Name: High-Throughput Capable Three-Dimensional Tissue Model for Quantification of Electroporation Thresholds (Video) | JoVE Journal
Uploaded: 2025-08-19T13:20:00.000+00:00
Duration: 8 min 23 s
Description: Discover computational modeling to quantify reversible and irreversible electroporation thresholds using three-dimensional tissue mimics. This high-throughput method optimizes transfection protocols for gene therapy applications.

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5 minutes

This work was performed at North Carolina State University and funded by the National Institutes of Health (R01CA272550).

The reagents and the equipment used in this study are listed in the Table of Materials.
1. Cell preparation
<ol>
	<li>In a biosafety cabinet, plate 1 &#215; 105 cells/mL of cell line HEK293 in a volume of 10 mL per tissue culture T75 flask in Eagle&#39;s Minimum Essential Medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin.</li>
	<li>Incubate HEK293 cells in a humidified incubator at 37 &#176;C with 5% CO2 environment.</li>
	<li>Harvest cells for experiments 2 days after seeding by removing the culture media and treating cells with 4 mL of 0.25% trypsin-EDTA.</li>
	<li>Incubate cells and trypsin for 3-5 min in a humidified incubator at 37 &#176;C with 5% CO2 environment or until the cells detach from the culture surface.</li>
	<li>Inactivate trypsin&#39;s enzymatic activity by adding 4 mL of the culture media.</li>
	<li>Pellet cells by centrifuging for 5 min at 180 &#215; g and resuspend in culture medium to a final concentration of 8 &#215; 106 cells/mL.</li>
</ol>
2. 3D tissue model creation
NOTE: Use reverse pipetting for these steps to prevent the introduction of air bubbles into the mixture.
<ol>
	<li>In a biosafety cabinet, thoroughly mix the prepared 8 &#215; 106 cells/mL cell suspension with Type I Bovine Collagen Solution (3 mg/mL) in a 1:1 ratio. Place on ice or in a cold bead bath to prevent premature polymerization while working with the mixture.</li>
	<li>Pipette 500 &#181;L of the combined solution to coat the bottom of each well of a 12-well plate. Ensure the gel coats the bottom evenly by starting to dispense the solution at the center of the well and then gently spiraling outwards.</li>
	<li>Gently swirl the plate to ensure the edges of the gel contact the walls of the well.</li>
	<li>Incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 6 h or until gels become firm and do not move when tilting the plate.</li>
	<li>Tilt the well plate and gently add 500 &#181;L of culture medium to each well by allowing it to slide down the wall of the plate at least 1 h before treatment. Gels should be treated within 48 h of creation.</li>
</ol>
3. Electrode fabrication
<ol>
	<li>Remove the plastic Luer connection on two 1.64 mm 304 stainless steel blunt tip syringe needles. Set one needle aside to serve as the pin electrode. For the other needle, flatten the last 5 mm of one end.</li>
	<li>Create the ring electrode by cutting a section of 19 mm outer diameter 316 stainless steel tubing that is long enough to sit flush against the bottom of the well plate.</li>
	<li>Design an electrode holder as seen in Figure 1 with CAD software. Ensure the holder has a central hole for the pin electrode, a groove for the ring electrode, and a hole that intersects the groove so that the flattened needle can be inserted to create a press fit that holds the ring electrode in place.
	<ol>
		<li>Also, ensure that the holder has a lip designed to snugly fit in a 12-well plate and to center the ring and pin electrodes in the well such that the electrodes and the well will all be coaxial38.</li>
	</ol>
	</li>
	<li>Fabricate the electrode holder from acrylic using established laser-cutting techniques40.</li>
	<li>Assemble the electrode by fitting the ring and pin electrodes into the electrode holder.</li>
	<li>Secure the ring electrode by press-fitting the needle with a flattened end into the holder.</li>
</ol>
4. Treatment of 3D tissue model with electroporation
<ol>
	<li>In a biosafety cabinet, tilt the plate and aspirate 400 &#181;L of culture media from each well. Take care not to contact the gel. 100 &#181;L of media should be left in each aspirated well to maintain gel hydration. If there is less than 400 &#181;L of fluid to aspirate (pipette leaves no fluid/draws air), add 100 &#181;L culture medium.</li>
	<li>Add 20 &#181;L of 5 &#181;g/&#181;L GFP plasmid solution to these aspirated wells (for a total volume of 120 &#181;L per well) by tilting the plate and adding the reagent to the fluid that accumulates against the wall of each well. Gently swirl to ensure it spreads evenly across the gel surface.</li>
	<li>Incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 10 min.</li>
	<li>Insert the fiber-optic temperature probe into the pin electrode and begin reading the temperature.</li>
	<li>Connect the positive lead of the electroporator to the pin electrode and the negative lead to the needle securing the ring electrode.</li>
	<li>Turn on the hot plate and heat the gels so that they maintain a temperature of 37 &#176;C.</li>
	<li>Insert the ring-and-pin electrode with fiber optic temperature probe in the pin into the well. Ensure that the pin is perpendicular to the plate and that both electrodes are fully contacting the gel.</li>
	<li>Ensure the gel is at 37 &#176;C.</li>
	<li>Deliver electroporation treatment.</li>
	<li>Add 100 &#181;L of media to any gels that appear dry.</li>
	<li>Repeat steps 4.7-4.10.</li>
