The authors would like to acknowledge financial support by the National Science Foundation (NSF CBET 0967598, DBI IDBR 1353918) and the U.S. Department of Education&#39;s Graduate Training in Emerging Areas of Precision and Personalized Medicine (P200A150131) for funding&#160;graduate student J.J.S. on fellowship.

NOTE: Users should review all MSDS for the materials and supplies used in this protocol. Appropriate PPE should be worn at each step and sterile technique used during experimentation. Sections 1-7 discuss the device fabrication.
1. Device fabrication- Mask design
NOTE: Refer to Figure 2 for an illustration of the microfabrication process. The microfabrication steps are to be carried out in a cleanroom environment. Additio...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+electroporation+device+for+cell+lysis.">A microfluidic electroporation device for cell lysis.</a> Lab on a Chip. 5 (1), 23-29 (2005).</li><li>Kar, S., 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:+current+trends,+applications+and+future+prospects.">Single-cell electroporation: current trends, applications and future prospects.</a> Journal of Micromechanics and Microengineering. 28 (12), (2018).</li><li>Shi, J. 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S., Kar, S., Chang, H. Y., Tseng, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-localized+single-cell+nano-electroporation.">Nano-localized single-cell nano-electroporation.</a> Lab on a Chip. 20 (22), 4194-4204 (2020).</li><li>Lee, W. G., Demirci, U., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+electroporation:+challenges+and+perspectives+for+clinical+applications.">Microscale electroporation: challenges and perspectives for clinical applications.</a> Integrative Biology. 1 (3), 242-251 (2009).</li><li>Santra, T. S., Chang, H. Y., Wang, P. C., Tseng, F. 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The authors have nothing to disclose.

The methodology presented within this protocol primarily focuses on the microfabrication of a microfluidic device that is then integrated into a specialized electroporation experimental setup. The term &#39;recipe&#39;, which is often used when describing the specifics of the microfabrication process, hints at the importance of following/optimizing each step to successfully fabricate a functioning device. However, certain critical steps within the process, when not optimized, such as UV exposure time/energy, PVD sputteri...

Current therapeutic innovations in biomedical research, such as CAR-T (Chimeric Antigen Receptor Engineered T cell) cell therapy and genetic editing using CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9, heavily rely on the ability to deliver exogenous material both successfully and efficiently into the intracellular space1. In CAR-T therapy, the gold standard to perform the gene delivery step in cell therapy manufacturing is using viral vectors2. Though viral-mediated gene delivery is an efficient delivery modality, it also has several drawbacks. These include manufacturing costs, cytotoxicity, immunogenicity, mutagenesis/tumorigenesis potential, and size limitations on the gene(s) to be delivered3. These limitations have led to the research and development of alternative, non-viral delivery technologies.
Electroporation, an alternative to viral-mediated gene delivery, relies on the application of an optimal electrical pulse waveform to perform DNA, RNA, and protein transfections of cells. Following the application of an external electric field, the cell membrane is briefly compromised, making the cell susceptible to the intracellular delivery of otherwise impermeable exogenous materials4. Compared to viral-mediated delivery, electroporation is advantageous as it is generally safe, easy to operate, and has low operating costs. Electroporation can deliver both small and large molecular cargo and can be efficient in transfecting cells regardless of lineage5. To achieve desirable outcomes following electroporation, i.e., good viability and good electro-transfection efficiency, a variety of experimental parameters need to be co-optimized. These include cell type6, cell density, molecule concentration7, electroporation buffer properties (e.g., molecular composition, conductivity, and osmolarity)8, electrode size/geometry9, and electrical pulse waveform (shape, polarity, number of pulses)10 (refer to Figure 1 for an illustration). Although each of these parameters can have a significant effect on the outcomes of electroporation experiments, pulse waveform has been especially studied in great detail, as the electrical energy of the applied pulse(s) is the root of the intrinsic trade-off between the resulting cell viability and electro-transfection efficiency8.
