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8-2011 Transfection of cells using flow-through electroporation based on constant voltage Tao Geng Birck Nanotechnology Center, Purdue University, [email protected]

Yihong Zhan Birck Nanotechnology Center, Purdue University, [email protected]

Jun Wang Birck Nanotechnology Center, Purdue University

Chang Lu Virginia Polytechnic Institute, [email protected]

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Geng, Tao; Zhan, Yihong; Wang, Jun; and Lu, Chang, "Transfection of cells using flow-through electroporation based on constant voltage" (2011). Birck and NCN Publications. Paper 979. http://dx.doi.org/10.1038/nprot.2011.360

This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. © 2011 Nature America, Inc. All rights reserved. a critical a threshold. critical is applied to cells and the potential transmembrane ( into cells nanoscale pores in the plasma membrane that allow DNA delivery those of viral or chemical transfection from different fundamentally is that mechanism a with transfer, gene expression altered induce also may reagents delivery Certain construct. of the size the and cells the of state the type, cell the on dependent delivery gene lipids cationic or havewith polymers also applied for been widely immunogenicity and Alternatively, virulence nonviral methods based on the complexing DNAof low have generally which and also limits tumorigenesis the viruses, adeno-associated use of patients because of the high titers required often nonintegrating, elicit strong although immune responses in mutagenesis insertional produce and genome ing oncoretroviruses mayand lentiviruses) into integrate the host high-efficiency gene transfer vectors. However,are virulence retroviruses disabled genetically with (includadenoviruses) and viruses retro (e.g., Viruses years. the over cells of modification genetic cells of at scales. various engineering These new clinical practices responses. demand immune advanced as methods for such genetic functions cellular desired stimulate or tissues restoredamaged either cells transfected implanted The modified and selected, before they are introduced into the patient. genetically donor, then a or patient a from isolated first are cells A substantial number of these therapies apply an have the potential to treat both inherited and acquired disorders for a wide range of emerging cell therapies and gene materials essential become have cells transfected recently,More gene products in cell development, differentiation and malignancy. and genes involvement of the regarding information critical vide research and applied biotechnology; genetically modified cells pro Gene delivery is an indispensable technique for both basic bi I uptake. migrations of cells during the flow, we also show ofpermeabilization the entire cell membrane and markedly increased D transfection of cell samples with rates ranging from 40 of cells, using disposable devices, a syringe pump and a low-cost power supply that provides a constant voltage. We show ofpermeabilization the cell membrane. Here we describe a flow-through electroporation protocol for continuous transfection limited by the small volume of cell samples processed (less than 10 E Published online 21 July 2011; doi:10.1038/nprot.2011.360 Correspondence should be addressed to C.L. ([email protected]). 1 Tao Geng electroporation based on constant voltage Transfection of cells using flow-through 1192 Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, USA. NTRO protocol lectroporation is a high-efficiency and low-toxicity physical gene transfer method. Electroporation is the most widely used physical method for gene for developed been have methods transfection of variety A

| VOL.6 NO.8VOL.6 D T UCT 24,2 he he fabrication of the devices takes 1 d and the flow-through electroporation typically takes 1–2 h. 1 5 . Electroporation occurs when an external electric field , Yihong Zhan I 15–1 ON 1 7 8 . However, results of these methods are strongly strongly are methods these However,. of results | . 2011 | natureprotocols 1 , Jun Wang 19–2 3 . Electroporation creates 8–1 1 & Chang Lu 0 . Random integration 6, ex ex vivo 7 . Adenoviruses, Adenoviruses, .

therapies therapies that ∆ ψ approach: E ) ) exceeds m 2 o l l min logical 11–1 1– 2

− 4 5 Department of Chemical Engineering, of Department Virginia Tech, Blacksburg, Virginia, USA. - - -

. . 1

to 20 ml min mixedDNAwith molecules flows through the appliesa constant voltage across a fluidic channel. Cell suspension that supply power common a use generator,we pulse a using of lished methods used in our laboratory troporation by constant voltage, which is based on previously pub Here we present a protocol for DNA delivery via flow-through elec based on constant Flow-through electroporation voltage efficiency. improving transfection small fraction of the cell surface area creates a profound hurdle for excessivedeath) cell prevent (which conditions electroporation optimized the under permeabilized not usually is membrane cell the of rest poles.The at occur the mostly gene and delivery membrane the of bilization tion given the pulse duration of milliseconds or shorter), permea electric field (this is typically the case for conventional electropora direction. When a cell remains static during the the application of (i.e., potential transmembrane the (1), Equation of basis the On electroporation. of practice the and physics the both by determined is limitation last surface.The cell the through area delivery limited and equipment the of cost high rate, death cell high relatively are electroporation with associated concerns. reducedoffers tion greatly safety limitations major The electropora transfection, chemical and viral with comparison In decreases the required voltage) markedly often (which miniaturization to amenable operate, to optimized. Because of its physical nature, electroporation is simple and pulse pattern), gene transfer efficiency and cell viability can be intensity,duration field field (e.g., parameters electric the tuning tion and intensity to a cuvette with embedded electrodes inside.dura defined By of pulses electric short exerts that generator pulse high-voltage a using conducted is electroporation Classical tion. direc field the and surface to membrane the normal the between a where g( 7 cells per reaction) and low D is the diameter of the cell, θ

→ λ ) is a function of the membrane and buffer conductivities, 0) where the surface normal is aligned with the field field the with aligned is normal surface the where 0)

−

1 with high efficiency. By inducing complex C lassical lassical electroporation protocols are 33,3 ∆ l y 4 . The fact that gene entry into cells is via a via is cells into entry gene that .fact The E = E 7 0 is the field intensity and ( . 26–3 ∆ NA 5 ψ g 1 and yields reproducible results E uptake due to partial is highest at the poles of a cell cell a of poles the at highest is c ) aE 35,3 6 os . In our protocol, instead q

channel,whichhas θ is the angle NA

(1) 3 2 ------.

© 2011 Nature America, Inc. All rights reserved. We showed a processing rate of up to 20 ml min ml 20 to up of rate processing a showed We 10 orderthe of However, materials). cal therapies, cell for ucts using cells from limited sources (e.g., animal or human clini genes and gene prodtests of functional permits amounts sample tiny with work to ability The samples. large-volume and scarce liters to ers format allows us to process sample volumes ranging from microlit Our protocol has several unique advantages. First, the flow-through Advantages and limitations have not tested the method with other plasmids or cell types. Chinesehamster (CHO-K1)ovary cells using thisprotocol of principle, pEGFP-C1 plasmid DNA was efficientlyelectroporation; transferred and into(vi) transfection and viability assays. As a proof device fabrication; (iv) cell and sample preparation; ing;(v) (ii)flow-through master mold fabrication; (iii) polydimethylsiloxane involved(PDMS) in the protocol, including (i) photomask design and print ing rate from the range of micrometers to millimeters to accommodate a process on its absolute size, the channel can be easily scaled up and down in soft lithography. Because the physics of the device does not depend to multiple pulses. The channels can be fabricated at low cost using channelrenders flowingcells subject tofield variations equivalent the in sections narrow multiple Havingsection(s).narrow the in duration electroporationof is determined by the residence time(s) electroporation while flowing through the narrow section(s) and wide sectionsthe does not affect membrane integrity. Cells experience beyondthe electroporation threshold, whereas the intensity inthe the in intensity field The sections. wide the in fieldyieldshighintensity thenarrowin sections andlow intensity area. Appropriate combination of the voltage and the channeland narrowdesign sections in order to create variation inchannel is thetypically made of uniformcross-sectional depth and composed of wide convenienceinverselytofabrication,proportional. the forOwing are section particular a of area cross-sectional the and intensity cross-sectionaldesigned multipleareas.sectionsof fieldlocal The The device fabrication process is depicted in the upper box. Figure 1 cells min cells Glass slide cleaning

| Flowchart of the flow-through electroporation protocol.

