenomena is essential
␴min : Conductivity of intact cells system at t ) 5␶ for optimal process design and for the characterization
␧m : Dielectric constant of cell membrane of critical process parameters of pulsed electric fields in
␧c : Dielectric constant of cell electrolyte
x: Volume fraction of cells
the food industry.
E0 : External field strength, obtained from voltage This paper summarizes part of our ongoing work,
peak amplitude aimed at the identification of crucial parameters of
E: External field strength, obtained from voltage pulsed electric field processing, such as critical external
decay electric field strength, the breakdown voltage of the cell
j: Current density through the sample
jc : Density of current flowing in an average cell
membrane and the voltage range of reversible mem-
ja: Current density through elementary non- brane permeabilization. The time sequence and the
cellular unit dynamics of the membrane electropermeabilization
jma x : Density of the peak current flowing in the process were also studied. We used the fact that the
sample at t ª 0 electric field-induced permeability variation of initially
jmin : Density of current flowing in the extracellular
part of the sample at t ) 5 ␶
insulating biological membranes due to pore formation
jp: Density of polarization current flowing in an is reflected in large conductivity changes in cell mem-
average cell branes and, consequently, in the tissues or cell suspen-
ic: Current flowing in an average cell sions.
i p: Polarization current flowing in an average cell By measuring current and voltage simultaneously
im: Current flowing through the permeabilized
membrane
with a high time resolution, it was possible to monitor
␸: Transmembrane voltage the process of pulse-induced membrane permeabiliza-
Cm : Specific membrane capacitance tion as a function of time. For a better understanding
Aw : Total area of pore surfaces Žporated membrane of these effects, the impact of relatively long, single
area. of an average cell electric field pulses was evaluated. These pulses were
Fw : Fraction of porated membrane surface
Gm : Membrane conductance
generated by storage capacitor discharges with varying
amplitude of the pulse voltage. The data obtained by
pulse repetition gave relevant information about the
recovery of the membrane structure after few pulses,
of microorganisms ŽDoevenspeck, 1961; Sale & Hamil- and of irreversible permeabilization with repeated
ton, 1967; Martin, Zhang, Castro, Barbosa-Canovas & pulses.
Swanson, 1994; Qin, Pothakamury, Vega, Martin, Bar-
bosa-Canovas & Swanson, 1995; Grahl & Markl, ¨ 1996.
The irreversible permeabilization of cell membranes in
plant tissues offers a wide range of process applications 2. Materials and methods
where cell membrane disruption is required, including
the expression, extraction or diffusion of plant 2.1. Cell materials
metabolites, as well as affecting the mass and heat
¨
transfer of the food products ŽDornenburg & Knorr, Tissue samples: Fresh potatoes ŽSpunta. and apples
1993; Knorr, Geulen, Grahl & Sitzmann, 1994; Angers- ŽJonagold. were purchased from a local market. Trout
bach & Knorr, 1997; Knorr & Angersbach, 1998.. The fillets were treated within 1 h after slaughter.
process development must be based upon the Cell culture: Potato cells Ž Solanum tuberosum, ‘De-
knowledge of the cell-specific critical transmembrane siree’. were grown in MS medium ŽMurashige & Skoog,
voltage, ␸c wfor many different types of cell and artifi- 1962. for 14 days at 25⬚C in the dark on a reciprocatory
cial membranes, ␸c is found to be approx. 1 V ŽHo & shaker. Cell suspensions in the exponential growth
Mittal, 1996.x, or of the critical external electric field phase were filtered and resuspended in fresh medium
strength Ec . with a conductivity of 3.5 mSrcm, which was equal to
Published information about Ec is limited and the the conductivity of the cell electrolyte. The volume
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 137

