Name: porous substrate-based electroporation with transepithelial electrical impedance monitoring
Uploaded: 2024-09-27T13:40:00.000+00:00
Duration: 8 min 6 s
Description: Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

We acknowledge the funding support from the NSF (Awards 1826135, 1936065, 2143997), the NIH National Institutes of General Medical Sciences P20GM113126 (Nebraska Center for Integrated Biomolecular Communication) and P30GM127200 (Nebraska Center for Nanomedicine), the Nebraska Collaborative Initiative and the Voelte-Keegan Bioengineering Support. The device was manufactured at the NanoEngineering Research Core Facility (NERCF), which is partially funded by the Nebraska Research Initiative.

The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of reagents and cell culture
<ol>
	<li>Prepare the cell culture media by adding 50 mL of fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin to a 500 mL container of Dulbecco&#39;s Modified Eagle Medium (DMEM). Produce eleven 50 mL aliquots to reduce the risk of contamination and refrigerate at 4 &#176;C.</li>
	<li>Create 1 mL of 25 &#181;g/mL human plasma fibronectin in phosphate-buffered saline (PBS) stock solution according to the manufacturer&#39;s instructions.</li>
	<li>Create 15 mL of 0.1 mg/mL propidium iodide in DMEM stock solution to allow for experiments with varying cargo concentrations.</li>
	<li>Culture A431 cells in a T75 flask containing 12 mL of the prepared cell culture media. Cells were passaged every 1-2 days to maintain 50% confluency.</li>
</ol>
2. Sample preparation
<ol>
	<li>Fibronectin coating
	<ol>
		<li>Select twelve inserts and two 24-well plates. Place the inserts into one well plate, creating two rows of six. Set the second well plate aside until later.</li>
		<li>Create 1,300 &#181;L of 1 &#181;g/mL fibronectin solution by mixing 52 &#181;L of fibronectin stock solution and 1,248 &#181;L of PBS in a 1.5 mL tube.</li>
		<li>Distribute 100 &#181;L of the fibronectin solution into each insert. Incubate the inserts in the well plate at 37 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Adjusting the cell concentration for optimized cell density
	<ol>
		<li>Around 1 h before the fibronectin incubation is complete, remove the T75 flask of A431 cells from the incubator for cell extraction.</li>
		<li>Remove the media in the flask with an aspirator and wash with 5 mL of PBS. Remove the PBS in the same fashion and add 3 mL of Trypsin. Incubate for 3-4 min before tapping the side of the flask to completely detach cells.</li>
		<li>Add 6 mL of cell culture media to the flask, mixing vigorously with a pipette to detach any remaining cells, and transfer contents to a 15 mL centrifuge tube. Centrifuge at 100 x g and 20 &#176;C for 5 min.</li>
		<li>Remove the cell culture media and Trypsin from the centrifuge tube using an aspirator, being careful not to disturb the cell pellet. Add 1 mL of media to the centrifuge tube and pipette back and forth (without producing bubbles) to break up the cell pellet and resuspend the cells.</li>
		<li>Pipette 10 &#181;L of the cell suspension, 40 &#181;L of cell culture media, and 50 &#181;L of trypan blue dye into a 200 &#181;L tube, using a pipette to mix thoroughly.</li>
		<li>Remove 10 &#181;L of the dye mixture and inject it into a hemocytometer. Count the cells using the 10% dilution of the dye mixture to estimate the total cell count in the 15 mL centrifuge tube. 
		NOTE: For this protocol, assume a concentration of 5,000,000 cells/mL.</li>
		<li>Multiply the desired seeding density by the insert membrane&#39;s surface area, divide by the counted cells/mL in the suspension, and multiply by 1,000 to calculate the required microliters of cell suspension per insert.
		<ol>
			<li>To find the total quantity of cell suspension required, multiply this figure by 10 (to ensure enough cells for 9 samples, as 3 of the 12 inserts will be cell-free controls), and round up to the nearest whole number. In this case, a total of 135 &#181;L of the cell suspension is required for this experiment.</li>
		</ol>
		</li>