− PDMS devicefabrication

1 3 ) with our flow-through electroporation protocol in in protocol electroporation flow-through our with ) 5 . This feature facilitates genetic modification of both both of modification genetic facilitates feature . This Cell andsample (Steps 25–31) (Steps 13–24) µ Plasmid DNA preparation preparation l min 9 cells per patient per trial is routinely required trial per patient per cells

Photomask design − Master fabrication

(Steps 1and2) 1 (Steps 3–12) to ml min and printin g

−

1 . electroporation Figure (Steps 32–34) Flow-through Wafer treatment 1 ex vivo ex shows the major steps

narrow sections is sections narrow transfection on transfection

−

1 Transfection and (or 10 (or fabrication (Steps 35–40) viability assa Devic

e 35,3 7 6 –10 ;we 37,3 y 8 - - - - - 8 .

an an extensive processing. of period over cell treatment and uniform age ensures also operation stable by variations in the design of the channel. The use of constant volt lithography, we are able to implement sophisticated pulse sequences current (DC) power supply and fluidic channels fabricated by soft direct low-cost a With electroporators. other all virtually in used generator, pulse a for need the featureeliminates unique is which tion technique that is based on application of constant voltage. This require expensive equipment. Our protocol is the only electropora transfection prerequisite high-efficiency a for as identified been has surface cell the at DNA of concentration viability.high cell A the on effect substantial a out flow the of exposed to the electric field for permeabilization during the course torise complex cell migrations, the entire surface area cells was of that, by creating and device geometry flow conditions, which gave recently We showed area. membrane cell the of fraction small a permeabilizes only suspension cell static to applied pulses with surface cell protocolelectroporation gene permits delivery through the entire a few milliliters. sizes of Second,sample typically handle flow-through our that electroporators cuvette-based conventional with work recent electroporation are field intensity, duration and pulse sequence. pulse and duration intensity, field are electroporation parameters. Electric design Experimental about these parameters. endless. Thus, to some guidelines it general establish is important tions of field intensities, durations and pulse patterns are practically other operational conditions is necessary. The potential combina and parameters electric the of optimization type, cell particular each for Furthermore, 10–20%. of rate cell-death a is still there and high cell viability. Typically, efficiency even transfection under optimized high conditions, between struck be to needs balance a example, For protocols. electroporation all of limitations mon com the of some shares also protocol Our microseconds. few a that the normally requiredoptimal transfection field durations of fast velocity (up to ~1 m s pulses (~10 permeabilization and delivery over the entire cell surface. produces that technique electroporation only the is protocol our (both undisclosed) optimized for each cell type. To our knowledge, fection efficiency due to the pulse sequence and buffer ingredients systems, the Amaxa Nucleofection system offers the highest trans and expensive apparatus than that of our technique. Among all the electroporate flowing cells, and it requires much more continuously sophisticatedto generator pulse high-performance a uses system troporationforlarge-volume processing limit our protocol from further scale-up. MaxCyte offers flow elec tical to transfect liters of cell samples. The physics involved does not with a constant output rate up to ~10 manner continuous a in protocolworks reaction.Our per less or work in batch mode and produce transfected samples of ~10 Pulser,NucleofectorGene Amaxa Cuvette, well-plate or capillary-based electroporators (e.g., Bio-Rad available commercial devices and techniques, as shown in One limitation of our protocol is that generating very short short very generating that is protocol our of limitation One Ourflow-through electroporation protocol compares wellwith 3 3 6 6 µ 3 . Such practice markedly improved the uptake with uptake the improved markedly practice Such . . As mentioned above, conventional electroporation electroporation conventional above, mentioned .As 5 s s or shorter) is challenging because this requires very . Such an operation is impossible or very difficult difficult very or impossible is operation an Such . The most relevant electric parameters for for parameters electric relevant most The natureprotocols

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1 ) in the channel. Fortunately, we found 3 9 . Finally, our protocol does not not does protocol our Finally, . 40–4 8 cells min 2 , Invitrogen Neon system Neon Invitrogen, 4

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cells 1193 4 1 3 ) ------. © 2011 Nature America, Inc. All rights reserved. ship ship ( sections two in intensities field Ohm’slaw, on the based areas, cross-sectional various with sections multiple has and buffer, conductive a with filled is that sections. ment various of arrange the by determined sequence, and pattern designed with sections in series, we are able to deliver a number pulses of to cells electroporation multiple having By duration. electroporation the determines sections) these of lengths the and velocity cell the by channel. The residence cells time in of (determined these sections the of geometry the and (V) channel the across applied voltage constant the by determined is intensity) field electroporation the cells) ovary hamster threshold the (e.g., electroporation ~300–400 V cm small cross-sectional area the of channel has higher intensity than with sections the in field electric the only designs, typical our In T 1194 respectively sections, these (this (this protocol) voltage constant based on electroporation Flow-through fection system scalable trans MaxCyte STX system transfection Neon Invitrogen Nucleofection Amaxa system electroporation Pulser Bio-Rad Gene T a protocol echnique Although a constant DC voltage is established across a channel channel a across established is voltage DC constant a Although b le

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A - j /A i | , where Disposable Disposable fluidic electroporation flow-through pulse generator; a pump and a pulsing chamber, Disposable flow pulse generator chamber with a electroporation Capillary-type generator with a pulse electroporation plate–based Cuvette or well- S electroporation flow-through constant voltage; that provides power supply and a common devices, a pump Cuvette Cuvette or well- each each cell type programmed for generator that is with a pulse electroporation plate–based 2011 etup 35,3 6 | . The field intensity in these sections (i.e., sections these in intensity field .The natureprotocols A i 4 and and 5 E . Because of the planar nature of soft soft of nature planar the of .Because i

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- - High Low High survival C High High ell the operational parameters for a device with known dimensions dimensions known with device a for parameters operational the ms. 5 Whendetermining of duration combined total a with each in ms 1 of time residence a with channel the in sections narrow such as notation use ‘ T as sections wide the in duration and length combined the sections wide the in integrity membrane the of compromise substantial no and sections narrow the in poration for 7–15 of ratio as sections) troporation sections as wide the of protocol,this intensity field and we refer towidth the In channel. one in sections narrow the for widths multiple have to possible also is it simplicity,although of sake the for sections) the same width among multiple narrow sections (or multiple wide where uniform uniform in all the sections and in that case we have channel the of depth the make often we fabrication, lithography 1 and those in the narrow section(s) as

W High each cell type required for and cuvette cific solution costs for spe with separate Very high High cost E Low pulses application of continuous erator for pulse gen sophisticated requiring Very high; quipment i and and W 1 and W - j are the widths of the sections. the Weof have widths are the typically W

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W L 1 j and and /W f i - - , © 2011 Nature America, Inc. All rights reserved. • fabrication Device REAGENTS M can be calculated by bilization took place at the entire cell surface. The two parameters permea the whether and migration, transverse experienced cells secondary flow lift forces inertial tive of magnitudes R number Reynolds flows.channel We the these in that cells found entrain and flows secondary generate to order in criteria certain bilized membrane area). difficulty with conventional electroporation (i.e., limited permea overcomeand field a cell to electric the major the areassurface of different expose migrations complex Such direction). flow main to the perpendicular is that (i.e., plane the direction in transverse secondary the in vortices the and path channel flow the the along viability cell the in decrease substantial a introducing without surface cell through electroporation generated permeabilization over the entire showed that by inducing transverse Dean flow in the channel, flow- the cell membrane area being permeabilized. In our recent work, we hydrodynamic manipulation of cell motions that strongly allows protocol influenceelectroporation our of format flow-through The Control of permeabilized membrane area using hydrodynamics. a few seconds to produce transfection high-efficiency the combined that, whereas the optimal combined showed we example, For nucleus. the into entry DNA facilitates and interior cell the into deeply DNA delivering for important is that drag electrophoretic exerts it electroporation, generate not the wide sections. Although the low field in the wide sections does by segmented are sections efficiency.electroporation narrow The sections electroporation has a major influence on the transfection devices with one single narrow section produce typically superior results compared with those created by where and the flow rate a of cell sample ( can use the following formulas to calculate the applied voltage ( ( intensity electroporation desired and densities. Choose anappropriate photoresist with devices for fabricating photoresists.SU-8 2000series Each photoresist hasdifferent and viscosities in aroom protected from light. photoresists of are sensitiveseries highly to light. Store at4°Candprocess appropriate personal protective equipment. resist istoxic and flammable. with onlyHandle ina chemical fume hood SU-8 2025and2150photoresist (MicroChem) c ATER and the dimensionless inertial force ratio The flow conditions and the channel geometry need to meet meet to need geometry channel the and conditions flow The sections narrow multiple having by created pulses Multiple H I 3 ALS is the channel depth. 6 . Under these flow conditions, cells are involved.are conditions,both Underflowcells in these T F 1 in the wide sections needed to be on the order of D ) were crucial measures for determining whether E V = W Q U R 1 2 m c =  / ( = W L

