Simple and Biocompatible Micropatterning of Multiple Cell Types on a

Nov 4, 2010 - Radiation Research Division for Industry and Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institut...
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Simple and Biocompatible Micropatterning of Multiple Cell Types on a Polymer Substrate by Using Ion Implantation In-Tae Hwang, Chan-Hee Jung, Jae-Hak Choi,* and Young-Chang Nho Radiation Research Division for Industry and Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si, Jeollabuk-do 580-185, Republic of Korea Received September 1, 2010. Revised Manuscript Received October 19, 2010 A noncytotoxic procedure for the spatial organization of multiple cell types remains as a major challenge in tissue engineering. In this study, a simple and biocompatible micropatterning method of multiple cell types on a polymer surface is developed by using ion implantation. The cell-resistant Pluronic surface can be converted into a cell-adhesive one by ion implantation. In addition, cells show different behaviors on the ion-implanted Pluronic surface. Thus this process enables the micropatterning of two different cell types on a polymer substrate. The micropatterns of the Pluronic were formed on a polystyrene surface. Primary cells adhered to the spaces of the bare polystyrene regions separated by the implanted Pluronic patterns. Secondary cells then adhered onto the implanted Pluronic patterns, resulting in micropatterns of two different cells on the polystyrene surface.

Introduction The fates and functions of cells critically depend on their diverse microenvironmental factors, including cell-cell interactions, cell-matrix interactions, and mechanical forces.1-3 To design and manipulate the cell microenvironment, surface micropatterning and engineering techniques have been extensively studied. However, the micropatterning of multiple cell types is essential for the creation of a more sophisticated cellular microenvironment that mimics the in vivo function.4-6 To date, a variety of surface micropatterning techniques, such as inkjet printing, photolithography, and microcontact printing, has been studied to generate patterned cell cocultures.7-10 However, these techniques require multiple steps to form the patterned surfaces and are not suitable for culture cells because of the use of toxic organic solvent, limitation of pattern size, and contamination of the stamp surface during the patterning process, even though they generate well-defined patterned surfaces. Therefore, a simple and biocompatible procedure for the micropatterning of multiple cell types on a substrate must be developed. Ion implantation through a pattern mask is an attractive method for the micropatterning of cells on a substrate because of its numerous advantages, including easy and precise process control, low temperature processing, reliability, and nontoxic processing without the use of harsh chemicals. Moreover, *To whom correspondence should be addressed. Phone: þ82-63-570-3062. Fax: þ82-63-570-3090. E-mail: [email protected].

(1) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063. (2) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2480–2487. (3) Ito, Y. Biomaterials 1999, 20, 2333–2342. (4) Buess, M.; Nuyten, D.; Hastie, T.; Nielsen, T. B.; Pesich, R.; Brown, P. GenomeBiology 2007, 8, R191. (5) Gurdon, J. B.; Lemaire, P.; Kato, K. Cell 1993, 75, 831–834. (6) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. FASEB J. 1999, 13, 1883–1900. (7) Yamazoe, H.; Okuyama, T.; Suzuki, H.; Fukuda, J. Acta Biomater. 2010, 6, 526–533. (8) Goubko, C. A.; Cao, X. Mater. Sci. Eng., C 2009, 29, 1855–1868. (9) Co, C. C.; Wang, Y. C.; Ho, C. C. J. Am. Chem. Soc. 2005, 127, 1598–1599. (10) Yousaf, M. N.; Houseman, H. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S. A. 2001, 98, 5992–5996.