		<li>Create 2,000 &#181;L of adjusted cell solution by mixing the previously calculated 135 &#181;L of the cell suspension with 1,865 &#181;L of cell culture media in a separate 15 mL centrifuge tube.</li>
	</ol>
	</li>
	<li>Seeding cells
	<ol>
		<li>Remove the excess fibronectin from each insert once the fibronectin incubation is complete.</li>
		<li>Wash the inserts twice by adding 100 &#181;L of sterile distilled water to each insert. Remove the water following the same order as it was added to ensure a consistent wash time between inserts.</li>
		<li>Wash the insert again by adding 100 &#181;L cell culture media to each insert. Remove the media following the same order as it was added to ensure a consistent wash time between inserts.</li>
		<li>Cell sample inserts
		<ol>
			<li>Seed cells by pipetting 200 &#181;L of adjusted cell solution into each insert. To ensure consistent confluency between inserts, mix the cell solution in the centrifuge tube prior to distribution and mix again within each insert after distribution.</li>
		</ol>
		</li>
		<li>Negative control inserts
		<ol>
			<li>Pipette 200 &#181;L of cell culture media into each insert. To remain consistent with the cell sample inserts, use the pipette to mix the cell culture media within each insert.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Labeling and incubation
	<ol>
		<li>Draw a line dividing the second well plate into two columns that are three wells wide (for conditions run in triplicate) using a permanent marker. Separate each column into rows. Label each region in the grid with relevant parameters.</li>
		<li>Add 1 mL of cell culture media to every well to receive an insert for the experiment. Transfer the inserts from the preparation well plate to their appropriate location in the labeled experiment well plate and incubate at 37 &#176;C for at least 12 h.</li>
	</ol>
	</li>
</ol>
3. Experimental procedure
<ol>
	<li>Delivery experiment
	<ol>
		<li>Pipette 1.5 mL of the 0.1 mg/mL PI solution into each well in the electrode array. Place an insert into each well in the electrode array, fitting the feet of the insert into the alignment grooves so the insert is flush with the upper surface of the well (Figure 1A,B).</li>
		<li>Screw the top electrode printed circuit board (PCB) to the top of the electrode array wells and connect the electrode array to the PSEP device.</li>
		<li>Place the electrode array in the 37 &#176;C incubator for at least 1 h to allow the temperature to equilibrate.</li>
		<li>Click the drop-down next to &#34;Membrane&#34; in the top left corner of the GUI and click on 400 nm GBO. Repeat this step for &#34;Electrolyte&#34;, &#34;Cells&#34;, &#34;Cell Seeding Density&#34;, and &#34;Cell Duration&#34;, selecting DMEM, A431, 200, and 12, respectively. 
		NOTE: These values are for record-keeping purposes only, and do not impact the function of the device. Please ensure to adjust these values as necessary for correct data tracking.</li>
		<li>Type 1 into the Post Pulse Time Duration (min) edit field on the right side of the GUI to change the default post-pulse measurement time to 1 min. Leave all other settings in the default state. 
		NOTE: Default pulse parameters create a square waveform with 30 volts, 20 Hz, 1 ms duration, and 200 pulses. Default TEEI measurement parameters are 0.5 volts and 100 Hz, 1,000 Hz, 10,000 Hz, and 100,000 Hz.</li>
		<li>Click on the Run button and enter appropriate names for wells 1-3 and 4-6 when prompted. Click on OK to start the experiment.</li>
		<li>Remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment well plate when the program has finished executing.</li>
		<li>Mix 2 &#181;L of Hoechst 33342 and 5 &#181;L of calcein AM with 123 &#181;L of cell culture media in a 200 &#181;L tube.</li>
		<li>Gently pipette 10 &#181;L of the stain solution into each post-pulse insert and place the inserts back into the incubator for 5 min.</li>
		<li>Transfer the well plate to the plate holder of a fluorescent microscope with a 5x magnification objective. Image using brightfield and the fluorescence of each stain. Center the insert over the objective before triggering the camera. 