Typically, electroporation experiments are performed on the macro-scale, where cells are suspended in 100s of microliters of buffer between a set of large, parallel-plate electrodes within an electroporation cuvette. The electrodes are commonly manufactured out of aluminum with an electrode distance of 1-4 mm. Once the cells are manually loaded via pipette, the cuvette is electrically connected to a bulky, electrical pulse generator where the user can set and apply the pulse waveform parameters to electroporate the cell suspension. Although macro-scale or bulk electroporation can process cell densities &#62;106 cells/mL, this feature can be wasteful when optimizing the electrical pulse waveform settings. This is particularly of concern when electroporating primary cell types where the cell population numbers can be limited. Additionally, due to the large distance between the electrodes, the pulse generator must be able to supply large voltages to achieve electric field strengths &#62;1kV/cm11. These high voltages cause resistive power dissipation through the electrolyte buffer resulting in Joule heating, which can be detrimental to the resulting cell viability12. Lastly, performing electroporation on a dense suspension of cells will consistently be burdened with an innate variability in the resulting electro-transfection efficiency and cell viability. Each cell in suspension could experience a different electric field strength due to the surrounding cells. Depending on whether the experienced electric field strength is either increased or decreased, the resulting cell viability or electro-transfection efficiency may each be negatively impacted11. These downsides to macro-scale electroporation have led to the pursuit and development of alternative technologies that operate on the micro-scale and allow for better control at the single-cell level.
The field of BioMEMS, or biomedical micro-electro-mechanical systems, stems from the technological advancements made in the microelectronics industry. Specifically, utilizing microfabrication processes to develop micro-devices for the advancement of biomedical research. These advancements include the development of micro-electrode arrays for in vivo electrical monitoring13, capacitive micro-electrodes for in situ electroporation14, miniaturized organ-on-a-chip devices15, microfluidic point-of-care diagnostics16, biosensors17, and drug delivery systems18, including nano- and micro-electroporation devices19,20,21. Due to the ability to design and manufacture devices at the same size scale as biological cells, nano- and micro-electroporation technologies are advantageous when compared to their macro-scale counterpart22,23. These electroporation devices eliminate the requirement of high voltage pulse applications, as electrode sets with spacings of 10s to 100s of micrometers are typically integrated. This feature drastically reduces the current through the electrolyte, which in turn reduces the accumulation of toxic electrolysis products and the effects of Joule heating in these systems. The micro-scale channels also ensure that a much more uniform electric field is reliably applied to the cells during pulse application, resulting in more consistent outcomes24. In addition, it is also commonplace for micro-electroporation devices to be integrated into a microfluidic platform which lends itself for future integration into a fully automated technology, a highly desirable capability in cell therapy manufacturing25. Lastly, micro-scale electroporation allows for the electrical interrogation of electroporation events. For example, the degree of cell membrane permeabilization can be monitored in real-time at a single cell level26,27. The purpose of this method is to describe the microfabrication, system operation, and analysis of a microfluidic, single-cell micro-electroporation device capable of measuring the degree of cell membrane permeabilization for optimizing electroporation protocols, yet increasing throughput over the previous state-of-the-art.
Performing single-cell level electroporation is no longer a novel technique, as it was first demonstrated by Rubinsky et al. in 2001 with the development of a static cell electroporation technology28. Their micro-device was innovative as they were the first to demonstrate the ability to electrically monitor the event of electroporation. This has further led to the development of static, single-cell electroporation technologies capable of electrically detecting the degree of cell membrane permeabilization in a parallelized manner to increase the throughputs of the devices. However, even with parallelization and batch processing, these devices severely lack the total number of cells they can process per unit time29,30. This limitation has led to the development of flow-through devices capable of performing single-cell level micro-electroporation at much greater throughputs31. This device transition, from static to flow-through environment, limits the capability of electrically monitoring the degree of cell membrane permeabilization following the application of the electroporation pulse. The method described in this work bridges the gap between these two technologies, a micro-electroporation technology capable of electrically detecting, pulsing, and monitoring the degree of cell membrane permeabilization of individual cells, in a continuous-flow, serial fashion.