	<li>Once all treatments are complete, incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 10 min.</li>
	<li>Tilt the well plate and gently add 500 &#181;L of culture medium to each well by allowing it to slide down the wall of the plate.</li>
	<li>Incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 24 h.</li>
</ol>
5. Washing and imaging
<ol>
	<li>Tilt the plate and aspirate the culture media from each well. Take care not to contact the gel.</li>
	<li>Tilt the plate and gently add 500 &#181;L of phosphate-buffered saline (PBS) to each well by allowing it to slide down the wall of the plate.</li>
	<li>Incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 5 min.</li>
	<li>Tilt the plate and aspirate the PBS from each well. Take care not to contact the gel.</li>
	<li>Tilt the plate and gently add 500 &#181;L of phosphate-buffered saline (PBS) to each well by allowing it to slide down the wall of the plate.</li>
	<li>Gently swirl the plate, tilt it, and aspirate the PBS from each well. Take care not to contact the gel.</li>
	<li>Add 100 &#181;L of fresh PBS to keep gels hydrated for imaging.</li>
	<li>Image plate using standard fluorescent microscopy techniques. This should yield images of gels with a distinct green, torus-shaped region where the reversible and irreversible electric field thresholds bound the outer and inner edges of the region, respectively.</li>
	<li>Tilt the well plate and gently add 500 &#181;L of culture medium to each well by allowing it to slide down the wall of the plate.</li>
	<li>Incubate the gels in a humidified incubator at 37 &#176;C with 5% CO2 environment for 24 h.</li>
	<li>Repeat steps 5.1-5.10 for each timepoint.</li>
</ol>
6. Computational model creation
<ol>
	<li>Open a physics simulation software and create a new 2D axisymmetric model that utilizes the Electric Currents, Heat Transfers in Solids, and Electromagnetic Heating physics.</li>
	<li>Create parameters for the voltage, ambient temperature, hot plate temperature, temperature setpoint, pulse delivery rate, integrated electrical dose, and dimensions of the ring and pin electrodes, the gel, and the well plate of the experimental setup.</li>
	<li>Create domain geometries representing a radial cross-section of the ring and pin electrodes, the gel, and the well plate of the experimental setup as seen in Figure 2A.</li>
	<li>Place a probe point inside the central electrode at half of the gel height to represent the fiber-optic temperature probe used during in vitro experimentation.</li>
	<li>Create a piecewise function that uses temperature as its input argument.</li>
	<li>Define the function such that its value is 1 when the temperature is less than the temperature setpoint parameter defined in step 6.2, and 0 otherwise.</li>
	<li>Apply a Voltage boundary condition to the topmost surface of the domain geometry representing the pin electrode and set the voltage value to the voltage parameter defined in step 6.2 multiplied by the piecewise function from step 6.6 evaluated at the temperature of the probe point created in step 6.4.</li>
	<li>Apply a Ground boundary condition to the topmost surface of the domain geometry representing the ring&#160;electrode.</li>
	<li>Apply an Electrical Insulation boundary condition to all remaining domain boundaries.</li>
	<li>Create a continuous function that uses temperature.</li>
	<li>Define the function using an established model27 of the temperature-dependent conductivity of the collagen gel as 0.8277 + 0.0323*T.</li>
	<li>Define a custom material and set the electrical conductivity to the continuous function defined in step 6.11. Apply this material to the gel domain.</li>
	<li>Define materials or use software defaults if available to assign 304 and 316 stainless-steel materials to the pin and ring electrode domains, respectively, and polystyrene to the well plate domains.</li>
	<li>Apply a Temperature boundary condition to the base of the model and set the temperature value to 37 &#176;C.</li>
	<li>Apply a Surface Ambient Radiation boundary condition to all other domain edges that do not interface with another domain and set the emissivity to 0.97, 0.9, and 0.075, depending on whether the domain was part of the plate, gel, or electrode, respectively27.</li>
	<li>Add a Free Triangular Mesh to the model. Set the element size to Finer.</li>
	<li>If evaluating a variety of parameter levels, create a parametric sweep that lists all values at which a parameter is to be evaluated.</li>
	<li>Run the simulation by clicking on the Compute button.</li>
	<li>Upon completion, create a 2D-Cutline Plot of the electric field in the Results section.</li>
	<li>Define the cutline to pass from the pin electrode to the ring electrode through the middle of the gel domain. This should result in an exponential decay from the edge of the pin electrode to the edge of the ring electrode.</li>
	<li>Export the plot data as a .csv or .xlsx file to create a lookup table.</li>
</ol>
7. Threshold derivation
<ol>
	<li>Using the microscope&#39;s software, measure the diameter of the outer and inner edges of the torus-shaped region along the vertical and horizontal axes. If the software does not have this capability, use ImageJ to do it.</li>
	<li>Average the outer and inner diameters, respectively, and divide by two to calculate the outer and inner radii of the torus-shaped region. The outer radius demarks the RE threshold. The inner radius demarks the IRE threshold.</li>
	<li>Using the lookup table created in step 6.21, derive the electric field intensity at the measured radii.</li>
</ol>