		NOTE: The excitation wavelengths for PI, calcein AM, and Hoechst 33342 are 558 nm, 495 nm, and 353 nm, respectively. The emission wavelengths are 575 nm, 519 nm, and 465 nm, respectively.</li>
	</ol>
	</li>
	<li>TEEI measurement experiment
	<ol>
		<li>Pipette 1.5 mL of the cell culture media into each well in the electrode array. Place cell sample inserts into wells 1-3 and control inserts into wells 4-6, fitting the feet of the insert into the alignment grooves so the insert is flush with the upper surface of the well.</li>
		<li>Screw the top electrode PCB to the top of the electrode array wells and connect the electrode array to the PSEP device.</li>
		<li>Place the electrode array in the 37 &#176;C incubator for at least 1 h to allow the temperature to equilibrate.</li>
		<li>Click on the drop-down next to &#34;Membrane&#34; in the top left corner of the GUI and click on 400 nm GBO. Repeat this step for &#34;Electrolyte&#34;, &#34;Cells&#34;, &#34;Cell Seeding Density&#34;, and &#34;Cell Duration&#34;, selecting DMEM, A431, 200, and 12, respectively. 
		NOTE: These values are for record-keeping purposes only, and do not impact the function of the device. Please ensure to adjust these values as necessary for correct data tracking.</li>
		<li>Leave all remaining settings in the default state. 
		NOTE: Default pulse parameters create a square waveform with 30 volts, 20 Hz, 1 ms duration, and 200 pulses. Default TEEI measurement parameters are 0.5 volts and 100 Hz, 1,000 Hz, 10,000 Hz, and 100,000 Hz.</li>
		<li>Click on the Run button and enter appropriate names for wells 1-3 and 4-6 when prompted. Click on OK to start the experiment.</li>
		<li>Remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment well plate when the program has finished executing.</li>
		<li>Mix 2 &#181;L of Hoechst 33342, 5 &#181;L of calcein AM, and 10 &#181;L of PI with 113 &#181;L of cell culture media in a 200 &#181;L reaction tube.</li>
		<li>Pipette 10 &#181;L of the stain solution into each post-pulse insert and place the inserts back into the incubator for 5 min.</li>
		<li>Transfer the well plate to the plate holder of a fluorescent imaging microscope with a 5x objective lens. Image using brightfield and the fluorescence of each stain. Center the insert over the lens before triggering the camera. 
		NOTE: The excitation wavelengths for PI, calcein AM, and Hoechst 33342 are 558 nm, 495 nm, and 353 nm, respectively. The emission wavelengths are 575 nm, 519 nm, and 465 nm, respectively.</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Analyzing image data with the CellProfiler pipeline
	<ol>
		<li>Use the custom CellProfiler workflow that is provided at GitHub:https://github.com/YangLabUNL/PSEP-TEEI to process the delivery and TEEI measurement experiment images.</li>
	</ol>
	</li>
	<li>TEEI analysis
	<ol>
		<li>Click on the Analysis tab in the GUI.</li>
		<li>Toggle the impedance type indicator to TEEI at the bottom of the GUI.</li>
		<li>Click on the arrow in the top left box to show all the experiment names in the data file. Select all cell sample data from the TEEI measurement experiment.</li>
		<li>Click on the arrow in the next box to the right to show all the experiment names in the data file. Select all control insert data from the TEEI measurement.</li>
		<li>Click on Run. A basic figure containing selected cell sample data at the lowest measurement frequency will appear.</li>
		<li>In the sample options box on the right-hand side of the GUI, click on the arrow to show all selected insert data. Outliers can be removed by selecting the appropriate data and clicking on Remove below. 
		NOTE: Any data that was removed from the analysis by the last click of the Remove button can be retrieved by the Undo button.</li>
		<li>Click on Done to move on to the next figure when the desired data is shown in the figure.</li>
		<li>Repeat steps 4.2.6 and 4.2.7 for the remaining cell sample data and control data. When the final dataset has been confirmed by clicking &#34;Done&#34;, the full analysis figure will appear.</li>
		<li>Save the analysis figure.</li>
	</ol>
	</li>
</ol>

