Arraying Heterotypic Single Cells on Photoactivatable Cell-Culturing

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Langmuir 2008, 24, 13084-13095

Arraying Heterotypic Single Cells on Photoactivatable Cell-Culturing Substrates Yukiko Kikuchi,†,‡ Jun Nakanishi,*,†,§ Takahiro Shimizu,| Hidekazu Nakayama,†,§ Satoshi Inoue,| Kazuo Yamaguchi,| Hideo Iwai,† Yasuhiko Yoshida,‡ Yasuhiro Horiike,† Tohru Takarada,*,⊥ and Mizuo Maeda⊥ World Premier International (WPI) Research Center InitiatiVe, International Center for Materials Nanoarchitectonics (MANA) and National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan, Graduate School of Engineering, Toyo UniVersity, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, Department of Materials Science, Faculty of Science, Kanagawa UniVersity, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan, and Bioengineering Laboratory, AdVanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ReceiVed July 29, 2008. ReVised Manuscript ReceiVed September 10, 2008 This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH2 substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.

Introduction The controlled assembly of heterotypic cells on a substrate is an important technology for a wide range of applications such as gaining fundamental biological insight into cell-cell interactions and engineering tissues in vitro for therapeutic application.1-4 In addition, heterotypic cell microarrays are expected to be a promising platform for high-throughput screening of drug candidates.5 Several methods have been developed to pattern heterotypic cells, which can be divided into two groups on the basis of strategy: (i) delivering cells to discrete regions of a substrate and (ii) switching the cell adhesiveness of discrete regions of a substrate. Region-selective cell delivery was * Corresponding authors. (J.N.) Phone: +81-29-860-4569. Fax: +8129-860-4706. E-mail: [email protected]. (T.T.) Phone: +81-48467-5489. Fax: +81-48-462-4658. E-mail: [email protected]. † MANA and NIMS. ‡ Toyo University. § JST. | Kanagawa University. ⊥ RIKEN. (1) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. FASEB. J. 1999, 13, 1883–1900. (2) Folch, A.; Toner, M. Annu. ReV. Biomed. Eng. 2000, 2, 227–256. (3) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Chen, G. P.; Okano, T. Biochem. Biophys. Res. Commun. 2006, 348, 937–944. (4) Nakanishi, J.; Takarada, T.; Yamaguchi, K.; Maeda, M. Anal. Sci. 2008, 24, 67–72. (5) Kapur, R.; Giuliano, K. A.; Campana, M.; Adams, T.; Olson, K.; Jung, D.; Mrksich, M.; Vasudevan, C.; Taylor, D. L. Biomed. MicrodeV. 1999, 2, 99–109.

established by using microfluidic devices,6,7 inkjet printing,8-10 micropipettes with agarose microchambers,11 dielectrophoresis,12,13 and optical tweezers.14 The placement of heterotypic cells was achieved by repeatedly changing the cell adhesiveness of selected regions on substrates, followed by seeding of different cell types. So far, these developed methods were based on a microelectrode,15 the layer-by-layer deposition of polymeric materials,16 and chemically modified substrates whose cell (6) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408– 2413. (7) Li, Y.; Yuan, B.; Ji, H.; Han, D.; Chen, S. Q.; Tian, F.; Jiang, X. Y. Angew. Chem., Int. Ed. 2007, 46, 1094–1096. (8) Xu, T.; Jin, J.; Gregory, C.; Hickman, J. J.; Boland, T. Biomaterials 2005, 26, 93–99. (9) Nakamura, M.; Kobayashi, A.; Takagi, F.; Watanabe, A.; Hiruma, Y.; Ohuchi, K.; Iwasaki, Y.; Horie, M.; Morita, I.; Takatani, S. Tissue Eng. 2005, 11, 1658–1666. (10) Jayasinghe, S. N.; Qureshi, A. N.; Eagles, P. A. M. Small 2006, 2, 216– 219. (11) Sugio, Y.; Kojima, K.; Moriguchi, H.; Takahashi, K.; Kaneko, T.; Yasuda, K. Sens. Actuators, B 2004, 99, 156–162. (12) Albrecht, D. R.; Underhill, G. H.; Wassermann, T. B.; Sah, R. L.; Bhatia, S. N. Nat. Methods 2006, 3, 369–375. (13) Taff, B. M.; Voldman, J. Anal. Chem. 2005, 77, 7976–7983. (14) Ozkan, M.; Pisanic, T.; Scheel, J.; Barlow, C.; Esener, S.; Bhatia, S. N. Langmuir 2003, 19, 1532–1538. (15) Kaji, H.; Kanada, M.; Oyamatsu, D.; Matsue, T.; Nishizawa, M. Langmuir 2004, 20, 16–19. (16) Kumar, G.; Meng, J. J.; Ip, W.; Co, C. C.; Ho, C. C. Langmuir 2005, 21, 9267–9273. (17) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem. Rapid Commun. 1990, 11, 571–576.

10.1021/la8024414 CCC: $40.75  2008 American Chemical Society Published on Web 10/17/2008

