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Optoelectrofluidic printing system for fabricating hydrogel sheets with on-demand patterned

cells and microparticles

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2017 Biofabrication 9 015011

(http://iopscience.iop.org/1758-5090/9/1/015011)

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Biofabrication 9 (2017) 015011 doi:10.1088/1758-5090/aa564c

PAPER

Optoelectrofluidic printing system for fabricating hydrogel sheetswith on-demand patterned cells andmicroparticles

Hyun JiGi, DongsikHan and Je-KyunParkDepartment of Bio andBrain Engineering, Korea Advanced Institute of Science andTechnology (KAIST), 291Daehak-ro, Yuseong-gu,Daejeon 34141, Republic of Korea

E-mail: jekyun@kaist.ac.kr

Keywords: dielectrophoresis, hydrogel sheet, on-demand patterning, optoelectrofluidics

Supplementarymaterial for this article is available online

AbstractThis paper presents a novel optoelectrofluidic printing system that facilitates not only theoptoelectrofluidic patterning ofmicroparticles andmammalian cells but also the harvesting of thepatternedmicroparticles encapsulatedwithin poly(ethylene glycol) dicarylate (PEGDA)hydrogelsheets. Although optoelectrofluidic technology has numerous advantages for programmable and on-demand patterning and the feasibility ofmanipulating singlemicroparticles, practical applicationsusing existing laboratory infrastructure in biological and clinical researchfields have been strictlyrestricted due to the impossibility of recovering thefinal patterned product. In order to address theseharvesting limitations, PEGDAwas employed to utilize optoelectrofluidic printing. TheClausius–Mossotti factor was calculated to investigate the dielectrophoreticmobility of themicroparticle andthe cell in the PEGDAprecursor solution. As a proof of concept, three basic controllabilities of theoptoelectrofluidic printing systemwere characterized: the number ofmicroparticles, the distancebetween themicroparticle columns, and the ratio of two differentmicroparticles. Furthermore, theoptoelectrofluidic patterning and printing of human liver carcinoma cells (HepG2)were demon-strated in 5minwith a single-cell level of resolution. The appropriate ranges of frequencywereexperimentally defined based on the calculated result of the dielectrophoreticmobility ofHepG2 cells.Finally, optoelectrofluidically cell-patterned hydrogel sheets were successfully recovered under ahighly viable physiological condition.

1. Introduction

A number of significant issues in manipulation (e.g.,transportation, trapping, concentration, sorting, andre-orientation) ofmultiplemicroparticles and biologi-cal objects have been made in the biological andclinical field because of the increasing needs forversatile and precise arrangement of microparticlesinto desired patterns: cell migration study [1] andcreation of in vivo-like cellular arrangements [2]. Todate, a variety of patterning technologies have beendeveloped in many biological research fields such asdrug screening [3, 4], tissue engineering [5, 6], micro-array [7, 8], and cell-based assays [9, 10]. To addresssuch patterning applications, many microfluidic tech-niques based on various forces such as mechanical[11], electrical [12], optical [13], and magnetic [14]

forces have been introduced. However, theseapproaches have disadvantages in practical applica-tions that require immobilization of patterned micro-particles and cells on the substrate surface or solidmatrices, and recover them to transfer from micro-device tomacroworld for further post processing.

Hydrogels have been widely used to immobilizethe patterned microparticles in biological applicationsdue to their biocompatibility [15, 16]. Moreover,hydrogels show a strong similarity in physicochemicaland mechanical characteristics of native extracellularmatrix structures. A simple immobilization procedurehas been employed for hydrogel-based cell encapsula-tion with high optical transparency [16]. To date, var-ious hydrogel-based patterning technologies havebeen reported. Micromolding, known as the mostgeneral patterning technique, employs a