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

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The authors declare no conflict of interest.

Figure 2C demonstrates that TEEI increases from minimum and decreases from baseline are plotted for each PSEP waveform voltage. The TEEI increase creates a parabolic arc, peaking around 20 volts before reducing, while the TEEI decrease from baseline increases exponentially as voltage increases. The delivery efficiency and death percentages in Figure 2D mirror these trends, with delivery efficiency arcing parabolically, peaking around 30 volts, and death increasing exponentially as waveform voltage is increased.
One hypothesis for the underlying mechanism causing the TEEI increase is electro-osmosis through the negatively charged substrate microchannels, a phenomenon caused by the application of an electric field18,19. Whether the TEEI response is due to mechanical stimulus from cell swelling due to electro-osmotic fluid flow, a factor known to occur with electroporation20, or due to the electrical stimulus of the waveform itself, it is clear health and completeness of the monolayer is paramount to achieving the proper voltage drop across the cell membrane required for electroporation. For this reason, the most critical steps in this method are the ones regarding cell seeding and ensuring proper cell monolayer formation. This can be confirmed by imaging the cell monolayer and by the baseline TEEI value. For A431 cells, the average TEEI is around 7 &#937;&#183;cm&#178;, whereas HEK293T cells average a slightly lower 5 &#937;&#183;cm&#178; (Figure 2Aii), likely due to morphological differences causing differences in cell-cell junction area.
Due to the electric field required for porous substrate electroporation, electrolysis will occur, causing the electrodes to corrode12,21. This was especially evident for the bottom electrode, as it was positively charged in this experiment to deliver positively charged PI. Through experimentation, it was determined that the bottom PCB could be used approximately 20 times before significant negative effects require replacement13. To clean the electrode array for reuse, remove the remaining cell culture or transfection media from the chambers using an aspirator. Fill each chamber three-quarters of the way full of 70% ethanol and place the top electrode PCB onto the electrode array so the top electrodes are submerged. Leave the ethanol in the electrode array for at least 10 min before removing the ethanol and setting the electrode array aside to dry.
It is possible to reuse the purchased inserts by removing the substrates, sterilizing the insert, and replacing the substrate with one taken from another source. 6-well inserts with the same pore density and diameter are available commercially and can be used to harvest four 24-well insert-sized replacement substrates. Once the previously used inserts are sterilized, add 10 &#181;L of ultraviolet-light-cured epoxy to a fresh Petri dish. Dip the substrate side of the insert into the pool of epoxy to coat the bottom surface, and carefully place a new substrate over the hole in the insert. Visually verify that the epoxy makes a complete ring to ensure there are no gaps in the connection. Cure under a UV light for 30 s and store the refurbished inserts in a clean 24-well plate to avoid damaging the new substrates before reuse.
As stated previously, while it is hypothesized that the observed TEEI increase will occur in multiple cell types, it has only been demonstrated with the A431 and HEK293T cell lines13 (Figure 1C), both of which are adherent cells. The method can be modified by selecting different cell lines by selecting membranes with different pore characteristics, replacing the fibronectin coating with another extracellular matrix protein by adjusting the concentration, or by changing the cargo. However, if any changes are made to the experiment's setup, it may be necessary to reoptimize the waveform. To optimize the waveform, a TEEI measurement experiment can be conducted in which only one waveform parameter, such as voltage, is changed between each group of three samples. Select the optimal voltage by identifying the largest increase in TEEI over at least nine healthy samples. Repeat this process for each waveform parameter, using the newly optimized values when moving on to the next one. Remember there may be multiple local optima for waveform parameters (i.e., the optimal voltage for one pulse duration may not be the optimal voltage for another pulse duration, and so forth).
The benefits of porous substrate electroporation are wide-reaching. While other methods of intracellular delivery have existed for a considerable time, few have combined high throughput with a high degree of control that PSEP possesses1,13. Additionally, the platform's use of TEEI measurements provides a glimpse into the intermediary steps of the electroporation process. The TEEI readings tell the condition of the cells, guide the selection of electroporation parameters, and allow further insight into specific cell behaviors and mechanisms13,17. Through the TEEI measurements, the platform is also capable of label-free delivery13, which allows for rapid optimization with a diminished need for expensive biomarkers and reagents every time an experiment is conducted. These contributions to the area of intracellular delivery make this a prime candidate as a delivery platform for fundamental biological research and biomedical applications.