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where where • • membrane membrane area. by the field intensity and than duration) by the cell permeabilized is much more affected by Joule heating (which is determined only smaller permeabilization area) channels (which generated no transverse vortices and led to much straight channels as spiral with with achieved cell similar viability wereefficiency markedly improvedtransfection permeabilized the entire cell membrane and both DNA uptake and flows secondary in a spiral channel, flow-through electroporation DNAdelivery enhanced enced were effectively in Dean the entrained flowsecondary and experi example, we showed that in the region of by inducing a high degree of entrainment in the secondary flow. electroporation Forvortex-assisted for conditions favorable most the diameter. In general, a combination of low where kinematic viscosity, and (with hydraulic diameter of the channel, which is defined as 2 When one narrowwere with channels section used, longer substantial when there was only one narrow section in the channel. size device the on dependence some We noticed that the transfection efficiency and cell viability showed sample accordingly in order to maintain the same field durations. cell the of rate flow the vary to Wechannel. need the in sections by increasing the cross-sectional area by the same factor for all the 40 between rates processing the with millimeters, to micrometers of able. We have tested feature channels fromwith sizes ranging tens size of the channel. Flow-through electroporation is thus fully scal actual the on dependent not is flows secondary the and poration Scaling up. performance performance can be different obtained using of devices sizes. In general, after optimization of the operational parameters, similar show a substantial loss in the transfection efficiency when scaled up. pulses multiple applying with associated efficiency improvedelectroporation the of result clear.However, a not as is this behind physics exact the ably in due a device,tolarger weakenedalthough electroporation device smaller a by yielded that to comparable required in a larger in device orderefficiency to transfection yield appropriate personal protective equipment. andisflammable.irritation with onlyHandle ina chemical fumehood Isopropanol (Fisher Scientific, cat. no. A516-4) appropriate personal protective equipment. toxic andflammable. with onlyHandle ina chemical fumehood SU-8 developer (1-methoxy-2-propyl acetate; MicroChem) inthis protocol.used (http://www.microchem.com/Prod-SU82000.htm). SU-82025and2150are desired channel depths. MicroChem See for datasheets process guidelines µ l min w R U and is the radius of curvature of the channel and and channel the of curvature of radius the is m

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© 2011 Nature America, Inc. All rights reserved. • • • • • Glass slide cleaning • • • • • • • • • • • • • • • Master fabrication EQUIPMENT • • • • • • andrelated assaysElectroporation • • • • • • • • • • • • cultureCell andsamplepreparation • • • • 1196 • • It iscorrosive. appropriate Handle with personal protective equipment. Hydrochloric (HCl; acid Fisher Scientific, cat. no. A144C-212) corrosive. appropriate Handle with personal protective equipment. hydroxideSodium (NaOH; Sigma-Aldrich, cat. no. S5881) appropriatewith personal protective equipment. EDTA (Sigma-Aldrich, cat. no. E6758) equipment. ! Tris(hydroxymethyl)aminomethane (Sigma-Aldrich, cat. no. 154563) (Sigma-Aldrich,PBS tablet cat. no. P4417) Trypsin-EDTA 4Na solution (0.05%(wt/vol); Invitrogen, cat. no. 25300) irritant. appropriate Handle with personal protective equipment. l Penicillin-streptomycin solution (Invitrogen, cat. no. 15140) FBS (Invitrogen, cat. no. 10082) DMEM (Invitrogen, cat. no. 11971)for CHO-K1cell culture pEGFP-C1 plasmid(Clontech), cat. no6084–1. CHO-K1 cells (ATCC) appropriateHandle with personal protective equipment. Silicones RTVElectric 615; MGchemicals) PDMS (Polydimethylsiloxane) agent (General prepolymer andcuring protective equipment. ingestion. Handle appropriate only inachemical with fumehood personal Scientific, cat. no. A669C-212) Ammonium hydroxide solution (27%(vol/vol); NH appropriatewith personal protective equipment. toxic by ingestion andacarcinogen. Handle only inachemical fumehood Sciences, cat. no. 26212-02) Hydrogen peroxide solution (30%(vol/vol); H personal protective equipment. and isflammable. with appropriate onlyHandle ina chemical fumehood Acetone (Fisher Scientific, cat. no. A494-4) protocol Hot plate stirrer bar stir Magnetic Aluminum foil Glass beaker Glass slide storage boxes Glass slide racks and 45×50mm, 170 Microscope slides (75×25mm, glass 1mmthick; 75×50mm, 1mmthick; Chemical fumehood Air line Vacuum line Nitrogen regulator Nitrogen line gas Petri dishes(100mmindiameter) Tweezers Wash bottles containersPlastic andtanks toolsAlignment Division) UV illuminator (OminiCure S1000; EXFOLife Science &Industrial Hot plates Spin coater (WS-400-6NPP-LITE; Laurell Technologies) Silicon wafers (3-inch diameter; University Wafer) Autodesk AutoCAD) Computer-aided software design (Macromedia Freehand MXand and a potent mutagen. Handle with appropriate personal protectivePropidium equipment. iodide (PI; Invitrogen, cat. no. P1304MP) equipment. ! YOYO-1 (1mMsolution inDMSO; Invitrogen, cat. no. Y3601) Sucrose (Sigma-Aldrich, cat. no. S9378) cat. no. 63138) Magnesium sulfate heptahydrate (MgSO phosphateSodium dibasic(Na Potassium phosphate monobasic (KH

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 system Electroporation • • • • • • • • • • cultureCell andsamplepreparation • • • Solution preparation • • • • • • • • • fabrication PDMS device store at room temperature. 8.0, from the respective stock solutions. Filter with a 0.2- adjust the volume to 100 ml. Prepare 10 mM Tris-HCl and 1 mM EDTA, pH water and adjusting pH to 8.0 with concentrated NaOH solution; add water to mM EDTA (pH 8.0) stock solution by dissolving 14.612 g of EDTA in 80 ml of centrated HCl solution; add water to adjust the volume to 100 ml. Prepare 500 oxymethyl)aminomethane in 80 ml water of and adjusting pH to 8.0 with con Prepare 1 M Tris-HCl (pH 8.0) stock solution by dissolving 12.14 g Tris(hydr TE buffer 24 hatroom temperature (25°C). cleaning theslides. cleaning The solution becomes stored when useless for and maintainat75°C. beaker. water ultrapure inaglass of Heat thesolution on ahotplate stirrer hydroxide 30%(vol/vol) and1volume of hydrogen peroxide to five volumes Glass slide solution cleaning REAGENT SETUP • • • • • • • Microscope • • • • • • • • • • • • • flow hood overnight prior to the experiment. Spectrophotometer Ice maker Hemocytometer Spray bottles Centrifuge (0.2, tubes Centrifuge 1, 15and50ml) Pipettes/tips cultureCell flasksandplateswell Tissue culture humidified, incubator at37°Cwith set 5%CO Laminar flow hood with 0.2 filters Sterile syringe Lure-Lock with Glass syringe (50ml) tip Milli-Q Direct system (Millipore) Plasma cleaner pump Plasma)andvacuum (Harrick Hole puncher Razor blades ovenLaboratory Vacuum desiccators rods Glass stirring pipettes transfer Disposable Polystyrene dishes weighing Balance ImageJ software (or similar) Stage micrometer Image acquisition software QCapture (QImaging Pro) a×63,with 1.4NA oil-immersion objective Confocal fluorescence microscope system (LSM510; Carl equipped Zeiss) emitter HQ645/75mandbeamsplitter Q565LP; Chroma Technology) emitter HQ535/50mandbeamsplitter Q505LP; for PI: exciter HQ535/50x, Fluorescence filter (For cubes EGFPand YOYO-1: exciter HQ480/40x, High-resolution (Hamamatsu, CCDcamera ORCA-285) ×10and×20objectives with equipped lamp(IX-71; Olympus) mercury Inverted phase-contrast andepifluorescence microscopewith 100 system W cat. no. 10957) Platinum (0.5mmindiameter, wire Premion, 99.997%; Alfa Aesar, Plastic-coated copper wire clip leads powerDC supply (PS350; Stanford Research System) alligator with Male flangeless nut, 1/8inch (IDEX Health &Science, cat. no. P-301) Flangeless ferrule, 1/8inch (IDEXHealth &Science, cat. no. P-300) Tubing, 1/8inch (IDEXHealth &Science, cat. no. 1641L) Male flangeless nut, 1/16inch (IDEX Health &Science, cat. no. P-201) Flangeless ferrule, 1/16inch (IDEXHealth &Science, cat. no. P-200) Tubing, 1/16inch (IDEXHealth &Science, cat. no. 1622L) Female Luer adaptor (IDEXHealth &Science, cat. no. P-678) Luer-Lock with Sterile syringes (6ml; tip Kendall) Luer with (1ml; sliptip Becton Dickinson)Sterile syringes pump(FusionSyringe 400; Chemyx)

