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 trypsi...

<ol>
	<li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+Past+present+and+future.">Electroporation Past present and future.</a> Dev Growth Diff. 55, 15-19 (2013).</li><li>Geng, T., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+electroporation+for+cellular+analysis+and+delivery.">Microfluidic electroporation for cellular analysis and delivery.</a> Lab Chip. 13, 3803-3821 (2013).</li><li>Shahini, M., van Wijngaarden, F., Yeow, J. T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+electro-microfluidic+channel+for+single+cell+electroporation.">Fabrication of electro-microfluidic channel for single cell electroporation.</a> Biomedical Microdevices. 15, 759-766 (2013).</li><li>Neumann, E., Toensing, K., Kakorin, S., Budde, P., Frey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+electroporative+dye+uptake+by+mouse+B+cells.">Mechanism of electroporative dye uptake by mouse B cells.</a> Biophysical Journal. 74, 98-108 (1998).</li><li>Jaroszeski, M. 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=Toxicity+of+anticancer+agents+mediated+by+electroporation+in+vitro.">Toxicity of anticancer agents mediated by electroporation in vitro.</a> Anti-Cancer Drugs. 11, 201-208 (2000).</li><li>Buntru, M., Gartner, S., Staib, L., Kreuzaler, F., Schlaich, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+multiple+transgenes+to+plant+cells+by+an+improved+version+of+MultiRound+Gateway+technology.">Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology.</a> Transgenic Res. 22, 153-167 (2013).</li><li>Mir, L. M., 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-efficiency+gene+transfer+into+skeletal+muscle+mediated+by+electric+pulses.">High-efficiency gene transfer into skeletal muscle mediated by electric pulses.</a> Proc Natl Acad Sci USA. 96, 4262-4267 (1999).</li><li>Heller, 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=Intradermal+delivery+of+interleukin-12+plasmid+DNA+by+in+vivo+electroporation.">Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation.</a> DNA Cell Biol. 20, 381-381 (2001).</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, 747-754 (2011).</li><li>Wang, 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=Synergistic+Effects+of+Nanosecond+Pulsed+Electric+Fields+Combined+with+Low+Concentration+of+Gemcitabine+on+Human+Oral+Squamous+Cell+Carcinoma.">Synergistic Effects of Nanosecond Pulsed Electric Fields Combined with Low Concentration of Gemcitabine on Human Oral Squamous Cell Carcinoma.</a> In Vitro PLoS One. 7, (2012).</li><li>Kim, M. J., Kim, T., Cho, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+electroporation+chip+using+multiple+electric+field+zones+in+a+single+channel.">Cell electroporation chip using multiple electric field zones in a single channel.</a> Appl Phys Lett. 101, (2012).</li><li>Wang, S. N., Lee, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-/nanofluidics+based+cell+electroporation.">Micro-/nanofluidics based cell electroporation.</a> Biomicrofluidics. 7, (2013).</li><li>Sharei, 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=A+vector-free+microfluidic+platform+for+intracellular+delivery.">A vector-free microfluidic platform for intracellular delivery.</a> Proc. Natl Acad Sci USA. 110, 2082-2087 (2013).</li><li>Kim, J. 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=Direct+reprogramming+of+human+neural+stem+cells+by+OCT4.">Direct reprogramming of human neural stem cells by OCT4.</a> Nature. 461, U649-U693 (2009).</li><li>Ozbas-Turan, S., Aral, C., Kabasakal, L., Keyer-Uysal, M., Akbuga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-encapsulation+of+two+plasmids+in+chitosan+microspheres+as+a+non-viral+gene+delivery+vehicle.">Co-encapsulation of two plasmids in chitosan microspheres as a non-viral gene delivery vehicle.</a> J Pharm Pharm Sci. 6, 27-32 (2003).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, U311-U313 (2007).</li><li>Hur, S. C., Mach, A. J., Di Carlo, D. <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+size-based+rare+cell+enrichment+using+microscale+vortices.">High-throughput size-based rare cell enrichment using microscale vortices.</a> Biomicrofluidics. 5, (2011).</li><li>Yun, H. Y., Hur, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+multi-molecule+delivery+using+vortex-assisted+electroporation.">Sequential multi-molecule delivery using vortex-assisted electroporation.</a> Lab Chip. 13, 2764-2772 (2013).</li><li>Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+theory+and+methods,+perspectives+for+drug+delivery,+gene+therapy+and+research.">Electroporation theory and methods, perspectives for drug delivery, gene therapy and research.</a> Acta Physiol Scand. 177, 437-447 (2003).</li><li>Wang, J., Zhan, Y. H., Ugaz, V. M., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vortex-assisted+DNA+delivery.">Vortex-assisted DNA delivery.</a> Lab Chip. 10, 2057-2061 (2010).</li><li>Jia, F. 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=A+nonviral+minicircle+vector+for+deriving+human+iPS+cells.">A nonviral minicircle vector for deriving human iPS cells.</a> Nature Methods. 7, U146-U197 (2010).</li><li>Dunbar, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+hematopoietic+stem+cells+Implications+for+gene+therapy+of+human+disease.">Gene transfer to hematopoietic stem cells Implications for gene therapy of human disease.</a> Annual Review of Medicine. 47, 11-20 (1996).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Angew Chem-Int Edit. 37, 551-575 (1998).</li><li>Graziadei, L., Burfeind, P., Barsagi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+Unlabeled+Proteins+into+Living+Cells+by+Electroporation+and+Isolation+of+Viable+Protein-Loaded+Cells+Using+Dextran+Fluorescein+Isothiocyanate+as+a+Marker+for+Protein-Uptake.">Introduction of Unlabeled Proteins into Living Cells by Electroporation and Isolation of Viable Protein-Loaded Cells Using Dextran Fluorescein Isothiocyanate as a Marker for Protein-Uptake.</a> Anal Biochem. 194, 198-203 (1991).</li><li>Dimitrov, D. S., Sowers, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Electroporation-Fast+Molecular-Exchange+by+Electroosmosis.">Membrane Electroporation-Fast Molecular-Exchange by Electroosmosis.</a> Biochimica Et Biophysica Acta. 1022, 381-392 (1990).</li><li>Sukharev, S. I., Klenchin, V. A., Serov, S. M., Chernomordik, L. V., Chizmadzhev Yu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+electrophoretic+DNA+transfer+into+cells.+The+effect+of+DNA+interaction+with+electropores.">Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.</a> Biophysical Journal. 63, 1320-1327 (1992).</li><li>Glogauer, M., McCulloch, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+large+molecules+into+viable+fibroblasts+by+electroporation+Optimization+of+loading+and+identification+of+labeled+cellular+compartments.">Introduction of large molecules into viable fibroblasts by electroporation Optimization of loading and identification of labeled cellular compartments.</a> Experimental Cell Research. 200, 227-234 (1992).</li><li>Verspohl, E. J., KaiserlingBuddemeier, I., Wienecke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+specific+antibodies+into+electropermeabilized+cells+is+a+valuable+tool+for+eliminating+specific+cell+functions.">Introducing specific antibodies into electropermeabilized cells is a valuable tool for eliminating specific cell functions.</a> Cell Biochemistry and Function. 15, 127-134 (1997).</li></ol>