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although other techniques based on electron beams, UV-light, γ-ray, and X-ray can be used in the structural modification of polymers, the ion beam technique is more effective in the structural modification of polymers compared to these techniques, given that an ion beam has greater linear energy transfer and its penetration trajectory is fairly straight in comparison to the other techniques.11,12 Therefore, microstructures formed by ion implantation have been widely used to spatially control the adhesion and proliferation of a single cell.13,14 However, a spatial distribution of multiple cell types by using ion implantation has not yet been studied. Poly(ethylene oxide) (PEO)-containing molecules, such as the Pluronic used in this study, have been widely used as cell-resistant polymers because of their simplicity and effectiveness in resisting the adsorption of cells through hydrophilicity, chain mobility, and high steric hindrance.15-18 It is well-known that PEO can be cross-linked by a high energy irradiation. Therefore, the patterning of PEO-containing molecules by irradiation such as electron beams for biological applications has been reported.19-23 However, the micropatterning of multiple cell types on a PEOpatterned substrate has not been reported because a noncytotoxic method has not yet been developed to alter cell-resistant PEO, (11) Dong, H.; Bell, T. Surf. Coat. Technol. 1999, 111, 29–40. (12) Watt, F.; Bettiol, A. A.; Van Kan, J. A.; Teo, E. J.; Breese, M. B. H. Int. J. Nanosci. 2005, 4, 269–286. (13) Amato, I.; Ciapetti, G.; Pagani, S.; Marletta, G.; Satriano, C.; Baldini, N.; Granchi, D. Biomaterials 2007, 28, 3668–3678. (14) Bacakova, L.; Mares, V.; Lisa, V.; Svorcik, V. Biomaterials 2000, 21, 1173– 1179. (15) Neff, J. A.; Caldwell, K. D.; Tresco, P. A. J. Biomed. Mater. Res. 1998, 40, 511–519. (16) Neff, J. A.; Tresco, P. A.; Caldwell, K. D. Biomaterials 1999, 20, 2377–2393. (17) Koegler, W. S.; Griffith, L. G. Biomaterials 2004, 25, 2819–2830. (18) Wang, Y. Q.; Su, Y. L.; Ma, X. L.; Sun, Q.; Jiang, Z. Y. J. Membr. Sci. 2006, 283, 440–447. (19) Kofinas, P.; Athanassiou, V.; Merrill, E. W. Biomaterials 1996, 17, 1547– 1550. (20) Rosiak, J. M.; Ulanski, P. Radiat. Phys. Chem. 1999, 55, 139–151. (21) Sofia, S. J.; Merrill, E. W. J. Biomed. Mater. Res. 1998, 40, 153–163. (22) Krsko, P.; Sukhishvili, S.; Mansfield, M.; Clancy, R.; Libera, M. Langmuir 2003, 19, 5618–5625. (23) Christman, K. L.; Schopf, E.; Broyer, R. M.; Li, R. C.; Chen, Y.; Maynard, H. D. J. Am. Chem. Soc. 2009, 131, 521–527.

Published on Web 11/04/2010

DOI: 10.1021/la103474s

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defining the arrangement of one cell type, into cell-adhesive PEO to enable the adhesion of the other cell types. In this study, a simple and biocompatible method for the control of the spatial distribution of multiple cell types on a Pluronicpatterned substrate by ion implantation is reported for the first time. The efficacy of this method is demonstrated by culturing two different cell types on a Pluronic-patterned substrate.

Experimental Section Materials. PEO-PPO-PEO triblock copolymer (PPO = poly(phenylene oxide); Pluronic F127, Mw: 12 600) and ethanol were purchased from the Aldrich Chemical Co. A tissue culture polystyrene Petri dish (TCPS) was obtained from SPL Life Science. Other chemicals were used as received. A pattern mask (100 μm spaces/300 μm pitches) was supplied by Youngjin Astech Co., Ltd. (Figure S1, Supporting Information).

Figure 1. Schematic diagram showing the preparation of the micropatterned Pluronic surface and the micropatterned cell coculture.