This technology was recently described in Zheng et al. In that work, the capabilities of this technology were introduced with the completion of a parametric study, where both the amplitude and duration of the electroporation pulse were varied, and the ensuing electrical signal, indicative of cell membrane permeabilization, was explored32. The results showed that an increase in the intensity of the electroporation pulse (i.e., increase in applied electric field or increase in pulse duration) caused an increase in the measured cell membrane permeabilization. To further validate the system, a common fluorescent indicator of successful electroporation, propidium iodide33, was added to the cell suspension, and a fluorescence image was captured immediately following the application of the electrical pulse. The optical signal, i.e., the fluorescence intensity of propidium iodide inside the cell, was strongly correlated with the electrical measurement of the degree of cell membrane permeabilization, verifying the reliability of this electrical measurement. However, this work only considered the delivery of the small molecule propidium iodide, which has little to no translatable significance.
In this work, a new application of this technology is introduced to improve upon the throughput of the system while delivering a biologically active plasmid DNA (pDNA) vector and assessing the electro-transfection efficiency of cells replated and cultured following electroporation. Though the previous work outperforms existing micro-electroporation technologies that are capable of electrically measuring the event of electroporation, the current state of the device still requires long cell transit times between the electrode set (~250 ms) to perform the cell detection, pulse application, and the cell membrane permeabilization measurement. With a single channel, this limits the throughput to 4 cells/s. To combat this limitation, a new concept of cell-population-based feedback-controlled electroporation is introduced to perform pDNA electro-transfection. By using a hypo-physiologic conductivity electroporation buffer, this system allows for the electrical interrogation of single cells across a multitude of electroporation pulse applications. Based on the electrical response, an &#39;optimal&#39; electroporation pulse is then determined. A &#39;high-throughput&#39; mode is then implemented where the cell membrane permeabilization determination is nullified, the flow rate is increased, and the electroporation pulse duty cycle is matched to the cell transit time to ensure one pulse per cell in transit between the electrodes. This work will provide extensive details into the microfabrication steps for the manufacturing of the micro-device, the material/equipment and their setup required to perform the experimentation, and the operation/analysis of the device and its electro-transfection efficiency (eTE).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63103/63103fig01.jpg" /> 
Figure 1: Experimental factors affecting electroporation outcomes. (Left) Cell Suspension-Important factors to consider prior to the onset of electroporation include: Payload (in this case, pDNA), concentration, cell density, and electroporation buffer properties. Electroporation buffer properties to consider are conductivity, osmolarity, and the exact molecular composition contributing to these values. (Middle) Pulse Application-The exact pulse-type (square wave vs. exponential decay) and pulse waveform (single pulse vs. pulse train) must be optimized to maximize both the resulting cell viability and electro-transfection efficiency. Common pulse trains implemented in electroporation processes are typically composed of a series of High Voltage (HV) pulses or series of pulses rotating between HV and Low Voltage (LV) pulse magnitudes. (Right) Cell Recovery-Down-stream processing steps, in particular, the recovery cell culture media that cells are transferred to, should be optimized. Not featured (Far Left), additional upstream cell processing steps can be implemented for overall electroporation process optimization. <a href="https://www.jove.com/files/ftp_upload/63103/63103fig1v3large.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>150-mm diameter petri dishes</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td>step 6.1.1 to secure wafer</td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td>step 10.3.6 to plate electroporated cells</td>
		</tr>
