ASSESSMENT OF SINGLE CELL VIABILITY FOLLOWING LIGHT-INDUCED ELECTROPORATION THROUGH USE OF ON-CHIP

MICROFLUIDICSJustin K. Valley', Hsan- Yin Hsu, Steven Neale, Aaron T Ohta, Arash Jamshidi4 and Ming C. Wu

IBerkeley Sensor & Actuator Center, Department of Electrical Engineering and Computer SciencesUniversity of California, Berkeley, USA

ABSTRACTThe high throughput electroporation of single

cells is important in applications ranging from genetictransfection to pharmaceutical development. Light-induced electroporation using optoelectronic tweezers(OET) shows promise towards achieving this goal.However, cell viability following light-inducedelectroporation has yet to be shown. Here we present anovel OET device which incorporates microfluidicchannels in order to assess the viability of single cellsfollowing light-induced electroporation. Monitoring ofsingle cell electroporation and viability is achievedthrough the use of fluorescent dyes which are exchangedusing the integrated fluidic channels. The successfulreversible electroporation of HeLa cells is shown.

INTRODUCTIONElectroporation is a widely used technique for

the introduction of exogenous molecules across the cellmembrane through the use of external electric fields. Ifthe field the cell is subjected to is large enough, the cell'smembrane will porate, allowing molecular exchange withthe external environment. The field can be properly tunedso that these pores can then reseal. This technique islargely used for genetic transfection and fluorescenttagging.

Conventional electroporation techniques are,however, limited by either low throughput or limitedselectivity. Due to this, there has been increasing interestin creating a system capable of performing highthroughput electroporation with single cell selectivity foron-chip transfection and cell-monitoring studies [1-3]. Wehave recently reported on the use of OET to achieve light-induced electroporation [4]. As shown in Figure 1, in thisscheme, we use light-induced dielectrophoresis tomanipulate individual cells in parallel and, then, byincreasing the bias, we selectively electroporate theilluminated cells. By combing OET with electroporation,a high throughput, high selectivity assay can beperformed.

Optoelectronic tweezers uses pattemed light toalter the conductivity of a photosensitive film to createlocalized electric field gradients. These gradients result ina dielectrophoretic (DEP) force on particles in thevicinity. Figure 2 shows the electric field distributioncreated by illuminating a region of the photosensitivefilm. Note the concentration of electric field and creationof localized electric field gradients at the center of the x-axis where the 20 [tm light spot is incident. Because of thelow light power necessary for actuation, compared to themore traditional optical tweezers, thousands ofsimultaneous traps can be created and manipulated inparallel [5].

A~~~II ~~ITOElectric Fieldr > | l ll ll lI+ . ...X | Nano-t:agsp ~~~~~~~~CellI~~~~~~~~~

Figure 1: Experimental setup and schematic showingelectroporation mechanism. Electric field is concentratedacross the illuminated cell resulting in electroporation. Aseries of filters are used to select the appropriatefluorescent dye to monitor. Fluid exchange occursthrough the use ofa syringepump (not pictured).

4 Max: 5.002 5

1.8 4

1.7 3,5

E 1.6 3

1X2111111...1111111.11111111................1111!111111.2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......1.1 ............

-1-0.8-0.6-0,4 -0~~~~~~~~~~~~~~~~~~~~~~~~~~~................2.2..0.4...0.6...0.....1..0

Figure 2. Electric field profile created by illumination inthe OET device at center ofx-axis. Arrows correspond toelectric field, while the surface plot corresponds toelectric potential for a 5 V amplitude signal. Note howlocalized electric field gradients and electric fieldconcentration occurs in the illuminated region.

When a cell is illuminated by the projected lightin the OET device, the electric field is concentrated acrossit. If the electric field is large enough, the cell's membranewill form nanoscopic pores allowing exogenousmolecules to enter the cell (Figure 1). In this manner, one

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MI TM UV

ITO on Glass Pattern 50 pm NOA-68

SU-8 and StampingDice

WY CombineIII I IDevices and

ITO on Glass Deposit 1 pm UV Expose

PECVD a-Si:H

Figure 4: Fabrication ofOETwith integrated microfluidic channels. Channels are defined in SU-8 on the topside OETsubstrate and bonded to the bottom OET substrate using a UV-curable epoxy.