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adhesiveness can be regulated by an external stimulus such as heat,3,17 voltage,7,18-21 or light.22-25 We have recently reported on a novel photoactivatable substrate, a glass coverslip modified with a silane coupling agent having a caged carboxyl group, where “caged” means “protected by a photocleavable protecting group.”26-28 First, we passivated this substrate against cell adhesion by coating the surface with a blocking agent, bovine serum albumin (BSA) or triblock copolymer Pluronic F108, and then irradiated the discrete regions with UV light at 365 nm under a standard fluorescence microscope. This irradiation removed hydrophobic protecting groups (2-nitrobenzyl groups) from hydrophilic carboxyl groups, leading to the dissociation of the blocking agents that had been adsorbed on the surface via hydrophobic interactions. Finally, we coated these irradiated regions with a cell adhesive protein, fibronectin, to make the regions cell-adhesive.26 Because the surface presenting the deprotected carboxyl groups revealed unsatisfactory cell adhesiveness, the addition of fibronectin was required after irradiation. However, this procedure not only limited the temporal resolution of the cell adhesiveness change but also potentially could have caused undesired hormonal effects by this protein on the cells. Therefore, it was necessary to develop an improved photoactivatable cell-culturing substrate where cell adhesion can be switched on only by UV irradiation without the assistance of fibronectin. In the present study, we newly synthesized silane coupling agents having different caged functional groups-amino (NH2), hydroxyl (OH), and thiol (SH)-because cell adhesion onto substrates is highly dependent on the surface chemistry.29-35 By the chemical modification of glass coverslips with these agents as well as one having a caged carboxyl group (COOH), four caged cell-culturing substrates were prepared to examine the effect of terminal functional groups on photoactivation for cell adhesion. We found that the caged NH2 substrate was highly photoactivatable without having to add fibronectin, whereas the other substrates were inert. Under optimized conditions, we succeeded in fabricating heterotypic single-cell microarrays on the caged NH2 substrate by alternating the UV irradiation and the cell seeding. Moreover, the present patterning method allowed us to control the relative positions and distances of heterotypic single cells precisely. It will offer a useful platform for dynamic (18) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992–5996. (19) Yeo, W. S.; Mrksich, M. Langmuir 2006, 22, 10816–10820. (20) Jiang, X. Y.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 2366–2367. (21) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Arango, D. C.; Brozik, S. M. Angew. Chem., Int. Ed. 2008, 47, 2631–2634. (22) Nakayama, Y.; Furumoto, A.; Kidoaki, S.; Matsuda, T. Photochem. Photobiol. 2003, 77, 480–486. (23) Edahiro, J.; Sumaru, K.; Tada, Y.; Ohi, K.; Takagi, T.; Kameda, M.; Shinbo, T.; Kanamori, T.; Yoshimi, Y. Biomacromolecules 2005, 6, 970–974. (24) Petersen, S.; Alonso, J. M.; Specht, A.; Duodu, P.; Goeldner, M.; der Campo, A. Angew. Chem., Int. Ed. 2008, 47, 3192–3195. (25) Park, S.; Yousaf, M. N. Langmuir 2008, 24, 6201–6207. (26) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. J. Am. Chem. Soc. 2004, 126, 16314–16315. (27) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. Anal. Chim. Acta 2006, 578, 100–104. (28) Nakanishi, J.; Kikuchi, Y.; Inoue, S.; Yamaguchi, K.; Takarada, T.; Maeda, M. J. Am. Chem. Soc. 2007, 129, 6694–6695. (29) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3–30. (30) Keselowsky, B. G.; Collard, D. M.; Garcia, A. J. J. Biomed. Mater. Res. A 2003, 66A, 247–259. (31) Faucheux, N.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Biomaterials 2004, 25, 2721–2730. (32) Liu, L.; Chen, S.; Giachelli, C. M.; Ratner, B. D.; Jiang, S. J. Biomed. Mater. Res. A 2005, 74A, 23–31. (33) Curran, J. M.; Chen, R.; Hunt, J. A. Biomaterials 2005, 26, 7057–7067. (34) Lee, M. H.; Ducheyne, P.; Lynch, L.; Boettiger, D.; Composto, R. J. Biomaterials 2006, 27, 1907–1916. (35) Arima, Y.; Iwata, H. Biomaterials 2007, 28, 3074–3082.

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single-cell analyses for quantitative biology36-40 and for cell biological studies on engineered extracellular matrices.41-45

Results and Discussion Synthesis of Silane Coupling Agents Having Caged Functional Groups. The synthesis of silane coupling agent having a caged COOH group [1-(2-nitrophenyl)ethyl 5-trichlorosilylpentanoate (1)] has been reported.46 Synthesis schemes of silane coupling agents having an NH2, OH, or SH group protected by a photocleavable 2-nitrobenzyl group are shown in Figure 1. The silane coupling agent with a caged NH2 group [1-(2nitrophenyl)ethyl N-(3-trimethoxysilylpropyl)carbamate (2)] was synthesized in three steps (Figure 1a). 2-Nitroacetophenone was reduced to obtain 1-(2-nitrophenyl)ethanol (5). Alcohol 5 was then reacted with di(N-succinimidyl) carbonate to provide activated ester 6. Amide bond formation with (3-aminopropyl)trimethoxysilane afforded desired silane coupling agent 2. The silane coupling agent with a caged OH group [2-nitrobenzyl 3-(trimethoxysilyl)propyl ether (3)] was synthesized in two steps (Figure 1b). Allyl alcohol was alkylated with 2-nitrobenzyl bromide by the Williamson reaction. Obtained ether 7 was then hydrosilylated to afford product 3. Finally, the silane coupling agent with a caged SH group [2-nitrobenzyl 3-(trimethoxysilyl)propyl sulfide (4)] was synthesized in one step by alkylating (3-mercaptopropyl)trimethoxysilane with 2-nitrobenzyl bromide (Figure 1c). Surface Characterization of Glass Coverslips Immobilized with the Silane Coupling Agents. A glass coverslip was chemically modified with the silane coupling agents (1-4) to form the corresponding alkylsiloxane monolayers on its surface (Figure 2). The obtained caged substrates were irradiated with UV light (365 nm) from a mercury lamp equipped on a conventional fluorescence microscope. Upon UV irradiation, COOH, OH, and SH groups were directly produced through photocleavage of the corresponding caged functional groups (Figure 2a), whereas uncaging of NH2 groups proceeded via decarboxylation after photocleavage of the 2-nitrophenylethyl carbamate (Figure 2b).47 The photochemical reactions were followed by an increase in surface wettability against water. As time for irradiation at 365 nm increased, all of the substrates exhibited a dose-dependent decrease in the contact angle (Figure 3), corresponding to the dissociation of hydrophobic 2-nitrobenzyl groups, as well as the exposure of hydrophilic functional groups. On all of the tested substrates, the contact angle reached a plateau at 7500 mJ/cm2, suggesting that the photoactivation was nearly completed with this exposure energy. (36) Di Carlo, D.; Lee, L. P. Anal. Chem. 2006, 78, 7918–7925. (37) Kuang, Y.; Walt, D. R. Anal. Biochem. 2005, 345, 320–325. (38) Yamamura, S.; Kishi, H.; Tokimitsu, Y.; Kondo, S.; Honda, R.; Rao, S. R.; Omori, M.; Tamiya, E.; Muraguchi, A. Anal. Chem. 2005, 77, 8050–8056. (39) Fukuda, T.; Shiraga, S.; Kato, M.; Suye, S. I.; Ueda, M. Biotechnol. Prog. 2006, 22, 944–948. (40) Kim, H.; Cohen, R. E.; Hammond, P. T.; Irvine, D. J. AdV. Funct. Mater. 2006, 16, 1313–1323. (41) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425–1428. (42) Brock, A.; Chang, E.; Ho, C. C.; LeDuc, P.; Jiang, X. Y.; Whitesides, G. M.; Ingber, D. E. Langmuir 2003, 19, 1611–1617. (43) Kandere-Grzybowska, K.; Campbell, C.; Komarova, Y.; Grzybowski, B. A.; Borisy, G. G. Nat. Methods 2005, 2, 739–741. (44) Thery, M.; Racine, V.; Pepin, A.; Piel, M.; Chen, Y.; Sibarita, J. B.; Bornens, M. Nat. Cell Biol. 2005, 7, 947–953. (45) Thery, M.; Racine, V.; Piel, M.; Pepin, A.; Dimitrov, A.; Chen, Y.; Sibarita, J. B.; Bornens, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19771–19776. (46) Yamaguchi, K.; Nakayama, H.; Futami, T.; Shimizu, T. J. Photopolym. Sci. Technol. 2008, 21, 519–524. (47) Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441–458.

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Figure 1. Synthesis scheme of silane coupling agents having a caged (a) NH2, (b) OH, or (c) SH group.

Figure 2. Photochemical reactions of glass coverslips modified with silane coupling agents having (a) caged COOH, OH, SH groups and (b) a caged NH2 group.