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- © 2011 Nature America, Inc. All rights reserved. 2000). As an example, for athickness of 75 the desired filmthickness. Determine the required spinspeedand time from spincurves(seeMicroChem data sheet on SU-8 5|  4| 3| Fabrication of masters than chrome masks.  (https://engineering.purdue.edu/NANO/Microfab). Department (http://130.126.4.33/scripts/contacts.cgi) and Purdue University Birck Nanotechnology Center suppliers provide aprinting service, such asUniversity of Illinois atUrbana-Champaign Facilities &Services Printing print the patternonglasswithalayerof chromium to generate chrome masks withhigher resolution. Many commercial 2| transparent area, allowing UV light to pass through the photomask and cross-link the SU-8 2000 photoresist.  and one spiral narrow section (din (a and bin Figure 1| Design andprintingof photomasks PROCE are maintained low in order to generate a solution conductivity ~0.14of S m 4 °C for up to 6 months. water,of andadjust pHto 7.4. a0.2- Filter with KH 0.272 g of MgSO buffer Electroporation for 20minto sterilize. Store at4°Cfor upto 1year. PBS Sterile ml 10% (vol/vol) FBS, 2mM Forcell types. CHO-K1cells, themedium isDMEMsupplemented with cultureCell medium DNA. plasmid the re-extract to advisable is it cases, UV absorbers, and a ratio of of ratio A 2.0. and 1.8 between be should (A260/280) nm 280 versus 260 at absorbance of ratio the purity, DNA reliable For viability. cell and efficiency transfection reduced in result could endotoxin and ethanol salts, as such Impurities chemicals. and acids nucleic contaminating other nucleases), (especially proteins and free of  observation. facilitate to model a as used is (Clontech) (EGFP) GFP enhanced plasmid encoding pEGFP-C1 degradation. marked without year 1 to up at them store and cycles freeze-thaw repeated aliquots avoid to multiple into solution DNA plasmid the Split nm. 280 and 260 at absorbance UV by purity and buffer concentration DNA TE the in Determine 8.0). DNA (pH Giga plasmid purified Plasmid Dissolve QIAfilter 12291). no. cat. e.g., (Qiagen, kit, Kit preparation plasmid available mercially DNA Plasmid equipment. protective personal appropriate with solution the Prepare corrosive. is acid Hydrochloric hydroxide irritants. EDTAare sodium and 30 s(

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CR CR CR

1 streptomycin. Store at4°Cfor upto 1year. I Pour ~5mlof SU-82000photoresist onthe center of the wafer. Spinthe waferwithanappropriate spinspeedtoobtain Place a3-inch silicon wafer onthe center of the spinner chuck and applyvacuum tohold the wafer. Turn onthe nitrogen cylinder, which isconnected tothe spin coater, and setthe pressure to60psi. For straight channels, print the design onhigh-resolution (5,080dpi)filmtransparencies. Forspiral channels, Draw patterns withacomputer-aided design software, such asMacromedia FreeHand MXorAutodesk AutoCAD. T 4 I I I ·7H I Fig. 3 T T T CAL D 2 I I I URE CAL CAL CAL 2 shows four examples of the designs: first, multiple alternating wide and narrow sections withdifferent sizes

O and250mMsucrose, pH 7.4. Na Dissolve 1.136gof For plasmid optimal DNAtransfection, should be of high quality Dissolve one PBS tablet in 200 ml of ultrapure water. ultrapure Dissolve one in200mlof PBStablet Autoclave 2 PO

a Fig.

rpr i avne sn ay tnad rtcl r com or protocol standard any using advance in Prepare STEP STEP STEP ). 4 , MgSO 0.2465 g of 2

The position of the wafer will need fine adjustments to ensure uniform spread of photoresist on the wafer. Film transparency is suitable for patterns with relatively large sizes ( SU-8 2000 is a series of epoxy-based negative photoresists. Channel patterns should be drawn as ); second, twowide sections and one straight narrow section (cin Use appropriate cell culture medium for specific

<1.8 suggests the contamination of proteins or other other or proteins of contamination the suggests <1.8

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PI stock solution voltage andcauseexcessive Joule thatlowers heating cell viability. may to lead bubble gas (hydrolysis) formation the uponapplication of whereas sucrose to isadded keep thebuffer isotonic. More conductive buffers residues such as alkanethiols from the glass surface by oxidization, during during oxidization, by surface glass the from alkanethiols as such residues box. storage slide a in slides glass cleaned the air. Place clean with them blow-dry thoroughly and water ultrapure with rinse tweezers, with slides glass remove h, 3 After bar. stir magnetic a with continuously solution the stir and plate, hot a on °C 75 at slides the Heat foil. aluminum of sheet a with beaker the cover and detail) for SETUP REAGENT (see solution cleaning the add racks, cleaning slide Glass equipment. protective personal appropriate with hood fume chemical a in only Handle ! environment. dry and clean a in store or coating, photoresist for perature tem room to down Cool surface. wafer from water adsorbed removethe to min 5 for °C 200 at plate air. hot a clean on Bake with water. ized Blow-dry deion with rinse min,and 5 for isopropanol in wafer silicon the Immerse isopropanol. with rinse min,and 5 for acetone in wafer silicon the Immerse wafers. the onto photoresist of adhesion the enhance to fabrication before Wafertreatment EQUIPMENT SETUP dilution from stock).of Store at 4°C, protected from light, for up to 1 month. PI working solution 4 °C, protected from light, for upto 1year. protective equipment. ­protective personal appropriate use and hood chemical a in step this Perform toxic. highly are peroxide hydrogen and hydroxide ammonium Both solution. the in vigorously generated are bubbles oxygen which

CAUT I ON Acetone and isopropanol cause irritation and are flammable. flammable. are and irritation cause isopropanol Acetoneand

Silicon wafers should be precleaned and dehydrated dehydrated and precleaned be should wafers Silicon Dissolve 1 mg of PI in 1 ml of PBSat1mgml PIDissolve in1mlof 1mgof 

Dilute PI stock solution to 1 Place the glass slides in a glass beaker with slide slide with beaker glass a in slides glass the Place

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CAL >20 >20 2 This process is used to remove organic organic remove to used is process This ); and, finally, twowide sections µ m), m), and is much cheaper

| VOL.6 NO.8VOL.6 µ g ml

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CAUT

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1197 I ON - -

© 2011 Nature America, Inc. All rights reserved.  adhesion betweenthe photoresist and wafer. Cooldown the waferbefore proceeding to the next step. 11| ? 10| ? protective equipment. !  tips ( unexposed photoresist isremoved. Briefly washthe tweezerswith developer to remove the photoresist remaining onthe 9|  photoresist ( 8| source during exposure. !  replicate the patterninto the photoresist ( density between585and 600mJcm on the transparency photomask. ExposetoUVlight (energy photomask ontopof the waferand placeablankglassplate 7| be uniform. the thickness of the photoresist on the wafer will not  no film photoresist thickness. Optimize the process to ensure that  harden the photoresist ( tweezers. Soft-bake at95°Ctoevaporate the solvent and 6| ? appropriate personal protective equipment. flammable. Handle only in a chemical fume hood with ! REAGENTS section for additional information). fabricating devices with the desired channel depth (see ate photoresist with the required viscosity and density for  H: the depth of the channel. width of the narrow sections; L width of the wide sections; L UV penetration. The table shows the dimensions of the channels. W proportion. The channel patterns are designed as transparent areas allowing electroporation devices. The narrow sections are not magnified by the same Figure 2 1198 protocol

CAUT CAUT CAUT TROU TROU TROU

CR CR CR CR CR CR CR When anultrathick photoresist such asSU-82150isused, hard-bake the wafer at150°Conahot plate toimprove the Briefly rinse the wafer with isopropanol several times and then blow-dry itwitha gentle stream of air(

Cool down the wafertoroom temperature. Submerge the waferinto SU-8developer and manually agitateuntil all Immediately afterexposure, bake the waferonahot plateat95°Ctocross-link the exposed areas of the Cool down the wafertoroom temperature. Align the Transfer the coatedwafer onto ahot platewith | I I I I I I I Fig. 3 VOL.6 NO.8VOL.6 T T T T T T T

I I I | I I I I I I I B B B ‘ ON ON ON Examples of photomasks used for fabricating flow-through CAL CAL CAL CAL CAL CAL CAL wrinkling LES LES LES SU-8 developer is toxic and flammable. Handle only in a chemical fume hood with appropriate personal UV radiation is harmful to eyes. Wear protective goggles or a face shield, and avoid directly looking at the UV SU-8 2000 series photoresists are toxic and e

H H H ). Fig. 3 STEP STEP STEP STEP STEP STEP STEP OOT OOT OOT | 2011 ’ The optimal hard-baking time depends on the film thickness. The optimal development time varies with film thickness. The post-baking time is determined by the photoresist thickness. The optimal exposure intensity and time depends on the photoresist thickness and the UV source. The hot plate must be leveled. Otherwise, The soft-baking time depends on the It is important to choose the appropri I I I occurs. d N N N ). G G G |

1 natureprotocols : the total length of wide sections; W Fig. 3 2 : the total length of the narrow sections; b ).

