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

<ol>
	<li>Brooks, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+and+highly+controllable+methods+for+in+vitro+intracellular+delivery.">High throughput and highly controllable methods for in vitro intracellular delivery.</a> Small. 16 (51), e2004917 (2020).</li><li>Stewart, M. P., Langer, R., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+by+membrane+disruption:+Mechanisms,+strategies,+and+concepts.">Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts.</a> Chem Rev. 118 (16), 7409-7531 (2018).</li><li>Canatella, P. J., Karr, J. F., Petros, J. A., Prausnitz, M. 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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 increasi...

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/files/ftp_upload/66971/66971fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 cm2 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 &#181;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 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (100-1000 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (20-200 &#181;L)</td>
			<td>fisherbrand Elite</td>
			<td>FBE00200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (2-20 &#181;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 &#181;m pore diameter, 2x106 cm-2 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 va...

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

Research

JoVE Journal

Bioengineering

339 Views.  University of Nebraska-Lincoln. 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.

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

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

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 ...

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned tissues due to convenience in the packaging of cells, improved cell retention, and minimal invasiveness. Moreover, the porous conformation of these microscale carriers could enhance the medium exchange and improve the level of nutrients and oxygen supplies. The present study illustrates the convenient fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (PLGA-HOPMs) by the facile microfluidic technology for cell delivery applications. The resultant monodispersed PLGA-HOPMs possessed particle sizes of ~400 &#181;m and open pores of ~50 &#181;m with interconnecting windows. Briefly, the emulsified oil droplets (PLGA solution in dichloromethane, DCM), wrapped with the 7.5% (w/v) gelatin aqueous phase, were introduced into the 1% (w/v) continuous flowing poly(vinyl alcohol) (PVA) aqueous solution through the coaxial nozzle in the customized microfluidic setup. Subsequently, the microspheres were subjected to solvent extraction and lyophilization procedures, resulting in the production of HOPMs. Notably, various formulations (concentrations of PLGA and porogen) and processing parameters (emulsifying power, needle gauge, and flow rate of dispersed phase) play crucial roles in the qualities and characteristics of the resulting PLGA HOPMs. Moreover, these architectures might potentially encapsulate various other biochemical cues, such as growth factors, for extended drug discovery and tissue regeneration applications.

Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned ...

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...