We further confirmed the progress of the photocleavage reaction by X-ray photoelectron spectroscopy (XPS) measurements. We focused on the chemical state of N 1s, which was supposed to exhibit a characteristic change during the removal of 2-nitrobenzyl groups. Figure 4 shows the N 1s spectrum of

each substrate before and after UV irradiation. In Figure 4a, the peak at 407 eV, which was observed for all of the four caged substrates, was assigned to the nitro (NO2) group of the 2-nitrobenzyl moiety.48 The strong peak at 400 eV, observed for the caged NH2 substrate, was assigned to the amide bond.49

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Figure 3. Effect of exposure energy on the water contact angle of the substrates having a caged COOH (b), NH2 (2), OH (9), and SH (() group. Error bars represent standard deviations of at least three different substrates. Exposure energy (mJ/cm2) was the product of the power (mW/cm2) and the time (s) of irradiation.

Upon irradiation of the substrates at 7500 mJ/cm2, the spectra changed drastically (Figure 4b). The peak assigned to the NO2 group disappeared for the four substrates, indicating that the photochemical removal of the 2-nitrobenzyl groups was almost completed with this exposure energy (Table 1). The strong, broad peak at 403-398 eV appeared for the uncaged NH2 substrate, which can actually be assigned as two peaks at 402 and 400 eV by curve fitting (Figure 4c), corresponding to the protonated NH3+ group and the unprotonated NH2 group, respectively.50 The coexistence of these two states in phosphate-buffered saline (PBS, pH 7.4) is reasonable because the pKa of the NH3+ groups on various self-assembled monolayers was determined to be much lower than that in solution (approximately from 4 to 7).51-53 Around the same region (403-398 eV), we found small peaks for the COOH, OH, and SH substrates before and after UV irradiation (Figures 4a,b). These peaks were likely to be decomposed products of the nitrobenzyl group by X-ray irradiation and/or contaminants in the air.48 Because we found no increase in the normalized peak intensity upon UV irradiation for the COOH and OH substrates (Table 1), we considered these small peaks to be negligible. For the SH substrate, however, we observed a slight increase in the peak intensity ratio around this binding energy upon UV irradiation, which was at least partially due to a recoupling reaction between the uncaged SH group on the surface and the NO group generated by the photocleavage reaction.54 Although unknown background peaks were observed in the XPS measurements as mentioned above, on the basis of a decrease in contact angle reaching to an almost constant value on each caged substrate (Figure 3) and the complete disappearance of the NO2 peak in the XPS measurements (Figure 4), we concluded that the photocleavage reactions proceeded almost completely on the caged substrates upon UV irradiation with 7500 mJ/cm2. On the basis of these results, we used this irradiation energy for all of the following experiments. (48) Zhao, B.; Moore, J. S.; Beebe, D. J. Science 2001, 291, 1023–1026. (49) Kilian, K. A.; Bocking, T.; Gaus, K.; Gal, M.; Gooding, J. J. ACS Nano 2007, 1, 355–361. (50) Nolting, D.; Aziz, E. F.; Ottosson, N.; Faubel, M.; Hertel, I. V.; Winter, B. J. Am. Chem. Soc. 2007, 129, 14068–14073. (51) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006–2015. (52) van der Vegte, E. W.; Hadziioannou, G. J. Phys. Chem. B 1997, 101, 9563–9569. (53) Abiman, P.; Wildgoose, G. G.; Crossley, A.; Jones, J. H.; Compton, R. G. Chem.sEur. J. 2007, 9663–9667. (54) Zuman, P.; Shah, B. Chem. ReV. 1994, 94, 1621–1641.

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Photoactivation of the Caged Substrates for Cell Adhesion. We next evaluated whether these caged substrates were photoactivatable for cell adhesion. The substrates were coated with BSA in order to make the entire surface non-cell-adhesive in the initial state. A circular region of each substrate was irradiated at 7500 mJ/cm2, to which COS7, NIH3T3, or HEK293 cells were seeded in a serum-free medium. Interestingly, only the caged NH2 substrate produced a clear, circular cell-adhesive region, corresponding to the UV-irradiated area (Figure 5). In marked contrast, we observed no cellular pattern on the caged OH and SH substrates but saw a small number of cells attached to the irradiated region of the COOH substrate (Figure 5). In our earlier study, such weak cell adhesiveness of the uncaged COOH substrate required us to use subsequent adsorption of fibronectin onto the UV-irradiated regions to attach cells.26 The caged NH2 substrate can acquire remarkably high cell adhesiveness just by UV irradiation without an additional fibronectin coating; this feature is effective, particularly in the preparation of heterotypic cell microarrays, which need repeated steps of photoactivation and cell seeding (vide infra). Preparation of Homotypic Single-Cell Microarrays. We next moved to the formation of homotypic single-cell microarrays on the caged NH2 substrate. For the efficient preparation of a single-cell microarray, it is important to optimize both the spot size of the irradiated regions and the cell seeding concentration. Furthermore, the optimal values of theses two parameters vary from one cell type to another. Okano and co-workers demonstrated that both the size of the array spots and the cell seeding concentration should be optimized to produce a good yield of homotypic single-cell microarrays,55 without having the majority of array spots being occupied with multiple cells or no cells. According to the previously reported procedure,26 a photomask of 40, 60, 80, or 120 micrometers square array spots every 800 µm was inserted at the field diaphragm of the microscope to obtain the UV-irradiated region of 10, 15, 21, or 30 micrometers square size, respectively. When employing the photomask of 60 micrometers square spots, the UV-irradiated areas were visualized by irradiating a glass coverslip painted with fluorescent ink (Figure 6a). The UV irradiation was conducted through the photomask onto the caged NH2 substrate with the BSA coating, and thereon the COS7, NIH3T3, or HEK293 cells were seeded in concentrations of 2 × 105, 4 × 105, 6 × 105, 8 × 105, and 10 × 105 cells/dish in a serum-free medium. Figure 6b-f illustrates the phase-contrast images of the COS7 cell microarray obtained by using the photomask of 60 micrometers square spots (the irradiated region was 15 micrometers square size) and seeding in concentrations of 2 × 105, 4 × 105, 6 × 105, 8 × 105, and 10 × 105 cells/dish, and this indicates that cell adhesion to the substrate was highly dependent on the cell seeding concentration. The number of adhered cells within each array spot was counted by using the magnified phase-contrast images of the cell outline (Figure 6b, insets). The dependence of the array spot occupancy for the single and multiple cells on the spot size and the cell concentration is summarized in Figure 7. The occupancy (%) is defined as the proportion of spots accommodating single or multiple cells to total spots. In COS7 cells, the occupancy of single cells increased approximately from 10 to 60% as the cell seeding concentration increased from 2 × 105 to 8 × 105 cells/dish on 10 and 15 micrometers square array spots simply as result of the increased probability of the cells suspended in the solution to contact the array spots (Figure 6b-e). However, when the cell seeding (55) Iwanaga, S.; Akiyama, Y.; Kikuchi, A.; Yamato, M.; Sakai, K.; Okano, T. Biomaterials 2005, 26, 5395–5404.