RECEIVED

13 July 2016

REVISED

16December 2016

ACCEPTED FOR PUBLICATION

3 January 2017

PUBLISHED

13 January 2017

© 2017 IOPPublishing Ltd

lithographically fabricated plastic mold that providesphysical barriers where the target particles are posi-tioned [17, 18]. However, critical problems, includingnot only the requirement of a complicated fabricationprocess but also the limitation of the precise control ofsingle microparticles, have remained significant chal-lenges in biological fields. Electrokinetics has also beenemployed in patterning microparticles using themicrofabricated electrodes [12, 19]. However,although microparticles could be precisely patternedbased on the electrokinetic phenomena, this methodstill requires complex fabrication processes and lacksflexibility. On the other hand, optoelectrofluidics,which is based on the electrokinetic motion of parti-cles and fluids under a nonuniform electric fieldinduced by light, has been explored to overcome thelimitation of conventional microparticle patterningplatforms [20–22]. Recently, this optoelectrofluidicmanipulation technique has been applied to manip-ulate various biological materials such as blood cells[23], oocytes [24], biomolecules [25], and colloidalparticles [26]. However, the practical applications ofon-demand patterned microparticles by an optoelec-trofluidic manner have been restricted due to theimpossibility of harvesting the final patterned productto integrate with common equipment in biologicallaboratories.

Here, we propose an optoelectrofluidic printingsystemwith patternedmicroparticles andmammalianliver cells utilizing a photopolymerizable hydrogel toovercome the limitations of optoelectrofluidic manip-ulation with respect to the harvesting issue. By calcu-lating the Clausius–Mossotti (CM) factor, behaviors oftarget materials under the optoelectrofluidic environ-ment were mathematically estimated. Based on theseresults, different types of microparticle were ondemand manipulated within the hydrogel precursorsolution by optically-induced dielectrophoresis (DEP)with the selected total number and the desired ratiousing a programed image from a conventional liquidcrystal display (LCD) module. Furthermore, wedemonstrate a successful harvest of patterned hydro-gel sheets with microparticles and cells from theoptoelectrofluidic printing system for further study inbiologicalfields.

2.Materials andmethods

2.1. Cell cultureHuman liver carcinoma cells (HepG2) were used foroptoelectrofluidic cell printing. HepG2 cells werecultured in Dulbecco’s modified Eagle’s medium(DMEM; Gibco, Grand Island, NY) including fetalbovine serum (FBS, 10% v/v) and penicillin–strepto-mycin (1% v/v), and incubated at 37 °C in 5% CO2 ina humidified water-jacketed incubator. The HepG2cells were split into two every four days and the

medium was exchanged every two days after washingwith phosphate-buffered saline (PBS).

2.2. Preparation of hydrogel precursorFor developing an optoelectrofluidic printing system,poly(ethylene glycol) diacrylate (PEGDA) Mw 700(Sigma-Aldrich Co., St Louis, MO) was used forsuspending medium with Irgacure 1173 (2-hydroxy-2-methyl-propiophenone; Sigma-Aldrich) as a photo-initiator. The PEGDA Mw 700 was diluted withdistilled water (ddH2O) for preparing the desiredconcentration. The photoinitiator was added to thedesired concentration of hydrogel precursor to form5% (w/v) of photoinitiator. Three types of polystyrenebeads were used; Polybead carboxylate 10.0 micronmicrospheres (Polyscience, Inc., Warrington, PA),FluoresbriteTM Plain YG 20.0 micron microspheres(Polyscience), and FluoSpheres polystyrene (10 μm,red; Life technologies). For cell patterning, the PEGDAMw 2000 (Sigma-Aldrich) with Irgacure 2959 (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one; BASF)was utilized as a photoinitiator.The PEGDAMw2000 was dissolved in the DEP bufferfor a suitable concentration in cell patterning. Thephotoinitiator was dissolved in dimethyl sulfoxide(DMSO; Sigma-Aldrich) to prepare a 10% (w/v) stocksolution. After the PEGDA precursor was diluted bythe DEP buffer to make a specific concentration, thephotoinitiator was added to prepare a desired concen-tration of photoinitiator. TheDEP buffer wasmade upof 8.5% sucrose (Sigma-Aldrich) and 0.3% dextrose(Sigma-Aldrich)dissolved in ddH2O.