−

2 ) for 17–20sto Fig. 3 c ). 2

1 : the : the

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a c,d b a W 8,730 7,500 1 500

( µ m) 4,000 +4 4,000 +4 Wide section 2 L × × × 1 20,000+4,000 30,000+4,000 3,000

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35 ( µ m) Fig. 3 c 1 5 5 L × × × 2

4,768 200 200 (

µ 1 cm m) f ). 1 cm

d H

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© 2011 Nature America, Inc. All rights reserved. transfection; ~1mmindiameter for large-volume transfection) using apuncher of the correct size( 19| ? slice onto aglassslide and measure the depth of the channel under a microscope withthe help of astage micrometer. 18| ? when cutting. ! aluminum foil. Cutthe PDMS mold into the desired size. Avoid touching the PDMS surface with the feature ( peel itfrom the master. Placeit,withthe feature side facing down, onasmooth bench surface covered byacleansheet of 17| 16| under the vacuumfor awhile. Fora100-mmPetri dish,~30gof the mixture isrequired togenerate a~5mmlayer( If there are stillsome bubblesremaining, remove them withadisposabletransfer pipetteorplacethe master/PDMS back 15| Degas under vacuumfor 2huntil allbubblesdisappear. Turn off the vacuumand slowlyvent the desiccator. 14| ! easily torn upon insertion of tubing and electrodes. may not be completely cross-linked; such PDMS devices are agent is not sufficient and/or not mixed thoroughly, PDMS components and thorough mixing are critical. If the curing  PDMS mixture. becomes milky. Avoid introducing dust particles into the vigorously withacleanglassstirring rod until the mixture curing agent isevenlydistributed, stirthe mixture disposable polystyrene weighing dish.To ensure thatthe of 10:1(RTV615Abase:RTV615Bcuring agent) ina 13| overnight sterilization Fabrication of ? environment indefinitely.  Place the master inaclosed100-mmPetri dishfor storage. sharp and the feature dimensions are the same as designed. wafer) under amicroscope tocheck whether the edges are 12| and rinse and dry with clean air (Step 10) ( agitate in SU-8 developer to remove the unexposed photoresist (Step 9) ( bake on a hot plate to fully cross-link the exposed photoresist (Step 8) ( the pattern on a photomask into the photoresist (Step 7) ( solvent and harden the photoresist (Step 6) ( silicon wafer (Steps 3–5) ( photolithography technique. ( Figure 3

CAUT CAUT TROU TROU TROU

PAUSE CR Punch aninlethole (~1mm indiameter) and anoutlet hole (~5mmto~10 indiameter for small-volume Cutathinslice of the PDMS device along the cross-section of the fluidic channel using asharp razor blade. Placethe Aftercooling PDMS toroom temperature, gently cutthe patterned area of the cured PDMS replica witharazor blade and Cure PDMS inanovenat80°Cfor 1–2htogenerate aPDMS layerthatistransparent and solid ( Slowlypourthe PDMS mixture overthe master inaPetri dish.Take care not tointroduce bubblesinto the Petri dish. Placethe weighing dishcontaining the PDMS mixture into avacuumdesiccator and evacuate the chamber. Weigh PDMS prepolymer and curing agent atthe ratio Inspect the master (i.e., photoresist patterned onthe I T

I I | I B B B ON ON Schematic illustration of master fabrication with standard CAL LES LES LES

PO The master is brittle and can be easily cracked by the razor blade. Do not exert excessive stress on the wafer Wear powder-free gloves when handling the PDMS prepolymer.

H H H I STEP NT OOT OOT OOT P The master can be stored in a dry and clean DM The ratio between the PDMS prepolymer I I I N N N S G G G a devices ); soft-bake on a hot plate to evaporate the a – f ) Spin-coat SU-8 2000 photoresist on a ●

T IMI f ). b ); UV exposure to replicate N G 4.5 h plus c ); post-exposure

d e );

); d c b a f e SU-8 developer natureprotocols Silicon wafer Photoresist Hot plate Fig. 4 Cross-linked Fig. 4

| Master Photomask VOL.6 NO.8VOL.6 c Post-exposure Rinse anddry UV exposure ). Photoresist b Soft bake spin coat Fig. 4 Develop ). bake protocol

c |

2011 ). Fig. 4 |

1199

a ). © 2011 Nature America, Inc. All rights reserved. 23| ? trapped atthe interface betweenthe PDMS and the glassifnecessary ( Immediately bring the PDMS into contact against the slide toform closedchannels. Press gently toremove airbubbles chamber. Remove the PDMS and glassslide withtweezersand avoid touching the surfaces thatare tobebonded. and vacuumpump.Openthe needle valveslowlytovent the 22| characteristics of sample surfaces. the pressure and flow rate of air, the treatment time and the determined by the intensity and frequency of the RF power, action between the plasma and the treated surfaces is groups on the PDMS surface into Si-OH groups. The inter  with plasma for 1min. is observed. Once itisstable, treat the PDMS and the slide to letairinto the chamberuntil abright pink-violet glow frequency) levelto 21| chamber for 5min. facing up,and turnonthe vacuumpumptoevacuate the the plasma cleaner chamberwiththe surfaces tobebonded tape. Use tweezerstoplacethe PDMS and glassslide into 20| temperature for several days in a clean and closed container.  waterproof seal under fluidic pressure. smaller than the outer diameter of the tubing to ensure a  20–22) ( oxidize by plasma treatment and bond to a clean glass slide (Steps oven at 80 °C (Step 16) ( over the master that is placed in a Petri dish (Step 15) ( wafer master. ( Figure 4 1200  transfection efficiency and cell viability.  cells (even immortalized cell lines) may change after repeated passages, leading to poor transfection.  in one 25 cm regularly subcultured. CHO-K1 cells are passaged every 2 d at a ratio of 1:10. Approximately 2 × 10 division improves transport of DNA into the nucleus. Each cell line has a characteristic optimal confluency and should be  cells). Count cellswithahemocytometer. ForCHO-K1 cells, culture to80%confluence. 25| C ethanol through the channel for atleast5minand subsequently flowing ultrapure water for atleast5min. 24|  protocol ell ell and sample preparation

TROU

PAUSE CR CR PAUSE CR CR CR CR Bake the whole device at 80 °C in the oven for another 1 h to enhance the bonding strength between PDMS and glass. ( Setthe RFlevelto Turn onthe plasma cleaner and setthe RF(radio Remove dust particles from PDMS surface withpaper Harvest cellsgrowing in exponential phaseaccording tostandard protocols (using trypsin-EDTA solution for adherent Sterilizethe devices prior toexperiments byUVirradiation inalaminar flow hood overnight or by flowing 70%(vol/vol)

| I I I I I I VOL.6 NO.8VOL.6 T T T T T T

d | I I I I I I B ); and bake to increase the bonding strength (Step 23) ( Schematic illustration of PDMS device fabrication using the silicon- CAL CAL CAL CAL CAL CAL LES

PO PO a

H – I I STEP STEP STEP STEP STEP STEP 2 NT e NT flask. OOT ) Dispense degassed PDMS prepolymer and curing agent | The PDMS devices can be stored at room temperature for several months in clean Petri dishes until use. The cured PDMS can be stored at room 2011 If multiple culture flasks are used, mix all cell suspensions in one tube for cell counting to avoid variability. The oxygen plasma transforms Si-CH The punched holes should be slightly Cells to be transfected should be healthy and actively proliferating for best results, because the rapid cell Each cell type has its own optimal digestion time. Avoid overdigestion of cells, which reduces both The number of cell passages is also essential for obtaining efficient gene transfer. The characteristics of I N ‘ b high G ); cut and punch access holes (Steps 17–19) ( |

natureprotocols ‘ off ’ and openthe needle valveslightly ’ . Turn off the plasma cleaner ●

T IMI N G ~30 min a ); cure PDMS in an e

).

3

­ c );

Fig. 4 d b c a e d ). Glass slide PDM S 6 CHO-K1 cells are yielded treatment dispense Cut and Plasm PDM PDM punch curing Bake

S S a

Fig. 4 e ) © 2011 Nature America, Inc. All rights reserved. ( surface needs tobeconfirmed. to stainthe DNAwithafluorescent dye before electroporation. Thisis necessary when DNA delivery overthe entire cell contamination. To observethe distribution of plasmid DNAonthe cellsurface afterdelivery, follow the procedure in to large volumes (>500 ing onthe transfected cellvolume. Option Aisfor processing small volumes ( 32| Flow-through electroporation incubation on ice, resulting in reduced transfection efficiency and viability. by electroporation. However, do not incubate cells on ice for longer than 1 h. Cells may become unhealthy after a long  31| of pEGFP-C1 plasmids (containing CMV promoter) and 2 × 10 promoters such as the human cytomegalovirus (CMV) immediate early promoter are toxic to cells. Thus, we use 40 concentration depends on the target cell line and the type of DNA. Excessive amount of plasmids containing potent  and cell viability or even clog the channel.  for cellsand plasmid DNA.Gently mixthe solution by pipetting several times. 30| to provide the required density of cellsfor experiments. 29| 28|  Wash the cells by centrifuging at 300 4 ml of the electroporation buffer, and gently disperse the cell pellet by pipetting up and down until the cells are resuspended. 27| 26| ( power supply. The plastic tubing is inserted into a punched hole for transfer of the cell sample. ( wire for connection with the wire leads of the A copper wire is prelinked with the platinum transfection) through elastic PDMS wall. the outlet in the case of large-volume an electrode and inserted into the inlet (and into the channel. A platinum wire is used as ( placed in a laminar flow hood during operation. voltage across the channel. The system is platinum wire electrodes establishes a constant pump. A power supply connected to two tubing affixed to the inlet, driven by a syringe into the PDMS fluidic channel through plastic DNA in the electroporation buffer is introduced operation. A mixture of cells and plasmid electroporation. ( Figure 5 ≤ b A ) Details of the platinum electrode insertion

