This work is supported by the Rowland Junior Fellow program. The authors would like to express gratitude to the scientists and staff at the Rowland Institute at Harvard: Chris Stokes for his help in the development of the custom-built, computer-assisted pressure control setup, Diane Schaak, Ph.D. for her input for biological sample handling, Winfield Hill for developing the electrical setup, Alavaro Sanchez, Ph.D. for granting access to the flow cytometer, Scott Bevis, Kenny Spencer and Don Rogers for machining mechanical plumbing components required for the pressure setup. Microfluidic masters were fabricated at the Center for Nanoscale Systems (CNS) at Harvard University.

1. Cell Preparation
<ol>
	<li>Plate 1&#215;105 cells/ml of metastatic breast cancer cell line MDA-MB-231 in a volume of 10 ml per tissue culture T75 flask in Leibovitz&#8217;s L-15 Medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin.</li>
	<li>Incubate MDA-MB-231 cells in a humidified incubator at 37 &#176;C with 0% CO2 environment.</li>
	<li>Harvest cells for experiments 2 days after seeding by treating cells with 0.25% trypsin-EDTA for 2 min and inactivate trypsin&#8217;s enzymatic activity by adding 8 ml of the growth media.</li>
	<li>Pellet cells by centrifuging for 5 min at 200 &#215; g and resuspend in Dulbecco&#8217;s phosphate buffered saline (DPBS, 1x, without Ca2+ and Mg2+) to a final concentration of 5 &#215; 105 cells/ml.</li>
</ol>
2. Device Design and Fabrication
NOTE: The mask, master mold fabrications and the microchannel enclosing process are to be conducted inside a clean room while the polydimethylsiloxane (PDMS) microchannel casting process can be performed on a regular laboratory benchtop.
<ol>
	<li>Design a microfluidic device as illustrated in Figure 1 with AutoCAD. The device should consist of an inlet with multiple injection ports, coarse filters and two parallel straight rectangular inertial focusing channels (L = 7 mm, W = 40 &#956;m, and H = 70 &#956;m). Individual straight channel consists of 5 electroporation chambers, which are placed 800 &#956;m apart (WC = 400 &#956;m) with two via holes for aluminum electrodes. Two transversally adjacent electroporation chambers share the hole for the negative electrode. Two straight channels merge into an outlet at the downstream of the electroporation region.</li>
	<li>Convert the CAD file with micro-patterns to a GDSII file using LinkCAD. Write the micro-patterns on a 5&#8221; x 5&#8221; photomask blank using a laser mask writer. Develop the mask by following the manufacturer&#8217;s protocol. NOTE: The mask development process includes photoresist developing, chrome etching and resist removal. Alternatively, micro-patterns printed on a high resolution transparency (&#62;20,000 dpi) can be purchased from a third party manufacturer and used as a photomask. Final microscale features on a photomask should be transparent to match the polarity of a negative photoresist.</li>
	<li>Fabricate the casting mold of the device design using standard soft lithography techniques23 with a negative photoresist spun coat on a 4 inch silicon wafer at 2,000 rpm to have the final thickness of 70 &#956;m.</li>
	<li>Prebake the wafer with negative photoresist for 20 min at 95 &#176;C.</li>
	<li>Contact the mask with the wafer and expose to a UV source (17.4 mJ/cm2) for 80 sec using a mask aligner.</li>
	<li>Develop the exposed wafer for 4 min in a SU-8 developer.</li>
	<li>Measure the developed photoresist thickness using a surface profilometer.</li>
	<li>Rigorously mix a 10:1 ratio of elastomer to curing agent (silicone elastomer kit) and pour the mixture on the casting mold to generate PDMS replicas. Degas the casting elastomer for 30 min and cure the casting mold with degassed elastomer in an oven at 70 &#176;C for 2 hr.</li>
	<li>Delaminate cured PDMS replica with microchannel patterns from the mold and punch holes for inlets, outlet and electrode insertions using a pin vise. NOTE: The normal cutting edge diameter of the pin vise should be 0.76 mm, adequate for tightly holding PEEK tubing (O.D. of 1/32&#8221;) and aluminum electrodes (O.D. of 0.029&#8221;) in place.</li>