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 mol...

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 border="0" cellpadding="0" cellspacing="0" width="1056">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:475px;">MDA-MB-231 cancer cell line</td>
			<td style="width:297px;">American Type Culture Collection (ATCC)</td>
			<td style="width:119px;">HTB-26</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Leibovitz&#8217;s L-15 Medium</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">10-045-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">fetal bovine serum (FBS)</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">penicillin-streptomycin</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:475px;">Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">21-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Trypsin</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Flow Cytometer easyCyte HT</td>
			<td style="width:297px;">Millipore</td>
			<td style="width:119px;">0500-4008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Oxygen Plasma Cleaner</td>
			<td style="width:297px;">Technics Micro-RIE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dektak 6M surface profiler</td>
			<td style="width:297px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">KMPR 1050</td>
			<td style="width:297px;">Microchem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td style="width:297px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Compressed Nitrogen gas</td>
			<td style="width:297px;">Airgas</td>
			<td style="width:119px;">NI 300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">High Pressure Regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td>6162K22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Downstream regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td style="width:119px;">4000K563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">high-speed 3/2way-8 valve manifold</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Inline Check Valve</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">CV3320</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">5/32&#34; OD x 3/32&#34;ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td>PU-156F-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/4&#34; OD X 0.17&#34; ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td style="width:119px;">PU-250PB-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/16&#34; PEEK tubings</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;">P1533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/32&#34; PEEK tubings</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P1569</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">PEEK tubing unions</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Pulse Generator</td>
			<td style="width:297px;">HP</td>
			<td style="width:119px;">8110A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Aluiminum Wire</td>
			<td style="width:297px;">Bob Martin Company</td>
			<td style="width:119px;">6061 ALUM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">oscilloscope</td>
			<td style="width:297px;">Agilent</td>
			<td style="width:119px;">DSO3062A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">50 mL centrifuge tubes</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">15 mL centrifuge tube</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">T75 culture flask</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">82050-862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 3000 MW, Anionic</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D3307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&#160;</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D1819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Texas Red, 3000 MW, Neutral</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">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 in...

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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

Research

JoVE Journal

Bioengineering

8.3K ビュー.  Harvard University. マイクロ流体渦補助エレクトロポレーション·プラットフォームは、正確かつ独立した投与量制御を備え​​た同一の細胞集団への複数の分子の順次配信のために開発されました。分子の送達効率および加工細胞の生存率を向上させるために支援エレクトロポレーションの前に、システムのサイズベースの標的細胞精製工程。 

ビデオ: Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

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

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

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

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...