<meta charset="utf8"></head><body>Método TEEI para análise de eletroporação e previsão de entrega em células A431

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+electroporation-mediated+molecular+uptake+and+cell+viability.">Quantitative study of electroporation-mediated molecular uptake and cell viability.</a> Biophys J. 80 (2), 755-764 (2001).</li><li>Pliquett, U., Gift, E. A., Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+electric+field+and+anomalous+heating+caused+by+exponential+pulses+with+aluminum+electrodes+in+electroporation+experiments.">Determination of the electric field and anomalous heating caused by exponential pulses with aluminum electrodes in electroporation experiments.</a> Bioelectrochem Bioenerg. 39 (1), 39-53 (1996).</li><li>Pan, 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=Cell+membrane+damage+and+cargo+delivery+in+nano-electroporation.">Cell membrane damage and cargo delivery in nano-electroporation.</a> Nanoscale. 15 (8), 4080-4089 (2023).</li><li>Boukany, P. E., 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=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nat Nanotechnol. 6 (11), 747-754 (2011).</li><li>Chang, L., 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=Micro-/nanoscale+electroporation.">Micro-/nanoscale electroporation.</a> Lab Chip. 16 (21), 4047-4062 (2016).</li><li>Patino, C. A., 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=Multiplexed+high-throughput+localized+electroporation+workflow+with+deep+learning-based+analysis+for+cell+engineering.">Multiplexed high-throughput localized electroporation workflow with deep learning-based analysis for cell engineering.</a> Sci Adv. 8 (29), eabn7637 (2022).</li><li>Sagvolden, G., Giaever, I., Pettersen, E. O., Feder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+force+microscopy.">Cell adhesion force microscopy.</a> Proc Natl Acad Sci U S A. 96 (2), 471-476 (1999).</li><li>Ishibashi, T., Takoh, K., Kaji, H., Abe, T., Nishizawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porous+membrane-based+culture+substrate+for+localized+in+situ+electroporation+of+adherent+mammalian+cells.">A porous membrane-based culture substrate for localized in situ electroporation of adherent mammalian cells.</a> Sensors Actuators B: Chem. 128 (1), 5-11 (2007).</li><li>Mukherjee, P., Nathamgari, S. S. P., Kessler, J. A., Espinosa, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+numerical+and+experimental+investigation+of+localized+electroporation-based+cell+transfection+and+sampling.">Combined numerical and experimental investigation of localized electroporation-based cell transfection and sampling.</a> ACS Nano. 12 (12), 12118-12128 (2018).</li><li>Brooks, J. R., 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=An+equivalent+circuit+model+for+localized+electroporation+on+porous+substrates.">An equivalent circuit model for localized electroporation on porous substrates.</a> Biosens Bioelectron. 199, 113862 (2022).</li><li>Brooks, J. R., 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=Transepithelial+electrical+impedance+increase+following+porous+substrate+electroporation+enables+label-free+delivery.">Transepithelial electrical impedance increase following porous substrate electroporation enables label-free delivery.</a> Small. 20 (25), 2310221 (2023).</li><li>Vindi&#353;, 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=Gene+electrotransfer+into+mammalian+cells+using+commercial+cell+culture+inserts+with+porous+substrate.">Gene electrotransfer into mammalian cells using commercial cell culture inserts with porous substrate.</a> Pharmaceutics. 14 (9), 1959 (2022).</li><li>Ye, 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=Single-cell+electroporation+with+real-time+impedance+assessment+using+a+constriction+microchannel.">Single-cell electroporation with real-time impedance assessment using a constriction microchannel.</a> Micromachines. 11 (9), 856 (2020).</li><li>Bednarek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+measuring+the+permeability+of+cell+monolayers.">In vitro methods for measuring the permeability of cell monolayers.</a> Methods Protoc. 5 (1), 17 (2022).</li><li>Harhaj, N. S., Antonetti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tight+junctions+and+loss+of+barrier+function+in+pathophysiology.">Regulation of tight junctions and loss of barrier function in pathophysiology.</a> Int J Biochem Cell Biol. 36 (7), 1206-1237 (2004).</li><li>Hunter, 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=.">.</a> Zeta potential in colloid science: Principles and applications. Vol. 2, (2013).</li><li>Wong, P. K., Wang, T. H., Deval, J. H., Ho, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetics+in+microdevices+for+biotechnology+applications.">Electrokinetics in microdevices for biotechnology applications.</a> IEEE/ASME Trans Mechatron. 9 (2), 366-376 (2004).</li><li>Qian, K., Wang, Y., Lei, Y., Yang, Q., Yao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+cell+swelling+for+osmotic+imbalance+induced+by+electroporation.">An experimental and theoretical study on cell swelling for osmotic imbalance induced by electroporation.</a> Bioelectrochemistry. 157, 108637 (2024).</li><li>Fox, M. B., 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+of+cells+in+microfluidic+devices:+A+review.">Electroporation of cells in microfluidic devices: A review.</a> Anal Bioanal Chem. 385 (3), 474-485 (2006).</li></ol>

<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

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>

<meta charset="utf8"></head><body>Autor em destaque: Otimizando a eletroporação de substrato poroso por meio de micro e nanocanais para monitoramento aprimorado e caracterização de estágio intermed

Eletroporação Baseada em Substrato Poroso com Monitoramento de Impedância Elétrica Transepitelial

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

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

225 visualizações.  University of Nebraska-Lincoln. Nossa pesquisa está focada em melhorar a eletroporação de substrato poroso. A pesquisa mostrou grandes benefícios do uso de micro e nano canais para localizar a eletroporação. Mas ainda há questões a serem respondidas, especialmente no que diz respeito ao monitoramento e caracterização dos efeitos durante as etapas intermediárias do processo.Alguns dos desenvolvimentos mais recentes incluem o uso de monitoramento de impedância elétrica tra...

Vídeo: Autor em destaque: Otimizando a eletroporação de substrato poroso por meio de micro e nanocanais para monitoramento aprimorado e caracterização de estágio intermed

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.

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.