ratio of the cells in the suspension was approximately insulator separating two conducting aqueous phases,
0.7. i.e. as a dielectric in a parallel plate capacitor ŽSchwan,
All samples were prepared for treatment as cylinders 1957, 1977; Pauly, 1964.. The application of an external
Žthe dimensions of the samples perpendicular to the electric field induced the charging process at the mem-
electrical field: 2 cm2 ; length: 1 cm.. brane interfaces. The relaxation time constant, ␶, is a
basic macroscopic characteristic for cell membrane
2.2. Electric field pulses protocol charge, which depends on cellular size, membrane ca-
pacitance, and the conductivity of cell and extracellular
electrolyte. Therefore, a model for examining systems
The exponential electric field pulses were applied
containing cells which is sufficiently described by the
with a high intensity pulsed electric field apparatus,
relaxation time ␶, should also reflect other electric
designed and constructed in our department. The cen-
properties of a cell.
tral components of the pulse generator consisted of a
Theoretical predictions of ␶ for a spherical cell model
capacitor charging unit ŽFUG, HCK 800M-20.000,
and for a planar bilayer membrane cell model Žcubic or
Rosenheim, D. with pre-selectable charging voltage
multilayer cell model. are shown in Table 1. The use of
and current, a 2-␮F storage capacitor ŽMaxwell,
噛30560, San Diego, CA, USA., a treatment chamber a cubic cell model for the analysis of electrical proper-
and a high voltage switch ŽBehlke, HTS 101-80-FI, ties assumes that the membrane regions involved are
FrankfurtrMain, D.. The pulse shape was monitored two planar areas on opposite sides of a cell. Consider-
on-line with a 100-MHz digital storage oscilloscope ing only the cell membrane geometry in the field direc-
ŽTektronix, TDS 220, Wilsonville, OR, USA.. The volt- tion is permissible for large cells in biological tissue
age and current at the treatment chamber were regis- which have a very small membrane-thicknessrcell-size
tered with a 75-MHz high voltage probe ŽTektronix, ratio Ž5 nm:50᎐100 ␮m. ŽHayden, Moyse, Calder,
P6015A, Wilsonville, OR, USA. and a 100-MHz cur- Crawford & Fensom, 1969; Zhang & Willison, 1991;
rent probe ŽTektronix, A6312, Wilsonville, OR, USA. Kuang & Nelson, 1998; Angersbach, Heinz & Knorr,
connected to an amplifier system probe ŽTektronix, AM 1999.. The application of the cubic cell model accu-
503B, Wilsonville, OR, USA.. The rise time of the rately describes a simulated behavior during the appli-
chamber voltage was approximately 200 ns. cation of electric field in the case of a yeast cell Ž S.
The treatment chamber was equipped with two cere¨ isiae. with a linear dimension of 8 ␮m ŽWeaver &
polished stainless steel cylindrical electrodes which were Barnett, 1992..
placed in the sample containing a PVC tube ŽHeinz, The comparison between theoretical and experimen-
Phillips, Zenker & Knorr, 1999.. The electrode area tally obtained charging time constants in Table 1 shows
was 2 cm2 . The gap was adjusted to 1 cm. The ohmic that the cell model with planar membrane areas pro-
resistance of the treatment chamber containing the cell vides a better description of the passive electrical be-
material varied in the order of 90᎐2500 ⍀ Žthe resis- havior of an individual cell, and, therefore, gives a
tance of the electrical leads was ; 1 ⍀ . in accordance more realistic basis for describing the charging process
with the degree of cell membrane permeabilization. and the breakdown of cell membranes during applica-
The peak field strength E0 was gradually increased tion of electric field pulses.
from 50 up to 2000 Vrcm, whereas the pulse duration In the following, the simplified cubic cell model with
was adjustable in a range between 0.9 and 25 ms. planar geometry is used for examination of cellular
The temperature increase in the treated samples due samples.
to Joule heating was low in all experiments. A single
pulse with a maximum field strength of 2 kVrcm
2.3.2. Discharging pulse and electrical properties of cells
caused a temperature increase of ⌬T s 0.55⬚C in the
system
sample Ženergy input 2 kJrkg.. Treatment with ten
A typical time course of the applied discharge pulse
repeated pulses Žfield strength amplitude E0 s 700
on tissues or suspensions with a high cell volume ratio
Vrcm. caused a temperature increase of ⌬T s 0.68⬚C
is presented in the Fig. 1. The relationship between
in the sample Žtotal energy input 2.45 kJrkg.. Hence,
current density, j, and field strength, E, gave the in-
temperature related alterations in conductivity were
stantaneous sample conductivity as ␴ s jrE. For the
not considered. The initial sample temperature was
calculation of the electrical properties of the cells, it
approximately 20⬚C in all experiments.
was assumed that the external electric field E0 in the
initial 8 ␮s after the start of the pulse Žouter rise time
2.3. Theoretical considerations period, approx. 0.2 ␮s. was constant.
A typical current flow across the cells in tissues or
2.3.1. Charging time constant and cell model suspensions within a short Ž␮s range. period after the
The intact cell membrane can be regarded as an application of an external electrical field Žassuming the
138
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
Table 1
Electrophysical properties of intact cell systems a

Sample Average Cell Relaxation time constant of the cell membrane ␶ Ž␮s. calculated according to: Relaxation time
cell size electrolyte Spherical model Multilayer model Cubic cell constant
d c Ž␮m. b conductivity ŽSchwan, 1957, 1977. c Žexperimental. e
ŽKuang & Nelson, 1998. modeld
␴c ŽmSrcm. dc 0.5⭈ ␧ m ⭈ d c q ␧ c ⭈ l m d c ⭈ Cm ␶ Ž␮s.
1 1
␶ s ⭈ Cm ⭈ Ž q . ␶ s ␧0 ␶s
2 ␴a 2 ⭈ ␴c ␴c l m 2 ⭈ ␴c

Potato tissue 100 5.5 1.40 0.40 0.90 0.70


Apple tissue 70 1.2 3.50 0.95 2.30 1.40
Fish tissue 75 6.0 0.94 0.26 0.62 0.63
Suspension cultured 50 3.5 1.10 0.30 0.66 0.45
potato cells
a
Cm is the specific capacitance, for biological membranes Cm is in the order of 1 ␮Frcm2 ŽSchwan, 1977.. Further note: ␧ 0 s 8.85= 10y1 2 Frm; ␧ m s 2.3; ␧ c s 78; l m s 5 nm.
b
Light microscopic examination of wet sections of the tissues investigated in this work showed that examined cells were roughly isometric.
c
Calculated under the assumption that the conductivity of cell electrolytes, ␴c , and extra cellular conductivity, ␴a , are identical.
d
According to a simplistic cell model with one-dimensional planar membrane.
e
Determined directly from experimental relaxation curves during DC pulse with subcritical field strength Žsee Figs. 2 and 4..
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 139

Fig. 1. Current density, j, field strength, E, and effective tissue conductivity, ␴, vs. time during a single discharge pulse of a 2-␮F storage
capacitor at various field strength amplitudes, E0 , through potato tissue. For E0 s 66 Vrcm, the peak current density was 0.27 Arcm2 and the
total pulse width was 15 ms.

