pubs.acs.org/Langmuir © 2010 American Chemical Society
Spatial Control of Cell Adhesion and Patterning through Mussel-Inspired Surface Modification by Polydopamine Sook Hee Ku, Joon Seok Lee, and Chan Beum Park* Department of Materials Science and Engineering, KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 335 Science Road, Daejeon 305-701, South Korea Received July 15, 2010. Revised Manuscript Received August 23, 2010 The spatial control and patterning of mammalian cells were achieved by using the universal adhesive property of mussel-inspired polydopamine (PDA). The self-polymerization of dopamine, a small molecule inspired by the DOPA motif of mussel foot proteins, resulted in the formation of a PDA adlayer when aqueous dopamine solution was continuously injected into poly(dimethylsiloxane) microchannels. We found that various cells (fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3) predominantly adhered to PDA-modified regions, maintaining their normal morphologies. The cells aligned in the direction of striped PDA patterns, and this tendency was not limited by the type of cell line. Because PDA modification does not require complex chemical reactions and is applicable to any type of material, it enables cell patterning in a simple and versatile manner as opposed to conventional methods based on the immobilization of adhesive proteins. The PDA-based method of cell patterning should be useful in many biomaterial research areas such as the fabrication of tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions.
Introduction The spatial organization and control of living cells are important for the development of tissue engineering scaffolds and biochips as well as for the investigation of cell behaviors according to different surface properties.1-3 To facilitate the patterning of cells, different substrates have been functionalized through the alteration of cell-adhesive and cell-nonadhesive regions. Adhesive domains are often achieved by the covalent immobilization or physical adsorption of extracellular matrix proteins on a defined area and surrounding surfaces are coated with nonfouling materials such as poly(ethylene glycol),4-6 but current methods for the fabrication of protein-patterned substrates have suffered from many problems. For example, protein immobilization can be applied only to limited types of supporting substrates and requires multiple complex reactions for covalent bonding. In the case of the noncovalent adsorption of proteins, cell patterns show low stability under serum-containing culture conditions.7 Thus, it is critical to develop an uncomplicated pattern fabrication method that is applicable to any type of material and shows long-term stability. Herein we introduce a biomimetic surface chemistry inspired by the mussel adhesion mechanism to develop a simple, versatile method for the fabrication of highly stable and cell-adhesive micropatterns. Mussels can attach to virtually any type of organic *Corresponding author. Tel: þ82 42 350 3340. Fax: þ82 42 350 3310. E-mail:
[email protected].
(1) Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863– 866. (2) Hatakeyama, H.; Kikuchi, A.; Yamato, M.; Okano, T. Biomaterials 2007, 28, 3632–3643. (3) Liu, W. F.; Chen, C. S. Adv. Drug Delivery Rev. 2007, 59, 1319–1328. (4) Ceriotti, L.; Buzanska, L.; Rauscher, H.; Mannelli, I.; Sirghi, L.; Gilliland, D.; Hasiwa, M.; Bretagnol, F.; Zychowicz, M.; Ruiz, A.; Bremer, S.; Coecke, S.; Colpo, P.; Rossi, F. Soft Matter 2009, 5, 1406–1416. (5) Lan, S.; Veiseh, M.; Zhang, M. Biosens. Bioelectron. 2005, 20, 1697–1708. (6) Takahashi, H.; Emoto, K.; Dubey, M.; Castner, D. G.; Grainger, D. W. Adv. Funct. Mater. 2008, 18, 2079–2088. (7) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063.