	<li>Create enclosed microfluidic channels by bonding PDMS replicas to a glass slide treated in an oxygen-plasma cleaner for 7 sec at a radio frequency power and oxygen partial pressure of 75 W and 500 mTorr, respectively.</li>
</ol>
3. Flow Experiments
<ol>
	<li>Insert aluminum electrodes (0.029&#8221; OD) and an outlet PEEK tubing (1/32&#8221; OD) into designated places via holes in the microchannel (see Figure 3B).</li>
	<li>Connect the electrical equipment18 responsible for generating high-voltage short square-wave pulses to the aluminum electrodes (see Figure 3C) that are in contact with flowing solutions in the PDMS mold. NOTE: The equipment should consist of a pulse generator and an in-house built high voltage amplifier.18</li>
	<li>Prepare four 50 ml centrifuge tubes individually containing DPBS, solutions with cells and biomolecules, and attach each tube to its respective vial holder connected to the pneumatic flow control system (see Figure 3A).18</li>
	<li>Connect inlet PEEK tubing from the vial holders into the respective inlet holes in the microfluidic device.</li>
	<li>Set the magnitude of square-wave pulses, V, to 100 V in order to have the electric field strength, E =V/Le, across the electroporation chamber be equivalent to 0.7 kV/cm. NOTE: Here, Le is the distance between two points where the positive and negative electrodes are in contact with the flowing solution (see Figure 1).</li>
	<li>Set the pressure regulator to 40 psi. NOTE: A single manually adjustable nitrogen source is used to uniformly pressurize all sample vials and a high-speed manifold is utilized to timely activate individual solution ports using the custom-built LabVIEW software.18</li>
	<li>For valve control, open the custom-built LabVIEW software labeled Valve Runner, and click run from the dropdown menu entitled operate.</li>
	<li>Turn on valves by clicking on each corresponding valve icon. NOTE: The valve icon should turn green when it is activated. Clicking the active icon will deactivate and close the valve. The closed valve icon should turn grey.</li>
	<li>Open the valve for the DPBS reservoir (i.e., washing solution) to prime the flow speed required for stable cell-trapping vortex generation for 1.5 min.</li>
	<li>Switch the active solution port from the washing solution to the cell solution to trap cells in the electroporation chamber for 30 sec (i.e., the cell-trapping step).</li>
	<li>Flow both washing and cell solutions through the device simultaneously for 10 sec prior to the cell-trapping step to ensure undisrupted flow during the solution-switching step. NOTE: This brief co-flow step should be repeated at each solution-switching step.</li>
	<li>Turn on the washing port and flush the device for 20 sec in order to remove non-trapped contaminating cells.</li>
	<li>Inject the solution containing the first molecule of interest (0.2 mg/ml of 3,000 Da neutral and anionic dextran molecules or 1.5 mg/ml of 70,000 Da neutral dextran molecule) into the device.</li>
	<li>Apply five short pulses (&#916;t= 30 msec with 2 sec intervals) promptly after injection of the molecular solution and monitor the magnitude and duration of the applied electrical pulses in real-time using an oscilloscope.</li>
	<li>Repeat step 3.10 as many times as the number of additional molecules to be delivered. For each molecular delivery, incubate cells for 100 s in the molecular solution then flush the device with the washing solution for 100 s to remove excess molecules.</li>
	<li>Release the cells in a 96-well plate for downstream analysis by lowering the operating pressure below 5 psi. NOTE: Approximately 100 &#956;l of solution with 100 cells is collected from each release.</li>
	<li>Run the experimental steps 3.9 through 3.16 three times to collect enough cells for flow cytometry.</li>
	<li>Centrifuge the 96-well plate containing processed cells for 5 min at 228 &#215; g.</li>
	<li>Remove the supernatant that contains excess fluorescent molecules.</li>
	<li>Resuspend cells in DPBS for flow cytometry analysis.</li>
	<li>Load cells in the flow cytometer for molecular uptake efficiency analysis.</li>
</ol>

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The authors have nothing to disclose.