Fig. 2. Time course of field strength Ža, b. and current density Ža⬘, b⬘. in a potato cylinder in the first 8 ␮s of a pulse with a field strength
amplitude of 66 Vrcm Ža, a⬘. or of 880 Vrcm Žb, b⬘.. The complete pulse course is shown in Fig. 1. The Roman numerals in Ža⬘. are defined
within the text ŽSection 2.3..
140 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

passive electrical properties do not change. is shown in The following equation is related to first order differ-
Fig. 2a. As for a heterogeneous dielectric, the current ential equations which describe the interaction of sev-
time course could be divided into three characteristic eral membrane properties, i.e. the parallel combination
periods. In the first instant, during the rise of the of a capacitance Cm and a resistance Rm in the equiva-
electric field, electrical polarization and charging of the lent circuit with cell current ic s jc. Am .
‘geometrical’ capacity caused the rapid development of
current peak jmax Žcurrent jump, period I.. During the d␸
second period ŽII., polarization effects arose from the i c s i p s Cm if t - tpore Ž3.
dt
induced charge accumulation at the interface of intact
membranes ŽMaxwell᎐Wagner effect.. The third period For t - tpore the polarization current charges the
related to a time range of 3᎐5 ␶. The polarization intact membrane. At t ) tpore , the formation of aqueous
process was terminated and the electrical current den- pores on the membrane interface occurred and the
sity decreased to a minimal value, jmin . When E0 s instantaneous current, im , flowing across the membrane
constant, jmin remained constant Žperiod III. and was developed. This depended on the fraction of the aque-
adjusted through the electrical conductance of the ex- ous pores and on decreased transmembrane voltages.
tracellular liquid. The cell current was given as:
The same effects for biological matter were postu-
lated in the frequency-domain as ␣- and ␤-dispersion
ic s i p q im if t ) t pore Ž4.
ŽSchwan, 1957, 1977.. The Laplace transformation may
be applied to translate the Maxwell᎐Wagner effect at
the membrane interface in the DC field Žtime-domain. For an intact cell, the current ic was equal to the
to an AC field Žfrequency-domain.. The analysis of polarization current ip as a result of the charging of the
conductance spectra within the ␤-dispersion range of insulated membrane through the cell electrolyte Žin
3᎐50 MHz ŽAngersbach et al., 1999. and of the conduc- case of high cell concentration . from the pulse genera-
tivity relaxation in the time range from 0.2 to 8 ␮s after tor. With this assumption, Eq. Ž3. could be transformed
the beginning of the pulse Žsee Fig. 4, curve number 1. to give the current density, and could be written as
provided identical information for all materials ex- follows:
amined according to the passive electrophysical proper-
ties of the cell system, such as volume ratio of intact jc s jp s ␴c ⭈ E0 ⭈ exp Ž ytr␶ . Ž5.
cells in the sample, charging time constant of the cells,
or maximum and minimum values of sample conductiv-
where t is the process time and ␴c is the specific
ity Ž ␴max , ␴min ..
electrical conductivity of the cell electrolyte. From Eqs.
The time dependent density of the direct current, j, Ž2. and Ž5. the transmembrane potential for an intact
flowing through the elementary layer Žorientated per-
membrane can be given as:
pendicular to the electric field. of the system contain-
ing cells after the application of an external electric
d
field E0 could be described as: ␸s E w 1 y exp Ž ytr␶ .x Ž6.
2 0
j s x⭈ jc q Ž 1 y x . ⭈ ja Ž1.
Eq. Ž1. shows the relationship between the instanta-
neous current density in the interior of the cells, jc ,
where x is the volume ratio of intact cells; jc is the and the total current density in the sample, j, which
density of current flowing in an average cell; and ja is could be measured. The values of x and ja in Eq. Ž1.
the current density, flowing through an elementary could be eliminated by analyzing the relationships given
non-cellular unit. by the exposure to a subcritical electrical field. For cells
Here, it was assumed that the layer thickness was with intact membranes at the onset and at the end of
equal to the average cell size and that the cells were the polarization process waccording to Eq. Ž5.x the fol-
uniformly distributed within the sample. lowing equations applied:
The field-induced time-dependent voltage drop across
a cell membrane Žneglecting the natural potential of a
At t ª 0 jmax s x⭈ ␴c ⭈ E0 q Ž 1 y x . ⭈ j a Ž 7a .
biological membrane, which was in the order of 10y2 V
ŽTsien, Hess, McClesky & Rosenberg, 1987; Tsong,
1996. can be expressed by Eq. Ž2.: At t G 5␶ jmin s Ž 1 y x . ⭈ ja Ž 7b .

d j where jmax and jmin denote the experimentally ob-


␸s E0 y c
ž / Ž2.
2 ␴c tained current density in the sample at the characteris-
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 141

tic time periods t ª 0 and t G 5 ␶, respectively Žsee Fig. where lm is the membrane thickness Ž; 5 nm.. Eq. Ž11.
2aX .. Hence, Eqs. Ž1., Ž7a. and Ž7b. give: is valid when the resistance of the lipid area is very
high and the solution conductivity in the individual
jmax y jmin pore is unchanged.
js ⭈ jc q jmin Ž8.
␴c ⭈ E0