The authors have no disclosures.

With this method, reversible and irreversible electroporation thresholds can be identified without the need to test multiple samples at discrete voltages. The use of a non-uniform electric field enables quantification based on the spatial distribution of transfected cells in the model. However, there are limitations associated with this non-uniform distribution. Foremost, the RE and IRE thresholds described herein are the thresholds for successful transfection and cell death, respectively, which may not be direct measure...

Reversible electroporation (RE) is a well-established methodology for delivering a variety of normally membrane-impermeable compounds and molecules into cells1,2,3. Here, pulsed electric fields (PEFs) are used to achieve a critical electric field intensity that induces transient, localized nanopores along portions of the cell membrane. RE has been used to deliver a variety of molecules and compounds ranging from dyes and nucleic acids to chemotherapeutics and other drugs4,5,6,7,8,9,10. However, if the electric field intensity is increased sufficiently, it can meet a higher critical threshold, which results in a rapid decline in cell viability1,2,11,12. This process is known as irreversible electroporation (IRE) and has been studied for soft-tissue ablation, including as a treatment for otherwise inoperable tumors and cardiac arrhythmias11,12,13,14,15,16. To understand treatment outcomes and inform future treatments, quantification of these RE and IRE thresholds is common practice in electroporation research12,17,18,19,20. However, RE and IRE thresholds vary depending on many factors, including cell type, applied voltage, pulse duration, number of pulses, and pulse delivery rate1,17,18,21,22.
Electroporation protocols have a number of parameters that result in varied RE and IRE thresholds1,17,18,21,22. Optimal treatment voltages and waveforms have long been investigated. Most commercially available electroporation technologies utilize protocols with monopolar PEFs on the order of 100 &#181;s-10 ms23. While these protocols achieve clinically relevant outcomes, they induce intense muscle contractions in vivo24,25,26. Because of this, shorter, bipolar microsecond pulses, which alleviate this stimulation, have become a recent topic of interest1,11,24. However, these pulses introduce new factors which affect RE and IRE thresholds, including pulse symmetry, burst duration, and temperature-dependence1,17,22,27. Additionally, cell type and tissue properties have also been shown to significantly affect both RE and IRE outcomes19,28,29. As the field continues to evolve, there is a need to further characterize how these factors and their interactions with each other and other possible environmental factors affect RE and IRE thresholds, making it increasingly necessary to improve experimental efficiency to enable higher-throughput experimentation.
Typical in vitro electroporation experiments test protocols by treating cells in a cuvette with parallel conductive plates, which results in a uniform electric field distribution within the cuvette during treatment19,30,31,32,33,34. As aforementioned, it is necessary to identify a protocol&#39;s RE and/or IRE thresholds to identify an optimal applied voltage for a given treatment. For this to be achieved in cuvette studies, multiple cuvettes must be treated, each with discrete applied voltages. This requires a significant number of samples that must be prepared, treated, and analyzed separately, limiting experimental throughput. To improve this process, both 2D35,36 and 3D13,20,27,37,38,39 culture models have been developed to evaluate a continuum of field strengths in a single sample by using electrodes that create non-uniform field distributions13,20,27,37,38,39. RE and IRE thresholds are then derived from computational models of the electric field distribution13,20,27,37,38. This greatly reduces the number of samples needed, enabling a more high-throughput approach to threshold quantification.
This article aims to demonstrate this approach for RE and IRE field quantification in a 3D in vitro model to demonstrate its potential as a means for efficient evaluation of electroporation protocols across several environments (e.g., cell types, treatment parameters, molecules to be delivered).