Electroporation is a technique in which cells are exposed to an electric field, creating temporary pores in the cell membrane through which cargos, including proteins, RNA, and DNA, can pass1,2. The most widely used version is bulk electroporation (BEP). BEP is performed by filling a cuvette with an electrolyte containing millions of cells, exposing the electrolyte to high voltage, and allowing cargo to enter the cells through diffusion or endocytosis1. There are many advantages to BEP, including high throughput and numerous commercially available systems. However, there are limitations to the BEP delivery. Inconsistent cell positioning relative to the electrodes and electric field shielding from adjacent cells causes significant variability in electric field exposure during BEP3,4. The high voltage required for BEP also has a significant negative impact on cell viability5. Since its inception in 20116, there has been growing interest in an electroporation method called porous substrate electroporation (PSEP), though it is sometimes referred to by other names, including localized electroporation and nano- or micro-electroporation1,7,8. In contrast to the cell suspension of BEP, PSEP is conducted on cells that are adherent to a porous substrate. Not only is an adherent state preferred for the majority of human cell lines9, but the pores in the substrate also focus on the electric current, localizing the transmembrane electrical potential (TMP) to specific regions of the cell membrane10,11. This localization allows for a significant reduction in applied voltage, decreasing damage and increasing cell viability. This combination of effects helps control cell membrane pore development, resulting in a more consistent and efficient delivery1,5,12.
A recent study introduced a PSEP device with a six-well, gold-plated electrode array for holding commercially available porous membrane inserts13 (Figure 1A,B), a practice that was first introduced by Vindis et al.14. The device can apply pulses and measure the electrical impedance across the cell monolayer, known as the transepithelial electrical impedance (TEEI), in real-time13. The user interface of the device allows complete control over the electroporation waveform and polarity. Importantly, real-time impedance measurements can be used to predict delivery outcomes without the need for expensive reagents or fluorescent markers, a concept known as label-free delivery15.
The PSEP platform consists of two major custom electrical components: the main body of the device, which houses the pulse generator and TEEI measurement equipment, and the electrode array, where the porous substrates are inserted, and the electroporation occurs. Diagrams for all custom electronics and 3D-printed components can be found at GitHub:&#160;https://github.com/YangLabUNL/PSEP-TEEI. In addition to the custom electronics, a computer is also required for the platform to function properly. The custom software requires MATLAB (version 2021a or later) to run, and Microsoft Excel to store and access data for analysis. The program controls the custom electronics and provides the graphical user interface (GUI) for adjusting settings. These programs were also made available at GitHub: https://github.com/YangLabUNL/PSEP-TEEI.
Preliminary data suggests this process is possible for different types of adherent cells (Figure 1C), but this article will only discuss the preparation of A431 cells using parameters that were found to be optimal for this cell line by Brooks et al.13. Additionally, because the propidium iodide (PI) cargo is cytotoxic, two experiments are performed, the first with a high concentration PI transfection media to quantify delivery efficiency, and the second with only cell culture media to measure TEEI over longer timescales. These experiments use identical electroporation waveforms, allowing the results to be correlated (Figure 1D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66971/66971fig01.jpg" /> 