Eq. Ž8. allowed the calculation of the current jc 3. Results and discussion
flowing in an average cell interior from one experimen-
tal current᎐time curve when the membrane is intact. In the present study, the pulse width of fully expo-
During the application of high field strength pulses, nential capacitor discharges through the material ex-
membrane rupture occurs, and the cell current jc can amined was strongly dependent on the effective sample
be determined as follows. Assuming that the contribu- resistancerconductivity, which varied in correspon-
tion of the current density jmin , flowing through the dence to with the applied field strength. In Fig. 1, this
extracellular part, to the total current density of the is illustrated using potato tissue.
cell system with intact or permeabilized membranes Obviously, in the experiment with a pulse amplitude
remained unchanged, the cell current density jc in the of E0 s 66 Vrcm, the field strength was not sufficiently
case of a ruptured membrane could be described as: high to initiate electropermeabilization of the cell
membranes. The discharge current only passed through
␴c ⭈ E0 ⭈ w j y j⬘min x the extracellular matrix of the potato tissue because
jc s Ž9.
jmax y j⬘min the intracellular electrolyte was isolated by intact mem-
branes. The effective conductivity remained at its lowest
with value wapprox. 0.33 mSrcm, which is common for the
measurement of passive electrical properties of intact
jmin ⭈ E0 potato tissue with DC or AC in the 1᎐5 kHz range
j⬘min s Ž 10.
Esub ŽShaw & Galvin, 1949; Hayden et al., 1969; Angersbach
et al., 1999.x. Consequently, the current density and
where jmin is obtained from experimental data by the field strength decayed exponentially, with the highest
application of a first pulse on a subcritical field strength time constant of ; 3 ms.
level, Esub Žfor examined cell materials, as a rule lower In contrast to the first test for the pulse application
than 100 Vrcm.. The values of jmax and j were ob- with an amplitude of 880 Vrcm, the field strength E
tained from recorded current᎐time curves by the appli- decayed exponentially with a very low time constant,
cation of a second pulse with critical or supercritical approximately 200 ␮s. The total sample conductivity
field strength Ec in the same sample. increased to 5.5 mSrcm as a result of the elec-
For the ruptured membrane, the relationship tropermeablization of previously insulating cell mem-
between a membrane current, im , and the transmem- branes.
brane voltage wEq. Ž2.x gave the membrane conduc- Fig. 2 illustrates the detailed time course of j and E
tance, Gm s im r␸, which could be attributed to the sum in potato tissue in the first 8 ␮s after the initiation of
of the instantaneous conductances of all individual the pulse with amplitudes of 66 and 880 Vrcm Žthe
transient pores. The determination of the membrane complete pulse sequence is shown in Fig. 1.. Through
current, im , deduced from Eq. Ž4., was based on the the application of a sub-critical field strength, the exis-
assumption that the membrane capacitance, Cm , tence of insulated membrane properties was clearly
changed only negligibly during the formation and detected by means of an exponentially decaying polar-
widening of the pores. This assumption holds because ization current in the cells within the first few mi-
only a small fraction of the membrane was occupied by croseconds. With a field strength level of E0 s 880
aqueous pores ŽFreeman, Wang & Weaver, 1994; Win- Vrcm, the current through the samples showed a devi-
terhalter, Klotz & Benz, 1996., and the polarization ation from exponential decay at 0.4 ␮s, indicating that
current ip exponentially decays even further after mem- electric breakdown in the cell membranes had oc-
brane breakdown. curred.
Assuming that the increase in membrane conductiv-
ity during field-induced breakdown resulted from the 3.1. Membrane polarization and electroporation
formation of cylindrical pores with ohmic behavior, the
time-dependent total Žaqueous. pore area, Aw , of the The changed conductivity of the total sample in the
membrane with cell liquid-filled pores was given by: pulse experiment yielded integral information about
the extent of the pulse-induced changes in the struc-
lm ⭈ Gm tural properties of the complete cell system. The time
Aw s Ž 11.
␴c sequence and dynamics of the electropermeabilization
142 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