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Figure 4. XPS N 1s spectra of the caged COOH, NH2, OH, and SH substrates (a) before and (b) after UV irradiation. (c) High-resolution spectrum of the caged NH2 substrate after UV irradiation. Deconvoluted peaks are shown in red. Table 1. Change in the Chemical State of N 1s during UV Irradiation peak intensity ratioa substrate COOH NH2 OH SH COOH NH2 OH SH

at 407 eV Before UV Irradiation 0.058 0.098 0.058 0.119 After UV Irradiationb n.d.c n.d. n.d. n.d.

at 403-398 eV 0.033 0.175 0.056 0.070 0.026 0.166 0.050 0.118

a Peak areas of N 1s were normalized to that of Si 2p. b The substrates were irradiated with 7500 mJ/cm2. c Not detectable.

concentration further increased to 10 × 105 cells/dish, a drastic decrease in single-cell occupancy was observed with each spot size (Figures 6f and 7). This is likely due to the aggregation of the COS7 cells in the bulk during seeding, which is unfavorable for stable cell adhesion onto such small array spots. In accordance with this interpretation, we observed a remarkable increase in the occupancy for multiple cells using a seeding concentration of 10 × 105 cells/dish on the largest 30 micrometers square array spots. Although the overall dependence of occupancy for NIH3T3 cells and HEK293 cells was roughly comparable to that of COS7 cells, the occupancy for single HEK 293 cells was lower than that for single COS7 and NIH3T3 cells under identical conditions. This difference can be attributed to a biological feature of HEK293 cells; this cell type forms aggregates relatively easily under the typical experimental conditions. On the basis of the results shown in Figure 7, we determined the optimal size of array spots as well as the most appropriate cell concentration for preparing each single-cell microarray using the caged NH2 substrate: 15 micrometers square spots and 8 × 105 cells/dish for COS7 cells (the occupancy for single cells was 69%); 10 micrometers square spots and 8 × 105 cells/dish for NIH3T3 cells (72%); and 10 micrometers square spots and 10 × 105 cells/dish for HEK293 cells (49%). Preparation of Heterotypic Single-Cell Microarrays. The principal advantage of a caged cell-culturing substrate over previously reported functional substrates is the successive fabrication of new cell-adhesive regions during cell cultivation. Our strategy for forming heterotypic single-cell microarrays is based on the sequential formation of each homotypic single-cell microarray on the same substrate by repeated UV irradiation and cell seeding. Figure 8 depicts each step for successively arraying single COS7 cells to prepare a quasi-heterotypic single-cell microarray. After seeding unstained COS7 cells for 0.5 h in a single-cell microarray format, we performed a second UV irradiation on other regions in proximity to the single cells attached

in advance (Figure 8a). Next we attached fluorescent COS7 cells stained with Cell Tracker Green CMFDA to the irradiated regions for another 0.5 h (Figure 8b). We then conducted a third UV irradiation on other regions (Figure 8c), seeded fluorescent COS7 cells stained with Mito Tracker Red CM-H2Xros and allowed the cells to attach for another 0.5 h (Figure 8d). This resulted in a quasi-heterotypic single-cell microarray with a single vacant site at the third blue cell (fourth row and third column) because of unsatisfactory occupancy for single COS7 cells under the conditions optimized in this study. Next, we prepared a heterotypic single-cell microarray. For COS7 and NIH3T3 cells, we employed the optimized conditions determined in the previous section. Figure 9a shows a heterotypic single COS7 and NIH3T3 cell microarray fabricated using the following procedure. First, we seeded unstained COS7 cells onto 15 micrometers square spots at a concentration of 8 × 105 cells/ dish, which were the optimized conditions mentioned above, to prepare a single COS7 cell microarray. After irradiating another 10 micrometers square array spot alongside the attached COS7 cells, we seeded NIH3T3 cells stained with Cell Tracker at a concentration of 8 × 105 cells/dish. The NIH3T3 cells were selectively attached to the secondary irradiated spots, leading to the formation of a heterotypic single-cell microarray (Figure 9a). Unfortunately, we observed three unoccupied spots (2B, 2D, and 4C) in the second NIH3T3 cell microarray. We also found two undesired adhesive spots in the secondary seeded NIH3T3 cell to the vacant sites of the first COS7 cell microarray (1B and 7D). The success probability of two-cell-type (COS7 and NIH3T3) patterning was observed to be 49% (196 of 400 array spots), which was quite similar to the value obtained by simply multiplying the highest occupancy of each cell type (69% (COS7) × 72% (NIH3T3) ) 50%). Therefore, it is reasonable to treat each patterning step as independent. It is expected that these imperfections and defects will be carefully avoided by further optimizing the conditions, including the shape of the spots and the distance between the spots. In practice, we can easily change the geometry of the spots and the distance between the spots by using a different photomask. Examples illustrated in Figure 9b demonstrate this feature. After preparing a heterotypic single COS7 and NIH3T3 cell microarray as described above, we further irradiated another 15 micrometers square spot alongside the single cells attached in advance, followed by seeding HEK293 cells stained with Mito Tracker at a concentration of 6 × 105 cells/dish. These were the secondbest conditions for HEK293 cells with an occupancy of 43% (Figure 7). We did not employ the best conditions with the highest cell seeding concentration (10 micrometers square spots and 10 × 105 cells/dish) because HEK293 cells tended to aggregate with other cell types to lower the success probability of heterotypic array formation. As shown in Figure 9b, we succeeded in correctly

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Figure 5. Photoactivation of the caged COOH, NH2, OH, and SH substrates for cell adhesion. BSA-adsorbed surfaces were irradiated in a circular pattern, whereupon COS7, NIH3T3, and HEK293 cells were seeded. Phase-contrast images at 1 h after cell seeding are shown.

Figure 6. (a) Pattern of UV irradiation according to the photomask having 60 micrometers square spots inserted at the field diaphragm of the fluorescence microscope to give UV-irradiated regions of 15 micrometers square size. A fluorescence image of a glass coverslip painted with fluorescent ink is shown. (b-f) phase-contrast images of a COS7 cell microarray on the caged NH2 substrate obtained by using the photomask of 60 micrometers square spots at 0.5 h after seeding at concentrations of 2 × 105, 4 × 105, 6 × 105, 8 × 105, and 10 × 105 cells/dish in a serum-free medium, respectively. (b, inset) Magnified phase-contrast images of a COS7 cell microarray showing cell adhesion with single (left) and multiple cell occupation (right).

positioning single COS7, NIH3T3, and HEK293 cells in the different geometries of an equilateral (Figure 9b, top) and a rightangled triangle (Figure 9b, middle). Moreover, we easily extended the distance between each spot (Figure 9b, bottom). The success probability of three-cell-type (COS7, NIH3T3, and HEK293) patterning was found to be 6.8% (26 of 380 array spots), which was lower than the value obtained by simple multiplication of

the occupancy of each cell type (50% × 43% (HEK293) ) 20%). The observed lower success probability arose from the undesired strong adhesion of the third seeded HEK293 cells onto preattached COS7 cells and NIH3T3 cells, as mentioned above. However, it should be noted that the probability of observing such a set of geometry is quite low when coculturing three different cell types on conventional, nonpatterned cell-

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Figure 7. Effect of the spot size and the cell seeding concentration on the occupancy of array spots by single (black) or multiple cells (white). COS7, NIH3T3, and HEK293 cells were seeded in various concentrations on the caged NH2 substrates having arrays of different sizes. Occupancy (%) was defined as the proportion of spots accommodating single or multiple cells to total spots (100). Error bars represent the standard deviation of three different substrates.

