This work was supported by the Slovenian Research and Innovation Agency (ARIS) within research program P2-0249, infrastructure program I0-0022, and research project J2-2503. The work was in part supported by ARIS and the University of Ljubljana through funding for Start-up Research Programs. The work was in part supported by the European Union and ARIS through NextGenerationEU and NOO funding within project MN-0023. The work was in part supported by the European Union through the ERC Starting Grant (No. 101115323 - REINCARNATION) and Marie Sk&#322;odowska-Curie fellowship (No. 893077 - EPmIC). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. The authors thank the group of Adam E. Cohen from Harvard University for kindly providing the tet-on spiking HEK cells and Du&#353;a Hod&#382;i&#263; for her help in the cell culture laboratory.

1. Preparation of reagents and cell culture
<ol>
	<li>Prepare the cell culture medium by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose with 10% fetal bovine serum, 2 mM glutamine, penicillin-streptomycin (penicillin 100 U/mL, streptomycin 100 &#181;g/mL), 2 &#181;g/mL puromycin, 5 &#181;g/mL blasticidin, and 200 &#181;g/ mL geneticin.</li>
	<li>Prepare phosphate-buffered saline (PBS) solution with 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4. Filter it through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare Tyrode solution with 2 mM KCl, 125 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 30 mM glucose, pH 7.3. Prepare low potassium Tyrode solution with 0.5 mM KCl, 126.5 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 30 mM glucose, pH 7.3. Filter both solutions through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare 1 mg/mL poly-L-lysine stock solution by adding 5 mL of PBS in a vial containing 5 mg of poly-L-lysine.</li>
	<li>Prepare 2 mg/mL doxycycline stock solution in distilled water and filter it through a sterilizing membrane filter with a 0.22 &#181;m pore size.</li>
	<li>Prepare 4 mM potentiometric dye ElectroFluor630 stock solution in pure ethanol (add 25 &#181;L of pure ethanol to the tube containing 100 nM - 73 &#181;g of the dye).</li>
	<li>Culture genetically engineered HEK cells in a T25 flask in 6 mL of the cell culture medium as previously described8. The ingredients of the culture medium are light sensitive; thus, keep the medium protected from light (wrap the medium flask in aluminum foil) and perform passaging in low light conditions.</li>
	<li>Passage the cells every 3-4 days by seeding 3 x 105 or 5 x 105 cells to a fresh T25 flask. Count the cells using a hemocytometer. Maintain cells in a master flask as non-excitable NS-HEK cells without doxycycline addition because they do not tolerate sustained Kir2.1 expression. Use cells from passages lower than 14 in the experiments.</li>
</ol>
2. Sample preparation
<ol>
	<li>Poly-L-lysine-coating of cover glass chamber slides
	<ol>
		<li>Pipette 1 mL of PBS to one well of the 2-well cover glass chamber slides, add 10 &#181;L of sterile 1 mg/mL poly-L-lysine solution and mix well with a pipette.</li>
		<li>Incubate for 1.5 h at room temperature in sterile conditions in a laminar flow hood. Remove the PBS with poly-L-lysine without any further washing.</li>
	</ol>
	</li>
	<li>Seeding the cells
	<ol>
		<li>Prepare a 1:10 dilution of doxycycline stock solution in a culture medium in a 1.5 mL tube. For each well 30 &#181;L of 1:10 doxycycline dilution is needed (3 &#181;L of doxycycline stock solution and 27 &#181;L of culture medium), prepare the final volume depending on the total number of experimental wells.</li>
		<li>Remove the culture medium from the T25 flask and trypsinize the cells with 2.5 mL of Trypsin-EDTA solution in a CO2 incubator at 37 &#176;C for 2 min.</li>