		<tr>
			<td>33220A Waveform/Function generator</td>
			<td>Agilent</td>
			<td></td>
			<td>step 9.2.3 electroporation pulse generator</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Si-wafers</td>
			<td>University Wafer</td>
			<td></td>
			<td>subsection 2.1 for microfluidic channel fabrication</td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td>step 8.1.8 to plate cells</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td>step 2.1.2 for cleaning and&#160; step 5.1 photoresist lift-off</td>
		</tr>
		<tr>
			<td>Allegra X-22R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>steps 8.1.4 , 8.3.2. and 8.3.3. to spin down cells</td>
		</tr>
		<tr>
			<td>AutoCAD 2018</td>
			<td>Autodesk</td>
			<td></td>
			<td>subsection 1.1. to design transparency masks</td>
		</tr>
		<tr>
			<td>Buffered oxide etchant 10:1</td>
			<td>VWR</td>
			<td>901621-1L</td>
			<td>subsection 3.1 for HF etching</td>
		</tr>
		<tr>
			<td>CCD Monochrome microscope camera</td>
			<td>Hamamatsu</td>
			<td>Orca 285 C4742-96-12G04</td>
			<td>step 11.2.3. for imaging</td>
		</tr>
		<tr>
			<td>CMOS camera- Sensicam QE 1.4MP</td>
			<td>PCO</td>
			<td></td>
			<td>subsection 9.3 part of the experimental setup</td>
		</tr>
		<tr>
			<td>Conductive Epoxy</td>
			<td>CircuitWorks</td>
			<td>CW2400</td>
			<td>subsection 7.6. for wire attachement</td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td>step 8.1.4. for cell centrifuging</td>
		</tr>
		<tr>
			<td>Dektak 3ST Surface Profilometer</td>
			<td>Veeco (Sloan/Dektak)</td>
			<td></td>
			<td>step 2.1.15 and 5.4 for surface profilometry</td>
		</tr>
		<tr>
			<td>Disposable biopsy punch, 0.75 mm</td>
			<td>Robbins Instruments</td>
			<td>RBP075</td>
			<td>step 6.2.3 for inlet access</td>
		</tr>
		<tr>
			<td>Disposable biopsy punch, 3 mm</td>
			<td>Robbins Instruments</td>
			<td>RBP30P</td>
			<td>step 6.2.3 for outlet access</td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td>abcam</td>
			<td>ab108410</td>
			<td>step 11.2.2. for live cell staining</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11885084</td>
			<td>step 8.1.2. part of media composition</td>
		</tr>
		<tr>
			<td>E3631A Bipolar Triple DC power supply</td>
			<td>Agilent</td>
			<td></td>
			<td>step 9.2.1.-9.2.2.part of the experimental setup</td>
		</tr>
		<tr>
			<td>Eclipse TE2000-U Inverted&#160; Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>&#160;subsection 9.3. part of the experimental setup</td>
		</tr>
		<tr>
			<td>EVG620 UV Lithography System</td>
			<td>EVG</td>
			<td></td>
			<td>&#160;step 2.1.9. and 2.2.7. for UV Exposure</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Neuromics</td>
			<td>FBS001</td>
			<td>step 8.1.2. part of media composition</td>
		</tr>
		<tr>
			<td>FS20 Ultrasonic Cleaner</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>subsection 5.1. for photoresist lift-off</td>
		</tr>
		<tr>
			<td>Glass Media Bottle with Cap, 100mL</td>
			<td>Fisher Scientific</td>
			<td>FB800100</td>
			<td>step 8.2.1. for buffer storage</td>
		</tr>
		<tr>
			<td>Glass Media Bottle with Cap, 500mL</td>
			<td>Fisher Scientific</td>
			<td>FB800500</td>
			<td>step 8.1.2.for media storage</td>
		</tr>
		<tr>
			<td>HEK-293 cell line</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td>subsection 8.1 for cell culturing</td>
		</tr>
		<tr>
			<td>HEPES buffer solution</td>
			<td>Sigma Aldrich</td>
			<td>83264-100ML-F</td>
			<td>step 8.2.1 part of electroporation buffer composition</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma Aldrich</td>
			<td>379212-25ML</td>
			<td>step 2.2.3 adhesion promoter</td>
		</tr>
		<tr>
			<td>HF2LI Lock-in Amplifier</td>
			<td>Zurich Instruments</td>
			<td></td>
			<td>subsection 9.2 part of the experimental setup</td>
		</tr>
		<tr>
			<td>HF2TA Current amplifier</td>
			<td>Zurich Instruments</td>
			<td></td>
			<td>subsection 9.2 part of the experimental setup</td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>A459-1</td>
			<td>step 2.1.2 for cleaning, step 2.1.14 for rinsing wafer following SU-8 development, and step 6.3.1 for cleaning PDMS</td>
		</tr>
		<tr>
			<td>IX81 fluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>step 11.2.3 for imaging</td>
		</tr>
		<tr>
			<td>L-Glutamine Solution</td>
			<td>Sigma Aldrich</td>
			<td>G7513-20ML</td>
			<td>step 8.1.2. part of media composition</td>
		</tr>
		<tr>
			<td>M16878/1BFA 22 gauge wire</td>
			<td>AWC</td>
