Biomaterials 25 (2004) 2047–2053

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*Correspondin

6046.

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doi:10.1016/j.bio

Cell micropatterning using photopolymerization with a liquid crystaldevice commercial projector

Kazuyoshi Itoga, Masayuki Yamato, Jun Kobayashi, Akihiko Kikuchi, Teruo Okano*

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-Cho, Shinjuku-ku, Tokyo 162-8666, Japan

Received 19 July 2003; accepted 15 August 2003

Abstract

Photopolymerization has been widely used for surface micropatterning. The technique often requires photomasks and light

sources with appropriate energies or filters. For rapid prototyping of surface photo-micropatterning, we have developed a novel

device by modifying a commercially available liquid crystal device projector. In place of the image expansion unit of the projector,

we attached an image reduction unit, an adjustable stage, and an optical monitoring unit. The device projected computer-generated

images onto surfaces and subjected these patterns to photopolymerization. Micropatterned images can be easily prepared with

various software run on personal computers. With the developed photopolymerization device, micropatterning of poly(ethylene

glycol) (PEG) was achieved with PEG-diacrylate and a visible light photopolymerization initiator, camphorquinone. Selective cell

adhesion control was also achieved on the micropatterned surfaces.

r 2003 Elsevier Ltd. All rights reserved.

Keywords: PEG; LCD projector; Cell patterning; Cell culture; Photopolymerization

1. Introduction

Surface micropatterning has been widely investigatedfor a wide range of biomedical applications from singlecell manipulations in basic cell biology [1,2] to complexmulti-patterned biochips [3]. Various methods have beenexploited to produce reliable patterns, including micro-contact printing [4,5], laser ablation [6,7], photolitho-graphy [8–11], inkjet printing [12–14], and microscaleself-assembly [15]. Except for inkjet printing and self-assembly, patterning has generally utilized photomasksand energy sources such as light and electron beams [16].Preparation of photomasks is often time-consuming andexpensive. Qin et al. showed the utilization of highquality laser printers and commercial transparencysheets for over-head projectors to prepare qualityphotomasks [17]. However, high resolution requires anexpensive high-end laser printer. In addition, adequatelight sources and optics are requisite for precisemicropatterning.

g author. Tel.: +81-3-3353-8111; fax: +81-3-3359-

s: tokano@abmes.twmu.ac.jp (T. Okano).

front matter r 2003 Elsevier Ltd. All rights reserved.

materials.2003.08.052

In order to overcome these shortcomings, we devel-oped a novel device by modifying a commerciallyavailable liquid crystal device projector (LCDP) forrapid photo-prototyping of micropatterned surfaces.Recent innovations have realized large-scale liquidcrystal panels with a high density and micrometer scaleresolution. A typical pixel size is approximately 20 mm.Such resolution can be useful for micropatterning inseveral biomedical fields, particularly those associatedwith cell patterning and arraying technologies. Further-more, LCDP has a well-controlled light source. Projec-tion images can be easily generated by a personalcomputer (PC) without any special software. Here wedescribe cell patterning on surfaces micropatterned withthis new device.

2. Materials and methods

2.1. Materials

Poly(ethylene glycol) (PEG)-diacrylate (MW ¼ 1000)was kindly provided by NOF Co. (Tokyo, Japan).

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3-methacryloxypropyltrimethoxysilane (MPTMS, Shin-Etsu Chemical Co., Tokyo, Japan), anhydrous methanol(Kanto Chemical Co., Tokyo, Japan), (7)-camphor-quinone, N,N-dimethyl-p-toluidine, anhydrous 1,4-di-oxane (Wako Pure Chemical Industries, Ltd., Osaka,Japan), and all other chemicals were used as received.An LCDP (LP-SG7) was kindly provided by SANYOElectric Co. (Osaka, Japan). A objective lens havinglong working distance (M Plan Apo, 2� , N.A. 0.055)and a tube lens (MT-40) were purchased from MitutoyoCo. (Kanagawa, Japan).

2.2. Functionalization of glass surfaces

Both sides of cover glass slips (24� 50mm, 0.2mm inthickness, Matsunami Glass Inc., Japan) were treated byoxygen plasma (irradiation intensity: 400W, oxygenpressure: 0.1mmHg) for 180 s in a plasma dry cleaner(PX-1000, SAMCO International, Kyoto, Japan) toclean the surfaces. Plasma-treated coverslips wereinstalled in a separable flask, and dried under vacuumfor 30min. Ten milliliters of MPTMS and 500ml ofanhydrous methanol were poured into the flask undernitrogen gas. The coupling reaction of MPTMS withclean, dry coverslip surfaces proceeded under reflux for24 h at 60�C. The modified coverslips were rinsedrepeatedly with methanol and distilled water, and driedfor 24 h at 70�C.