500

) For small-volume ( CR CR CR CR (i) Slowly aspirate and discard the supernatant, retain ~50 Aliquot the mixture into microcentrifuge tubes with the volume for one experimental run in each tube. Leave them on ice. Forflow-through electroporation (Seesystemsetup in Combine the cellsuspension withanappropriate amount of plasmid DNAsolution toachieve the desired concentrations Slowly aspirate and discard the supernatant and resuspend cells with an appropriate volume of the electroporation buffer Repeatthe washstep(Step27)once. Centrifuge the required number of cellsat300 I I I I µ laminar flowhood overnight before the experiment.  tubing inorder. Finger-tighten the assembly into the female Lueradaptor. Insert the syringe into the Lueradaptor. system and afemale Lueradaptor. Slide a1/16-inch male flangeless nut and a1/16-inch flangeless ferruleover the T T T T Connect alength of 1/16-inch tubing toa1-mlsterile Luer-tippedsyringe withthe help of the flangeless fittings l) transfection. (

| I I I I The setup for flow-through CAL CAL CAL CAL

CR I

T STEP STEP STEP STEP I a CAL ) The entire apparatus in In our hands, incubation of cells (suspended in the electroporation buffer) on ice decreases later cell death The concentration of plasmid DNA affects transfection efficiency and cell viability. The optimal Single cell suspension must be obtained at this point. Cell aggregates may reduce transfection efficiency High shear stress induced by vigorous pipetting may damage cells and negatively affect cell transfection.

STEP d ) Schematic illustration of the setup for large-volume ( µ The tubing assembly isreusable and easy toreplace. It should besterilizedbyUVirradiation ina ≤ l) of cells( 500 m ● l) celltransfection

T

IMI

Fig. 5 g for 5 min at room temperature and then resuspending in the electroporation buffer. N G 1–2 h d ). The electroporation should beperformed inalaminar flow hood to reduce b a Syringe pum g for 5minatroom temperature. p PDMS device Tubing Platinum electrode Fig. 5a, µ 6 cells ml l of the liquid in the centrifuge tube to avoid cell damage. Add PDMS device

>

500 DC powersupply b ), twoslightly different procedures canbeused, depend µ

− l) transfection.

1 of cells in the electroporation buffer. c ) Schematic illustration of the setup for small-volume ≤ 500 c Platinum d wire Tubing µ l) of cells( Air natureprotocols Cell Power supply – Fig. 5 – Plasmid Glass slide Tubing PDM c S + +

). Option Bisapplied | VOL.6 NO.8VOL.6 protocol Pipette tip

µ | Platinum Reservoir 2011 g g ml wire

Box Container

− |

1 1201

1

-

© 2011 Nature America, Inc. All rights reserved. (B) For large-volume (>500 1202 protocol (vii) Box 1 (For YOYO-1: excitation maximum 11. View the samplewithaconfocal fluorescence microscope equippedwithan×63,1.4NAoil-immersion objective. 10. Add adrop of cellsuspension toacleanglasscoverslip(~170 9. Resuspend the cellswithanappropriate volume of PBStoachieve aconcentration of 5×10 8. Repeatthe wash(Step7)once. and then discarding the supernatant. The washremoves nonspecifically adsorbed DNAfrom the cellsurface. 7. Slowlyaspirate and discard the supernatant. Wash the cellswith4mlof PBSbycentrifuging at300 6. Centrifuge the cellsat300 5. Immediately afterelectroporation, transfer the processed cellstoacentrifuge tube. 4. Perform flow-through electroporation as described inStep32 of the PROCEDURE. concentration of 500 3. Add the fluorescently labeledDNAtocellsuspension toachieve afinal cellconcentration of2×10  2. Mixthe DNAand YOYO-1 solutions bypipetting and incubate the mixture at room temperature for 1hinthe dark. ! provided bythe manufacturer (Invitrogen). 1. Labelplasmid DNAusing the fluorescent dye YOYO-1 ata ratio of 1 dye molecule per5bp of the DNAaccording tothe protocol (iii) (vi) (iv)

(ii) Draw the electroporation bufferinto the syringe and tubing, and fix the syringe inasyringe pump. (ii) Draw the electroporation bufferinto the syringe and tubing, and fixthe syringe inasyringe pump.

(v) (i) CAUT

CR

| VOL.6 NO.8VOL.6 ? corners of the wide sections. The presence of bubblesmay alterthe electroporation conditions.  the electroporation buffer. Gently pulloutthe tubing from the inlet. the electroporation bufferfor atleast5mintocondition the channel and remove impurities. Prime the channel with  Insert alength of 1/16-inch tubing into the other end of 1/8-inch tubing tofacilitate affixing tothe inlet. the tubing inorder. Finger-tighten the assemblyinto the female Lueradaptor. Insert the syringe into the Lueradaptor. fittings systemand afemale Lueradaptor. Slide a1/8-inch male flangeless nut and a 1/8-inch flangeless ferruleover !  turn off the pumpand power supplypromptly. electroporation, and collectcellsfrom the outletreservoir withapipette. When the airplugreaches the channel inlet, Turn onthe DCpowersupply and the syringe pumpsimultaneously. Flowthe cell/DNAsolution through the device for of the electric fieldinthe channel orevendisconnect the current. air bubblesinto the channel. Bubblesmay migrate withthe flow, blockthe channel, and thus change the distribution   suspension tothe end of the tubing and insert the end of the tubing into the inletof the channel. the tubing sothatitisseparated from the electroporation bufferinthe tubing bythe airplug.Fillthe cell/DNA ? platinum wire, asshown in negative terminal of the powersupplybyclipping the wire leadtothe supporting copperwire prelinked withthe Carefully insert one platinum wire into the inletthrough the elastic PDMS wall.Connect the platinum wire tothe Insert the tubing filledwithliquid into the device inletasillustrated in Connect alength of 1/8-inch tubing toa6-ml(orlarger) sterileLuer-tippedsyringe withthe help of the flangeless Set the pumpparameters (syringe diameter, infusion volume and flow rate) and the electric parameter (voltage). Draw ashort plugof airinto the tubing bypulling the syringe pistoncarefully. Then draw the cell/DNAmixture into Aspirate and discard the liquid inthe outletreservoir. Placethe other platinum wire connected tothe positive platinum wire isincontact withthe solution. (depending onthe sizeof the outletreservoir) of the electroporation bufferinto the outletreservoir sothatthe terminal of the powersupplyinto the outletreservoir (punched during fabrication, Step19).Add acertainvolume I T

I CAUT

TROU I

TROU ON CR CR CR CR CR CAL | YOYO-1 isanirritant. Handle withappropriate personal protective equipment. I I I I I T T T

T T

STEP I O I I I I I B B ON CAL CAL CAL CAL CAL LES LES BSERVING PLASMID DNA DISTRIBUTI | 2011 Donot touch the leadsof the powersupplyinorder toavoid electric shock when the powerison. The incubation stepshould beperformed inthe dark toprevent photobleaching of the fluorescent dye.

H H STEP STEP STEP STEP STEP OOT OOT µ |

g ml natureprotocols Use amicroscope tocheck whether there are airbubblestrapped inthe channel, especially atthe Use asyringe withthe appropriate volume when working withlarge-volume cellsamples. The applied voltage and flow rate canbe determined using Equations (2)and (3). Before inserting tubing, ensure thatthe liquid reaches the end of the tubing toavoid introducing The volume of the airplugshould besmall enough toensure accurate flow rate. I I N N

− G G

1 . Gently mixthe solution bypipetting several times. g for 5minatroom temperature. m l) celltransfection

=

Figure 5 491nm;emission maximum b .

=

509nm.) µ m inthickness) and allowthe cellstosettlefor 10min. O N

O N CELL SURFACE Figure 5 6 b cellsml . Extensively flushthe channel with 6 cellsml g for 5minatroom temperature

−

1 .