With the new parallelized electroporation platform, 10-fold enhancement in throughput and efficiency of multi-molecule delivery was achieved in addition to all the merits that the previously developed single-chamber system provides.18 Previously available merits include (i) pre-purification of target cells with uniform size distribution for viability enhancement, (ii) precise and individual molecular dosage control, and (iii) low operational electrical current. Fluorescently labeled dextrans were chosen as molecules of interest because they are readily available in a wide range of molecular weights, conjugated fluorophore types, and electrical charges. These macro-molecules are good candidates for such a study since they are inherently membrane impermeable for living cells and do not exhibit cytotoxicity.24 Optimum concentration of fluorescent dextran, incubation time, and electrical parameters were identified for the current system prior to the electroporation experiments. The optimization process was rather simple and efficient due to the platform&#8217;s ability to monitor the molecular uptake process in real-time. Visualization of this process is very beneficial if a molecule or cell type with little-to-no knowledge of electroporation parameters is being tested. The incubation time for all molecules tested was set to be 100 s by which cells trapped in all 10 chambers exhibit detectable fluorescent intensity signals in real time, indicating the completion of successful molecular delivery. Note that this is not the minimum incubation duration required for molecular delivery.
There has been a long-standing debate regarding the molecular delivery process using electroporation, whether it is solely mediated by diffusion4,19,25 or by electrophoresis.26 Identification of the dominant mechanism of molecular delivery involves a series of laborious individual tests, comparing the outcomes depending on molecular sizes, electrical charges and electroporation parameters. To the best of the authors&#8217; knowledge, these laborious optimization processes were not reported with conventional electroporation systems.4,26-28 The developed system&#8217;s simple parameter optimization capability allowed for identification of the dominant molecular delivery mechanism, by examining variations in the electroporation efficiency depending on molecular and electric parameters. Results showed that the efficiency of anionic 3,000 Da dextran was not significantly different than that of its neutral counterpart (83 and 75%, respectively), suggesting that the molecular uptake observed was possibly mediated by diffusion. Unlike other electroporation systems, processed cells were continuously agitated throughout the electroporation process in this system. Gradual increase in fluorescent signals of orbiting cells was observed, suggesting that diffusion would be the dominant delivery mechanism. High efficiency molecular uptake (90%) of neutral 70,000 Da dextran molecules further implies that even at high molecular weights the delivery process occurs as a result of diffusion. Lastly, successful sequential delivery of 3,000 Da anionic and neutral dextran molecules solidifies the claim that molecular delivery occurs despite the molecules charge. This result also suggests that membrane resealing is not completed within 3 min of electroporation (100 sec per molecular delivery).
The presented molecular delivery platform can sequentially deliver wide ranges of molecular sizes regardless of their electrical charges. Timely fine-tuning and modification of individual parameters can be achieved with little effort aided by the real-time process monitoring capability. The system&#8217;s size-based cell purification process eliminates the need for laborious sample preparation and time-consuming centrifugation pre- and post-electroporation steps. Furthermore, the system&#8217;s low operational current substantially reduces cell mortality.