2.3.Optoelectrofluidic printing device andexperimental systemAn optoelectrofluidic printing device consists of twoelectrodes: a bare indium tin oxide (ITO)-coated glassand a-Si: H photoconductive layer-deposited ITOglass [23]. The distance between two electrodes wasdetermined by the gap spacer which was fabricated onthe bare ITO wafer (Samsung-Corning PrecisionGlass, Korea) with photoresist (PR) using conven-tional UV photolithography. The optoelectrofluidicdevices with a height of 60 and 80 μm were used forprinting microparticles and cells, respectively. Theprinting device was placed on an upright microscope(Zeiss Axioskop 40; Carl Zeiss, Germany) integratedwith an LCD module (LCX034CNB8; SONY, Japan).The light passed through the LCD module in whichpatterns were programmably controlled by a comp-uter software (Microsoft PowerPointTM) was focusedon the photoconductive layer of the device by acondenser lens. Subsequently, the electric field gener-ated froma function generator (AFG-2005;GWInstek,Taiwan) can be applied to the two electrodes of theprinting chip. The microscopic images were thencaptured by a charge-coupled device camera (DS-U1;Nikon Co., Japan) and they were recorded by a

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Biofabrication 9 (2017) 015011 H JGi et al

commercialized image processing software (NIS-Ele-ments; Nikon Instruments Inc.). The fluorescentimages of final printed hydrogel sheets were capturedby a fluorescence microscope (IX72; Olympus, Japan).Anultraviolet (UV) source (SUV-2010-S; SMT;Korea)with a wavelength of 365 nm was used for thepolymerization of the PEGDA precursor encapsulat-ing the patternedmicro-sizedmaterials.

2.4.Optoelectrofluidic printing of on-demandhydrogel sheetThe proposed optoelectrofluidic printing system pro-ceeds in the following steps: injection of liquid sample,on-demand optoelectrofluidic manipulation ofmicroparticles, UV exposure to polymerize the hydro-gel precursors, and harvesting the microparticle-patterned hydrogel sheets (figure 1). 2.5 μl of liquidsample containing microparticles suspended in thePEGDA hydrogel precursor is loaded into the optoe-lectrofluidic device. After loading the liquid sample,spacer-fabricated ITO glass and a-Si:H deposited ITOglass are assembled asfigure 1(A). Subsequently, a lightpattern passed through an LCD is illuminated to thephotoconductive layer, which forms a virtual electrodedue to the decrease in its resistance. An AC voltage isapplied to two electrodes, which induces a non-uniform electric field in the liquid sample(figure 1(B)). The DEP force is exerted to microparti-cles to locate them to the desired location in aprogrammable manner, which can make on-demandpatterned microparticles (figure 1(E)). The negativeDEP force which makes microparticles move towardthe region of the lower electric field is used for printingmicrobeads and cells in this study due to the

polarizability of target materials and medium. Then,UV is used to polymerize the hydrogel precursor toharvest hydrogel sheets with encapsulating printedmicroparticles (figure 1(C)). After disassembling theITOglass of optoelectrofluidic device, the polymerizedhydrogel sheets are immersed in ddH2O or PBS for5 min to harvest intact hydrogel sheets. Finally, we canharvest on-demand microparticle-patterned hydrogelsheets using tweezers or cell lifter (figure 1(D)).

2.5. Cell viability testAfter printing hydrogel sheets encapsulating HepG2cells, cell viability was measured by live/dead cellstaining. The hydrogel sheets were immersed in thesolution with 4 μM calcein-AM and 2 μM ethidiumhomodimer-1 for 5 min. The microscopic and fluor-escent images of printed hydrogel sheets with pat-terned cells were captured by a fluorescencemicroscope (IX72) and were analyzed by ImageJ(https://imagej.nih.gov/ij/) to calculate cell viability.

3. Results and discussion

3.1.Optoelectrofluidic patterning ofmicroparticlesThe dielectrophoretic force exerted on the dielectricparticles is described as

[ ]p e= F r f E2 Re ,DEP3

m CM2

where r is the radius of particles; εm is the permittivityofmedia; E is the local electric field; and Re [fCM] is thereal part of the CM factor. The CM factor is the valueof indicating the direction of the DEP force and isdescribed as

Figure 1. Fabrication of on-demand hydrogel sheets using an optoelectrofluidic system. (A)–(D) Schematic illustration ofoptoelectrofluidic printing processes. (E)Adetailed process of optoelectrofluidicmanipulation in panel (B). Schematic side views ofan optoelectrofluidic device showing an on-demand patterning process ofmicroparticles. A light pattern passed through an LCD isilluminated to the photoconductive layer, which induces a non-uniform electric field under the applied potential. The optically-inducedDEP force is exerted tomicroparticles andmoves them to the desired location in a programmablemanner.