Figure 8. Coculturing quasi-heterotypic single COS7 cells on the caged NH2 substrate by repeated UV irradiation and cell seeding. (a) A phase-contrast image of an unstained single COS7 cell microarray at 0.5 h after seeding. White squares represent the outlines of the second irradiated regions. (b) Fluorescence image (red) merged with the corresponding phase-contrast image (colorless) of a microarray accommodating unstained COS7 single cells (colorless) and those stained with Cell Tracker Green CMFDA (red) 0.5 h after the second cell seeding. (c) The outlines of the third irradiated regions are represented by white squares in image b. (d) Fluorescence image (red and blue) merged with the corresponding phase-contrast image (colorless) of a microarray accommodating the first unstained COS7 single cells (colorless), with the second one stained with Cell Tracker Green CMFDA (red) and the third one stained with Mito Tracker Red CM-H2Xros (blue) 0.5 h after the third cell seeding.

culturing dishes and even on surfaces having predetermined array patterns. Furthermore, because we have already succeeded in inducing arrayed cells to migrate and/or proliferate by irradiating regions alongside the patterned cells in our previous studies,27,28 this treatment will also be technically feasible with the caged NH2 substrate to make heterotypic single cells interact with and/ or grow into one another. Therefore, the present technology will be quite useful in various cell biological studies on cell-cell interactions at the single-cell level.

Improvements over the Previous Study Based on the Caged COOH Substrate. The previously reported caged COOH substrates with the additional adsorption of fibronectin26,27 did not allow for the efficient production of a microarray of heterotypic single cells, owing to the difficulty in spatial control of adhesion of the secondary seeded cells. The unsatisfactory spatial control can probably be attributed to (i) a gradual replacement of BSA with fibronectin in the nonirradiated regions, resulting in undesired cell adhesion to these regions, and (ii) an unavoidable adsorption

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Figure 9. Coculturing heterotypic single cells on the caged NH2 substrate by repeated UV irradiation and cell seeding. (a) Single COS7 (colorless) and NIH3T3 cells (red) were arrayed with a given separation. After the preparation of the single COS7 cell microarray, the NIH3T3 cell microarray was subsequently constructed. (b) Single COS7 (colorless), NIH3T3 (red), and HEK293 cells (blue) were positioned with different arrangements. NIH3T3 and HEK293 cells were stained with Cell Tracker Green CMFDA and Mito Tracker Red CM-H2Xros, respectively. The order of cell seeding was COS7, NIH3T3, and then HEK293 cells. Merged phase-contrast and fluorescence images are shown.

of fibronectin on the first seeded cell surface where the secondary seeded cells were allowed to attach. As shown in Figure 9a, we succeeded in arraying a number of different single cells on the caged NH2 substrate. Omitting the fibronectin-coating steps eliminated the probability of attaching cells at nonirradiated regions via undesired fibronectin deposition. In addition, the optimization of the spot size and the cell seeding concentration for each kind of cell was conducted in this study. It should be pointed out that efficient and precise arraying of different single cells on cell-culturing substrates will be a very important tool for quantitative single-cell analysis in future cell biology. Moreover, the caged NH2 substrates have made it possible to position three different single cells in a spatiotemporally controlled manner (Figure 9b). This has never been achieved by the previous caged COOH substrate with fibronectin for the same reasons suggested above. Expected future applications in this area include network formation between several kinds of neural and glia cells positioned in any desired arrangements on the caged cell-culturing substrates through successive bypass formation between the cells by photoirradiation.11 Origin of Efficient Photoactivation for Cell Adhesion on the Caged NH2 Substrate. As shown in Figure 5, only the caged NH2 substrate was photoactivatable for cell adhesion, whereas the other substrates were not. Interestingly, when we seeded cells onto the preirradiated substrates, namely, “uncaged” substrates in serum-free medium, the cells adhered well to the four substrates, and we found no difference in cell adhesiveness between them (Figure 10a). Therefore, if the surface-adsorbed BSA on the UV-irradiated regions had been completely dissociated from the surface, then the cell adhesive regions would have been obtained on all of the caged substrates, which is in conflict with the experimental results shown in Figure 5. To elucidate the origin of efficient photoactivation on the caged NH2 substrate, we performed quantitative and qualitative analysis of the surfaceadsorbed BSA. To estimate the difference in the amount of surface-adsorbed BSA before and after UV irradiation, we visualized surfaceadsorbed BSA by in situ staining with SYPRO Ruby protein blot stain or by prelabeling with Alexa 488, and measured the fluorescence intensity in nonirradiated and irradiated regions on

the caged substrates (states I and II in Figure 11a). After UV irradiation, the fluorescence intensity of the irradiated region decreased by only 22 and 17% on the caged COOH and NH2 substrates, respectively, and negligible changes were observed on the caged OH and SH substrates (FI and FII in Figure 11b). These results strongly indicate that a large portion of BSA remained on the surface of all of the substrates even after UV irradiation. In addition, for the caged COOH and NH2 substrates, we found no correlation between the efficiency of photoactivation for cell adhesion (Figure 5) and the amount of BSA dissociated from the surface after irradiation (FI and FII in Figure 11b). To focus on the difference in the passivation activity between the BSA on the four uncaged substrates, we next examined the cell adhesiveness of the BSA-adsorbed, preirradiated (uncaged) substrates (state III in Figure 11a). The four caged substrates were first irradiated to produce the uncaged functional groups on the surfaces, and then BSA was allowed to adsorb on the four uncaged surfaces. The amount of BSA adsorbed on the uncaged substrates (state III) was confirmed to be almost the same as that of BSA remaining on the irradiated surface (state II) for all of the substrates (compare FII and FIII in Figure 11b). When COS7 cells were seeded on the substrates, a large number of COS7 cells adhered to the uncaged NH2 substrate even after BSA adsorption (Figure 10b,c). However, no cell adhesion was observed on the BSA-adsorbed, uncaged COOH, OH, and SH substrates. Similar results were also obtained with NIH3T3 and HEK293 cells (data not shown). As a reference, we also evaluated the cell adhesiveness of a BSA-adsorbed poly-L-lysine-coated coverslip and found that this substrate also remained cell-adhesive after BSA adsorption (Figure 10b,c). It should be noted that there was the possibility for the replacement of BSA with extracellular matrix (ECM) proteins secreted by the cells even though we had seeded the cells in the serum-free medium. Therefore, the obtained results, shown in Figure 10b,c, might reflect a marked difference in the exchange efficiency of the surface-adsorbed BSA with ECM proteins among the four substrates and a poly-L-lysine-coated coverslip. On the basis of quantitative and qualitative analysis of the surface-adsorbed BSA, we propose a mechanism for the efficient photoactivation of cell adhesion on the caged NH2 substrate

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Figure 10. (a) Phase-contrast images of COS7 cells attached to the preirradiated (uncaged) substrates and a poly-L-lysine-coated coverslip 1 h after seeding. (b) Phase-contrast images of COS7 cells attached to the BSA-adsorbed uncaged substrates and the BSA-adsorbed poly-L-lysine-coated coverslip 1 h after seeding. Each substrate was soaked in PBS containing 10 mg/mL BSA for 1 h at room temperature. (c) Number of COS7 cells (per unit area) attached to BSA-adsorbed uncaged substrates and a poly-L-lysine-coated coverslip 1 h after seeding. Caged substrates were irradiated to produce uncaged substrates and then soaked in PBS containing 10 mg/mL BSA for 1 h at room temperature. PLL denotes a poly-L-lysine-coated coverslip. Error bars represent the standard deviation of five independent experiments.