2.3. Surface patterning of PEG derivatives by

photopolymerization

Surface patterning of PEG derivatives on the cover-slip was performed by irradiation of visible lightthrough patterned images on the liquid crystal panel.A typical preparation procedure follows: PEG-diacry-late (0.60 g), camphorquinone (20mg) as a photopoly-merization initiator [18,19], and N,N-dimethyl-p-toluidine (2.0 ml) as photosensitizer were dissolved in0.80 g of 1,4-dioxane. The solution (4.0 ml) was droppedon the MPTMS-immobilized coverslip. This solutionwas then covered with an untreated coverslip(24� 24mm), creating a liquid film spread uniformlybetween the coverslips. The approximate thickness ofthe solution between coverslips is B7 mm. The set ofcoverslips was placed in the LCDP apparatus where theMPTMS-immobilized coverslip was the front sidetoward the light source. Visible light irradiation wasperformed directly through the top of the MPTMS-immobilized coverslip for 20min. After irradiation, theuntreated coverslip was stripped off from the micro-fabricated PEG immobilized on the MPTMS-immobi-lized coverslip. The resulting PEG-micropatternedcoverslip was repeatedly washed with acetone andfurther characterized.

2.4. Characterization of LCDP photo-patterned surfaces

Micropatterned surfaces were examined under aphase contrast microscope (ET-300, Nikon, Tokyo,Japan) and a scanning electron microscope (S-800,Hitachi, Tokyo, Japan). For electron microscopy,micropatterned surfaces were deposited by a thinconducting gold sputtered layer. Three-dimensionalprofiles of the surfaces were obtained by reflectiveconfocal laser scanning microscopy (ICM-1000, LeicaMicrosystems, Wetzlar, Germany) and Tapping modes

atomic force microscopy (AFM) (Nano Scope IIIa,Digital Instruments, Santa Barbara, CA). Micropat-terned surfaces were stained with a hydrophobicfluorescent dye, DiIC18 (Molecular Probes, Eugene,OR), at concentration of 25 mg/ml for 20min, andwashed with distilled water. Fluorescence images onDiIC18-stained micropatterned surfaces were obtainedwith a microscope equipped with an epifluorescenceunit.

2.5. Cell culture

Bovine aortic endothelial cells (JCRB0099) wereprovided from Japan Health Science Foundation andcultured in Dulbecco’s modified Eagle’s minimumessential medium (DMEM) supplemented with 10%fetal bovine serum (FBS), 100 units/ml penicillin and100 mg/ml streptomycin. Before cell seeding, micropat-terned surfaces were incubated with fibronectin solution(50 mg/ml in Dulbecco’s phosphate buffered saline, pH7.4) at 37�C overnight and then washed. Endothelialcells (5.0� 104 cells/ml) were seeded on these treatedglass coverslips. Cell morphology in culture wasmonitored under a phase contrast microscope (ET300,Nikon, Japan).

3. Results

3.1. PC-controlled photopolymerization system

Typically a commercial LCDP connected to compu-ters generating images will automatically expand theimages to project them onto a large screen. Attachedadjustable lenses permit focus of the projected image.Fig. 1a shows the optical diagram of typical projectors.Light irradiated from the lamp (120W) is decomposedthough the dichroic mirror to three colors: red, greenand blue. After passing through each liquid crystalpanel, the three-color light beams are re-integrated againby the second dichroic mirror. Then, the integrated lightis expanded with a projection lens and projected onto asurface external to the projector. For our purposes, theoptical path was modified as follows (see Figs. 1b and c).In the modified LCDP unit, light illuminated from the

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lamp

dichroic mirror

mirror

mirror

G

B

scope for focus

adjustment

beam splitter

sample

lamp

stage

liquid crystal panel

(a)

(b)

(c)

(d)

R

Fig. 1. Photopolymerization device. Schematic optical diagrams of LCD (a) and the modified device (b). The overall view of the device (c) and the

magnified view of additional optics (d).

K. Itoga et al. / Biomaterials 25 (2004) 2047–2053 2049

lamp passed through only one liquid crystal panelwithout light decomposition. The emitted light wasdown-sized by the projection lens, turned downward bythe beam splitter, then irradiated onto the sample stage.For focus adjustment, the position of the sample stagewas monitored and controlled (Fig. 1d). The LCDPused here has three liquid crystal panels each of 1.78 cmin diagonal consisting of 480,000 pixels (800� 600).Each square pixel has sides of 18 mm on the liquid crystalpanel, but was reduced through the projection optics. Inthe present study, the reduced individual pixel size wasfixed to 10 mm, so that the final projection area was8� 6mm. Mask patterns were generated on a typicalinterfaced PC with commercially available software suchas Microsoft Words and Microsoft Power Points. Thesame images appearing on the PC monitor wereprojected onto the sample stage in a reduced size bythe re-arranged internal LCDP optical path.