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>1000 uL pipette tips</td><td>Fisher Scientific</td><td>13-811-164</td><td></td></tr><tr><td>15 mL conical tube</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>200 uL pipette tips</td><td>Fisher Scientific</td><td>13-811-139</td><td></td></tr><tr><td>304 stainless steel blunt tip needles</td><td>McMaster Carr</td><td>75165A753</td><td></td></tr><tr><td>316 Stainless steel tubing</td><td>McMaster Carr</td><td>89785K319</td><td></td></tr><tr><td>Acrylic</td><td>McMaster Carr</td><td>8505K754</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Fisher Scientific</td><td>13-261-312</td><td></td></tr><tr><td>CAD software</td><td>Dassault Systems</td><td>SolidWorks</td><td></td></tr><tr><td>Centrifuge</td><td>Nuaire</td><td>NU-C200R</td><td></td></tr><tr><td>Electroporator</td><td>Hbio</td><td>45-0662</td><td>A custom built device and a BTX ECM 830 system were used in this study</td></tr><tr><td>EMEM</td><td>Fisher Scientific</td><td>MT10009CV</td><td>This may vary&amp;nbsp; if using a cell line other than HEK293</td></tr><tr><td>Falcon 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid</td><td>Corning</td><td>353043</td><td>Any 12 well plate can be used, just ensure that well dimensions are taken into consideration when fabricating the electrode holder.</td></tr><tr><td>FBS</td><td>Fisher Scientific</td><td>A5670701</td><td></td></tr><tr><td>Fiber Optic Temperature Probe</td><td>Micronor Inc.</td><td>TS5-20MM-02</td><td></td></tr><tr><td>GFP-Plasmid</td><td>Aldeveron</td><td>gWiz-GFP</td><td></td></tr><tr><td>HEK293 Cells</td><td>ATCC</td><td>CRL-1573</td><td>Other cell lines can be used, just ensure the correct culture medium is used</td></tr><tr><td>Hot Plate</td><td>Thermoelectric Cooling America Corporation</td><td>AHP-301CPV</td><td></td></tr><tr><td>Ice or Refrigerated Bead Bath</td><td>Fisher Scientific</td><td>10-876-001</td><td></td></tr><tr><td>Image analysis software</td><td>Leica</td><td>LAS X</td><td></td></tr><tr><td>Incubator</td><td>Fisher Scientific</td><td>15-015-2633</td><td></td></tr><tr><td>Inverted Fluorescence Microscope</td><td>Leica</td><td>DMi8</td><td></td></tr><tr><td>Laser Cutter</td><td>McMaster Carr</td><td>7344N11</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Pipette Kit</td><td>Fisher Scientific</td><td>14-388-100</td><td></td></tr><tr><td>Simulation Software</td><td>COMSOL</td><td>COMSOL Multiphysics 6.2</td><td></td></tr><tr><td>T75 Flask</td><td>Fisher Scientific</td><td>156499</td><td></td></tr><tr><td>Type I Bovine Collagen Solution</td><td>Advanced Biomatrix</td><td>5005</td><td>3 mg/mL</td></tr></tbody></table>

high-throughput capable three-dimensional tissue model for quantification of electroporation thresholds

High-Throughput Capable Three-Dimensional Tissue Model for Quantification of Electroporation Thresholds

Electroporation is a promising technology utilizing electrical pulses for macromolecule delivery and soft-tissue ablation, with applications that include next-generation prophylactics and the treatment of genetic diseases such as cancer. This study demonstrates a high-throughput capable 3D tissue culture model for quantification of the reversible and irreversible electroporation thresholds for a given electroporation protocol. By using a non-uniform electric field and analyzing the spatial distribution of transfected cells, both reversible and irreversible thresholds can be identified within a single sample, increasing the efficiency at which electroporation protocols can be characterized, especially for in vivo translation. To show this capability, 3D tissue mimics containing HEK293 cells were transfected using a ring and pin electrode to deliver a GFP-encoding plasmid. Electroporation thresholds were then derived based on fluorescent microscopy images of the transfected samples. This model demonstrates potential for use as a means for high-throughput evaluation of electroporation protocols, a key advantage over current methods to evaluate these thresholds, which tend to be time-intensive and are less representative of in vivo conditions.

The ring and pin electrode successfully delivered a DNA plasmid encoding a green fluorescent protein (GFP) into 3D collagen tissue mimics embedded with HEK293 cells. Successful RE threshold quantification was achieved by measuring the outer radius of the transfected region and deriving the corresponding field intensity from the computational model of the experimental setup, as seen in Figure 2. The IRE threshold was similarly quantified by measuring the inner...

This protocol uses computational modeling to quantify reversible and irreversible electroporation thresholds using the spatial distribution of transfected cells within a three-dimensional tissue mimic for high-throughput analysis of electroporation protocols.

Electroporation is a promising technology utilizing electrical pulses for macromolecule delivery and soft-tissue ablation, with applications that include ...

high-throughput-capable-three-dimensional-tissue-model-for

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Discover computational modeling to quantify reversible and irreversible electroporation thresholds using three-dimensional tissue mimics. This high-throughput method optimizes transfection protocols for gene therapy applications.

High-Throughput Capable Three-Dimensional Tissue Model for Quantification of Electroporation Thresholds

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