Figure 11. Quantitative analysis of surface-adsorbed BSA on the substrates having different caged functional groups. (a) Schematic illustrations of procedures for sample preparation. (Left) A caged substrate was coated with BSA (state I) and then irradiated (state II). (Right) A caged substrate was preirradiated to produce an uncaged substrate and then coated with BSA (state III). (b) Fluorescence intensity of the surface-adsorbed BSA visualized by in situ staining with SYPRO Ruby protein blot stain or by prelabeling with Alexa 488. FI, FII, and FIII are the observed and calculated fluorescence intensities for the substrate in states I, II, and III, respectively (see text). Error bars represent the standard deviation for five independent experiments.

(Figure 12). (i) Upon irradiation, BSA adsorbed in advance on the caged groups via hydrophobic interaction is readsorbed onto the uncaged COOH, NH2, OH, and SH groups. (ii) BSA readsorbed onto the uncaged NH2 groups becomes unable to passivate the surface against cell adhesion, whereas BSA readsorbed onto the uncaged COOH, OH, and SH groups was still allowed to passivate the surface as usual. (iii) Consequently, only the caged NH2 substrates can exhibit the high efficiency of photoactivation for cell adhesion. Some research groups have reported on the passivation effect of BSA adsorbed on the surface presenting the NH2 group.32,34,35 However, their conclusions disagree with one another; the surface was found to be noncell-adhesive32 or cell-adhesive,34,35 probably because of the differences in the various surface properties, including the

wettability of the surface and the density of the NH2 group, as well as conditions of the employed buffer such as pH and ionic strength. All of these factors could cause significant differences in the conformation, orientation, and density of surface-adsorbed BSA. In the present work, we have actually observed no passivation activity of BSA on the NH2 group. One of the plausible mechanisms is described as follows. Negatively charged BSA (pI 4.8) in PBS (pH 7.4) should be subject to readsorption to the NH3+ groups of the substrates through electrostatic interactions. The readsorbed BSA might be liable to undergo an exchange with ECM proteins secreted by the seeded cells. In contrast, BSA readsorbed on the other functional groups via hydrophobic interactions might be inert to the exchange. Elucidation of the molecular mechanism for the disappearance of the passivation

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Figure 12. Schematic illustrations of a proposed mechanism for the photoactivation of the caged NH2 substrate for cell adhesion. (a) Side and (b) bird’s eye pictures are depicted. The change in color for BSA molecules from red to blue corresponds to the property change from passivating for cell adhesion to nonpassivating. The possible exchange of BSA adsorbed onto the uncaged NH2 groups with the ECM protein secreted by cells and CO2 molecules produced from the alkylsiloxane after photocleavage are omitted for clarity. The cells and BSA molecules are not drawn to scale.

effect of BSA adsorbed on the uncaged NH2 groups requires detailed information on the structure and density of the surfaceadsorbed BSA, which will be obtained using various analytical methods, including circular dichroism spectroscopy, differential scanning calorimetry, Fourier transform infrared spectroscopy, XPS, and atomic force microscopy. Such attempts are currently under way.

corresponding photomasks. Because the present method is employed under a conventional fluorescence microscope without additional instruments, it is expected to be compatible with advanced fluorescence imaging technologies and hence will be useful in cell biological studies on various cell-cell interactions at the single-cell level.

Conclusions

Experimental Section

In this study, we developed a method for arraying heterotypic cells on a functional cell-culturing substrate where cell adhesion can be spatiotemporally controlled by UV irradiation. The photoactivatable substrate was prepared by chemical modification of a glass coverslip with a silane coupling agent having a caged NH2 group. The substrate was non-cell-adhesive after being coated with BSA but became cell-adhesive by UV irradiation under a fluorescence microscope. In contrast to the caged NH2 substrate, the caged COOH, OH, and SH substrates prepared by similar methods were nonphotoactivatable for cell adhesion. Highly efficient photoactivation on the caged NH2 substrate was attributed to the properties of surface-adsorbed BSA, which practically lost its passivation activity against cell adhesion for only the NH2 groups. Accordingly, the principle of the present methodology is based on the indirect phototriggered property change of a blocking agent adsorbed on the surface of cell-culturing substrates. On the caged NH2 substrate, we succeeded in arraying heterotypic single cells by repeating the UV irradiation in array patterns and the cell seeding, which were optimized for different cell types. In addition, we demonstrated precise control of the geometry of spots and the distance between spots by using the

Reagents and Materials. BSA, paraformaldehyde, casein, and organic solvents were purchased from Wako (Osaka, Japan). Di(Nsuccinimidyl) carbonate, 3-aminopropyl-trimethoxysilane, tetramethoxylsilane, nitrobenzyl bromide, trimethoxysilane, and (3mercaptopropyl)trimethoxysilane were purchased from TCI (Tokyo, Japan). Platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex, as a solution in xylenes (2% Pt) (Karstedt’s catalyst), and poly(allylamine hydrochloride) were purchased from Sigma-Aldrich (St. Louis, MO). Eagle’s minimum essential medium (MEM), Dulbecco’s modified eagle’s medium (DMEM), and tripsin-EDTA were purchased from Sigma (St. Louis, MO). Newborn calf serum (NBCS), horse serum (HS), fetal bovine serum (FBS), SYPRO Ruby protein blot stain, Cell Tracker Green CMFDA, Mito Tracker Red CM-H2Xros, and penicillin-streptomycin solution were purchased from Invitrogen (Carlsbad, CA). HEK293 cells were obtained from JCRB, and COS7 and NIH3T3 cells were obtained from the RIKEN cell bank. Glass coverslips were purchased from Matsunami (0.12-0.17 mm thick, Osaka, Japan), and glass-bottom dishes were obtained from MatTek (Ashland, MA). Poly-L-lysine-coated coverslips were obtained from Iwaki (Funabashi, Japan). The silane coupling agent having a caged COOH group [1-(2-nitrophenyl)ethyl 5-trichlorosilylpentanoate (1)] was synthesized by the method reported previously.46