−

1 ● and DNA

T

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N

G 2 h

© 2011 Nature America, Inc. All rights reserved. ?  (the percentage of viable cellsamong the totalcells processed). 40| efficiency and the cell viability.  39| maximum phase-contrast/epifluorescence microscope equippedwithan×20 dry objectiveand aCCDcamera. (ForEGFP: excitation 38| ! dark for 20mintostaindead cells. 37| 36| characteristics of the transfected gene. The expression of EGFP in CHO-K1 cells reaches its peak at 48 h after electroporation.  settle atthe bottomof wellsorculture flasks. cell viability bystaining withPI.Centrifuge the sampleat300 35| T  34| ? current for eachrunof electroporation for comparisons betweenexperiments. Astablecurrent indicates smooth operation. 33| ransfection ransfection and viability assays

(iii) CAUT (vi) (iv) TROU TROU

(v) CR CR CR CR Calculatethe transfection efficiency (the percentage of cells expressing EGFPamong viable cells)and cellviability Use image analysis software such asImageJ toenumerate apopulation of atleast1,500–2,000cells ineachsample. Capture bothphase-contrast and fluorescent images of cellsat10different locations ineachsample using aninverted Aspirate and discard the PBS.Add PIworking solution (1 Gently aspirate and discard the supernatant. Rinse the cellswithprewarmed PBS. Fortransient expression, examine cellsat24–72hpost-transfection toevaluate expression of the transfected gene and Ifnecessary, reload the syringe and repeat Steps32and 33until the entire cellsampleistreated. Immediately transfer transfected cellstoprewarmed culture medium inaculture plateorflask.Record the electric I I I I ? tubing, and cellsare collectedinto acontainer.  ing from the inletand outlet. the electroporation buffer. The presence of bubblesmay alterthe electroporation conditions. Gently pulloutthe tub the electroporation bufferfor atleast5mintocondition the channel and remove impurities. Prime the channel with containers) atthe other end of the outlettubing tocollectthe transfected sample. Extensively flushthe channel with 1/16-inch tubing tothe outlet(which hasasmall punched hole instead of areservoir), and placeatube (orother T T T T forth outside the syringe. Thispractice prevents settling of cellsinthe syringe. small magnetic stirbarinside the syringe sothatthe solution canbeperiodically stirred bymoving amagnet backand If the cell/DNAmixture hasasubstantial volume inthe syringe and the processing time islonger than5min,placea electroporation, and collectcellswithatube. When allliquid isinfused, turnoff the pumpand powersupplypromptly. Turn onthe DCpowersupplyand the syringe pumpsimultaneously. Flowthe cell/DNAsolution through the device for ? as shown in positive terminal byclipping the wire leadstothe supporting copperwires prelinked withthe platinum wires, the platinum wire inthe inlettothe negative terminal of the powersupplyand the one inthe outlettothe Set the pumpparameters (syringe diameter, infusion volume and flow rate) and the electrical parameter (voltage). Draw the cell/DNAmixture into the tubing and the syringe. Fillthe suspension tothe end of the tubing and insert it Carefully insert twoplatinum wires into the inletand outlet,separately, through the elastic PDMS wall.Connect Insert the tubing filledwiththe bufferinto the device inletasillustrated in into the inlet.Then alsoinsert the other tubing into the outlet. I I I I I B B

ON

CAL CAL CAL CAL TROU TROU LES LES CR

= PI is a suspected carcinogen. Wear safety equipment when working with this dye and dispose of it properly. I

488nm;emission maximum

T H H STEP STEP STEP STEP I B B OOT OOT CAL LES LES Every data point should be based on at least three experimental trials, and expressed as percentage ± s.d. When working with a large number of cells, flow cytometry can also be used to analyze the transfection The optimal assay time depends on the growth rate of the cell line and the specific expression It is advisable to use a new device for each run especially when a different DNA or cell type is used. I I

H Figure 5 H N N STEP OOT OOT G G Forlarge-volume transfection, the device outletshould haveasmall punched hole for affixing I I N N G G b .

●

T IMI

= N

507nm.ForPI:excitation maximum G 0.5 h per sample

µ g g ml for 5minatroom temperature sothatthe floating cells

−

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=

493nm;emission maximum Figure 5 natureprotocols b . Affixanadditional length of

| VOL.6 NO.8VOL.6 protocol

=

632nm.) | 2011

|

1203 - © 2011 Nature America, Inc. All rights reserved. T Troubleshooting advice canbefound in ? 1204 a 18 12 10 9, 17 5 S 22 protocol

TROU tep b le

| VOL.6 NO.8VOL.6 2 B | LES

Troubleshooting table. PDMS and glass do not bond expected PDMS channel depth is not as ent in depth the same master are inconsist PDMS channels fabricated using (e.g., SU-8 2150) for thick photoresist film Pattern distortion and cracks White traces form on the wafer Patterns on the wafer lift off coating photoresist film after spin Air bubbles trapped in P roblem H OOT | 2011 I N G | natureprotocols

- T able optimized Plasma treatment time is not plasma cleaner chamber Plasma does not form in the making photoresist more viscous Photoresist solvent evaporates, photoresist are not optimized Spin-coating conditions for coated Photoresist is not uniformly during fabrication Great internal stress is produced Photoresist is underdeveloped Photoresist is overdeveloped cross-linked Photoresist is not sufficiently by UV Photoresist is underexposed dispensing Bubbles are generated during container that stores photoresist Bubbles are trapped in the P ossible reason 2 .

Reduce the development time Increase the post-exposure bake time Increase the exposure dose Remove air bubbles with a clean and thin tip 50-ml centrifuge tube Use a dispenser with a large orifice, i.e., a viscosity Heat the container at 50–60 °C to reduce the S condition Finely tune plasma treatment time to find the best cleaner door evacuated. Check the vacuum system and plasma Make sure the plasma cleaner chamber is adequately Use a new bottle of photoresist Increase the spin speed selected photoresist Obtain a thickness versus spin speed curve for the Level the hot plate before soft baking coating, especially for SU-8 2150 Relax the photoresist on a leveled surface after spin Dispense the photoresist on the center of the wafer chuck Carefully center the silicon wafer on the spinner Allow the wafer to slowly cool after each baking step create uniform cross-linking for the photoresist film Optimize UV exposure and post-exposure bake to the photoresist film is thick Vigorously agitate the wafer during development if little bit longer until no white traces are left Immerse the wafer back into SU-8 developer for a Reduce the agitation during development olution

(continued)

© 2011 Nature America, Inc. All rights reserved. T a 32B(iii) 32A(iii), S 40 33 32B(iv) 32A(iv), tep b le 2 |

Troubleshooting table (continued). Low Low transfection efficiency between experiments Inconsistent current reading platinum wire electrode Solution leakage along Solution leakage P Low Low cell viability roblem

to to cells Excessive plasmid DNA is toxic times Devices are reused for multiple is not optimized Post-electroporation culture time Channel geometry is not optimized residence time are inappropriate Electrical field intensity and Cell/DNA ratio is inappropriate Cell quality is poor Plasmid DNA is of poor quality is low The amount of plasmid DNA used different PDMS devices The channel depth varies among row sections of the channel Cells or PDMS scraps clog the nar Air bubbles block the channel too thick The platinum wire is bent or to the channel Too much pressure is applied with the tubing device is not properly connected The inlet (or outlet) of the PDMS is not strong Bonding between PDMS and glass Bonding surfaces are not clean P ossible reason

-

Use a straight platinum wire with a diameter Decrease the flow rate movement of the tubing Carefully insert the tubing and avoid unnecessary smaller than the tubing Make sure that the diameter of the puncher is slightly noncircular hole avoid creating small tears around the access hole or a Punch the inlet (or outlet) with a sharp puncher to See TROUBLESHOOTING for Step 22 gloves or tweezers tape or gas blow. Do not touch the surfaces with Remove dust particles from the PDMS surface with S Reduce the concentration of plasmid DNA Use a new device for each run to determine the optimal time Observe cells at different times post-electroporation Design a new channel optimal combination of electric parameters Treat cells with different conditions to obtain an plasmid DNA concentrations to optimize the ratio Transfect the same number of cells using various longer than 1 h Do not incubate cell/DNA mixture on ice for grown actively in the exponential phase Ensure that cells are uncontaminated, healthy and Prepare a new batch of plasmid Increase the plasmid DNA concentration See TROUBLESHOOTING for Step 18 Use a new device to perform electroporation before flowing the cell/DNA mixture Prime the channel with the electroporation buffer inserting tubing Make sure that no air bubbles are introduced while of 0.5 mm olution natureprotocols ’ s s outer diameter

| VOL.6 NO.8VOL.6 protocol | 2011 (continued)

|

1205 © 2011 Nature America, Inc. All rights reserved. (a microscale channel withone straight narrow section) and achieved is36%. from 5.5to1.1ms). The cellviability under these conditions isveryhigh (93–98%)and the highest transfection efficiency in the wide sections), whereas the flow rates are varied inthe range of 3.94–19.7mlmin depicted in 1.87 mlmin reducing the cellviability. Anoptimal transfection efficiency ashigh as75%isachieved with 1.87 to4.36mlmin (leading to800Vcm nel withitsdesign in DNA. alternating wide and narrow section(s). Here weshow typical results for transfection of CHO-K1 cellsbypEGFP-C1plasmid This protocol enables continuous transfection of cellsbyflow-through electroporation inPDMS fluidic channels with ANT Box Steps 35–40,Transfection and viability assays:0.5hpersample Steps 32–34,Flow-through electroporation: typically 1–2h,depending onthe sampleamount Steps 25–31,Celland samplepreparation: ~30min Step 24,Sterilization of PDMS devices: overnight Steps 13–23,Fabrication of PDMS devices: 4.5h Steps 3–12,Fabrication of masters: 0.5–3.5h,depending onthe photoresist thickness Steps 1and 2,Design and printing of photomasks: 1hfor design and 1–4dfor printing ● 1206 T a protocol S