		<li>Add 2.5 mL of fresh culture medium and, by gentle pipetting, detach the cells from the surface. Mix the cell suspension well.</li>
		<li>Count the cells with a hemocytometer and determine the density of cells in suspension.</li>
		<li>Calculate the volume of trypsinized cell suspension needed to establish the culture. For 2-day culture, seed 2.5 x 105 cells, and for 3-day culture, seed 1 x 105 cells per well. Adjust the exact number of cells if needed.</li>
		<li>Calculate the volume of the culture medium by subtracting the previously calculated cell suspension volume from 1470 &#181;L. The remaining 30 &#181;L represents the volume of 1:10 doxycycline dilution. The total volume in each well will be 1500 &#181;L (cells, doxycycline, and culture medium).</li>
		<li>For preparing samples with excitable S-HEK cells, pipette the calculated volume of culture medium and trypsinized cell suspension into one well and add 30 &#181;L of 1:10 doxycycline dilution. Use only one well of the two-well chambers since the experiment takes a long time (around 40 min). Mix well by gentle pipetting just before placing the chambers into the CO2 incubator.</li>
		<li>For preparing samples with non-excitable NS-HEK cells, pipette the calculated volume of culture medium into a well and add 90% of the calculated volume of cell suspension for S-HEK cells without doxycycline. NS-HEK cells grow slightly faster than S-HEK cells.</li>
		<li>Incubate the chambers in a 5% CO2 incubator at 37 &#176;C for 2-3 days prior to the experiment. For samples with doxycycline, this incubation time is sufficient to render the cells excitable.</li>
	</ol>
	</li>
	<li>Labeling the cells
	<ol>
		<li>Before the experiment, pipette 3 &#181;L of potentiometric dye stock solution to a 1.5 mL tube and leave the open tube at room temperature in a laminar flow hood to allow the ethanol to evaporate and the potentiometric dye to dry (approximately 15 min). Add 997 &#181;L of cold culture medium (from a refrigerator at 4 &#176;C) to the tube and dissolve the dye to a final concentration of 12 &#181;M.</li>
		<li>Remove the culture medium from the well and add 1 mL of potentiometric dye solution to the cells. Refrigerate them at 4 &#176;C and incubate for 20 min.</li>
		<li>Remove the potentiometric dye solution and save it in the tube for further reuse (for up to six sequential experiments in 1 day).</li>
		<li>Gently wash the cells 3x with Tyrode solution, and after the last wash, pipette 1 mL of low potassium Tyrode solution into the well.</li>
	</ol>
	</li>
</ol>
3. Electrostimulation and electroporation
<ol>
	<li>Configure synchronization of the pulse delivery with image acquisition using a TTL signal from the microscope system that triggers the pulse generator. For generation of high-voltage pulses, use a custom pulse generator11 or any pulse generator that can be triggered externally with a TTL signal. 
	NOTE: In the presented experimental configuration, LAS X software is used for image acquisition, which can be configured to send a TTL signal at a preselected time during a time-lapse acquisition.</li>
	<li>Place two parallel Pt/Ir wire electrodes, with 0.8 mm diameter and 5 mm distance between inner edges, at the bottom of the chamber and fix the chamber under the microscope.</li>
	<li>Connect the electrodes to the pulse generator. Connect the output of the pulse generator to an oscilloscope using a voltage and a current probe to enable verification that the pulses were correctly delivered during the experiment.</li>
	<li>Focus the cells in brightfield. Image the cells that are located roughly in the middle between the electrodes.</li>