13094 Langmuir, Vol. 24, No. 22, 2008 1-(2-Nitrophenyl)ethanol (5). Sodium tetrahydroborate (7.0 g, 190 mmol) was slowly added with stirring to a solution of 2-nitroacetophenone (10 g, 61 mmol) in methanol (150 mL) on ice. The mixture was continuously stirred on ice for 30 min and then at room temperature for 2.5 h. The reaction mixture was concentrated by evaporation, and 150 mL of water was added. The product was extracted with chloroform five times, dried with anhydrous MgSO4, filtered, evaporated, and dried in vacuo to give a yellow oil (10 g, 98%). 1H NMR (CDCl3, 400 MHz): δ 1.57-1.59 (d, 3H, J ) 6.4 Hz, CH3), 2.36 (br, 1H, OH), 5.40-5.45 (m, 1H, CH), 7.40-7.45 (t, 1H, ArH), 7.64-7.67 (t, 1H, ArH), 7.84-7.91 (t, 2H, ArH). 1-(2-Nitrophenyl)ethyl N-Succinimidyl Carbonate (6). To a solution of 5 (4.08 g, 24 mmol) in dry acetonitrile (60 mL) containing triethylamine (6 mL) was added di(N-succinimidyl) carbonate (13 g, 50 mmol) under nitrogen. After the solution was mixed at room temperature for 3 h, water (100 mL) and 2 M NaCl (8 mL) were added. The product was extracted with ethyl acetate five times, dried with anhydrous MgSO4, filtered, and purified by chromatography on silica (4:1 hexane/ethyl acetate). Purified fractions were evaporated and dried under vaccum to yield a white solid product (4.1 g, 78%). 1H NMR (CDCl , 400 MHz): δ 1.80 (d, 3H, J ) 6.4 Hz, CH ), 2.80 3 3 (s, 4H, CH2), 6.40 (q, 1H, J ) 6.4 Hz, ArCH), 7.51 (m, 1H, ArH), 7.74 (t, 2H, ArH), 8.03 (d, 1H, J ) 8.6 Hz, ArH). 1-(2-Nitrophenyl)ethyl N-(3-Trimethoxysilylpropyl)-carbamate (2). A solution of 6 (0.58 g, 3.3 mmol) and 3-aminopropyltrimethoxysilane (0.99 g, 3.2 mmol) in dry THF (15 mL) containing triethylamine (3 mL) was stirred under nitrogen at room temperature overnight. The reaction mixture was concentrated and applied to a silica gel chromatography column (8:2:0.025 hexane/ethyl acetate/ tetramethoxylsilane). Purified fractions were evaporated and dried under vaccum to yield a yellow oil (0.67 g, 68%). 1H NMR (CDCl3, 400 MHz): δ 0.61 (t, 2H, J ) 8.2 Hz, SiCH2), 1.61 (m, 5H, CH3, NCH2), 3.13 (m, 2H, CH2), 3.56 (s, 9H, OCH3), 4.95 (br, 1H, NH), 6.25 (q, 1H, J ) 6.4 Hz, ArCH), 7.41 (m, 1H, ArH), 7.61 (m, 2H, ArH), 7.92 (d, 1H, J ) 8.4 Hz, ArH). Allyl 2-Nitrobenzyl Ether (7). Sodium hydride (1.3 g, 54 mmol) was suspended in dry hexane (50 mL) and stirred for 10 min. After decantation, fresh dry hexane (30 mL) was added to the reaction vessel. Allyl alcohol (18 mL) was added to the suspension on ice, and then 2-nitrobenzyl bromide dissolved in allyl alcohol (46 mL) was added. The reaction mixture was stirred at room temperature for 19 h. Then 2 M HCl (10 mL) and water (50 mL) were added, and the product was extracted with chloroform five times, dried with anhydrous MgSO4, concentrated, and vacuum distilled (0.15 mmHg, bp 82 °C) to obtain a yellow liquid (2.32 g, 74%). 1H NMR (CDCl3, 400 MHz): δ 4.12 (m, 2H, OCH2), 4.90 (s, 2H, ArCH2), 5.22-5.38 (m, 2H, CHdCH2), 5.93-6.02 (m, 1H, CHdCH2), 7.41-7.46 (m, 1H, ArH), 7.63-7.67 (m, 1H, ArH), 7.82-7.84 (m, 1H, ArH), 8.05-8.08 (m, 1H, ArH). IR (NaCl) νmax (cm-1): 3081, 2921, 2856 (CH), 1526, 1344 (NO2). 2-Nitrobenzyl 3-(Trimethoxysilyl)propyl Ether (3). To a mixture of 7 (0.55 g, 2.9 mmol) and trimethoxysilane (0.53 g, 4.3 mmol) was added 3 drops of Karstedt’s catalyst under nitrogen. The mixture was stirred at room temperature for 1.5 h and then at 60 °C for 1.5 h. The product was vacuum distilled (0.2 mmHg, bp 160 °C) to obtain a yellow liquid (0.24 g, 27%). 1H NMR (CDCl3, 400 MHz): δ 0.72-0.76 (m, 2H, SiCH2), 1.75-1.82 (m, 2H, CH2), 3.54-3.58 (m, 11H, OCH3, OCH2), 4.88 (s, 2H, ArCH2), 7.41-7.45 (m, 1H, ArH), 7.62-7.66 (m, 1H, ArH), 7.81-7.82 (m, 1H, ArH), 8.05-8.07 (m, 1H, ArH). IR (NaCl) νmax (cm-1): 2942, 2843 (CH), 1527, 1344 (NO2). 2-Nitrobenzyl 3-(Trimethoxysilyl)propyl Sulfide (4). A mixture of (3-mercaptopropyl)trimethoxysilane (1.0 g, 5.1 mmol), 2-nitrobynzyl bromide (1.1 g, 5.0 mmol), and K2CO3 (0.69 g, 5.0 mmol) in 40 mL of dry acetone was refluxed at 80 °C for 7 h under nitrogen. Ethyl acetate (100 mL) was added to the concentrated reaction mixture, and then the precipitate was removed by filtration. The product was purified by silica gel chromatography (6:1:0.025 hexane/ ethyl acetate/tetramethoxylsilane). Purified fractions were evaporated and dried under vaccum to yield a yellow oil (0.71 g, 43%). 1H NMR (CDCl3, 400 MHz): δ 0.70 (t, 2H, J ) 8.0 Hz, SiCH2), 1.62-1.70