Micropatterning of Cell-Resistant Pluronic Films on a TCPS. To obtain Pluronic micropatterns, thin Pluronic films were prepared by spin-coating a 10 wt % Pluronic solution in distilled water on a TCPS and drying in a vacuum oven for 24 h. The prepared Pluronic films were implanted with 200 keV Hþ ions at fluences ranging from 1  1014 (14 s) to 7.5  1014 ions/cm2 (1308 s) through a pattern mask at room temperature. Ion implantation was executed by using a 300-keV ion implanter at the Advanced Radiation Technology Institute (ARTI, Republic of Korea). The ion current density was less than 1.0 μA/cm2 to prevent a thermal effect on the samples. The pressure in the implanter’s target changer was 10-5 to 10-6 Torr. Afterward, to generate the patterns of the Pluronic, the implanted films were developed with ethanol and then dried in a N2 stream. The Pluronic micropatterns on the TCPS were investigated using an optical microscope (Type 020-519, Leica, Germany). Characterizations. The surface profiles of the Pluronic patterns were measured with a Nanosurface 3D Optical Profiler (NanoSystem, Korea). The chemical structure of the Pluronic was investigated by using an attenuated total reflectance Fourier transform infrared spectroscope (ATR-FTIR, Tensor 37, Bruker Co., USA). The effect of ion implantation on the chemical compositions of the Pluronic was analyzed by an X-ray photoelectron spectrometer (XPS, MultiLab 2000, ThermoElectron Corporation, England) employing MgKR radiation. The applied power was 14.5 keV and 20 mA, and the base pressure in the analysis chamber was less than 10-9 mbar. In Vitro Cell Culture on Pluronic-Patterned TCPS. In this study, NIH 3T3 (mouse fibroblast), L929 (murine fibroblast), and HaCaT (human keratinocyte) were used. The preconfluent cells were trypsinized and then pipetted several times to disperse them into a single cell. Prior to cell culture, the Pluronic-patterned TCPS was sterilized with 70% ethanol. Cells were seeded on the Pluronic-patterned TCPS at a density of 1  105 cells/well and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO2 in a humidified incubator. During cell culturing, cell adhesion and growth were observed with an optical microscope (Type 020-519, Leica, Germany). Cell Proliferation Assay. NIH3T3 cells (5  104 cells/well) were seeded onto the Pluronic-patterned substrates implanted at fluences ranging from 11014 ions/cm2 to 7.51014 ions/cm2. The cells were incubated for 4 days and harvested daily. The cells were stained with trypan blue (Gibco-Invitrogen) and counted using a hematocytometer. This experiment was performed in triplicate. Cell Staining and Patterned Cell Coculture. NIH3T3 and HaCaT cells were stained with PKH26 dye (Sigma Aldrich). L929 cells were distinguished by staining with CFSE dye (carboxyfluorescein diacetate succinimidyl ester, Sigma Aldrich). The staining procedure involves detaching cells from cell culture dishes, 18438 DOI: 10.1021/la103474s

Figure 2. Optical microscopic images of Pluronic patterns on a TCPS formed by ion implantation at fluences of 1  1014 (a), 2.5  1014 (b), 5  1014 (c), and 7.5  1014 ions/cm2 (d). The insets in b and c magnify the dotted rectangle in each respective figure. centrifuging them to obtain cell pellets, washing them in a serum-free medium, centrifuging them again to remove the medium, resuspending them within the staining solution, and incubating them for 10 min at room temperature. To terminate the staining, a volume of the culture medium equal to that of the cell solution was added to the cell suspension and incubated for 1 min at room temperature. After centrifugation for 3 min at 3000 rpm, the supernatant was removed and replaced with fresh medium for washing the cells. The stained cells were washed three times with fresh medium. A Pluronicpatterned TCPS was used for the patterned coculture with NIH3T3, HaCaT, and L929 cells. First, the red-stained NIH3T3 or HaCaT cells with a density of 1  105 cells/well were seeded on the Pluronicpatterned TCPS and then incubated for 2 days. The medium was replaced with fresh DMEM including 1  105 cells/well of the greenstained L929 cells on the primary cell prepatterned TCPS as the secondary cell. The resulting cell cocultures were visualized with a fluorescence microscope (BX61, Olympus). The fluorescence images of cells were merged with Image-J software.

