Two-dimensional nanosecond electric field mapping based on cell electropermeabilization
© Chen et al 2009
Received: 27 April 2009
Accepted: 11 November 2009
Published: 11 November 2009
Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 μm from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration of electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. PACS Codes: 87.85.M-
Ultra-short (< 100 ns), high-field (MV/m) electric pulses produce a variety of effects , including release of intracellular calcium [2, 3], eosinophil disruption , vacuole permeabilization , mitochondrial release of cytochrome c , caspase activation [7, 8], and phosphatidylserine (PS) externalization [9, 10]. Nanosecond electric pulses have been shown to kill a wide variety of human cancer cells in vitro, including basal cell carcinoma and pancreatic cancer cells, and to induce tumor regression in vivo [11, 12], and nanoelectropulse therapy is under development for skin cancer treatment. Some studies of nanosecond pulse effects on tumors have been carried out with parallel-plate electrodes, like those in commercial electroporation cuvettes, where fringing effects are negligible and the electric field distribution can be assumed to be homogeneous. In published [11, 12] and ongoing efforts directed at tumor therapy, however, needle-array electrodes are employed, for which the electric field distribution is not as simple. Magnetic resonance current density imaging and three-dimensional finite modeling were employed to qualitatively evaluate the electric field distribution of different electrode configurations in a prior study of in vivo electroporation . In the present work we demonstrate, using live cell responses, a qualitative mapping of the electric field around three electrode configurations, and we show the correspondence of these electric field profiles with those expected from electromagnetic modeling. Extension of this method can lead to a better and more rigorously quantitative analysis of electric field distributions around electrodes in biological systems, leading to an increased understanding of the in vivo electroporation process and also contributing to evaluations of the efficacy of nanoelectropulse exposure in clinical applications.
In this paper we report the use of living cell monolayers as nanoelectroporation-based, two-dimensional electric field sensors. Fluorescence imaging patterns from the pulse-induced influx of YO-PRO-1 are used to construct two-dimensional maps of the electric field applied with three electrode assemblies -- single-needle, five-needle array, and flat-cut coaxial cable -- immersed in biological media over the monolayers. The field distributions from the different electrode configurations and the responses of different types of cells to nanosecond pulses are compared. In addition, finite element method-based software, COMSOL Multiphysics, was used to calculate the electric field distribution for an electrostatic model. Modeling results and measurements are compared.
2. Materials and Methods
2.1 Experimental setup
2.1.1 Pulse generation and measurement
A solid-state, opening-switch-based pulse generator, generating 15 ns, 10 kV pulses at repetition rates up to 50 Hz, was designed and fabricated at the University of Southern California . A built-in resistive voltage divider based on cascaded attenuation stages with a total attenuation of -54 dB (1:500) was used to measure the pulse voltage delivered to the load . A current transformer with a ratio of 1 to 5 was used to measure the pulse current. A high saturation flux density Finemet® Metglas core (ID = 0.8 cm, OD = 1.5 cm, h = 0.6 cm) provides fast response and linearity for the current measurement. The attenuated pulse current was converted to a voltage signal with a 50 ohm, surface-mount, low-inductance resistor, terminated at the secondary winding of the transformer, to give a total current-to-voltage conversion of 20 V/A. A 50 ohm-terminated digital oscilloscope (Tektronix TDS 5104) was connected with 50 ohm coaxial cables to record the output from the voltage divider and the current sensor.
A 50 ohm SHV coaxial cable assembly was used to deliver nanosecond electric pulses to the electrodes. The losses in the cable are about 3%, single transit. Since the pulse generator represents an open circuit for the reflected pulses and the impedance of the electrodes is 100 ohms or less, depending on the electrode configuration, the reflected pulse amplitude is always less than 50%. Because of the complexity and variable impedance of biological loads precise matching is not possible, but since nanosecond biolectric effects are primarily dependent on the applied electric field, power transfer is not a critical consideration.
2.1.2 Electrode configurations
2.1.3 Adjustable stage
An adjustable stage incorporating a screw-driven micrometer with a resolution of 25 μm was used to adjust the distance between the cells and the electrodes. The culture dish containing the experimental samples was placed on a rigid, level horizontal surface. The electrode assembly was fixed to an arm of the mechanical stage, and the center electrode distance was adjusted with the center axis normal to the bottom surface of the culture dish. The cell monolayer has a thickness of 5-10 μm. After identifying the location where the electrodes touch the bottom of the culture dish, we retracted the electrodes upward 25 μm to obtain a monolayer-to-electrode tip spacing of 15-20 μm.