			<td>B22-1</td>
			<td>subsection 7.5 for device fabrication</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>208337-100G</td>
			<td>step 8.1.2 part of electroporation buffer composition</td>
		</tr>
		<tr>
			<td>MF 319 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>10018042</td>
			<td>step 2.2.9. photoresist developer</td>
		</tr>
		<tr>
			<td>Microposit S1818 photoresist</td>
			<td>Kayaku Advanced Materials</td>
			<td>1136925</td>
			<td>step 2.2.4 positive photoresist for electrode patterning</td>
		</tr>
		<tr>
			<td>Microscope slides, 75 x 25 mm</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td>step 2.2.1 electrode soda lime glass substrate</td>
		</tr>
		<tr>
			<td>Model 2350 High voltage amplifier</td>
			<td>TEGAM</td>
			<td>2350</td>
			<td>step 9.2.5. part of the experimental setup</td>
		</tr>
		<tr>
			<td>National Instruments LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>data acquisition</td>
		</tr>
		<tr>
			<td>Needle, 30G x 1 in</td>
			<td>BD Scientific</td>
			<td>305128</td>
			<td>step 10.1.1. part of the system priming</td>
		</tr>
		<tr>
			<td>PA90 IC OPAMP Power circuit</td>
			<td>Digi-key</td>
			<td>598-1330-ND</td>
			<td>Part of the custom circuit</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P4458-20ML</td>
			<td>step 8.1.2. part of media composition</td>
		</tr>
		<tr>
			<td>Plasmid pMAX-GFP</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>step 8.3.4. for intracellular delivery</td>
		</tr>
		<tr>
			<td>Plastic tubing, 0.010&#39;&#39; x 0.030&#34;</td>
			<td>VWR</td>
			<td>89404-300</td>
			<td>step 10.1.2. for system priming</td>
		</tr>
		<tr>
			<td>Platinum targets</td>
			<td>Kurt J. Lesker</td>
			<td></td>
			<td>subsection 4.2. for physical vapor deposition</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9333-500G</td>
			<td>step 8.2.1. part of electroporation buffer composition</td>
		</tr>
		<tr>
			<td>Pump 11 PicoPlus microfluidic syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>MA1 70-2213</td>
			<td>step 10.1.4. for system priming</td>
		</tr>
		<tr>
			<td>PVD75 Physical vapor deposition system</td>
			<td>Kurt J. Lesker</td>
			<td></td>
			<td>subsection 4.1. for physical vapor deposition</td>
		</tr>
		<tr>
			<td>PWM32 Spinner System</td>
			<td>Headway Research</td>
			<td></td>
			<td>steps 2.1.6 and 2.2.2. for substrate coating with photoresist</td>
		</tr>
		<tr>
			<td>PX-250 Plasma treatment system</td>
			<td>March Instruments</td>
			<td></td>
			<td>subsection 7.2 for PDMS and glass substrate bonding</td>
		</tr>
		<tr>
			<td>SDG1025 Function/Waveform generator</td>
			<td>Siglent</td>
			<td></td>
			<td>step 9.2.2. part of the experimental setup</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S8045-500G</td>
			<td>step 8.2.1. part of electroporation buffer composition</td>
		</tr>
		<tr>
			<td>SU-8 2010 negative photoresist</td>
			<td>Kayaku Advanced Materials</td>
			<td>Y111053</td>
			<td>step 2.1.7. for microfluidic channel patterning</td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Microchem</td>
			<td>Y010200</td>
			<td>step 2.1.12. for photoresist developing</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903-1KG</td>
			<td>step 8.2.1. part of electroporation buffer composition</td>
		</tr>
		<tr>
			<td>Sylgard 184 elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>step 6.2.1&#160; 10 : 1 mixture of PDMS polymer and hardening agent</td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>step 8.3.4. part of system priming</td>
		</tr>
		<tr>
			<td>SZ61 Stereomicroscope System</td>
			<td>Olympus</td>
			<td></td>
			<td>subsection 7.3. for channel and electrode alignment</td>
		</tr>
		<tr>
			<td>Tissue Culture Treated T25 Flasks</td>
			<td>Falcon</td>
			<td>353108</td>
			<td>step 8.1.2 for cell culturing</td>
		</tr>
		<tr>
			<td>Titanium targets</td>
			<td>Kurt J. Lesker</td>
			<td></td>
			<td>subsection 4.2. for physical vapor deposition</td>
		</tr>
		<tr>
			<td>Transparency masks</td>
			<td>CAD/ART Services</td>
			<td></td>
			<td>steps 2.1.9. and 2.2.7. for photolithography</td>
		</tr>
		<tr>
			<td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>step 6.1.2. for wafer silanization</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma Aldrich</td>
			<td>T4049-100ML</td>
			<td>steps 8.1.3. and 8.3.1. for cell harvesting</td>
		</tr>
	</tbody>
</table>

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.