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or inorganic material; this universal adhesive property is attributed to the repeated 3,4-dihydroxy-L-phenylalanine (DOPA) motif in mussel foot proteins.8,9 Inspired by this motif, small molecules containing catecholamine functional groups, such as dopamine and norepinephrine, have been used to mimic mussel adhesion.10,11 We fabricated chemically patterned substrates for cell patterning by using mussel-inspired surface modification. To avoid cell adhesion onto unmodified sites, nonadhesive poly(dimethylsiloxane) (PDMS) was used as a model substrate. The chemical patterns were prepared via the self-polymerization process of dopamine to polydopamine (PDA) and were formed on the PDMS surface by continuously injecting aqueous dopamine solution through microchannels; the resulting PDA layer served as a cell-adhesive region. The formation of a PDA adlayer was characterized by using optical microscopy and Raman spectroscopy. To investigate the applicability of the PDA pattern to different mammalian cells, the following cell lines were tested in this study: human fibrosarcoma HT1080, which has been used for the high-throughput screening of anticancer drugs;12 mouse preosteoblast MC3T3-E1, which has been intensively studied with regard to bone tissue engineering and cell-material interaction;13,14 and mouse fibroblast NIH-3T3, which has been widely used as a model cell line for cell-based devices.15,16 We found that the cells selectively adhered only to the PDA-modified regions when they were seeded onto PDA-patterned PDMS (8) Waite, J. H. Integr. Comp. Biol. 2002, 42, 1172–1180. (9) Waite, J. H.; Qin, X. Biochemistry 2001, 40, 2887–2893. (10) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426–430. (11) Kang, S. M.; Rho, J.; Choi, I. S.; Messersmith, P. B.; Lee, H. J. Am. Chem. Soc. 2009, 131, 13224–13225. (12) Nair, R. R.; Avila, H.; Ma, X.; Wang, Z.; Lennartz, M.; Darnay, B. G.; Boyd, D. D.; Yan, C. Mol. Pharmacol. 2008, 73, 919–929. (13) Jiang, T.; Abdel-Fattah, W. I.; Laurencin, C. T. Biomaterials 2006, 27, 4894–4903. (14) Das, K.; Bose, S.; Bandyopadhyay, A. Acta Biomater. 2007, 3, 573–585. (15) Jain, T.; Muthuswamy, J. Lab Chip 2007, 7, 1004–1011. (16) Yu, Z. T. F.; Kamei, K.-I.; Takahashi, H.; Shu, C. J.; Wang, X.; He, G. W.; Silverman, R.; Radu, C. G.; Witte, O. N.; Lee, K.-B.; Tseng, H.-R. Biomed. Microdev. 2009, 11, 547–555.
Published on Web 08/31/2010
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substrates. According to our results, adhered cells exhibited wellorganized cytoskeletons with normal morphology and were aligned in the direction of the PDA patterns. The mussel adhesion-inspired cell patterning approach could contribute to the fabrication of 3D tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions.
Experimental Section Materials. Dopamine and negative photoresist (SU-8) were purchased from Sigma-Aldrich (St Louis, MO) and MicroChem (Newton, MA), respectively. PDMS (DowCorning, Midland, MI) elastomer was prepared by mixing a precursor and a curing agent in a ratio of 10:1 (w/w) and curing at 60 °C for 12 h. Fabrication of the Polydopamine (PDA) Micropattern. Microchannels were fabricated using the replica molding process in the literature.17-19 Briefly, a negative photoresist (SU-8) was spun onto a silicon wafer and patterned using conventional photolithography. Microchannels were then obtained by PDMS molding on the patterned silicon wafer. The dimension of each microchannel was 100 μm (width) 100 μm (height) 15 000 μm (length). A flat PDMS substrate was also prepared and ultrasonically cleaned in ethanol before PDA coating. PDMS microchannels were physically pressed onto the PDMS substrate with aluminum slabs and screws for easy detachment. For the PDA coating, an aqueous dopamine solution (2 mg/mL in 10 mM Tris buffer, pH 8.5) was continuously introduced into the microchannels using an 11 Plus syringe pump (Harvard Apparatus, Holliston, MA) at a flow rate of 10 μL/h for 24 h at room temperature. After the PDA coating, the PDMS substrate was separated from the microchanneled PDMS film and washed with Tris buffer and water. The PDA coating was characterized using optical microscopy (Eclipse TS100, Nikon, Japan) and Raman spectroscopy (LabRAM HR UV/vis/NIR, Horiba Jobin Yvon, France). Raman spectra were obtained by accumulating 50 scans with a resolution of 2 cm-1 in the range of 1200-1800 cm-1. Cell Culture. Three different types of cell lines were tested in this study: human fibrosarcoma HT1080 (Korean cell line bank, Korea), mouse preosteoblast MC3T3-E1 (ATCC, Manassas, VA), and mouse fibroblast NIH-3T3 (Korean cell line bank, Korea). The cell lines were maintained in MEM (Welgene, Korea), a-MEM (Gibco, Carlsbad, CA), and DMEM (Welgene, Korea) medium, respectively. All media contained 10% FBS (Welgene, Korea) and 1% antibiotics (Gibco, Carlsbad, CA). Cells were subcultured at least twice a week and were maintained in a humidified atmosphere of 95% air and 5% CO2. Plating Cells on PDA-Micropatterned PDMS. For the cell patterning, the PDA-patterned PDMS substrates were placed in a 12-well plate and cells were seeded at a specified cell density. The cell density and culture time were as follows: for HT1080, 9 104 cells/well, 4 days; for MC3T3-E1, 1.5 105 cells/well, 2 days; and for NIH-3T3, 1.5 105 cells/well, 2 days. The grown cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with rhodamine-phalloidin and Hoechst. The actin filaments and nuclei were observed using a laser scanning confocal microscope (LSM510, Carl Zeiss, Germany) or fluorescence microscope (Eclipse 80i, Nikon, Japan). The nucleus angle and the relative intensity of fluorescence from actin staining were analyzed using Image J software (freely available at http://rsb.info.nih.gov/ij/).