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Biofabrication 9 (2017) 015011 H JGi et al

* *

* *

e e

e e=

-

+f

2,CM

p m

p m

where *ep and *em are the complex permittivities ofthe particle and the medium, respectively, andε*=ε−(σ/ω) j, where ε is the permittivity; σ is theconductivity;ω is the angular frequency (=2πf, where fis the applied frequency); and = -j 1 .The real partof the CM factor was calculated to predict the move-ment of the microparticles within the PEGDA pre-cursor. The relative permittivity of the PEGDAprecursor is varied according to its proportion. Thepermittivity of heterogeneous mixtures of two differ-entmaterials is described as

e e e e= - +⎡⎣⎢

⎛⎝⎜

⎞⎠⎟

⎤⎦⎥v ,2

13

1

13

2 1

13

3

where ε1 and ε2 are the relative permittivities of eachmaterial, respectively, and v2 is the volume fraction ofthe second material [27]. The relative permittivity of25%PEGDAprecursorwas calculated by this equationusing the relative permittivities of ddH2O and PEGDAwhich are 78 and 10, respectively [28]. The relativepermittivity and conductivity of polystyrene micro-particles are 2.55 and 2.3×10−4 S m−1. The real partof the CM factor had negative values under thefrequency range from 1 kHz to 100MHz at theexperimental condition σm=4.3×10−4 S m−1

(figure S1(A)). In this condition, the microparticlesexhibited negative DEP within the concentration ofthe PEGDA precursor since the polarizability para-meter has a negative value. This DEP force could beutilized to manipulate microparticles by an opticalmanner.

Based on the manipulable condition of the optoe-lectrofluidic system, two significant abilities of theoptoelectrofluidic printing system were characterized:controlling the number of microparticles and control-ling distance between microparticle columns. First,the number of concentrated microparticles within acircular region was controlled via dynamic right-typedlight patterns. Since microparticles were repelled fromthe irradiated region under this condition, micro-particles could be concentrated at the desired region asthe dynamic light pattern was gradually shrunk fromthe initial diameter of light pattern (Di) to the final dia-meter (Df) (figure 2(A)). Herein, the number of con-centrated microparticles could be controlled by theinitial concentration of the microparticles and theinitial diameter of the light patterns. Hence, we ana-lyzed optoelectrofluidic concentration of micro-particles according to the initial concentration of themicroparticles and initial diameter of the circular lightpatterns. These experiments were conducted in thesame condition of the simulation as 20 Vpeak-to-peak

bias at 20 kHz with an identical final diameter (Df) ofthe light pattern, 400 μm. Three initial concentrationsof microparticles were used: 1.0×107, 5.0×106,and 2.5×106 ml−1. As shown in figures 2(B) and (C),the experimental results showed that the number of

concentrated microparticles was determined by boththe initial diameter of the light pattern and the initialconcentration of the microparticles. The larger initialdiameter of the light patterns could concentrate morenumbers of microparticles into the designated region.In addition, more microparticles could be gatheredwith the higher initial concentration of the micro-particles. In other words, we have verified the possibi-lity of controlling the number of microparticles in thisprinting systemwith a designated concentration of themicroparticles and initial diameter of the light pattern.

Furthermore, the distance between concentratedgroups could be adjusted by the light patterns. Whenstripe-shaped light pattern made columnar groups ofmicroparticles, the width of a single stripe determinesthe distance between the groups of microparticles.Figure 2(D) illustrates that microparticle columnswere arranged by repelling from the stripe-patternedlight due to the negative DEP force. As shown infigures 2(E) and (F), the experimental results showedthat the gap between the microparticle columns (dc;dca<dcb) could be decided by the width of the lightpatterns (dl; dla<dlb). Accordingly, the arrangementofmanipulated groups could be precisely controlled inthe optoelectrofluidic printing system. Therefore, wehave verified that this printing system could controlthe number of microparticles and the distancebetween microparticle groups in 1 min (figure S2(A)and supporting video 1).