Figure 1: Electrode array assembly diagram and foundational data. (A) CAD model of an insert inside a well of the electrode array. (B) CAD model of the electrode array. (C) Impedance increase due to PSEP for select cell lines, n = 3 per cell line. Error bar: standard error of the mean. (D) Delivery efficiency vs. TEEI increase correlation data. Delivery efficiency was calculated by dividing the number of cells labeled in both PI and calcein images from delivery experiments by the total number of cells identified with Hoechst. Cell count was determined using a custom CellProfiler pipeline, n = 6 per voltage. Error bar: (x- and y-axis) standard error of the mean. This figure is reproduced from Brooks et al.13 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66971/66971fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>15 mL Conical Centrifuge Tube</td><td>Thermo Scientific</td><td>339651</td><td></td></tr><tr><td>2-Chip Disposable Hemocytometer</td><td>Bulldog Bio</td><td>DHC-N01</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask</td><td>fisherbrand</td><td>FB012937</td><td></td></tr><tr><td>A431 Cells</td><td>ATCC</td><td>CRL-1555</td><td></td></tr><tr><td>Calcein AM</td><td>Invitrogen</td><td>C3099</td><td></td></tr><tr><td>Class II Type A2 Biosafety Cabinet</td><td>Labgard</td><td>NU-543-600</td><td></td></tr><tr><td>Custom Components</td><td>YangLab</td><td></td><td>https://github.com/YangLabUNL/PSEP-TEEI</td></tr><tr><td>Disposable Centrifuge Tube (50 mL)</td><td>fisherbrand</td><td>05-539-6</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A5670401</td><td></td></tr><tr><td>Fluid Aspiration System</td><td>vacuubrand</td><td>20727403</td><td></td></tr><tr><td>HERACELL 240i</td><td>Thermo Scientific</td><td>51026331</td><td></td></tr><tr><td>Hoechst 33342</td><td>Thermo Scientific</td><td>62249</td><td></td></tr><tr><td>Human Plasma Fibronectin</td><td>Sigma-Aldrich</td><td>FIBRP-RO</td><td></td></tr><tr><td>Inverted Fluorescent Microscope</td><td>Zeiss</td><td>491916-0001-000</td><td></td></tr><tr><td>Inverted Microscope</td><td>Labomed</td><td>TCM 400</td><td></td></tr><tr><td>PBS</td><td>cytiva</td><td>SH30256.02</td><td></td></tr><tr><td>PCR Tube 200 &amp;micro;L</td><td>Sarstedt</td><td>72.737</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Gibco</td><td>15140148</td><td></td></tr><tr><td>Pipette (0.2-2 &amp;micro;L)</td><td>fisherbrand Elite</td><td>FBE00002</td><td></td></tr><tr><td>Pipette (100-1000 &amp;micro;L)</td><td>fisherbrand Elite</td><td>FBE01000</td><td></td></tr><tr><td>Pipette (20-200 &amp;micro;L)</td><td>fisherbrand Elite</td><td>FBE00200</td><td></td></tr><tr><td>Pipette (2-20 &amp;micro;L)</td><td>fisherbrand Elite</td><td>FBE00020</td><td></td></tr><tr><td>Propidium Iodide</td><td>Invitrogen</td><td>P1304MP</td><td></td></tr><tr><td>Reaction Tube 1.5 mL</td><td>Sarstedt</td><td>72.690.300</td><td></td></tr><tr><td>Sorvall ST 16R Centrifuge</td><td>Thermo Scientific</td><td>75004240</td><td></td></tr><tr><td>Thincert (24-well)</td><td>Greiner Bio-One</td><td>662 641</td><td>0.4 &amp;micro;m pore diameter, 2x10&lt;sup&gt;6&lt;/sup&gt; cm&lt;sup&gt;-2&lt;/sup&gt; pore density, transparent PET</td></tr><tr><td>Tissue Culture Plate (24-well)</td><td>fisherbrand</td><td>FB012929</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Sigma-Aldrich</td><td>T8154-20mL</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090046</td><td></td></tr></tbody></table>