Results and Discussion For the spatial control of cell adhesion, we fabricated PDA micropatterns on a hydrophobic PDMS surface that is resistant to (17) Lee, J. S.; Ryu, J.; Park, C. B. Anal. Chem. 2009, 81, 2751–2759. (18) Lee, J. S.; Um, E.; Park, J. K.; Park, C. B. Langmuir 2008, 24, 7068–7071. (19) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575.
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the attachment of cells (Figure 1A). PDMS itself has a low surface energy with a hydrophobic nature, thus it is highly resistant to cell adhesion.20,21 After surface modification, such as oxygen plasma treatment, PDMS becomes hydrophilic and possesses a cell-adherent property.20,22 In this work, we used PDMS as a nonadherent substrate to repel cell attachment and modified it with a PDA coating to enhance cell adhesion. When an aqueous dopamine solution (2 mg/mL in Tris buffer, pH 8.5) was introduced into microchannels at room temperature, PDA micropatterns having a 100 μm width with 120 μm spacing were formed via the self-polymerization of dopamine. The polymerization of dopamine is known to occur via oxidation and intramolecular rearrangement, resulting in a melanin-like structure (Figure S1 in the Supporting Information).10,23-25 According to our observation using an optical microscope (Figure 1B), there was a color change (i.e., from transparent to dark) in the regions over which the dopamine solution passed, indicating that PDA patterns were successfully formed on the PDMS substrate. This dark color of the PDA is attributed to the chemical structure of PDA that is similar to that of biopigment melanin.10 The formation of the PDA adlayer was also confirmed by using Raman spectroscopy (Figure 1C); after the PDA coating, two new peaks appeared at 1340 cm-1 (by the stretching of catechol) and 1600 cm-1 (by the deformation of catechol), in addition to the intrinsic peaks of PDMS at 1265 cm-1 (δCH bend) and 1414 cm-1 (δCH bend).26,27 We investigated the behavior of fibrosarcoma HT1080 cells after their seeding onto PDMS substrates with and without a PDA coating. We observed that only a small number of HT1080 cells were attached to unmodified PDMS substrates, and these displayed a spherical morphology with poorly organized actin bundles. This indicates that PDMS repels cell attachment. After surface modification with the PDA adlayer, the efficiency of cell adhesion remarkably increased; the whole substrate surface was covered with attached cells that exhibited normal spindle-shaped morphology similar to that of cells cultured on a glass substrate (Figure S2). The increased cell adhesion on the PDA region is attributed to the adsorption of serum proteins on the PDA adlayer.28 When protein adsorption occurs on highly hydrophobic surfaces such as PDMS, the adsorbed proteins are easily denatured, thus they do not support cell adhesion. However, the adsorbed proteins on PDA adlayers maintain their natural form and activity,29,30 thus the PDA coating can serve as a celladhesion site. In addition, the critical surface tension of PDA was in a suitable range for cell adhesion.28 By combining the nonadhesive property of PDMS and the adhesive property of PDA, we successfully developed a selectively patterned system of HT1080 cells. As shown in Figure 2, HT1080 cells predominantly adhered to and grew on PDA-coated regions, forming strip arrays (20) Johann, R. M.; Baiotto, C. H.; Renaud, P. H. Biomed. Microdev. 2007, 9, 475–485. (21) Patrito, N.; McCague, C.; Norton, P. R.; Petersen, N. O. Langmuir 2007, 23, 715–719. (22) Achyuta, A. K. H.; Stephens, K. D.; Lewis, H. G. P.; Murthy, S. K. Langmuir 2009, 4160–4167. (23) Li, Y.; Liu, M.; Xiang, C.; Xie, Q.; Yao, S. Thin Solid Films 2006, 497, 270– 278. (24) Chen, C.; Fu, Y.; Xiang, C.; Xie, Q.; Zhang, Q.; Su, Y.; Wang, L.; Yao, S. Biosens. Bioelectron. 2009, 24, 2726–2729. (25) Tan, Y.; Deng, W.; Li, Y.; Huang, Z.; Meng, Y.; Xie, Q.; Ma, M.; Yao, S. J. Phys. Chem. B 2010, 114, 5016–5024. (26) Fei, B.; Qian, B.; Yang, Z.; Wang, R.; Liu, W. C.; Mak, C. L.; et al. Carbon 2008, 46, 1792–828. (27) Smith, A. L.; Anderson, D. R. Appl. Spectrosc. 1984, 38, 822–834. (28) Ku, S. H.; Ryu, J.; Hong, S. K.; Lee, H.; Park, C. B. Biomaterials 2010, 31, 2535–2541. (29) Lee, H.; Rho, J.; Messersmith, P. B. Adv. Mater. 2008, 21, 431–434. (30) Poh, C. K.; Shi, Z.; Lim, T. Y.; Neoh, K. G.; Wang, W. Biomaterials 2010, 31, 1578–1585.