at the level of a cell membrane can be characterized by


the present model of cell systems, which is expressed in
Eqs. Ž1. ᎐ Ž11.. The equations were obtained with the
assumption that within this small time period, the ex-
ternal field strength, which is equivalent to the pulse
amplitude, is constant Žsee Fig. 2a,b.. By the applica-
tion of a pulse with a field strength amplitude of 66
Vrcm, the membrane was exponentially polarized to
␸ s 0.3 V without an influence on its structural state.
At higher pulse amplitudes Žsuch as 880 Vrcm., a
current flowed towards the membrane through the
intracellular electrolyte and the transmembrane poten-
tial, ␸, increased exponentially with time Žtime constant
0.7 ␮s.. Close to 0.4 ␮s, ␸ reached a value of 1.7 V, and
later decreased to a residual value of 0.1 V, as a
consequence of pore formation and a drastic increase
in membrane conductance ŽFig. 3.. As soon as the
membrane was ruptured, the membrane current, im ,
was also initiated, which in turn resulted in the sudden
observed increase in cell current, ic .
The decay of the membrane voltage and the simulta-
neous increase of the membrane current, im , indicated
that the membrane conductance, Gm , had drastically
increased. There was a sudden rise in the membrane
conductance from approximately 10y8 S wthe initial
specific conductivity of a cell membrane was of the
Fig. 3. Typical current, voltage and membrane property changes in
order of 10y4 Srcm2 ŽWinterhalter et al., 1996; an individual cell Že.g. a potato cell in tissue. induced from an
Zimmermann, 1996.; the membrane area of the potato external electrical field with supercritical field strength: Ža. currents
cell in the field direction was 10y4 cm2 x to 5 = 10y3 S and Žb. transmembrane potential in an average potato cell; Žc.
in less than 5 ␮s. Such a massive conductivity increase membrane conductance, Gm , and total pore area, Aw , increase in the
membrane surface in the field direction. Field strength amplitude
could be explained by the formation of an aqueous
E0 s 880 Vrcm Žtime course of field strength and current density in
phase at the membrane interface which has been the sample is shown in Fig. 2b,b⬘.. The intracellular current, ic , was
observed for artificial lipids as well as for cell mem- calculated as ic s jc ⭈ Ac by using the Eq. Ž9. in which j⬘min s 0.32
branes ŽZimmermann, 1982; Chernomordik, Sukharev, Arcm2 Žobtained according to Eq. Ž10... im s current through the
Popov, Pastushenko, Sikorko, Abodor & Chizmadzhev, membrane. The polarization current, ip , was calculated as ip s jp ⭈ Ac
with polarization current density, jp , obtained from Eq. Ž5. using
1987; Weaver & Barnett, 1992; Ho & Mittal, 1996.. A
␶ s 0.7 ␮s.
rapid rupture of limited areas of the cell membrane,
but not of the entire membrane, may be possible
ŽChernomordik et al., 1987; Freeman et al., 1994; Ho & aqueous area of the membrane, Fw s Aw rAm , was
Mittal, 1996.. In the case discussed above, the total found to be small and never exceeded 10y2 Ži.e. ap-
area of aqueous pores, Aw , reached a maximum value prox. 1% of the membrane area.. Similarly, 0.01᎐1% of
of up to 46 ␮m2 ŽFig. 3c., suggesting that approxi- lipid bilayer surface was affected by such defects
mately 0.46% of the membrane area in the field direc- ŽChernomordik, Sukharev, Abidor & Chizmadzhev,
tion was filled with an aqueous phase. This small maxi- 1983; Tsong, 1990..
mum value of the fraction of aqueous area of the A better comparison of experimental data through
membrane supports the assumption that the structural the application of various field strengths is given by the
changes within the entire membrane can have a negli- presentation of j᎐E data as effective conductivity ␴ s
gible change in membrane capacitance ŽFreeman et al. jrE0 ŽFig. 4.. For all cell materials examined, the shape
1994; Winterhalter et al., 1996.. of the time behavior of ␴ indicated excellent repro-
The data analysis for all cell materials examined ducibility for the membrane charging processes before
indicated that only a small fraction of the membrane the start of the membrane rupture and also for various
was occupied by pores. This is typical for reversible external field strength E0 . The polarization time up to
electroporation phenomena in biological and artificial the moment of membrane breakdown and the dy-
membrane breakdown ŽHo & Mittal, 1996; Freeman et namics of pore formation were strongly influenced by
al., 1994.. Although dramatic changes occurred in the the field strength. When higher amplitudes than the
membrane conductivity during the pulse, the fractional critical level were used, the conductivity changes of the
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 143

Fig. 4. The field induced permeabilization in the first 12 ␮s after pulse start with various field strength amplitude E0 ŽVrcm. for: potato tissue
Ža.: 1᎐100, 2᎐250, 3᎐400, 4᎐680, 5᎐1000, 6᎐1700; apple tissue Žb.: 1᎐100, 2᎐270, 3᎐550, 4᎐750, 5᎐1100, 6᎐1700; culture cell suspension Žc.:
1᎐110, 2᎐250, 3᎐330, 4᎐590, 5᎐1100, 6᎐1800; fish tissue Žd.: 1᎐80, 2᎐300, 3᎐500, 4᎐900. The effective conductivity course was calculated from
monitored current and voltage variation with time. The shortest total pulse width in all experiment was 900 ␮s.

sample show a deviation from exponential decay in the speed of pore formation suggests that this action was
short time scale after pulse initiation. This indicated located at the lipid domain of the cell membrane
that electric breakdown in the membranes of individual rather than at protein channels. Pore initiation was
cells occurred during the charging process, i.e. in the most likely triggered by defects in the lipid structure
time period of 3᎐5 ␶. In all experiments, the charging and occurred in the range of nanoseconds ŽCoster &
process was followed by a rapid development of the Zimmermann, 1975; Benz, Beckers & Zimmermann,
membrane current which was caused by a rupture and 1979; Gruenwald, Frisch & Holzwarth, 1981; Zimmer-
the evolution of high conductance membranes, initi- mann, 1996.. The pore widening of the lipid bilayer
ated by the critical or supercritical electrical field. happened in the microsecond range ŽSerpersu, Kinosita
With increasing field strength, the charging time up & Tsong, 1985; Ho & Mittal, 1996.. Other issues also
to the moment of membrane rupture was reduced, and needed to be considered. If a protein channel was
the rate of pore widening and the total area of the punctured by an electrical potential, the local heating
pores of the membrane increased. This was reflected by due to excessive membrane current might thermally
the rise in conductivity of the samples after apparent denature the protein Žor modify it electrically ., a reac-
membrane permeabilization ŽFig. 4.. tion known to occur in the range of milliseconds᎐sec-
onds ŽKim & Baldwin, 1990; Tsong, 1996.. Within a cell
3.2. Biological aspects of the membrane permeabilization membrane, the opening and closing of protein channels
could be controlled by the transmembrane electrical
Several hypotheses have been established that rea- potential. The gating potential of a typical ion channel
son that in real cell membranes, pore initiation may is in the range of 50 mV ŽTsien et al., 1987; Tsong,
occur in lipid domains or at protein channels 1996.. The argument that an intense pulse may force
ŽChernomordik et al., 1987; Glaser, Leikin, Cher- these channels to open before the breakdown of the
nomordik, Pastushenko & Sokirko, 1988; Tsong, 1990; lipid bilayer occurs may not be valid, because the
Zimmermann, 1996; Ho & Mittal, 1996.. In all our charging curve up to the breakdown point Žand also by
experiments it was observed that the immediate initia- super-critical field intensity . showed an exact exponen-
tion of conductive channels across the membrane and tial course with a time constant ␶, which was character-
the rapid formation of a high conductance membrane istic of an intact unchanged membrane Žsee Figs. 2 and
due to their expansion took place in less than 1 ␮s. The 4.. The simultaneous opening of protein channels and
144 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