	<li>Start the first acquisition of fluorescence images in time-lapse mode with a 40x objective, recording the fluorescence signal in the whole field of view. The camera should be fast enough to acquire a clear time course of an action potential (ideally higher than 25 frames/s).
	<ol>
		<li>Acquire 80 images in a time-lapse mode, with at least 25 frames/s to resolve the real-time course of the action potential. The total duration of image acquisition results in around 3&#160;s. During image acquisition, illuminate the cells with an excitation wavelength of 635 nm, exposure time of 10 ms, and detect the emission at around 700 nm. In this first acquisition, do not apply any pulse - this acquisition will be used to correct the subsequent acquisitions for dye photobleaching. After this acquisition, wait for 2 min. 
		NOTE: This experiment uses 27.8 frames/s, i.e., 36 ms period, which is the time from the beginning of the first image acquisition to the beginning of the next image acquisition in the time-lapse recording).</li>
	</ol>
	</li>
	<li>Every 2 min, record a time-lapse in the same way as in step 3.5. For these acquisitions, apply a single 100 &#181;s pulse at the time of acquisition of the 10th image (at 324 ms). Acquire images with the following pulse voltages: 63 V, 75 V, 88 V, 100 V, 125 V, 150 V, 175 V, and 200 V. Estimate the electric field to which the cells are exposed as the applied voltage-to-electrode-distance ratio (E = 126, 150, 176, 200, 250, 300, 350, and 400 V/cm). 
	NOTE: At lower electric fields, the pulses will trigger action potentials; at higher electric fields, they will trigger electroporation, which manifests as a prolonged depolarization. The duration of the experiment on each sample of cells is approximately 40 min (20 min for labeling and 20 min for microscopy).</li>
</ol>
4. Data analysis
<ol>
	<li>Export the time-lapse recordings to tiff format.</li>
	<li>Place time-lapse recordings of all the applied voltages during an individual experiment (one sample of cells) into a single folder. Rename the time-lapse recordings in such a form that electric field E can be recognized by the MATLAB application: Vcm (e.g., 126Vcm for 126 V/cm). The remaining parts of the name can be arbitrary. For the acquisition at the beginning of the experiment where no pulse is applied, rename using the phrase 000Vcm. An example of the time-lapse recording names: 2022_1_20 1DOX 000Vcm, 2022_1_20 1DOX 126Vcm, 2022_1_20 1DOX 150Vcm, etc.</li>
	<li>Open the application, which can be downloaded from 
	https://github.com/bor-kos/fluorescenceActionPotential. Click on&#160;Select Data Folder to choose the folder containing time-lapse recordings of one experiment.</li>
	<li>Select the pixels to be used for further data analysis. To do this, use two sliders to determine the low threshold for a clear image of the membranes (in purple) and the high threshold for eliminating debris (large spots with high fluorescence) from the analysis (in green).</li>
	<li>Select Analyze Data. In the Analysis tab, a table with extracted parameters appears. On the right side of the table, graphs of relative fluorescence in time appear for each E used. Select graphs for a given E from the drop-down menu on the left side above the table.</li>
	<li>Examine all the graphs and perform manual correction in the case of false peak detection. Select Clear AUTO Peaks at the bottom in the Analysis tab, and the peaks that were detected automatically are cleared. Click on the Graph to manually determine a new peak time point.</li>
	<li>Select Rerun AUTO Peak Detection at the bottom in the Analysis tab to perform automatic peak detection again. The extracted parameters will now be adjusted to the new peak.</li>
	<li>Select Export Data at the bottom right in the Analysis tab to export data to the same folder in the form of a .mat file, spreadsheet file, and PNG images for each E used.</li>
</ol>