Kikuchi et al. (m, 2H, CH2), 2.47 (m, 2H, SiCH2CH2), 3.55 (s, 9H, Si(OCH3)), 4.05 (s, 2H, ArCH2S), 7.41 (t, 1H, J ) 7.4 Hz, ArH), 7.47 (d, 1H, J ) 7.8 Hz, ArH), 7.55 (t, 1H, J ) 7.6 Hz, ArH), 7.95 (d, 1H, J ) 8.2 Hz, ArH). Preparation of Photoactivatable Substrates. Glass coverslips were cleaned in piranha solution (3:1 sulfuric acid/hydrogen peroxide) for 1 h at 100 °C. Caution! Piranha is a Vigorous oxidant and should be used with extreme caution. After being cooled at room temperature, the glass coverslips were rinsed with distilled water and then dried with nitrogen. Alkylsiloxane monolayers with caged functional groups were formed on these cleaned coverslips by refluxing with the corresponding silane coupling agents in dry benzene for 1 h. The substrates were rinsed with methanol and chloroform, sonicated in chloroform for 10 min, and then dried with nitrogen. UV Irradiation. UV irradiation was performed using either an Axiovert 200 fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 10× Plan Apochromat objective lens (Carl Zeiss) or an IX81 fluorescence microscope (Olympus, Tokyo, Japan) with a UPLSAPO objective lens (Olympus). Both microscopes were equipped with a 100 W mercury arc lamp and a 330WB80 (330 ( 40 nm) excitation filter. Emission and excitation filters and dichroic mirrors were purchased from Omega (Brattleboro, VT) or Semrock (Rochester, NY). For patterned irradiation under the fluorescence microscope, a photomask was inserted at the field diaphragm slider of the microscope.26,27 The photomask was prepared by printing a pattern on a transparency. The power of the UV light was measured by using a UIT-150 power meter equipped with a UVD-S365 (Ushio, Tokyo, Japan). Contact Angle Measurements. A piece of the substrate was placed on a glass-bottom dish, irradiated in PBS under the fluorescence microscope, and then rinsed with water, methanol, and dichloromethane. After being sonicated in dichloromethane for 15 min, it was dried with nitrogen. Contact angle measurements were conducted on a DropMaster 500 (Kyowa Interface Science, Saitama, Japan) contact angle meter with a constant drop volume (l µL) of ultrapure water at room temperature. The obtained images were analyzed using FAMAS software (Kyowa Interface Science). XPS Spectroscopy. XPS spectra were taken on a PHI Quantera SXM (ULVAC-PHI, Kanagawa, Japan) equipped with a monochromatized A1 KR focused X-ray source. The binding energy was calibrated to the Au 4f7/2 line at 83.96 eV, and a 26 eV pass energy was used. The energies of all spectra were shifted by correcting with the C 1s peak at 285 eV for energy calibration. Survey spectra, in a binding energy range from 1380 to 0 eV, were obtained at a 280 eV pass energy to evaluate the atomic concentration of the observed elements, and then high-resolution N 1s and Si 2p spectra at a 55 eV pass energy were recorded to evaluate the binding energy. To determine the change in the chemical state of N 1s, we normalized the peak intensity of N 1s to that of Si 2p. The sample preparation procedure was the same as that for the contact angle measurements. Cell Culture. Cells were maintained at continuous growth in an appropriate medium at 37 °C under a humidified atmosphere containing 5% CO2 and subcultured every 2 or 3 days by using 0.25% trypsin-EDTA. The growth medium used for each cell line was DMEM containing 10% FBS for COS7 cells, DMEM containing 10% NBCS for NIH3T3 cells, and MEM containing 10% HS for HEK293 cells. All of the media contained 100 U/mL penicillin and 100 µg/mL streptomycin. Cell Patterning. A cut piece of the substrate (ca. 25 mm2) was placed on the bottom of a glass-bottom dish (φ 35 mm) and soaked in PBS containing 10 mg/mL BSA for 1 h at room temperature. The substrate was then irradiated in a certain pattern in PBS under the fluorescence microscope (λ ) 365 nm, 7600 mJ), as mentioned above. Cells were seeded onto the substrates in a serum-free medium and were allowed to attach for 30 or 60 min at 37 °C in 5% CO2. Unattached cells were removed by washing with serum-free medium, and then the cells were cultured in a serum-containing medium. In coculturing experiments, the substrates were again irradiated just after the washing step, and the second (or third) cell type was seeded with the optimized concentrations. To distinguish each cell type, NIH3T3 and HEK293 cells were fluorescently labeled in advance

PhotoactiVatable Cell-Culturing Substrates with Cell Tracker and Mito Tracker, respectively. All of the microscopic images were captured with a Cool Snap HQ (Photometrics, Tucson, AZ) or a Retiga-Exi (Q-Imaging, Burnaby, BC, Canada) cooled charge-coupled device camera and analyzed by the MetaMorph image processing system (Molecular Devices, Downingtown, PA). Quantitative Analysis of Surface-Adsorbed BSA. The amount of surface-adsorbed BSA was determined from direct observation using the fluorescence microscope. BSA adsorbed on the surfaces was visualized in two different ways-in situ fluorescent staining and fluorescently labeled BSA-to avoid the photobleaching of fluorescently labeled BSA by irradiation and because of the variation in the number of fluorophores attached to BSA in different conformations and/or orientations. In Situ Fluorescent Staining. To determine the change in the amount of surface-adsorbed BSA during the irradiation step (from state I to state II in Figure 11a), we stained BSA with SYPRO Ruby protein blot stain. The substrates coated with BSA were irradiated in a striped pattern. After the BSA remaining on the surface was fixed with 2% paraformaldehyde, the substrate was soaked in a SYPRO Ruby protein blot stain solution for 30 min at room temperature and washed with PBS five times prior to microscopic observation. The filter settings for fluorescence microscopy were FF438/24, 455DRLP, and 595AF60. The amount of surface-adsorbed BSA before and after UV irradiation was determined from the fluorescence intensity in nonirradiated (FSYPRO, before) and irradiated regions (FSYPRO, after irr) after background subtraction. Use of Fluorescently Labeled BSA. The amount of adsorbed BSA in state I (FI) and state III (FIII) in Figure 11 was determined by using fluorescently labeled BSA, which was obtained by mixing BSA (25 mg/mL in PBS, pH 8.3) and Alexa 488 succimidyl ester (10 mg/mL in DMSO) in a 3:1 ratio (v/v) in the dark for 1 h at room temperature with gentle stirring. The coupling reaction was stopped by the addition of 100 µL of a 1.5 M hydroxyl amine solution. The reaction mixture

Langmuir, Vol. 24, No. 22, 2008 13095 was gel filtered with a NAP-10 column (GE Healthcare, Buckinghamshire, U.K.) to give BSA labeled with an average of 1.3 dye molecules/protein. The substrates irradiated in advance under the fluorescence microscope with a striped pattern were soaked in PBS containing the labeled BSA (10 mg/mL BSA) for 1 h at room temperature. After washing with PBS five times, the fluorescence intensity in irradiated (FAlexa, irr) and nonirradiated regions (FAlexa, nonirr) was used to determine the surface-adsorbed BSA after background subtraction. The filter settings were 485DF15, 505DRLPXR, and FF542/27. To compare the fluorescence intensity obtained with Alexa 488 in the second method to that obtained with SYPRO Ruby protein blot stain in the first method, we did the same experiment with nonlabeled BSA, stained the substrate with SYPRO dyes, and measured the fluorescence signal in irradiated (FSYPRO, irr) and nonirradiated regions (FSYPRO, nonirr). The bar graphs shown in Figure 11b were calculated using the following equations:

FI ) FAlexa, nonirr FII ) FSYPRO, after irr ×

FAlexa irr FSYPRO irr

FIII ) FAlexa, irr Acknowledgment. This work was supported by a grant from Ecomolecular Science Research, provided by RIKEN (to T.T. and M.M.) and by the High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.Y.). Y.K. also received support from a research fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. LA8024414