Žapprox. 1 V., at which membrane breakdown was first


observed in a series of simulations in which E0 was
varied. When a slightly smaller transmembrane poten-
tial Že.g. ␸ - 0.7 V. was used, no membrane rupture
occurred. Similar results were found with cell mem-
branes in potato and in the corresponding cell suspen-
sions, as well as in fish tissues ŽFig. 5..
The critical transmembrane voltage for most eukary-
otic cells has been reported to be approximately 1 V
Ž0.5᎐1.5 V., independent of the method used ŽSale &
Hamilton, 1967; Coster & Zimmermann, 1975; Ho &
Mittal, 1996; Lynch & Davey, 1996; Zimmermann,
1996.. In our experiments the lowest breakdown volt-
age was approximately 0.7 V Žapple tissue, E0 s 200
Vrcm and potato cultures suspension, E0 s 250 Vrcm..
Fig. 5. Critical transmembrane voltage for membranes of cells in However, such a value was only observed for the exper-
potato tissue Ž䢇., a potato cell culture Ž⽧., apple tissue ŽB. and fish iments when the charging time of the membrane was
tissue Ž^. as a function of field strength amplitude. Critical voltage is approximately 3᎐5 ␶ Žfor apple tissue, at 5.0 ␮s; for
defined as the maximal electrical potential across the cell membrane. potato cell suspension cultures, at 1.9 ␮s., i.e. at the
end of the exponential charging process. In this case,
initiation of pore formation in the lipid matrix is quite the external field strength could be defined as critical.
unlikely. Furthermore, by the application of a single For potato tissues, the first indication of membrane
pulse, we observed only reversible field-induced changes breakdown was observed at E0 s 180 Vrcm Žinduced
of the membrane structures. An application of a sec- breakdown voltages 1.2 V, charging time s 3.0 ␮s..
ond pulse with a field strength of 700 Vrcm to potato By the application of super-critical amplitude pulses,
tissue ŽFig. 7. showed that in the time period between the cell membranes were charged to substantially higher
the pulses, the properties of the sample before perme- voltages within a shorter time. For a pulse of 1700
abilization was repeated were almost identical to the Vrcm amplitude, the membrane in potato cells was
intact state. This suggests an almost complete recovery polarized up to 2.2 V within 0.23 ␮s. Although the
of the membrane structure after the first pulse. How- classical electroporation theory assumes a breakdown
ever, a recovery of protein channels within 1 s is membrane voltage in the critical or super-critical range,
unlikely. a tendency towards a rise in the breakdown voltage
Several studies also suggested that the early stages of with the increasing external field was observed for all
electroporation of biomembranes are primarily associ- cell materials examined ŽFig. 5.. Similar relationships
ated with the appearance of pores in the lipid matrix, were also observed for artificial lipid membranes ŽBenz
rather than in the protein-rich domains of membranes & Zimmermann, 1980; Winterhalter et al., 1996.. The
ŽChernomordik et al., 1987; Glaser et al., 1988; Neu- application of Eq. Ž6. for the planar model Žor ␸ s
mann, 1989; Chernomordik, 1992.. 1.5⭈ d⭈ E⭈ cos␣ for the spherical membrane model. to
calculate the membrane potential induced and to op-
3.3. Critical transmembrane potential timize the relation between field intensity and pulse
duration should be used cautiously, because these
Although various cell materials have different mor- equations assume a non-variable breakdown voltage.
phological structures and electrophysical properties, the
similarity between the voltage dependent membrane 3.4. Lifetime of the perforated membrane
ruptures suggested that the same mechanism was re-
sponsible for the electropermeabilization of the cell The data in Fig. 6 show the monitoring of simultane-
membranes, namely, charging of the insulated mem- ously measured current density and field strength
brane up to a critical value. Thus, the direct field changes during the first 80 ␮s of the exposure of potato
effects on the membrane structure were expressed as tissue to an electric field as previously shown in Fig. 1.
interfacial polarization up to strong transmembrane The changes in the conductivity of potato tissues indi-
fields Žin our experiments in the order of 1400᎐4400 cated that, within this period, the cell conductance did
kVrcm, considering a membrane thickness of 5 nm. not increase appreciably during the intensive pulse.
which, in turn, induced structural changes ŽNeumann, This supported the conclusion that, by the application
1989; Ho & Mittal 1996.. of a pulse with super-critical field strength, the forma-
For apple cells, the maximum transmembrane volt- tion of the conductive state of the membrane was
age achieved Ž1.3 V. was close to the threshold value completed within a few microseconds after the start of
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 145