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

<ol>
	<li>Kotnik, T., Rems, L., Tarek, M., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+and+electropermeabilization:+Mechanisms+and+models.">Membrane electroporation and electropermeabilization: Mechanisms and models.</a> Ann Rev Biophys. 48 (1), 63-91 (2019).</li><li>Yarmush, M. L., Golberg, A., Ser&#353;a, G., Kotnik, T., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+technologies+for+medicine:+principles,+applications,+and+challenges.">Electroporation-based technologies for medicine: principles, applications, and challenges.</a> Ann Rev Biomed Eng. 16, 295-320 (2014).</li><li>Kotnik, T., 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=Electroporation-based+applications+in+biotechnology.">Electroporation-based applications in biotechnology.</a> Trend Biotechnol. 33 (8), 480-488 (2015).</li><li>Mahni&#269;-Kalamiza, S., Vorobiev, E., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+in+food+processing+and+biorefinery.">Electroporation in food processing and biorefinery.</a> J Memb Biol. 247 (12), 1279-1304 (2014).</li><li>Reddy, V. Y., 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=Pulsed+field+or+conventional+thermal+ablation+for+paroxysmal+atrial+fibrillation.">Pulsed field or conventional thermal ablation for paroxysmal atrial fibrillation.</a> New Engl J Med. 389 (18), 1660-1671 (2023).</li><li>Cvetkoska, A., Ma&#269;ek-Lebar, A., Trdina, P., Miklav&#269;i&#269;, D., Reber&#353;ek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+contractions+and+pain+sensation+accompanying+high-frequency+electroporation+pulses.">Muscle contractions and pain sensation accompanying high-frequency electroporation pulses.</a> Sci Rep. 12 (1), 8019 (2022).</li><li>Tung, L., Tovar, O., Neunlist, M., Jain, S. K., O'neill, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+strong+electrical+shock+on+cardiac+muscle+tissue.">Effects of strong electrical shock on cardiac muscle tissue.</a> Ann New York Acad Sci. 720 (1), 160-175 (1994).</li><li>Batista Napotnik, T., 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=Genetically+engineered+HEK+cells+as+a+valuable+tool+for+studying+electroporation+in+excitable+cells.">Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells.</a> Sci Rep. 14 (1), 720 (2024).</li><li>McNamara, H. M., Zhang, H., Werley, C. A., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+controlled+oscillators+in+an+engineered+bioelectric+tissue.">Optically controlled oscillators in an engineered bioelectric tissue.</a> Phys Rev X. 6 (3), 031001 (2016).</li><li>Tian, H., 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=Video-based+pooled+screening+yields+improved+far-red+genetically+encoded+voltage+indicators.">Video-based pooled screening yields improved far-red genetically encoded voltage indicators.</a> Nat Methods. 20 (7), 1082-1094 (2023).</li><li>Sweeney, D. C., 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=Quantification+of+cell+membrane+permeability+induced+by+monopolar+and+high-frequency+bipolar+bursts+of+electrical+pulses.">Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses.</a> Biochim Biophys Acta Biomembranes. 1858 (11), 2689-2698 (2016).</li><li>Pakhomov, A. G., 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=Long-lasting+plasma+membrane+permeabilization+in+mammalian+cells+by+nanosecond+pulsed+electric+field+(nsPEF).">Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF).</a> Bioelectromagnetics. 28 (8), 655-663 (2007).</li><li>Lee, P., 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=In+vivo+ratiometric+optical+mapping+enables+high-resolution+cardiac+electrophysiology+in+pig+models.">In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models.</a> Cardiovasc Res. 115 (11), 1659-1671 (2019).</li></ol>

The authors declare no conflict of interest.

S-HEK cells are a simple excitable cell model with well-defined sodium and potassium channels, NaV1.5 and Kir2.19. This enables us to perform in-depth studies of how excitable cells respond to applied electric pulses and link the experimental data with mathematical models based on the well-known theoretical characteristics of the channels expressed in S-HEK cells. In our previous study8, we described the responses of these cells to 100 &#181;s pulses o...