3.2.Optoelectrofluidic printing of on-demandpatterned hydrogel sheetwithmicroparticlesIn biological fields, the control of a heterogeneousmixture of several types of micro-sized materials hasbeen emphasized for various applications, such asmultiple bead-based assays and co-culture of cells. Inthis optoelectrofluidic printing system, different typesof microparticles could be manipulated and concen-trated with the selected total number and the desiredratio of the particles. Each microparticle could betransferred toward a designated location or removedfrom the location. Figure 3(A) illustrates that differentmicroparticles were manipulated by ring-shaped lightpatterns toward a designated location (dashed circle)with a desired ratio of two different microparticles.After locating microparticles into a designated loca-tion, a ring-shaped light pattern covering all targetedmicroparticles was projected and gradually shrunk toconcentrate them. As a result, different microparticlescould be located and concentrated into a local regionwith the targeted ratio. Furthermore, we demonstratedthat the desired ratio of two different microparticlescould be controlled with a constant total number of 10infigure 3(B).

Meanwhile, since the patterning of complicatedstructures has been required for biological studiessuch as drug assay on patterned sheets and tissuereconstruction, on-demand arrangements of

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Biofabrication 9 (2017) 015011 H JGi et al

microparticle groups within hydrogel sheets weredemonstrated by combining the presented functionsof the optoelectrofluidic system for manipulatingmicroparticles. First, serially different numbers ofmicroparticles with columnar shapes were patternedby controlling the initial width of rectangular light pat-terns with an identical final size. Simultaneously, thedistance between microparticle groups can also beadjusted by the dynamic light patterns. As a result, dif-ferent numbers of microparticle columns at regularintervals were serially located in figure 4(A). Addition-ally, circular shaped groups with controlling both theratio of different microparticles and the total numbersof microparticles were patterned in a 2×3 arraybased on the previously presented method(figure 4(B)). We not only generated the spots,

including the fixed ratio and the total numbers ofmicroparticles, but also controlled the distance of thesix spots to confine the targeted microparticles. Sincethese controllabilities facilitate the easy fabrication ofon-demand patterned hydrogel sheets, the optoelec-trofluidic printing system is considered to be appliedin various biological studies such as cell-to-cell inter-actions [29] and co-culture of cells [30]. Furthermore,on-demand patterned hydrogel sheets are also advan-tageous for applying biological studies such as tissueengineering and cellular engineering.

3.3.Optoelectrofluidic printing of on-demandpatterned hydrogel sheetswithmammalian cellsFor the manipulation of cells, the real part of the CMfactor was calculated to predict the movement of cells

Figure 2.Controlling the number ofmicroparticles and the distance betweenmicroparticle columns based on dynamic light patterns.(A) Schematic illustration of themanipulation ofmicroparticles by shrinking circular light patterns. (B)Microscopic images ofmanipulatedmicroparticles by adjusting different initial diameters of light patternswith afixedfinal diameter of 400 μm (scale bar:100 μm). (C)The graph shows the number ofmicroparticles according to the different initial size of the light patterns. (D) Schematicillustration of the arrangement ofmicroparticle columnswith stripe-patterned light. The distance between themicroparticle columns(dc; dca<dcb) is controlled by thewidth of the light patterns (dl; dla<dlb). (E)Microscopic images ofmicroparticle columnswithselected distances of light patterns (scale bar: 100 μm). (F)The graph shows the distance betweenmicroparticle columns according todifferent widths of striped light patterns.

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Biofabrication 9 (2017) 015011 H JGi et al

Figure 3. Interactivemanipulation of two types ofmicroparticles to form a desired concentration ratio using two different-sizedmicroparticles. (A) Schematic illustration of themanipulation of single particles to form a desired ratio of two differentmicroparticlesat a desired location (dashed circle). Thick circular light pattern is illuminated and shrinking to concentrate the desired ratio ofmicroparticles. (B)Microscopic and fluorescent images for controlling the ratio of two differentmicroparticles with a constant totalnumber of 10 (scale bar: 100 μm). The diameters of themicroparticles are 20 μm (green) and 10 μm (red), respectively.

Figure 4.Microscopic and fluorescent images showing two different applications ofmicroparticlemanipulationwith dynamic lightpatterns. (A) Serial arrangement of different numbers ofmicroparticles. (B)Array-patternedmicroparticles with controlling the ratioof the differentmicroparticles (vertical) and the total numbers (horizontal) (scale bar: 100 μm).