3.2. Micropatterned PEG graft on glass surfaces

PEG was covalently grafted onto silanated glasssurfaces in order to confirm the fidelity of the surfacemicropatterning using the novel device. An imagecomprising black and white domains was used as the

projected mask, although the device could also projectimages in an eight-bit gray scale. After photopolymer-ization, the resultant micropattern was easily observedunder a phase contrast microscope, with generally highfidelity with the mask image generated by the PC(Fig. 2a). Each micropattern comprised small squaredots having 10 mm sides. Because of the shading fromembedded electronic wires aligned between pixels on theliquid crystal panel to switch each pixel on and off, smallgaps were created among the square dots (arrow inFig. 2c). Scanning electron microscopy revealed that themicro-patterned surfaces had micrometer-scale three-dimensional structures (Fig. 3). Three-dimensionalprofiles of the micropatterned surfaces were obtainedby means of reflective confocal laser scanning micro-scopy and AFM (Fig. 4). The height difference wasaround 0.9 mm between the convex and concavedomains, corresponding to white and black domains inPC-generated images, respectively.

In the present method, glass surfaces were at firsttreated with a surface-reactive cleaning plasma andintroduced with silane-coupled double bonds priorto photopolymerization. After plasma treatment, glasssurfaces were highly hydrophilic but changed tohydrophobic by the reactive silane coupling. After

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(a)

(b)

(c)

Fig. 2. Mask image generated by the PC (a), and phase contrast

microscopy of micropatterned surfaces (b, c). Note small gaps among

square dots (circle in (c)) Scale bar=200mm (b), 50mm (c).

Fig. 3. Scanning electron microscopy of micropatterned surfaces

Scale bar=50 mm.

Fig. 4. Atomic force microscopy of micropatterned surface. Three-

dimensional (a) and two-dimensional profiles (b).

K. Itoga et al. / Biomaterials 25 (2004) 2047–20532050

hydrophobic fluorescent dye (DiIC18) staining, convexpatterned polymer domains corresponding to whitedomains in mask images showed a weak red fluorescencederived from the dye, but the concave domainscorresponding to black domains in mask images didnot exhibit fluorescence derived from the dye (Fig. 5).This observation strongly suggested that convex do-mains were slightly hydrophobic, while the concavedomains were highly hydrophilic.

3.3. Micropatterned cell culture

Cell culture and in vitro adhesion experiments clearlyconfirmed that the surfaces formed PEG micropattern-ing according to exactly the original images designed

.

with PC. On the silane-introduced glass surfaces, celladhesion was hardly observed, even in the presence ofserum or fibronectin (data not shown). On the micro-patterned PEG surfaces, endothelial cells specificallyadhered to the convex domains (Fig. 5). On the concavedomains, cell adhesion was completely inhibited. Thus,micropattened cell seeding was achieved with a relatively

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Fig. 5. Hydrophobic dye staining of micropatterned surfaces and cell culture. Micropatterned surfaces were subjected to either fluorescent staining

with DiIC18 (a, b) or cells seeding (c, d). Micropatterned cell culture was microphotographed after one-day culture. Scale bar=100 mm.

K. Itoga et al. / Biomaterials 25 (2004) 2047–2053 2051

simple fabrication technique. The cell micropatternfidelity was maintained intact more than one month inthe presence of serum culture. No cytotoxicity wasobserved during the culture (data not shown).

4. Discussion

Micro-patterned surfaces were fabricated easily andquickly with the present LCDP device without a needfor expensive pattern masks, an additional light source,or other optical accessories. Even with the presenttypical LCDP, pattern fidelity and resolution is relevantto several biomedical applications in diagnostic arrayingand microfluidics. Recent progress in high resolutionand high density LCDP devices will achieve much finermicro-patterning required for other more advancedapplications.

Results of the photopatterning prompt several ques-tions about the spatial photochemistry occurring on thesilane-coupled glass surfaces. From the following threelines of observation, we concluded that PEG was photo-grafted on both patterned (e.g., white and black)domains but at different degrees of polymerizationunder the present conditions. First, three-dimensionalprofiles were obtained with micropatterned surfaces.These profiles reflected the PC-generated images withhigh fidelity, where the white (photo-exposed) domainsproduced convex surface-grafted domains (Figs. 2–4)within a matrix of concave domains (non-photo-