can individually select and electroporate cells in parallel.However, until now, the reversible

electroporation of cells has not been demonstrated in thisdevice. Reversible electroporation refers to the ability ofthe pores in an electroporated cell to reseal. This process

typically takes on the order of minutes to tens of minutes[6] and is highly dependent on the electric field doseapplied during the electroporation process. A common

method for the investigation of reversible electroporationis the use of fluorescent dyes [7]. First a cell iselectroporated in the presence of a dye which causes

successfully electroported cells to fluoresce. Next, thesolution surrounding the cells is replaced with a solutioncontaining another dye which indicates whether theelectroporated cell is viable. For cells not adhered to thesurface, this procedure requires the use of on-chipmicrofluidic channels. Here we present a process bywhich the OET device is integrated with lithographicallydefined channels and demonstrate its capability byshowing that reversible electroporation can be obtained.

,ion MediaInlet

Loading Channr

Electroporationi

Cell Culture<Uloading Channels

U-8 ChannelSidewalls

photosensitive bottom substrate. Additionally, in order toaccommodate cells, the channel height must be relativelythick (-50 pm) and the channel material must be non-

conductive. SU-8 lends itself well to this task due to itsability to easily form high-aspect ratio, permanent,structures. The process is depicted in Figure 4.

Indium tin oxide (ITO) (300 nm) coated glassserves as the top and bottom OET surface. The bottomsubstrate is coated with a 1 ptm layer of PECVD a-Si:H(100 sccm 10%SiH4:Ar, 400 sccm Ar, 900 mTorr, 350°C,200 W). The top substrate is patterned with 50 pm of SU-8 (Microchem, SU-8 2050) which defines the channelgeometry. Both substrates are then diced into 2x2 cm

chips using a dicing saw (Esec 8003). Fluidic input andoutput ports are then drilled into the topside device with a

drill press and 0.75 mm drillbit.A 20 pm layer of UV-curable epoxy (Norland,

OA-68) is then spin coated (4000 RPM, 2 min.) on a

dummy wafer. A block of polydimethlysiloxane is used tostamp the NOA-68 from the dummy wafer to the SU-8channels. This results in a -15 pm layer of NOA-68 on

the top of the SU-8 channel sidewalls. Finally, the top andbottom OET substrates are combined and the epoxy iscured using a handheld UV gun (Norland, Opticure-4,10sec). No alignment is needed in this case as the bottomsubstrate is featureless. The combination of the twosubstrates can be accomplished by hand or through the use

of a flip-chip bonder. Fluidic access connections are thenattached to the ports on the topside of the device withadditional NOA-68.

Figure 3: Concept overview of OET-basedelectroporation assay. Cells are electroporated in parallelwith single cell selectivity in lithographically definedcompartments and then moved into perfusion chambersallowingfor subsequent cell culture.

FABRICATIONThe integration of microfluidic circuits with the

OET device will allow for applications ranging from on-

chip sorting to on-chip cell culture (Figure 3). We havedeveloped a process that allows lithographically definedchannels to be integrated with the OET electroporationdevice. This process needs to allow for arbitrary top andbottom substrates. This is because the OET devicerequires a transparent conductive top surface and a

Figure 5. SEM of cross section of OET device withintegrated SU-8 microfluidic channels.

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Figure 5 shows an SEM cross-section of thedescribed device showing the top and bottom substrates,SU-8, a-Si:E, and NOA-68. Figure 6 is a picture of thecompleted device.

Figure 6: Picture of completed device. The top andbottom OET substrates are bonded together using a UVcurable epoxy resulting in sealed micro/luidic channels.Fluids are introduced through fluidic ports.

EXPERIMENTALFor many applications (e.g. transfection), the cell

must remain viable following electroporation (i.e.reversible electroporation). For a demonstration of theintegrated device, we assess the viability of a single cellfollowing light-induced electroporation. In order to showthis, we use the technique mentioned before using twocellular dyes. The first dye, Propidium Iodide (PI)(Invitrogen), is a membrane impermeant dye. However, ifthe dye enters the cell, it binds to DNA and fluoresces red.It is an indicator of successful electroporation. The seconddye, Calcein AM (CaAM) (Invitrogen), passively diffusesacross the cell membrane. Once in the cytosol, it interactswith enzymes in the cell and produces a green fluorescentmolecule which is membrane impermeant. Strong greenfluorescence in the presence of CaAM means that thecell's membrane is intact and that the intracellularcontents contain the necessary enzymes to catabolize theCaAM. If these two conditions are met, it is a strongindicator of cellular viability.