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Biofabrication 9 (2017) 015011 H JGi et al

within the optimized hydrogel precursor. First, therelative permittivity of the mammalian cells wascalculated using a single-shell model. The single-shellmodel is the simplest and most widely used modelamong them because the mammalian cell can usuallybe modeled electrically as a shelled particle [31, 32].The permittivity and conductivity of cell membraneand cytoplasm should be considered, respectively, inthis model. The effective complex permittivity of thecells can be expressed as

( )*e

wtw t t

=+

+ +

⎡⎣⎢

⎤⎦⎥C a

j

j

1

1,eff m

cp

m cp

where Cm is the membrane capacitance, τcp (=εcp/σcp) and τm (=Cm·r/σcp) is the relaxation time ofcytoplasm and media, r is the radius of cells, εcp andσcp are the permittivity and conductivity of cytoplasm,respectively [32]. Constants which were used in thissimulation are as follows [31–33]: the relative permit-tivities of DEP buffer and PEGDA are 80 and 10,respectively [28, 34]. The membrane capacitance is0.0015 F m−2, the radius of the liver cells is 6 μm, therelative permittivity and conductivity of the cytoplasm

is 50 and 0.4 S m−1, respectively. At the frequencyrange below 100 kHz, the real part of the CM factor isalmost−0.5, which means that a strong negative DEPforce is exerted to cells in 20%PEGDA (figure S1(B)).

Finally, HepG2 cells in the PEGDA precursor werepatterned with circular and hexagonal shape under themanipulable conditions using the optically inducednegative DEP. A number of HepG2 cells were repelledfrom the bright area projected through the LCD form-ing a circular and a hexagonal shape under a voltage of20 V at 20 kHz. At first, no voltage was applied and theHepG2 cells were randomly distributed regardless ofthe projected image, but when the voltage was appliedand the projected image was shrunk or enlarged, theHepG2 cells were arranged in the opposite direction ofthe electric field in 5 min (figures 5(A), S2(B), and sup-porting video 2). Additionally, the viability of encap-sulated HepG2 cells within a hydrogel sheet wasverified after harvesting from the optoelectrofluidicdevice. Figure 5(B) shows the fluorescence images ofhydrogel sheets containing patterned live HepG2 cells,which emit bright green fluorescence due to calceinAM staining. Some of dead HepG2 cells stained by

Figure 5.On-demand patterned hydrogel sheets withmammalian cells. (A)Microscopic images of patterned cells with circular andhexagonal type of dynamic light patterns. (B) Fluorescent images for verifying cell viability in the harvested hydrogel sheet. The livecells were stained green by calcein-AM and the dead cells were stained red by EthD-1 (scale bars: 100 μm).

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Biofabrication 9 (2017) 015011 H JGi et al

EthD-1 were observed because of free radicals. Theviability of the cells within the PEGDA hydrogel sheetcould be further improved by lowering the concentra-tion of the photoinitiator (figure S3) [35, 36].

4. Conclusions

This paper presented a novel optoelectrofluidic print-ing system for harvesting the patterned microparticlesencapsulated within PEGDA hydrogel sheets. On-demand arrangements of microparticle groups withina hydrogel sheet were demonstrated by combiningbasic functions of the optoelectrofluidic system.Although optoelectrofluidic technologies haveattracted interest in the patterning of microparticlesdue to their on-demand and programmable manip-ulationwith a high resolution, practical applications ofpatterned microparticles have been limited due to theimpossibility of harvesting. This optoelectrofluidicprinting system facilitates not only the on-demandpatterning but also the harvesting of the patternedhydrogel sheets for further research. The basic abilitiesof this system for cell patterning can be utilized:in vivo-like tissue reconstruction and the study of cell-to-cell interactions with changing the ratio of severaldifferent cells.We believe this system could be usefullyemployed for many biological applications by match-less on-demand patterning techniques.

Acknowledgments

This research was supported by a Mid-CareerResearcher Program (NRF-2016R1A2B3015986) anda Bio & Medical Technology Development Program(NRF-2015M3A9B3028685) through the NationalResearch Foundation of Korea funded by the Ministryof Science, ICT and Future Planning. The authors alsoacknowledge aKAIST SystemsHealthcare Program.

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