Results and Discussion Formation of Well-Organized Pluronic Patterns on the TCPS Surface. Figure 1 shows the schematic representation for the micropatterned cell-coculture. Thin Pluronic films were prepared by spin-coating, and the micropatterns of the Pluronic were generated by a selective ion implantation. Primary cells were cultured on a Pluronic-patterned TCPS for the patterning of primary cells. Secondary cells were then cultured on the primary cell-patterned surface for the micropatterned cell coculture. Micropatterns of Pluronic were formed on a TCPS by proton irradiation through a pattern mask (100 μm space and 300 μm pitch) and followed by development in ethanol. Figure 2 shows the formed Pluronic patterns on the TCPS. It was clearly observed from Figure 2b-e that well-defined 100 μm patterns of the Pluronic were formed on the TCPS at all fluences. Furthermore, Langmuir 2010, 26(23), 18437–18441

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Figure 3. Surface profiles of the Pluronic patterns as a function of the fluence.

as shown in Figure 3, the remaining thickness of the Pluronic after the development was quite similar at fluences of 1 and 2.5  1014 ions/cm2. However, at a fluence above 5  1014 ions/cm2, the thickness decreased gradually. This reduction in the thickness of the Pluronic can be attributed to the fact that the gaseous species such as H2 as generated by ion implantation-induced cleavage of the covalent bonds in Pluronic were liberated. Therefore, the thickness of the Pluronic decreased.24,25 These results indicate that Pluronic was effectively cross-linked even at a fluence of 1  1014 ions/cm2, thereby resulting in the formation of the wellorganized Pluronic patterns on the TCPS surface. The ATR-FTIR spectra of the pristine and implanted Pluronic films are shown in Figure 4. In the spectrum of the pristine Pluronic (Figure 4a), the peaks corresponding to the C-H and C-O of the Pluronic backbone were identified at 2871 and 1087 cm-1, respectively.26-28 In the case of the implanted Pluronic surfaces, the peaks assigned to -OH and CdO groups were newly generated in the ATR-FTIR spectra, and their intensities were gradually increased with an increasing fluence (Figure 4b-e). This formation of the functional groups on implanted surfaces such as -OH and CdO can be attributed to oxidation after ion implantation. The ion implantation effect on the chemical compositions of the Pluronic surface was investigated by XPS analysis. The surface chemical compositions of the pristine and implanted Pluronic films are presented in Figure 5. As shown in Figure 5a, the C1s spectra of the pristine Pluronic surface showed the characteristic peaks at 283.8 eV (hydrocarbon (-C-CH3) bond of methyl group), 285.0 eV (hydrocarbon (-C-C-)), and 286.0 eV (ether carbon -C-O-) .29,30 After ion implantation, the intensities of these peaks were noticeably changed with the fluence, as shown in Figure 5b-e. The intensity of the peak for the C-C bond was decreased with an increasing fluence, while those of the peaks for C-O bond and the -C-CH3 bonds were increased, indicating that the chemical environment was changed by ion implantation. This chemical change in the implanted Pluronic surfaces could be ascribed to the formation of a network structure in the Pluronic caused by hydrogen abstraction and interpolymer radical coupling during implantation and oxidation (24) Wang, J.; Rangel, E. C.; Cruz, N. C.; Swart, J. W.; Moraes, M. A. B. Nucl. Instrum. Methods Phys. Res., Sect. B 2000, 166-167, 420–425. (25) Wang, Y. Q. Nucl. Instrum. Methods Phys. Res., Sect. B 2000, 161-163, 1027–1032. (26) Su, Y.-L.; Wang, J.; Liu, H. Z. Macromolecules 2002, 35, 6426–6431. (27) Wang, Y.; Su, Y.; Sun, Q.; Ma, X.; Ma, X.; Jiang, Z. J. Membr. Sci. 2006, 282, 44–51. (28) Morent, R.; De Geyter, N.; Leys, C.; Gengembre, L.; Payen, E. Surf. Interface Anal. 2008, 40, 597–600. (29) Zhang, C.; Easteal, A. J. Macromol. Chem. Phys. 2008, 209, 1220–1231. (30) Mao, C.; Liang, C. X.; Mao, Y. Q.; Li, L.; Hou, X. M.; Shen, J. Colloids Surf., B: Biointerfaces 2009, 74, 362–365.