the pulse. Under these conditions, pulse parameters branes. Electrical breakdown experiments on cell mem-
such as magnitude, the shape of the decaying electric branes have also led to the conclusion that local heat-
field, and the total pulse duration determined only the ing may play an important role after the primary process
secondary pulse effects, such as lifetime of the pores of electrical breakdown ŽCoster & Zimmermann, 1975;
already formed. Benz et al., 1979..
Once a membrane breakdown occurs, a current, im , The high conductance of the membrane during the
flows through the aqueous pore. Joule energy is gener- voltage relaxation following a pulse with supercritical
ated during the field application. An integration of field strength amplitude remained unchanged even
i2m rGm over the full pulse duration wassuming that at 8 when the current and voltage started to decay by the
␮s, the value of Gm is approx. 5 = 10y3 S Žsee Fig. 3c., experiment with E0 s 880 Vrcm ŽFig. 1.. With this
and that further conductance of the perforated mem- amplitude, the sample conductivity during the total
brane changes during the pulse proportionally with pulse width did not change significantly. However, it
decreasing sample conductivityx showed that for the showed a tendency towards the recovery of the initial
pulse experiment with E0 s 880 Vrcm presented in low conductivity state. In experiments with an ampli-
Fig. 1, the transmembrane electric field energy gener- tude of 440 Vrcm, the significant decay of the sample
ated during a pulse of 1 ms duration was approximately conductivity began only when the external field was at
2.5= 10y9 J. The dissipated energy in the small aque- half of this level ŽFig. 1.. Furthermore, the decay of the
ous zones of the porated membrane Žat approx. 8 ␮s sample conductivity to its initial state Žthe conductivity
after the start of a pulse, considering a total pore area course extrapolated to the end of the pulse was approx.
of 46 = 10y1 2 m2 and a membrane thickness of 5 nm, 0.3 mSrcm. indicated that the membrane rapidly re-
yields a volume of approx. 2.3= 10y1 9 m3 . resulted in sealed during the voltage relaxation. Also, in experi-
an enormous specific energy input, of the order of ments where the maximum changes of the membrane
MJrkg. In comparison, the energy input into potato conductance were reached Žan experiment of this type
samples was approximately 0.5 kJrkg and the resulting is shown in Fig. 1, by Eo s 880 Vrcm., the measure-
temperature increase was 0.14⬚C. Obviously, the enor- ment of the passive electrical properties Žstarted 2 s
mous energy concentration on the pores zone could after pulse termination. indicated that the sample con-
produce local thermal fluctuations and some secondary ductivity was identical to the measurement before the
effects on the elastic modulus of the biological mem- pulse was applied Ži.e. the insulation properties of cell
membrane were completely recovered.. A reversible
breakdown of the cell membranes was observed. This
phenomenon is established experimentally for a wide
range of biological cells and is used as a basis for the
electromanipulation of cells in the fields of medicine
and biotechnology ŽChang et al., 1992; Zimmermann &
Neil, 1996; Lynch & Davey, 1996; Ho & Mittal, 1996..
Monitoring of the current during the first 8 ␮s after
starting the repeated supercritical pulse applications
resulted in reversibility after the first pulse and irre-
versible cell membrane rupture after several pulses
ŽFig. 7.. At repeated pulses, with intervals of 1 s between
them, the oscillographic tracking of the charging cur-
rent of the cell membranes for the first and the second
pulse were reproduced with high accuracy. This meant
that the structural properties of the membrane after
the first pulse were identical to those of an intact
membrane. This finding is essential for the appropriate
use of pulsed electric fields. Only if the membrane
structure is restored, does the induction of critical
membrane voltage and membrane breakdown occur
again. Also, for the third pulse, the current course
showed only a slight deviation from the exponential
decaying process. This meant that the membrane struc-
Fig. 6. Variation of Ža. field strength, E, Žb. current density, j, and ture was restored to a greater extent within 1 s after
Žc. effective tissue conductivity, ␴, with time for potato tissue during
first 80 ␮s after pulse start with pulse amplitude 440 Vrcm Ž1. and
the second pulse. The membrane current was observed
880 Vrcm Ž2.. Complete pulse sequence is shown in Fig. 1. Effective for pulse number 5 in addition to the polarization
tissue conductivity is obtained as ␴ s jrE. current at the start of the pulse. At 0.5 ␮s, a slight
146 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

increase in the current course Žcaused by an increase in


the membrane conductance. was also observed. The
current course of the pulse indicated that an irre-
versible membrane permeabilization had occurred.
Detailed information about irreversible membrane
permeabilization in the cell materials examined, as well
as the dynamics of the process, will be presented in
future publications.

3.5. Engineering aspects

The data presented previously show a general rela-


tionship between field-induced transmembrane voltage
and structural changes in biological membranes. From
an engineering standpoint, it is important to under-
stand how the entire cell system responds to electrical
field pulses of different field strength levels.
The relationship between the pulse amplitude and Fig. 8. Critical field strength range for cell systems examined. Rela-
the effective conductivity of the examined cell system is tive permeability is obtained as w ␴ y ␴min xrw ␴max y ␴min x, where ␴ is
given in Fig. 8. This suggests the existence of typically the average effective sample conductivity during the pulse. The
three characteristic ranges of external field strength. In characteristic conductivities for intact sample, ␴ma x and ␴min , are
the sub-critical range of field strength Žless than ap- taken from Fig. 4.
prox. 150 Vrcm. the electrical behavior is determined
by passive electrical parameters of the cell system,
tropermeabilization of the cells. This range of field
which results in no detectable change. At E0 f Ec ,
strength can be proposed as the transitional range. The
electrical membrane breakdown occurs and no further
reasons for the different conductivity values of the
fundamental increase of E0 causes an incomplete elec-
various samples are: the dependence of the membrane
rupture on the transmembrane voltage Žwith decreasing
voltage across the membrane, a significant drop in the
membrane conductance occurs. as well as the uneven
cell size distribution in the samples. Compared to the
field strength usually used for irreversible membrane
ruptures of up to several kVrcm, the transitional E0
range is small.
At the super-critical field strength, the formation of
the aqueous phase in the membrane section in the field
direction was so large that the contribution of the cell
membranes to the total electrical conductivity of the
cells system was not significant, and the effective sam-
ple conductivity reached the finite maximum value. The
sample conductivity was determined only by the extra-
and intracellular electrolyte conductivity. These find-
ings led to the conclusion that when a supercritical
field is applied, the maximum conductivity value of the
cell system should be used for the calculation of the
electric field and the energy distribution in the treat-
ment chamber containing cellular material and non-
cellular medium.
Fig. 7. Kinetics of permeabilization of potato cells in tissue during
the application of ten pulses with constant amplitude of field strength This conclusion has been based on tests for plant
E0 s 700 Vrcm. Ža.: time dependency of field strength, E. Žb.: tissues Žpotato cubes; size 1 cm. regularly distributed in
current density in the tissue in the first 8 ␮s after pulse start. Pulse media with different conductivities Ž0.5᎐8 mSrcm. in a
frequency s 1 sy1 ; n s pulse number. Current density trace for ex- large treatment chamber Ždistance between electrodes
ample without membrane permeabilization Žintact., j⬘, is recon-
structed from relation Ž10., where jsub is registered by application in
s 3 cm; volumes 500 ml; liquid ratio s 0.8. using the
the same sample of a single pulse with subcritical field strength level, field strength measurement with two isolated needle
Esub s 80 Vrcm. electrodes and the detection of temperature increase
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149 147