Figure 4 highlights the operating principles behind the single-cell-level membrane permeabilization detection for a single pulse amplitude. Following the initiation of the electroporation experiment, the cell detection algorithm determines an optimal threshold for cell detection via a point-by-point, slope-based detection method. The system then continuously monitors (1) for a significant negative change in the measured electrical current, which is indicative of the entry of a cell. This is ...

Watch this Scientific Journal Video about The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection at JoVE.com

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

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 ...

This method allows researchers to determine optimal electroporation pulsing conditions for the desired cell type, while minimizing the need for the traditional empirical experimental approach. This method utilizes micro-electroporation techniques to manufacture a scalable microfluidic actuation device that is capable of electrically monitoring the degree of cell membrane permeabilization throughout the electroporation process. Demonstrating the procedure will be Kishankumar Busha, a master's student from my laboratory.To begin, secure the silicon wafer to the chuck of the wafer spin coater using the spin coater's vacuum system. Then program the spinner. Next, dispense four milliliters of SU-8 2010 photo resist onto the center of the silicon wafer and run the program.Once the system comes to a halt, turn off the vacuum. Then secure the photo mask with the 2D microfluidic channel designs onto the mask holder and insert the silicon wafer with the SU-8 coating facing upwards onto the wafer chuck. Set the exposure settings for 150 millijoules per square centimeter and run the machine.After the UV exposure, submerge the silicon wafer in the SU-8 developer solution with gentle agitation for three to four minutes. Then remove the wafer from the solution and rinse the surface with isopropanol. Next, place the silicon wafer into a 150 degrees Celsius oven for 30 minutes for a hard bake.Then allow it to cool down to room temperature before using stylus profilometry to measure the exact height and slope of the channel side walls. Next, dispense one milliliter of photo resist onto the surface of the glass slide and run the program once again. Once the system comes to a halt, turn off the vacuum and remove the glass slide.After securing the photo mask with the 2D electrode designs onto the mask holder, insert and align the glass wafer with the S-1818 coating facing upwards onto the wafer chuck. Set the exposure settings for 215 millijoules per square centimeter and run the machine. After the exposure, submerged the glass slide in MF-319 developer solution for two minutes, applying gentle agitation.Then remove the glass wafer and rinse its surface with deionized water. Finally, place the glass slide into the 150 degree Celsius oven for a hard bake, ensuring that the substrates surface of interest is facing up. After 30 minutes, remove the slide from the oven and protect it from light.To etch the glass slide submerge it in a 10 to 1 buffered hydrofluoric acid solution for one minute in a polytetrafluoroethylene container. Then transfer and wash the glass slides in deionized water three times. Next, using a physical vapor deposition system sputter titanium for eight minutes at a rate of 100 angstroms per minute and platinum for 10 minutes at 200 angstroms per minute.Then to lift off the photo resist, submerge the metal coated glass slides in an acetone bath for 10 minutes and sonicate the bath to introduce agitation. If necessary use an acetone soaked wipe to remove any residues. Next for polydimethylsiloxane replica molding makes the PDMS elastomer base with a hardener at a 10 to 1 weight ratio in a disposable container placed on top of an electronic balance.Then, pour the PDMS solution over the silicon wafer and place the mixture under a vacuum to remove air bubbles. After curing the mixture at 65 degrees Celsius for a minimum of four hours, use the tip of a razor blade to cut out the molded PDMS and peel it from the silicon wafer. Then using a sharpened biopsy punch, remove the PDMS from the inlet and outlets of the device.Next, program the plasma generator for PDMS bonding. Then place the PDMS and electrode glass slide into the system with the features facing up and run the program. After the program is complete, remove the devices and