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Figure 1. (A) Schematic illustration of polydopamine (PDA)-based cell patterning using microfluidics. (B) Optical micrograph of a PDA-patterned PDMS substrate. (C) Raman spectra of PDA-unmodified and PDA-modified regions. The color change from transparent to dark and the appearance of new peaks at 1340 and 1600 cm-1 indicate the PDA adlayer formation.
Figure 2. HT1080 cells grown on PDA-micropatterned PDMS. Cells were seeded at a density of 9 104 cells/well and cultured for 4 days on the substrate.
corresponding to PDA micropatterns. On the PDA micropatterns, spherical/ellipsoidal nuclei and well-stretched cytoskel15106 DOI: 10.1021/la102825p
etons were observed. We investigated the effects of micropatterns on cell attachment and spreading by initially adhering cells at a Langmuir 2010, 26(19), 15104–15108
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Figure 3. Morphology of HT1080 cells cultured on a PDA micropattern for 2, 5, and 24 h after the initial adhesion of a small number of cells (3 104 cells/well) onto the strips. Cells attached to the strips well and show normal spindle shapes. Table 1. Quantitative Analysis of Cell Alignment and Average Alignment Angle (Absolute Value) and Percentage of Cells Aligned within (20° of the Direction of the PDA Micropattern average alignment angle (absolute value)
percentage cells aligned (within (20°)
cell line
w/o PDA
w/PDA
w/o PDA
w/PDA
HT1080 MC3T3-E1 NIH-3T3
44.55 45.29 45.16
36.25 21.60 22.81
22.50 20.08 23.44
34.59 62.56 56.59
low density (3 104 cells/well) for a short time. As shown in Figure 3, HT1080 cells attached to the strips well and had a normal spindle shape after cultivation for 2 and 5 h. Most cells were viable after 24 h of cultivation, and those at the interface between PDA-modified and PDA-unmodified regions showed an elongated morphology in the direction of the micropattern. Our results indicate that spatial restriction on the 100 μm scale did not influence the cell adhesion and growth process very much. We observed the cell alignment on PDA micropatterns to investigate the effect of the striped PDA pattern on the orientation of cells. Whereas HT1080 cells were randomly oriented on an unpatterned PDMS substrate (Figure S2), their actin cytoskeletons and nuclei tended to align in the direction of the stripes when grown on PDA micropatterns (Figure 2). We quantitatively analyzed the cell alignment by the measurement of nuclear orientation according to the literature.31 On a bulk PDA-coated substrate (i.e., without PDA micropatterning), the nuclei did not show any noticeable alignment in a specific direction (Figure S3). The average angle of the nuclei of approximately 45° also demonstrated the random orientation (Table 1). In contrast, significantly more cells exhibited x-axis aligned nuclei on PDA micropatterns, parallel to the direction of the PDA layer, and approximately 35% of cells were aligned within (20° of the PDA pattern direction (Figure S3 and Table 1). Our results suggest that cells are spatially confined within PDA micropatterns and aligned (31) Charest, J. L.; Eliason, M. T.; Garcia, A. J.; King, W. P. Biomaterials 2006, 27, 2487–2494. (32) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1996, 32, 165–173. (33) Thomson, D. M.; Buettner, H. M. Tissue Eng. 2001, 7, 247–265.