directly in the tissue. A field intensity with an ampli- applied, the membrane rupture occurred within 1
tude of more than 1 kVrcm was induced directly in the ␮s after pulse initiation. At a very high field
cell material. strength, the membrane breakdown should occur
The tests in mixed systems showed that when the during the voltage rise time. For all of the observed
supercritical field intensity is applied, the specific en- electroporation behaviors, it was found that the
ergy input into particulate tissues per pulse can be only attainment of critical transmembrane poten-
calculated as: tial did not explain all essential aspects of electro-
poration. By the application of a pulse with super-
␴max T 2 critical field strength, the membrane was substan-
Qs H0 E Ž t . dt Ž 12 .
␳ tially charged to higher voltages as compared to
critical field strength. This clearly emphasized the
where ␳ is the tissue density; EŽ t . is the effective field need to consider the dynamics of the membrane
strength in the particulate as a function of time Žmea- voltage build up.
sured or predicted from electric field distribution in the 2. The formation of a conductive membrane due to
treatment chamber with regards to high conductivity the widening of pores was not an instantaneous
state of the cell material.; T is the total pulse width; process and was strongly dependent on the field
and ␴max is the maximum tissue conductivity. This strength amplitude, if the pulse duration was a few
consideration has been confirmed with suspended cell times more than ␶. The larger the field strength,
agglomerates and individual cells. the more rapid the dynamics of the pore formation.
The maximum conductivity value, ␴max , could also be The initiation of conductive channels across the
determined experimentally prior to the supercritical membrane occurred in nanoseconds, while the for-
pulse application via the measurement of passive elec- mation of high conductance membranes due to
trical properties by a DC pulse experiment with subcrit- their expansion took place in a few microseconds.
ical field strength Žas represented in Fig. 4, ␴max s A rapid formation of pores in a lipid domain of the
jmaxrE0 . or by a high frequency AC field yielding the cell membrane might be possible. Although a dras-
so-called ␤-dispersion. For plant or animal tissue, and tic change in the membrane conductivity during the
for large plant cells in suspension, the electrical pulsation occurred Žin the order of up to 105 ., the
properties of intact or totally ruptured membranes are fractional aqueous area of the membrane was found
identical within the characteristic frequency range from to be small and never exceeded 1% of the mem-
5 to 50 MHz ŽAngersbach et al., 1999.. brane area in the field direction.
3. The originally low conductance state of the cell
material could be reached in all cell materials
4. Conclusion examined within a couple of seconds after termina-
tion of the pulse Ži.e. the insulating properties of
The time sequence of the electropermeabilization at cell membrane was completely recovered.. When
the cell membrane level showed similar breakdown critical or slightly lower amplitude pulses were used,
phenomena for different cell systems. When a single the original low conductance of the membrane
pulse with a critical or super-critical field amplitude could be reached, even during the pulse. The appli-
was applied, three different phases were typically in- cation of a single pulse with super-critical ampli-
volved: Ži. the development of a critical transmembrane tude caused the reversible breakdown of the cell
potential due to charge accumulation at the intact membranes. The lifetime of the high-conductance
membrane surface; Žii. the termination of the insulat- state of the membrane and the reversibility of the
ing properties of the membrane Žmembrane break- structural changes was found to be the secondary
down. resulting in development of a conductive mem- effect of electric field interactions with the cells
brane; and Žiii. the opening time of the pores and the and was also influenced by the energy concentra-
tendency towards complete recovery of the initial insu- tion on the pores zone of the membrane.
lating properties of the membrane.
These results are in accordance with the observa-
1. Classical membrane breakdown phenomena oc- tions reported for biological and artificial membranes
curred for large cells at field strength levels in the in classical electroporation phenomena.
range of 150᎐200 Vrcm. When the electrical po- From an engineering perspective, the electric field
tential over the cell membrane reached a critical interactions with real food cell systems can be summa-
value of approximately 0.7᎐2.2 V, it resulted in rized as follows:
membrane breakdown. The polarization time up to
membrane breakdown was influenced by the field 1. The field strength of electric pulses directly applied
strength. When supercritical electrical fields were to the materials with a cell size of 50᎐120 ␮m must
148 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149

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