2.2 Cell lines and cell preparations
Human Jurkat T lymphoblasts (ATCC TIB-152) were cultured in suspension with RPMI 1640 medium (Irvine Scientific, Irvine, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA), 2 mM L-glutamine (Gibco), 45 units/mL penicillin (Gibco), and 45 μg/mL streptomycin (Gibco). Human prostate cancer PC3 cells (ATCC CRL-1435), U251 human glioblastoma cells (RCB-0461, RIKEN CELL BANK), and human keratoacanthoma cells (skin, mixed morphology, ATCC CRL-7630) were grown in Dulbecco's Modified Eagle's Medium (DMEM, ATCC) with 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, 10% FBS, 45 units/mL penicillin, and 45 μg/mL streptomycin. All cells were grown at 37°C in a humidified, 5% CO2 atmosphere. Before an experiment, the PC3, U251, and keratoacanthoma cells were detached with 0.05% trypsin/0.53 mM EDTA in Hank's Buffered Salt Solution (HBSS) without sodium bicarbonate, calcium and magnesium (Cellgro, Herndon, VA) and washed with DMEM growth medium. 1 mL of PC3, U251, and keratoacanthoma cell suspensions (1 × 106 cells/mL) was added to appropriate flat-bottomed wells of a 24-well culture plate, and the cells were incubated until they reached confluence (about 24 hours).
2.3 Fluorescence microscopy and imaging processing
YO-PRO-1 (Molecular Probes, Invitrogen; λex = 491 nm, λem = 509 nm) is a membrane-impermeant fluorescent probe. A permeabilized cell can be identified by the greatly increased fluorescence resulting from YO-PRO-1 influx and binding to nucleic acid material in the cell interior. A Zeiss AxioVert 200 M fluorescence microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY) and AxioVision 3.1 imaging software were used to capture and analyze fluorescence images. Low-power (10× objective) images of cell monolayers were taken 15 minutes after pulse exposure. Since the total area of the pulse-exposed cells is greater than the imaging region of the 10× objective, composite images were generated from a sequence of overlapping images that covered the entire area under and around the electrodes. Each experiment was performed three times, with similar results in each case.
2.4 Electrostatic calculation of the electric field distribution
where c is the speed of light in vacuum, ω = 2πf is the angular frequency, and ε is the permittivity of free space. The rise and fall times of the 15 ns voltage pulses are 5 ns or longer so that the primary frequency of the pulses is expected to be 200 MHz or lower. For DMEM (approximate conductivity: σ = 1.4 S/m, and dielectric constant: ε r ≈ 80 ), the minimum wavelength and the skin depth of the electromagnetic fields are 14 cm and 4 cm, respectively. Since both the wavelength and the skin depth are large compared to the geometry of interest, we can use an electrostatic model, as implemented in the electrostatics module of COMSOL Multiphysics http://www.comsol.com/, for the electric field distribution calculation.
2.5 Temperature measurement
3. Results and Discussion
3.1 Nanoelectropulse-induced membrane permeabilization depends on pulse amplitude
3.2 Electrical measurement
3.3 Electric field mapping
3.3.1 Fluorescence images of three electrode configurations
3.3.2 Comparisons of electric field distribution between electrostatic simulation and fluorescence integration analysis
Three-dimensional electrostatic models for each of the three electrode configurations (Figure 2) immersed in water were generated, considering the electrodes to be perfect conductors. Gauss's law, -∇·ε r ∈ 0 ∇V = ρ (with ρ = 0), where V is the electric potential, was solved for a 1 V potential difference between the center electrodes and the ground electrodes. Zero space charge and zero electric displacement were assumed at the dielectric boundaries.
Maximum electric field intensity of electrostatic simulation and the maximum fluorescence intensity of permeabilized PC3 cell monolayers.
Field Intensity (MV/m)
Maximum Fluorescence Intensity (Arbitrary Unit)
Single needle electrode
3.3 ± 0.8
Five-needle array electrode
6.5 ± 0.8
15.1 ± 3.0
3.3.3 Fluorescence images of various cell types at different spacing distances
3.4 Thermal effect induced by electric pulses
3.4.1 Experimental measurement
Temperature increase and energy per pulse of three different electrode configurations form measurement.
(°C/15 000 pulses)
Single needle electrode
Five-needle array electrode
3.4.2 Numerical calculation
A time constant, = 280 μs, can be assumed for the time required to conduct the electric pulse-induced heat away. This means that the time for temperature to drop to one third of maximum temperature (e-1 = 0.368) is 280 μs. After 20 ms, the increased temperature induced by the first electric pulse will be is negligible. For 2D and 3D calculations, we expect the time constants will be in the same order of magnitude. In addition, there are other forms of heat dissipation including convection and radiation which will help equalize the temperature even faster. Therefore, based on the above experimental measurements and thermal conduction calculation, the thermal effect induced by the nanosecond pulses are negligible.