exposed regions). Vertical line topological resolutionbetween each domain at their interface was not square,indicating some interfacial zone of continually changingtopology that never reached to the glass-silane primedsurface. This suggests that concave domains are coveredby some photochemistry. Second, a hydrophobicfluorescent dye, DiIC18, specifically bound to theconvex domains (Fig. 5). Such hydrophobic dyepartitioning has been observed in many interfacialchemistry situations and is indicative of a preferreddye binding environment. Third, seeded cells selectivelyadhered the convex domains (Fig. 5). The concavedomains repelled cells for a long time in serum-containing culture. Taken together, we assert thatPEG was photo-grafted even onto the concave domains,which were masked with black domains in PC-generatedimages. While liquid crystal panels equipped inside theprojector can completely shield emitted light to achieveblack color, the irradiated light can be readily diffractedor scattered at the glass surface on both faces, within theglass substrate and between the glass sandwichingplates. Such effects could compromise the maskingfidelity, permitting some light access to black-maskedregions and permitting photochemistry here as well, butto a limited extend. Therefore, PEG is likely also graftedto some limited extent onto the black domains wheredirectly irradiated light was otherwise completelyshielded. Under these conditions, grafted PEG in theseregions was much thinner (i.e., in the concave domains)than in the convex domains where emitted light was

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irradiated directly through the liquid crystal panel atsubstantially higher intensity and photochemical effi-ciency. Molecular weight of the PEG-diacrylate used inthe present study was 1000. In previous studies, proteinand/or cell adhesion on PEG-grafted materials werereduced with increasing PEG molecular weight [20–22].However, adhesion behavior on PEG-grafted surfaceshas not been solely explained by PEG molecular weighteffects [23]. Typical hydrogels prepared by chemical orradiation-induced procedures have cross-linking pointsdistributed throughout the hydrogel where distancesbetween relatively hydrophobic (e.g., acrylate or acry-lamide linkages) in the gel network are spanned byrelatively hydrophilic ethylene oxide chains of differentlengths. By sharp contrast to previous work with longerPEG acrylates, the network structure of current graftedPEG hydrogels is considered to comprise shorter oligo-PEG acrylates where the crosslink points (hydrophobicdomains) are more chemically dominant over theshorter spanning PEG chains (hydrophilic domains).Hydrophobic oligoacrylate sequences with lower PEGmolecular weights would therefore be predictablyexposed to adsorbing serum proteins [24,25], resultingin possibly selective adsorption of fibronectin on theconvex domains. In support of this claim, hydrophobicfluorescent dye (DiIC18) was observed to selectivelyadsorb onto or within the convex domains. Thesecorrelative data imply that convex domains subject tointense direct LCDP irradiation are highly polymerizedacrylate domains with less PEG mobility, reduced watercontent and generally increased local hydrophobicitycapable of serum protein and dye interactions. It isplausible that the majority of the PEG was photo-immobilized at the both terminal ends to allow sufficientserum protein adsorption and subsequent cell adhesionon the convex domains. By contrast, our contention isthat indirectly irradiated, less reacted PEG immobilizedon the masked black regions (concave domains) main-tains less crosslinking, more free terminal PEG ends,and more single-point grafting with hydrated mobilitythat reduces protein adsorption, and repels cells inculture.

As shown in Fig. 5, seeded cells were confined withinthe convex cell adhesive domains. Cells in the vicinity ofedges of cell adhesive domains showed highly elongatedshapes, and the cell long axis was completely parallel tothe edge. To the contrary, cells in the center of celladhesive domains did not show such a cell orientation.These findings imply that the micropattern fidelityachieved by the present method is sufficient forbiomedical applications utilizing biomolecules and cells.The present LCDP device has both a liquid crystal panelimaging PC-generated patterns and a strong lightsource. Because of availability, cost and ease ofmodification to accommodate photo-patterning, suchan all-in-one device should prove useful for the

preparation of micro-patterned surfaces for biomedicalapplications in a rapid prototyping manner.

5. Conclusion

We developed an all-in-one device for photopolymer-ization-based surface micropatterning by modifying acommercially available liquid crystal device projector.Micropatterned surfaces were fabricated from theimages prepared with various softwares run on personalcomputers. With PEG-diacrylate and a visible lightphotopolymerization initiator, camphorquinone, 10-mmresolution, which seems sufficient for biomedical appli-cations, was obtained. On the micropatterned surfaces,selective cell adhesion control was also achieved.

Acknowledgements

The present work was supported by Grants-in-Aid forScientific Research (13308055) of Japan Society forPromotion of Science, The Promotion and Mutual AidCorporation for Private School of Japan, and CoreResearch for Evolution Science and Technology fromJapan Science Technology Corporation. We also ap-preciate the useful comments and technical criticismfrom Prof. D.W. Grainger (Colorado State University,USA). We are thankful for the cooperation of DigitalSystems Development Center of Sanyo Electric Co.,Ltd.

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