In electroporation, high-voltage electric pulses cause an increase in plasma membrane permeability for molecules that are otherwise poorly permeant1. Electroporation-based techniques are nowadays widely used in applications in medicine2, biotechnology3, and food technology4.
Excitable cells such as&#160;neuronal and muscle cells are among the primary targets in rapidly emerging electroporation-based treatments in the heart, brain, and skeletal muscles5. Moreover, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects such as pain, muscle twitching, and nerve damage6. Namely, electric pulses can cause electrical stimulation of excitable cells: they activate voltage-gated ion channels and trigger action potentials (AP). However, at higher electric fields, the cells&#39; plasma membrane becomes permeabilized, and additional ionic current through pores/defects occurs7. This additional ionic current affects cell excitability in a complex interplay between excitation and electroporation. 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.
In a recent study8, we explored genetically engineered human embryonic kidney (HEK) cells developed by Cohen et al.9,10, as a valuable tool for studying electroporation in excitable cells. In these otherwise non-excitable cells, a minimal complement of sodium and potassium channels (NaV1.5 and Kir2.1) needed for excitability is expressed. The expression of Kir2.1 in these cells is controlled by a doxycycline-induced tet-on system that allows the establishment of two cell variants: excitable spiking S-HEK (containing NaV1.5 and Kir2.1) and non-excitable non-spiking NS-HEK cells (containing only NaV1.5 channels). This allows us to compare the effects of electric pulses on excitable and non-excitable variants of the same type of cells. Compared to isolated primary excitable cells, excitable cells differentiated from embryonic or induced pluripotent stem cells, and cell lines derived from excitable tissues, S-HEK cells have several advantages: they are easy to handle, cultivate and propagate, have well-defined sodium and potassium channels, and generate robust APs8.
We designed a protocol for monitoring changes in transmembrane voltage (TMV) triggered by electric pulses of different amplitudes with the use of a fluorescent potentiometric dye. This way, we can study how electroporation affects AP generation in S-HEK cells and other changes in the TMV in both S-HEK and NS-HEK cells. Briefly, the S-HEK or NS-HEK cells are exposed to a single 100 &#181;s electric pulse every 2 min, and relative changes in fluorescence are recorded for roughly 3 s around each pulse application. The applied pulses are of increasing voltage, first triggering APs&#160;in cells and, at a higher electric field, electroporation. The 2 min interval between pulse application was chosen to minimize the possible cumulative effects of consecutive pulses while keeping experiments short enough not to deteriorate the cells. The image processing was automated with a custom MATLAB application to extract relevant parameters of the fluorescence responses of the potentiometric dye, hence evaluating the changes in TMV (APs and&#160;sustained depolarization due to electroporation) in S-HEK and NS-HEK&#160;cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube</td>
			<td>Ratiolab&#160;</td>
			<td>5615000</td>
			<td></td>
		</tr>
		<tr>
			<td>2-well Nunc Lab-Tek II Chambered #1.5 German Coverglass System</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP305 Differential voltage probe</td>
			<td>Teledyne LeCroy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AP015 Current probe</td>
			<td>Teledyne LeCroy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator&#160;</td>
			<td>PHCbi&#160;</td>
			<td>MCO -230AICUV</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline&#160;</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modifed Eagle&#8217;s Medium high glucose growth medium</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>ElectroFluor630 (Di-4-ANEQ(F)PTEA)</td>
			<td>Potentiometric Probes</td>
			<td>31795</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10131035</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Iskra Pio&#160;</td>
			<td>MC 15-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>P07681</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide&#160;</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>P9155-5MG&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse generator&#160;</td>
			<td>Custom made</td>
			<td></td>
			<td>see reference11</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 25cm&#178; Tissue culture flask</td>
			<td>TPP&#160;</td>
			<td>90026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tet-on spiking HEK cells</td>
			<td>American Type Culture Collection ATCC</td>
			<td>CRL-3479</td>
			<td></td>
		</tr>
		<tr>
			<td>Thunder Imager Live Cell system for fluorescence microscopy</td>
			<td>Leica Microsystems&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich/Merck</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>WavePro 7300A Oscilloscope</td>
			<td>Teledyne LeCroy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Changes in TMV in excitable cells in vitro triggered by electric pulses can be optically monitored with this protocol, and the&#160;fluorescence signals can be analyzed to extract their relevant parameters.
The experimental setup is shown in Figure 1A. A typical signal of 100 &#181;s electric pulse used in experiments is shown in Figure 1B. Cells in the chambers that are placed under the microscope typically look like the cel...

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

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 ...

monitoring-electroporation-induced-changes-action-potential

Research

JoVE Journal

Bioengineering

191 Views.  University of Ljubljana. 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.

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

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...