Figure tep b T le IMI I

1 C | Figure VOL.6 NO.8VOL.6 2 , Observing plasmid DNAdistribution oncellsurface: 2h I PATE N | G

7 Troubleshooting table (continued). shows the effectsof hydrodynamics onflow-through electroporation. Using the devices shown in P results Nonreproducible transfection D Figure 2

− roblem 6

1 ). RESULTS , panel | Figure 6 2011

− b

1 − | c (corresponding to . The totalvoltage

Figure 2 1

inthe narrow sections and 53Vcm shows the transfection efficiency and cellviability when DNA delivery isconducted using achan natureprotocols d shows the results obtained withthe device having adepth of 412 a (having adepth of 480 T V 2 of 784Visapplied (generating 800Vcm devices varies Channel depth of different PDMS over time Cells Residence time is too long too high Electrical field intensity is Cells are damaged taminants bacterial endotoxin and other con Plasmid DNA is contaminated with P ranging from 7.7to3.3ms). Longer ossible reason ’ characteristics change µ m and 5narrow sections). The totalvoltage

−

1 inthe wide sections), whereas the flow rate Figure 2

d (achannel with the same dimensions asthe one - Increase the flow rate Reduce the field intensity immediately after electroporation Transfer cells into prewarmed culture medium than 1 h Do not incubate cell/DNA mixture on ice for more Centrifuge cells at lower speed Pipette cells smoothly and gently to avoid air bubbles Avoid overdigestion of cells by trypsin-EDTA solution methods involving phenol-chloroform Use transfection-grade plasmid DNA. Do not use S See See TROUBLESHOOTING for Step 18 excessively passaged growing. Use a new vial of frozen cells if cells are Subculture cells regularly and maintain active olution T 2 increases the transfection efficiency while

−

1 inthe narrow sections and 80Vcm

−

1 (corresponding to µ T m and five narrow sections as 2 of 7.7ms (with V of 763Visapplied Q changes from Figure 2 T 2 ranging Q of

c

-

−

1

© 2011 Nature America, Inc. All rights reserved. difference at experiments, and two-tailed Student All data points are generated by triplicate flow rate cell viability ( plasmid. The transfection efficiency ( results on CHO-K1 cell transfection by pEGFP-C1 (see procedure detailed in are taken immediately after electroporation section ( cm ( narrow section ( processed in the device ( cell populations shown in (i). ( on representative individual cells from the pEGFP-C1 plasmids. The images in (ii) focus CHO-K1 cells delivered with YOYO-1–labeled by confocal fluorescence imaging (ii) of and 3D reconstruction images generated in one of the same dimensions. (See designs shown with a straight narrow section and a spiral delivery into CHO-K1 cells in the devices Figure 7 efficiency without seriously compromising cellviability. and are used under the same operational parameters ( efficiency and cellviability when the devices in narrow section. parameters usedasinthe device withthe straight delivered withDNA( that render the entire cellsurface permeabilized and flow givesrisetocombined vortex motion and rotation (shown in Furthermore, when the device withaspiral narrow section limited tothe cellpolesdue tothe cellrotation. revealing the fact thatthe permeabilization isno longer delivery occursvia acircular striparound the cell( when in flow-through electroporation isconducted inthe device that the delivery islimitedtoone poleof acellwhen the reconstructed 3Dconfocal images in difference inthe uptake. The epifluorescence and labeled DNAmolecules are delivered for observing the in triplicate experiments. Modified from ref. identical field intensity ( viability vary with the total duration in the narrow sections ( taken at 48 h post-electroporation. The transfection efficiency and cell The data in protocol. The data shown in results ( ( Figure 6 a b – ) ) with a straight narrow section ( Fig. Fig. 2c,

− d Figure 2 Figure 2

1 ) The typical phase-contrast image ( ; ; E T 2 2 T of 400–700Vcm

c

2 E = , | | Q isincreased to5ms withthe same device, the d 2

Flow-through electroporation for large-volume cell transfection. Comparison of plasmid DNA d 5 ms); and (

of 150 ) of CHO-K1 cell transfection by pEGFP-C1 plasmids following our

). ). ( = d

are obtained using the device in 700 V cm Fig. 2 P e c c

a ) ) vary with

withashort except aspiral narrow section), fluorescently < – E

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) ) Epifluorescence images (i) µ

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1 T ). The data indicate thatthe device withaspiral narrow section yields much higher transfection – ; ; T , ). ). ( 2 c e c of 5 ms. T 2 ) ) Cells are of 0.5ms. Incomparison, ), withthe same electric 2 shows the transfection E

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35 with permission from Elsevier. ), fluorescent image ( 36 36 with permission from the Royal Society of Chemistry.

Figure 2 Figure 7

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207–363Vand these conditions generate d c a Transfection efficiency (%) Transfection efficiency (%) 100 100 100 µ 60 70 80 90 10 20 30 40 50 60 70 80 90 10 20 30 40 50 m 0 0 i i Viability (% Transfection efficiency(% Viability (% Transfection efficiency(% 7 6 5 4 3 6 5 4 3 2 1 natureprotocols Residence time Residence time ) ) P d

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0.05 and (**) indicates significant b 10 20 40 60 80 10 15 20 25 30 35 40 0 0 0 5 ( ( T T ) ) 2 2 , ms , ms 40 40 ** Spiral Spiral

) ) 5 0 5 0 | Field intensity VOL.6 NO.8VOL.6 Field intensity 8 ** 00 00 protocol Straight Straight T 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 2 ( of 5ms E

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2 2011 E ** Viability (%) Viability , Vcm (%) Viability 2, 7 0 7 0 Vcm –1 |

–1 ) 1207 ** 00 * 00 ) © 2011 Nature America, Inc. All rights reserved. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.htm at online available is information permissions and Reprints Published online at interests. CO gene delivery and analyzed the data. T.G., Y.Z. and C.L. wrote the manuscript. and analyzed the data. J.W. and Y.Z. performed experiments on vortex-assisted experiments. T.G. and Y.Z. performed experiments on large-volume transfection electroporation with constant voltage. T.G., J.W., Y.Z. and C.L. designed the AUT technical assistance. 35603-05059. We thank K.G. Cornetta and V. Ugaz for helpful discussion and 0967069, and United States Department of Agriculture grant USDA-NRI 2009- Early Career Award, National Science Foundation grants CBET 1016547 and CBET A 1208 protocol ckno

M

H PET Electroporation and Electrofusion and Electroporation Methods Nat. challenges. and Res.Mater. 18 carcinoma. liver. to delivery vector AAV without liver.mouse in integration vector virus (2002). integration. vector virus adeno-associated transfer. gene adenoviral following patient deficient transcarbamylase ornithine Med. fibrosis. cystic with patients of epithelium nasal the in transfer (1994). therapy.gene for adenoviruses (2008). 3132–3142 SCID-X1. of therapy generetrovirus-mediated (2003). SCID-X1. for therapy gene after patients two Adv.Biotechnol. treatment. disease monogenic in applications cells: stem pluripotent (2008). cells. stem mesenchymal modified genetically medicine.regenerativeskeletal (2005). 928–940 therapy. cancer for cells T of modification genetic masses. the Chang, D.C., Chassy, B.M., Saunders, J.A. & Sowers, A.E. (eds). A.E. Sowers, & J.A. Saunders,B.M., Chassy, D.C., Chang, Fedorov,Y. Montier,T. delivery.gene non-viral for Materials L.D. Shea, & T.Segura, Luo, D. & Saltzman, W.M. Synthetic DNA delivery systems. delivery DNA W.M.Synthetic Saltzman, & D. Luo, A. Donsante, P.Bell, H. Nakai, of effects D.W.Chromosomal Russell, & E.A. Rutledge, D.G., Miller, S.E. Raper, M.R. Knowles, Y.Yang, S. Hacein-Bey-Abina, S. Hacein-Bey-Abina, induced and targetingtherapy,gene GeneA.T. Chiu, & G.K. Wong, of potentialTherapeutic S. Ponnazhagan, & D. Chanda, Kumar,S., for engineeringGenetic A.J. Garcia, & J.E. Phillips, C.A., Gersbach, cells: T Darcy,P.K.Supernatural & M.J. M.W.,Smyth,Teng, Kershaw,M.H., for treatment a immunotherapy:P.D.Cancer Greenberg, & J.N. Blattman, OR

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