In recent years, the use of electric pulses to facilitate cytosolic delivery of extracellular molecules has become an attractive means of manipulating mammalian cells.1 This process, also known as electroporation, reversibly permeabilizes the cellular membrane, allowing for inherently membrane impermeable molecules to gain access to the cells&#8217; intracellular milieu. Because virtually any molecule can be introduced into the cytosol via temporary created pores in the membrane of any type of cells using electroporation, the technique has been reported as being more reproducible, universally applicable, and more efficient than other methods including virus-mediated, chemical and optical approaches.2-3 This technique has been utilized to introduce fluorescent molecules,4 drugs5 and nucleic acids6-7 while keeping cells viable and intact. Given these benefits, electroporation has been adopted as a common laboratory technique for DNA transfection, in vivo gene therapy8 and cell vaccination studies. It is, however, still difficult for conventional electroporation systems to simultaneously achieve practical efficiency and viability for samples with large heterogeneity in size because the electric field strength required for successful electroporation closely correlates with the cell&#8217;s diameter. Moreover, those systems do not allow precise control of the multiple molecular amounts being delivered due to reliance on bulk stochastic molecular delivery process.9 In order to address these issues, many groups have developed microfluidic electroporation platforms, offering the advantage of lower poration voltages, better transfection efficiency, a large reduction in cell mortality, and ability to deliver multiple molecules.10-13 These advantages were made possible owing to the small footprints of microscale electroporation systems whose electrode pitch lengths are sub-millimeters, dramatically decreasing voltages required for successful delivery. Moreover, these microscale electroporation systems can achieve uniform electric field distribution and rapidly dissipate generated heat, yielding reduced cell mortality while enhancing delivery efficiency. The utilization of transparent materials for these microchips further allows in situ observation of the electroporation process for prompt parameter modifications.2,12 However, precise dosage control and molecular- and cellular-dependent parameter control, required for emerging research and therapeutic applications,6,14-16 still remain unresolved.
This work presents a microfluidic vortex-assisted electroporation system, capable of delivering multiple molecules sequentially into a pre-selected identical population of target cells. Cells with uniform size distribution are isolated prior to electroporation using previously reported size-selective trapping mechanism.17-18 By having a uniform size distribution, less variation in electroporation efficiency and enhanced viability per given electric field strength were achieved.19 Furthermore, continuously agitating trapped cells using microscale vortices allowed for uniform delivery of molecules across the entire cytosol, in agreement with the results previously reported using another vortex-assisted electroporation platform.20 To demonstrate that this system would be applicable to a broad range of molecules commonly utilized in biological applications, macromolecules with a wide range of molecular weights were delivered to metastatic breast cancer cells. In addition, with the aid of real-time process monitoring, this work provides more evidence to put an end to the long standing debate regarding the mechanism of molecular delivery during electrporation, being predominantly electrophoresis-mediated versus diffusion-mediated.14 Unlike other electroporation systems, this platform uniquely provides the combined advantages of precise multi-molecule delivery, high molecular delivery efficiency, minimal cell mortality, a wide span of size and charges of delivered molecules, as well as real-time visualization of the electroporation process. Given these capabilities, the developed electroporation system has practical potential as a versatile tool for cellular reprogramming studies,6,14,21-22 drug delivery applications10,19 and applications requiring for in-depth understanding of electroporation molecular delivery mechanisms.

<table><tbody><tr><td>MDA-MB-231 cancer cell line</td><td>American Type Culture Collection (ATCC)</td><td>HTB-26</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 Medium</td><td>Cellgro, Mediatech, Inc.</td><td>10-045-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Cellgro, Mediatech, Inc.</td><td>21-030</td><td></td></tr><tr><td>Trypsin</td><td>Gibco, Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Flow Cytometer easyCyte HT</td><td>Millipore</td><td>0500-4008</td><td></td></tr><tr><td>Oxygen Plasma Cleaner</td><td>Technics Micro-RIE</td><td></td><td></td></tr><tr><td>Dektak 6M surface profiler</td><td>Veeco</td><td></td><td></td></tr><tr><td>KMPR 1050</td><td>Microchem</td><td></td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Compressed Nitrogen gas</td><td>Airgas</td><td>NI 300</td><td></td></tr><tr><td>High Pressure Regulator</td><td>McMaster-Carr</td><td>6162K22</td><td></td></tr><tr><td>Downstream regulator</td><td>McMaster-Carr</td><td>4000K563</td><td></td></tr><tr><td>High-speed 3/2way-8 valve manifold</td><td>Festo</td><td></td><td></td></tr><tr><td>Inline Check Valve</td><td>Idex Health and Science</td><td>CV3320</td><td></td></tr><tr><td>5/32&quot; OD x 3/32&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-156F-0</td><td></td></tr><tr><td>1/4&quot; OD X 0.17&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-250PB-4</td><td></td></tr><tr><td>1/16&quot; PEEK tubings</td><td>Festo</td><td>P1533</td><td></td></tr><tr><td>1/32&quot; PEEK tubings</td><td>Idex Health and Science</td><td>P1569</td><td></td></tr><tr><td>PEEK tubing unions</td><td>Idex Health and Science</td><td>P881</td><td></td></tr><tr><td>Pulse Generator</td><td>HP</td><td>8110A</td><td></td></tr><tr><td>Aluminum Wire</td><td>Bob Martin Company</td><td>6061 ALUM</td><td></td></tr><tr><td>Oscilloscope</td><td>Agilent</td><td>DSO3062A</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>VWR</td><td>21008-216</td><td></td></tr><tr><td>T75 culture flask</td><td>VWR</td><td>82050-862</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 3,000 MW, Anionic</td><td>Gibco, Life Technologies</td><td>D3307</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>D1819</td><td></td></tr><tr><td>Dextran, Texas Red, 3,000 MW, Neutral</td><td>Gibco, Life Technologies</td><td>D3329</td><td></td></tr></tbody></table>

microscale vortex-assisted electroporator for sequential molecular delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