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Figure 4. ATR-FTIR spectra of the pristine Pluronic film (a) and the Pluronic films implanted at fluences of 1  1014 (b), 2.5  1014 (c), 5  1014 (d), and 7.5  1014 ions/cm2 (e).

reaction occurring between the generated radicals and the oxygen present in the air.31,32 The characterization results of the patterned Pluronic revealed that the Pluronic was effectively cross-linked and hydrophilic functional groups were generated on the surface of the implanted Pluronic. The plausible mechanism for the chemical changes in the Pluronic induced by ion implantation is illustrated in Figure S2 in the Supporting Information. In Vitro Cell Culture on Pluronic-Patterned TCPS. In order to investigate cell behaviors, two types of cells (NIH3T3: mouse fibroblast; HaCaT: human keratinocyte) were in vitro cultured on the Pluronic-patterned TCPS substrates prepared at ion fluences between 1  1014 and 7.5  1014 ions/cm2. Cell attachment and proliferation behaviors were observed by an optical microscope, and the results are shown in Figure 6. As a result, all the cultured cells adhered predominantly onto the 200 μm spaces of the bare TCPS regions separated by 100 μm lines of cell-resistant Pluronic patterns prepared at a fluence below 2.5  1014 ions/cm2. However, at a fluence above 5  1014 ions/cm2, the cells began to randomly adhere and grow onto the Pluronic lines. This fluence-dependent phenomenon could be explained as follows: at fluences of 1  1014 and 2.5  1014 ions/cm2, cell adhesion could be effectively inhibited by the PEO units in the Pluronic, which is well-known as a cell-resistant material and, therefore, resulted in the formation of well-defined cell patterns. However, at fluences of 5  1014 and 7.5  1014 ions/cm2, the chemical changes and cross-linking of the Pluronic caused by ion implantation could deteriorate the cell-resistant property of the PEO units in the Pluronic backbone, thereby resulting in cell adhesion and growth onto the entire Pluronic surface. Therefore, well-defined cell micropatterns were not obtained at fluences above 5  1014 ions/cm2. These results mean that the ion implantation altered the cell-resistant Pluronic surface into a cell-adhesive one due to the formation of a cross-linked network and hydrophilic functional groups in the implanted Pluronic, and indicate that a Pluronicpatterned TCPS can provide a suitable template for the spatial control of cell adhesion and cell growth. To further examine the cell response to the implanted Pluronic surfaces, a cell proliferation assay was carried out on the implanted Pluronic at various fluences (Figure 7). The proliferation rate of cells was increased with an increasing fluence from 1  1014 to 1  1015 ions/cm2. In the case of the Pluronic implanted at a (31) Krsko, P.; Sukhishvili, S.; Mansfield, M.; Clancy, R.; Libera, M. Langmuir 2003, 19, 5618–5625. (32) Christman, K. L.; Dorbatt, V. V.; Schopf, E.; Kolodziej, C. M.; Li, R. C.; Broyer, R. M.; Chen, Y.; Maynard, H. D. J. Am. Chem. Soc. 2008, 130, 16585– 16591.

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Figure 5. C1s spectra of the pristine Pluronic film (a) and the Pluronic films implanted at fluences of 1  1014 (b), 2.5  1014 (c), 5  1014 (d), and 7.5  1014 ions/cm2 (e).

Figure 6. Optical microscopic images of the NIH3T3 (a) and HaCaT (b) cells on pristine TCPS and Pluronic-patterned TCPS at different fluences.

fluence of 1  1015 ions/cm2, the proliferation rate was almost the same as that of the TCPS surface, indicating that the implanted Pluronic surface showed almost the same affinity as the pristine TCPS surface to the cells. Interestingly, the Pluronic surface implanted at a fluence of 2.5  1014 ions/cm2 initially exhibited a cell-resistant property with a culturing time of up to 2 days after cell seeding, during which time it was gradually changed into a cell-adhesive property. Therefore, these fluence-dependent behaviors of the cells on the implanted Pluronic surfaces revealed that the cell-resistant surface of the Pluronic could be alterable to a cell-adhesive surface with the fluence, which could suggest a fascinating cue for the feasibility of multiple cell patterning. Patterned Cell Coculture. By utilizing these fluence- and time-dependent responses of the cells on the implanted Pluronic, cocultures of two different cell lines were constructed where the two types of cells were spatially separated. Fluorescence microscopic images show the results of cocultures with two different types of cells in Figure 8. The primary cells stained with PKH26 (red), NIH3T3, or HaCaT were cultured on the Pluronic-patterned TCPS for about 3 days to generate well-defined 200 μm cell patterns. Subsequently, the secondary cells, L929, were seeded and cultured to adhere and grow onto the remaining Pluronic surfaces between the precultured cell patterns. Red-stained primary cells were preferentially attached and proliferated onto the bare TCPS surfaces separated by the implanted Pluronic lines, resulting in well-defined patterns of the primary cells. 18440 DOI: 10.1021/la103474s