porous substrate-based electroporation with transepithelial electrical impedance monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

The given protocol establishes a method for using TEEI measurements to examine the intermediate processes of electroporation and make delivery predictions, specifically for the A431 cell line and PI cargo. While modification of this protocol is discussed further in the article, it is important to note now that while the specific values may change, general trends in the response remain consistent. For example, TEEI data that dips below the initial baseline corresponds with cell death, while the maximum increase in TEEI value above the minimum corresponds with delivery efficiency13. These general trends and their implications are explored below.
As shown in Figure 2A, a range of TEEI measurement and cell imaging trends can arise while using the PSEP platform. The ideal outcome of this protocol is to produce a curve similar to the optimized healthy data shown in Figure 2Ai. This is characterized as the ideal outcome, as there is no dip below baseline, indicating very little, if any, cell death. Additionally, the optimized healthy curve has the largest increase in TEEI from minimum, indicating a high degree of delivery efficiency13. These inferences are supported by the post-PSEP imaging of the cell monolayer, which reveals negligible cell death and a healthy, fully confluent cell monolayer (Figure 2Aiii). Furthermore, a successful delivery experiment using identical PSEP waveforms can be characterized by the images shown in Figure 2B. Proper electroporation waveform application and cargo concentration result in a high degree of delivery consistency and cell viability.
The health and confluency of the cell monolayer are critical to the successful application of TEEI-based delivery predictions16,17. Even with an optimized waveform, an unhealthy or incomplete cell monolayer results in a diminished TEEI response, as illustrated by the optimized unhealthy data in Figure 2Aii. However, note that the images for this outcome (Figure 2Avi) still correspond to the interpretation of the TEEI response given by Brooks et al.13. There is no dip below the baseline, indicating near-zero cell death (Figure 2C,D). Additionally, the reduced increase from the minimum corresponds to a negative impact on the delivery efficiency, as fewer cells in the monolayer reduce total PI delivery (Figure 2C,D).
If an unoptimized waveform is applied, it is possible to see even more significant decreases in TEEI response. Depending on the total energy and the timeframe in which it is applied, unoptimized waveforms can produce results ranging from decreased efficiency to near-total annihilation of the cell monolayer (Figure 2Ai,iii,v). Both the unoptimized and very unoptimized curves show a significant decrease from baseline, indicating substantial cell death. However, increasingly unoptimized waveforms impede cell recovery, resulting in diminished delivery efficiency.
Delivery efficiency was calculated by dividing the number of cells labeled in both PI and calcein images from delivery experiments by the total number of cells identified with Hoechst. Death was calculated by taking the cells marked with PI in TEEI measurement experiments and dividing by the total number of cells identified with Hoechst. Cell count was determined using a custom CellProfiler pipeline for both metrics.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66971/66971fig02.jpg" /> 
Figure 2: TEEI response curves and imaging for common conditions. (A) (i) TEEI response data illustrating percent TEEI change for optimized and unoptimized conditions. (ii) TEEI response comparison between healthy and unhealthy monolayers under optimized waveform conditions. (iii-v) Representative imaging of potential outcomes for optimized and unoptimized waveform conditions showing cell death (red) and living cells (green). (vi) Representative imaging of unhealthy monolayer after applying optimized waveform conditions showing cell death (red) living cells (green). (B) Images showing successful PI delivery (red), living cells (green), and nuclei locations (blue). All images brightened for clarity. Scale bars: 1000 &#181;m. (C) TEEI decrease from pre-PSEP baseline to minimum post-PSEP and increase from post-PSEP minimum to post-PSEP peak for given voltages. (D) Delivery efficiency and cell death percentages for given voltages. Error bars represent the SEM (n = 6). (C,D) reproduced from Brooks et al.13 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66971/66971fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

porous-substrate-based-electroporation-with-transepithelial

Nanyang Technological University

Research

JoVE Journal

Bioengineering

387 Views.  University of Nebraska-Lincoln. Ons onderzoek is gericht op het verbeteren van de elektroporatie van poreuze substraten. Onderzoek heeft grote voordelen aangetoond van het gebruik van micro- en nanokanalen om elektroporatie te lokaliseren. Maar er zijn nog vragen die beantwoord moeten worden, vooral met betrekking tot het monitoren en karakteriseren van effecten tijdens de tussenstadia van het proces.Enkele van de meest recente ontwikkelingen zijn ons gebruik van trans-epitheliale el...

Video: Auteur in de schijnwerpers: Optimalisatie van elektroporeuze substraat door middel van micro- en nanokanalen voor verbeterde monitoring en karakterisering in de tu

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Biology

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

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