Electric field distributions for three different electrode configurations have been evaluated based on nanoelectropulse-induced YO-PRO-1 influx and electrostatic models. The measurement method was also used to gauge the electropermeabilization sensitivity of different cell lines. The visualization of the two-dimensional pattern of permeabilization in living cell monolayer allows us to map the electric field distribution with nanoelectropulses in a biological system for different kinds of electrode configurations. More important, we have proved that a diagnostic tool based on electropermeabilization of cells can be used to test invasive, minimum invasive and noninvasive electrodes for nanoelectropulse therapy. This method can be expected to test the sensitivity of tissues from patients, animals or plants to nanoelectropulses for ex-vivo studies. It also has potential to construct a three-dimensional nanosecond electric field distribution mapping by combining a series of fluorescence images taken with sequential spacing distance between the cell monolayers and electrode.
This work was supported by grants from the Alfred Mann Institute at the University of Southern California and Air Force Office of Scientific Research. The authors gratefully acknowledge Tao Tang and Andras Kuthi for pulse generator engineering expertise.
- Schoenbach KH, Joshi RP, Kolb JF, Chen NY, Stacey M, Blackmore PF, Buescher ES, Beebe SJ: Proceedings of the IEEE. 2004, 92: 1122-1137. 10.1109/JPROC.2004.829009.View ArticleGoogle Scholar
- Vernier PT, Sun YH, Marcu L, Salemi S, Craft CM, Gundersen MA: Biochem Biophys Res Commun. 2003, 310: 286-295. 10.1016/j.bbrc.2003.08.140.View ArticleGoogle Scholar
- White JA, Blackmore PF, Schoenbach KH, Beebe SJ: J Biol Chem. 2004, 279: 22964-22972. 10.1074/jbc.M311135200.View ArticleGoogle Scholar
- Schoenbach KH, Beebe SJ, Buescher ES: Bioelectromagnetics. 2001, 22: 440-448. 10.1002/bem.71.View ArticleGoogle Scholar
- Tekle E, Oubrahim H, Dzekunov SM, Kolb JF, Schoenbach KH, Chock PB: Biophys J. 2005, 89: 274-284. 10.1529/biophysj.104.054494.View ArticleGoogle Scholar
- Beebe SJ, Fox PM, Rec LJ, Somers K, Stark RH, Schoenbach KH: IEEE Trans Plasma Sci. 2002, 30: 286-292. 10.1109/TPS.2002.1003872.View ArticleADSGoogle Scholar
- Beebe SJ, Fox PM, Rec LJ, Willis LK, Schoenbach KH: FASEB J. 2003, 17: 1493-1495.Google Scholar
- Vernier PT, Li AM, Marcu L, Craft CM, Gundersen MA: IEEE Trans Dielectr Electr Insul. 2003, 10: 795-809. 10.1109/TDEI.2003.1237329.View ArticleGoogle Scholar
- Vernier PT, Sun YH, Marcu L, Craft CM, Gundersen MA: Biophys J. 2004, 86: 4040-4048. 10.1529/biophysj.103.037945.View ArticleGoogle Scholar
- Vernier PT, Sun YH, Marcu L, Craft CM, Gundersen MA: FEBS Lett. 2004, 572: 103-108. 10.1016/j.febslet.2004.07.021.View ArticleGoogle Scholar
- Nuccitelli R, Pliquett U, Chen XH, Ford W, Swanson RJ, Beebe SJ, Kolb JF, Schoenbach KH: Biochem Biophys Res Commun. 2006, 343: 351-360. 10.1016/j.bbrc.2006.02.181.View ArticleGoogle Scholar
- Garon EB, Sawcer D, Vernier PT, Tang T, Sun YH, Marcu L, Gundersen MA, Koeffler HP: Int J Cancer. 2007, 121: 675-682. 10.1002/ijc.22723.View ArticleGoogle Scholar
- Miklavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G: Biophys J. 1998, 74: 2152-2158. 10.1016/S0006-3495(98)77924-X.View ArticleGoogle Scholar
- Tang T, Wang F, Kuthi A, Gundersen MA: IEEE Trans Dielectr Electr Insul. 2007, 14: 878-883. 10.1109/TDEI.2007.4286519.View ArticleGoogle Scholar
- Kuthi A, Gabrielsson P, Behrend MR, Vernier PT, Gundersen MA: IEEE Trans Plasma Sci. 2005, 33: 1192-1197. 10.1109/TPS.2005.852403.View ArticleADSGoogle Scholar
- Arnold WM, Fuhr G: Industry Applications Society Annual Meeting, 1994, Conference Record of the 1994 IEEE. 1994, 2: 1470-1476.Google Scholar
- Vernier PT, Sun YH, Gundersen MA: BMC Cell Biol. 2006, 7: 37-52. 10.1186/1471-2121-7-37.View ArticleGoogle Scholar
- Tanabe KK, Curley SA, Dodd GD, Siperstein AE, Goldberg SN: Cancer. 2004, 100: 641-650. 10.1002/cncr.11919.View ArticleGoogle Scholar
- Haemmerich D, Laeseke PF: Int J Hyperthermia. 2005, 21: 755-760. 10.1080/02656730500226423.View ArticleGoogle Scholar
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