Since CaAM is membrane permient it isnecessary to introduce it following electroporation,necessitating the use of microfluidic channels. Todemonstrate successful reversible electroporation, we firstselect a cell, electroporate it in the presence of PI, andthen exchange the media with a solution containingCaAM. If the cell fluoresces both red and green at thecompletion of the experiment, the cell has been reversiblyelectroporated.

HeLa cells were washed three times in andsuspended in electroporation buffer (CytopulselCytoporation Medium, 10 mS/m) at a cellularconcentration of 2xl06/mL. Due to the presence of largeelectrical fields during electroporation (kV/cm), joule

heating in the liquid is an issue. Small temperaturechanges can reduce cellular viability significantly. Sincejoule heating is directly proportional to liquidconductivity, it is advantageous to use lowly conductingmedia.

PI dye was added to the cellular solution toachieve a concentration of 2 1M. CaAM dye at 6 tM wasalso prepared to be introduced later. The cell and PIsolution was then introduced into the device via a syringepump. The CaAM solution was also connected to thedevice to be introduced later.

Figure 7: Bright-field image of HeLa cell under test inOET device with applied optical pattern. Cells aresuspended in Cytopulseo® Electroporation media (8 mS In).

An individual HeLa cell is selected and a lightpattern is generated to illuminate it (Figure 7). After initialpositioning at low voltage (4 Vppk), the cell does notexhibit any fluorescence (Figure 8). This is because thefields the cell experiences during movement are less thanthat required to porate the cell membrane. However, oncethe electroporation bias is applied (20 Vppk), the criticalfield required to porate the membrane is surpassed and thecell uptakes P1 and fluoresces accordingly. No CaAMfluorescence is seen at this stage as this dye is notcurrently present in the solution (Figure 8). Next themedia surrounding the cells is exchanged, via the fluidicchannels using a syringe pump, with the CaAM solution.After fluid exchange, the cells are incubated for 15minutes to allow the CaAM to diffuse into the cells. Afterincubation, the cell now exhibits both PI and CaAMuptake (Figure 8). This means that the cell's membranehas resealed following electroporation and that it containsthe appropriate enzymes necessary to catabolize theCaAM into its fluorescent derivative. Therefore,successful reversible electroporation has occurred.

CONCLUSIONWe have demonstrated the integration of

microfluidic chambers onto the OET device todemonstrate reversible light-induced electroporation. Thisprocess can be extended to allow for on-chip gene

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After Movement4Vppk I

After Poration

20 Vppk

After Media

Exchange

Figure 8: Fluorescent response ofcell in Figure 7for both PI and CaAM Initially, the cell is suspended in a solution onlycontaining P1 (6pM). In the first panel, no dye uptake is observedfollowing positioning of the cell with OET at 4 Vppk. Inthe secondpanel, the cell is subjected to the electroporation bias resulting in P1 dye uptake. In the thirdpanel, the media isexchanged using microfluidic channels with a solution containing CaAM (24M). The cell now exhibits CaAM and P1response verifying successful reversible electroporation.

transfection and cell culture, eventually leading to a highthroughput electroporation assay with single cellselectivity.

ACKNOWLEDGEMENTSThe authors would like to thank Ann Fischer of

the UC Berkeley Cell Culture Facility for providing thecells. This work was funded by the Center for CellControl, a National Institute of Health NanomedicineDevelopment Center, Grant # PN2 EY0 18228.

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[4] J. K. Valley, H. Y. Hsu, A. T. Ohta, S. Neale, A.Jamshidi, and M. C. Wu, "In-situ Single CellElectroporation Using Optoelectronic Tweezers,"in IEEEILEOS Optical MEMS, Freiburg,Germany, 2008.

[5] P. Y. Chiou, A. T. Ohta, and M. C. Wu,"Massively parallel manipulation of single cellsand microparticles using optical images," Nature,vol. 436, pp. 370-372, Jul 21 2005.

[6] S. W. Hui, "Effects of Pulse Length and Strengthon Electroporation Efficiency," in Animal CellElectroporation and Electrofusion Protocols.vol. 48, J. A. Nickoloff, Ed.: Humana Press,1995, pp. 29-40.

[7] J. S. Soughayer, T. Krasieva, S. C. Jacobson, J.M. Ramsey, B. J. Tromberg, and N. L.Allbritton, "Characterization of CellularOptoporation with Distance," Anal. Chem., vol.72, pp. 1342-1347, 2000.

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