use a stereoscope to quickly align channel features to the electrodes.Firmly apply pressure from the center of the PDMS towards the sides to remove any unwanted air bubbles at the bonding interface. Harvest the HEK293 cells, centrifuge them, and resuspend the pellet in electroporation buffer at approximately five million cells per milliliter. Next, add plasma DNA encoding for GFP to a final concentration of 20 micrograms per milliliter and mixed gently.Then transfer the plasma-DNA cell suspension into a one cubic centimeter syringe for experimentation. Place the microdevice onto the stage of the microscope via a slide holder. Then turn on the charged coupled device, CCD camera, and bring the microfluidic channel into focus.Next for single cell electroporation, set the syringe pump flow rate to 0.1 to 0.3 microliters per minute to ensure the flow of single cells through the electrode set. Then set the pulse parameters for the initial and lowest electrical energy electroporation pulse. Follow a predetermined number of cell detections per pulse application.At the end of each tested condition, aspirate cells from the microdevice outlet and replenish the outlet with recovery media. Iterate to the next electroporation pulse condition and repeat until all electroporation pulse conditions are tested. Then, determine the electroporation pulse parameters for high throughput population-based feedback.For population-based feedback controlled electroporation, set the syringe pump flow rate to one to three microliters per minute and set the pulse amplitude to the optimized condition. Then turn off the trigger mode and set the pulse width to match the cell transit time. After the desired number of cells have been electroporated turn off both the syringe pump and function generator.Then transfer the cells from the outlet reservoir into an appropriately sized cell culture flask or plate filled with pre-warmed recovery media and transfer the culture flask or plate into an incubator. For data analysis, Load the data into an analysis software and generate a plot of current versus time for each pulsing condition. Then determine the degree of cell membrane permeabilization, generate the cell membrane permeabilization map over all tested pulse conditions, and verify the optimal pulsing condition.After the incubation, capture epi-fluorescence images using FITC and far red filters, and analyze the image sets manually or via an algorithm. Operating principles behind the single cell level permeabilization detection for a single pulse amplitude are highlighted here. After each electroporation pulse parameter, the next highest energy electroporation pulse is tested.The cell membrane permeabilization map for HEK293 cells showed a distinct correlation between applied electrical energy and the degree of cell membrane permeabilization. In this experiment, an electric field strength of 1.8 kilovolts per centimeter and a 670 microsecond pulse was determined as optimal. At these values, a 70%electro-transfection efficiency was achieved.In contrast with an electric field strength of 0.4 kilovolts per centimeter and a three millisecond pulse duration, the electro-transfection efficiency at 24 hours was less than 5%The term recipe, which is often used when describing the specific of the micro fabrication process insert the importance of following or optimizing each step to successfully fabricate a functioning device. This electroporation technology can be utilized in additional biomedical research projects such as the generation and testing of CAR T-cells or the optimization and testing of CRISPR-Cas9 gene editing techniques. This micro-electroporation technology allows for the electrical interrogation of different cell types'responses to apply the electrical pulsing conditions and correlating that information to the delivery of clinically relevant molecular cargo.

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JoVE Journal

Bioengineering

2.6K visualizações.  Rutgers, The State University of New Jersey. Este método permite aos pesquisadores determinar as condições ideais de pulsação de eletroporação para o tipo de célula desejada, minimizando a necessidade da abordagem experimental empírica tradicional. Este método utiliza técnicas de micro-eletroporação para fabricar um dispositivo de atuação microfluídica escalável que é capaz de monitorar eletricamente o grau de permeabilização da membrana celular durante todo o processo de eletroporação. Demonstrando o procedimento ...