The developed parallel microfluidic electroporator delivered macromolecules with varied sizes and electrical charges into living metastatic breast cancer cells. Successful molecular delivery was qualitatively determined by monitoring changes in fluorescent intensity of electroporated orbiting cells in situ and confirmed by quantitative measurements via flow cytometry analysis. Figure 4A&#160;shows that 90% of treated cells uptake the 70,000 Da neutral dextran. For the statistical analysis, an intensity threshold for each fluorophore was established such that the majority (&#62;99%) of unprocessed living cells is counted below the threshold (see Figure 4 (c)). The efficiency is defined as the ratio of the number of cells successfully taking up the molecules of interest to the total number of processed cells. Figure 4B&#160;illustrates that the efficiency does not substantially vary depending on molecular weight or electrical charges (P &#62;0.1). All tested dextran molecules were delivered into the cytosol with efficiency greater than 70%. In addition, Figure 4D&#160;exhibits that sequential molecular delivery was successfully performed with a dual molecule delivery efficiency of 56% using the anionic and neutral dextrans with identical molecular weight (MW = 3,000 Da). The current system can process cells with 10-fold higher throughput and multi-molecule delivery efficiency than the previously reported system18 and this improvement does not affect single-molecule delivery (82% and 70% for anionic and neutral dextran, respectively).
<img alt="Figure 1" src="/files/ftp_upload/51702/51702fig1highres.jpg" /> 
Figure 1. (A) A schematic of the microfluidic electroporation system, consisting of inlets for cells (denoted as C), molecules (denoted as M1 and M2) and a flush solution (denoted as F), two straight channels where inertial focusing occurs, 10 electroporation chambers with electrodes and an outlet. (B) Solution exchange demonstration at the inlet using a 1&#956;M FITC solution and a flush solution (DPBS) indicates that the active solution can be uniformly injected to all arrays of chambers downstream. Image contrast is enhanced by adjusting look-up table (LUT). Scale bars are 250 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51702/51702fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51702/51702fig2highres.jpg" /> 
Figure 2. A photograph of the 15-pin electrode utilized for short-pulse high voltage application, consisting of 10 positive (+) and five negative electrodes (-). Each positive electrode is spaced 2 mm apart from a negative electrode, and each electrode of the same polarity is spaced 1.35 mm apart. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51702/51702fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51702/51702fig3highres.jpg" /> 
Figure 3. Schematic of the experimental apparatus, consisting of (A) the fluid control unit, (B) the microfluidic electroporator, and (C) the electrical equipment. (A) Vials containing solutions with molecules, cells and clean buffer are individually pressurized on demand using the LabView controlled pneumatic flow system. The solution from the pressurized vial is injected into the microfluidic electroporator through PEEK tubing with a check valve installed. (B)&#160;and&#160;(C) The electric signals are sent to 15-pin electrodes in contact with the flowing solution in the microfluidic system during the electroporation step. The electric pulses with the programmed duration are generated using the pulse generator and the magnitude of the electric pulse is amplified to 100 V by the high voltage amplifier. All applied electrical parameters are monitored in real time using an oscilloscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51702/51702fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/51702/51702fig4highres.jpg" /> 
Figure 4. Representative flow cytometry data. (A) Fluorescent signals of MDA-MB-231 cells, which successfully took up the 70,000 Da anionic dextran molecule, compared to that of the control counterpart. (B) The efficiencies for each transferred dextran molecule do not exhibit significant molecular dependent variation (p &#62;0.1). (C) Representative flow cytometry profiles for cells, which were not treated with electroporation (control). The fluorescent threshold indicating successful molecular delivery is set from the data such that the signals from control samples are found below the threshold. (D) Representative flow cytometry data for sequentially electroporated cells. Green, red and yellow boxes in the flow cytometry plot and fluorescent streak images on the right-side represent fluorescent signals from cells uptaking 3,000 Da neutral dextran-only, anionic dextran-only and both dextran molecules, respectively. Scale bar is 100 m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51702/51702fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery at JoVE.com

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

microscale-vortex-assisted-electroporator-for-sequential-molecular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.5K Views.  Harvard University. A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system’s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Video: Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

Engineering

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetics

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Biology

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

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

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

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

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

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...