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in the orientation of the patterns. According to the literature,32-34 the nature of adherent cells, which is to avoid the nonadhesive region, results in the cell orientation in narrow domains, and the interface between adhesive and nonadhesive regions provides the directional cue. We further investigated the patterning efficiency of PDA for other cell lines such as preosteoblast MC3T3-E1 and fibroblast NIH-3T3. According to confocal microscope images and fluorescence intensity analysis (Figures 4 and S4), both cell lines selectively adhered only to PDA micropatterns and showed well-organized actin cytoskeletons. For the measurement of cell alignment, the nucleus orientation of these cell lines was quantitatively analyzed (Table 1). When the cells were grown on a bulk PDA-coated substrate, the average angle of the nuclei was approximately 45°, indicating the nuclei distributed in all directions. In the case of cells grown on PDA micropatterns, the average angle decreased to approximately 22° and over 50% of the cells were oriented within (20° of the striped pattern direction. These results indicate that both MC3T3-E1 and NIH-3T3 cells align with PDA patterns better than HT1080 cells. The difference in the degree of cell orientation is attributed to the different cytoskeleton organizations of tested cell lines, which are known to be dependent on cell types.35,36 Our results suggest that PDA can guide cell patterning irrespective of the cell lines and that cells align well with the direction of PDA patterns, although the degree of orientation is dependent on the cell line. The PDA adlayer can serve as a cell-adherent region for a long time; we observed that HT1080 cells were stable for (at least) up to 4 days on PDA-micropatterned PDMS (Figure 2) and 8 days on PDA-coated PDMS (Figure S5). Furthermore, the PDA layer was very stable under harsh conditions (e.g., with organic solvents, strong acids, sonication, and heat treatment) (Figure S6). The cell-adherent property of PDA remained unchanged under the extreme environments, with cells displaying their normal (34) Thomson, D. M.; Buettner, H. M. Ann. Biomed. Eng. 2004, 32, 1120–1130. (35) Clark, P.; Connolly, P.; Curtis, A. S. G.; Dow, J. A. T.; Wilkinson, C. D. W. Development 1992, 108, 635–644. (36) Neidelinger-Wilke, C.; Grood, E. S.; Wang, J. H.-C.; Brand, R. A.; Claes, L. J. Orthop. Res. 2001, 19, 286–293.
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Figure 4. Cell patterning via PDA modification with a different cell line, MC3T3-E1, and its fluorescence intensity analysis. The cells selectively adhere to PDA regions and show well-stretched actin filaments and x-axis oriented nuclei.
morphology. The cell adhesiveness of PDA can be affected by the physicochemical properties of PDA, such as its molecular weight, and the thickness of the PDA adlayer. According to a previous report,10 the thickness of the PDA layer temporally increased up to approximately 50 nm and PDA had a molecular weight of approximately several million Daltons. The effects of PDA thickness and molecular weight on cell attachment remain to be investigated in a further study. In the present work, mammalian cell patterns were achieved by using mussel-inspired PDA surface chemistry. Material independency is one of most important properties of PDA.10 PDA can modify any material surface, including organic and inorganic materials, thus the applicability of PDA patterning is not limited to the type of substrate. According to our recent study, such mussel-inspired surface chemistry was universally applicable to the cell adhesion of various nonwetting materials such as polyethylene, poly(tetrafluoroethylene), silicone rubber, and PDMS,28 which suggests that any material with highly nonadhesive properties can be used as a substrate for cell patterning. In addition, PDA can serve as an immobilization site for amine- or thiol-functionalized molecules via Schiff base or Michael addition chemistries, respectively,10 thus the PDA coating can easily change the surface properties by using amine-/thiol-functionalized reactants. Taken together, PDA patterning is applicable to the study of cell responses against specific surface properties while not requiring the complicated chemical reactions that are unavoidable in conventional protein-immobilization methods.
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Conclusions We developed a simple, versatile method for cell patterning via mussel-inspired PDA surface modification. PDA patterns were formed on highly nonadhesive substrates using a microfluidic system. The cells were patterned on the PDA-micropatterned substrates through selective adhesion to the PDA-coated region. We found that different mammalian cells, such as fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3, preferred to adhere to PDA-coated regions and they aligned well with the direction of striped PDA patterns. Considering that a PDA coating can be applied to any type of material, it is expected that cells can be patterned on a wide variety of nonwetting surfaces through the micropatterning of PDA. Acknowledgment. This study was supported by the National Research Foundation (NRF) via National Research Laboratory (NRL) (R0A-2008-000-20041-0), Converging Research Center (2009-0082276), and Engineering Research Center (20080062205) programs. This research was also partially supported by the BioGreen 21 program (20070301034038), Republic of Korea. Supporting Information Available: Possible mechanism of dopamine polymerization; cell adhesion on glass, bare PDMS, and PDA-coated PDMS; cell morphology on PDA micropatterns at low cell density; and the stability of the PDA adlayer. This material is available free of charge via the Internet at http://pubs.acs.org.
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