Figure 7. Proliferation curves of the NIH3T3 cells cultured on the Pluronic surfaces implanted at different fluences.

In coculturing with secondary cells, green-stained L929 cells adhered and proliferated predominantly onto the surface of the Pluronic lines. Therefore, as shown in Figure 8, well-organized micropatterns of two different cell types were generated on the Pluronic-patterned surface at a fluence of 2.5  1014 ions/cm2. Well-defined patterns of two different cell types were formed on the Pluronic-patterned TCPS at lower fluences than 2.5  1014 ions/cm2, but the cell population of cells on the Pluronicpatterned TCPS at a fluence of 2.5  1014 ions/cm2 was much higher than that at a fluence of 1.5  1014 ions/cm2. However, the Langmuir 2010, 26(23), 18437–18441

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Figure 8. Fluorescence images of the patterned cell coculture on the Pluronic-patterned TCPS surfaces.

patterns of cocultured cells formed at fluences of 3.5 and 4.5  1014 ions/cm2 became merged and more obscure due to the random attachment of the cells onto the implanted Pluronic lines during the initial seeding of the primary cells (Figure S3, Supporting Information). Therefore, these results demonstrate that this approach based on the Pluronic-patterned surface is effective for the creation of the micropatterned cell coculture. The plausible mechanism for the formation of the coculture on the Pluronic-patterned TCPS surface could be ascribed to the change in the antibiofouling property of the Pluronic with the fluence. Thus, especially for a Pluronic-patterned surface at a fluence of 2.5  1014 ions/cm2, the reason for the most distinct micropatterned cocultures could be speculated as follows. The proteins present in the culture medium and secreted by the cells were adsorbed onto the implanted Pluronic surface during cell culturing. The proteins adsorbed by the Pluronic surfaces provided a better environment for cell adhesion, and, therefore, the Pluronic surfaces were more cell-adhesive than the primary cellpreoccupied regions owing to the weakened nonfouling nature of the Pluronic by ion implantation.33,34

Conclusions The micropatterns of a cell-resistant Pluronic were formed by using a selective ion implantation. Primary cells were then (33) Fukuda, J.; Khademhosseimi, A.; Yeo, Y.; Yang, X.; Yeh, J.; Eng, G.; Blumling, J.; Wang, C. F.; Kohane, D. S.; Langer, R. Biomaterials 2006, 27, 5259–5267. (34) Jesus, M.; Fuente, D. L.; Andar, A.; Gadegaard, N.; Berry, C. C.; Kingshott, P.; Riehle, M. O. Langmuir 2006, 22, 5528–5532.

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cultured on the Pluronic-patterned TCPS and, subsequently, secondary cells were seeded and proliferated on the primary cell-patterned TCPS. Finally, the micropatterns of two different cells were formed on a substrate using fluence-dependent cell behaviors. In comparison to other techniques for micropatterning two different cells, this procedure offers the merits such as easy and reliable processing and high biocompatibility without harsh chemicals, which make it quite beneficial in the preparation of a platform for the formation of well-defined patterns of multiple cell types. Thus, this convenient and biocompatible technique can be potentially employed as a useful tool for the study of cell biology, tissue regeneration, and the development of cell-based biosensors and drug discovery. Acknowledgment. This research was supported by the Nuclear R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology, Korea. Supporting Information Available: The layout and photograph of the stainless steel mask used in the ion implantation process, the plausible mechanism of the ion implantationinduced changes in the chemical structure of the Pluronic, and fluorescent microscopic images of micropatterned cell coculture with two different cells at different fluences. This material is available free of charge via the Internet at http:// pubs.acs.org.

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