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Fluorescent Aromatic Platforms for Cell Patterning Jesus M. de la Fuente,*,† Abhay Andar,† Nikolaj Gadegaard,† Catherine C. Berry,† Peter Kingshott,‡ and Mathis O. Riehle† Centre for Cell Engineering, Institute of Biomedical and Life Science, Joseph Black Building, UniVersity of Glasgow, Glasgow, G12 8QQ, UK, and Danish Polymer Centre, FrederiksborgVej 399, Roskilde DK-4000, Denmark ReceiVed NoVember 11, 2005. In Final Form: March 13, 2006 This paper describes a simple experimental method of patterning fluorescent organic dyes, fluorescein, and rhodamine on gold substrates by microcontact printing techniques. The development of this step-by-step protocol has allowed us to prepare striped and squared micropatterns with poly(ethylene glycol) (PEG) linkers terminated by these fluorophores using a fast, easy, and inexpensive technique. Although the rest of the surface was covered with aliphatic molecules (methyl terminated), human fibroblasts demonstrated an unexpected response, aligning themselves according to the aromatic patterns, despite the presence of PEG, which is a cell resistant molecule, in the fluorescent regions.
Introduction The study of cell adhesion to biomaterials is a very important topic in tissue engineering.1 The understanding of complex biological adhesion phenomena requires the development of biological materials and novel technologies.2 The ideal biological material should be amenable to design, versatile, and easy to fabricate and analyze. In this regard, chemically microstructured surfaces have gained increasing attention in the cell biological biomaterial community in the past few years.3 There is a broad field of applications of microstructured substrates, ranging from tissue engineering through miniaturized biosensors, artificially designed neuronal networks, and hybrid molecular electronics to microseparation.4 Several microfabrication methods for biological surface modification have been developed. For example, photolithography has often been used, and, although it is a common method for microfabrication, its use for cell biologists is limited by the equipment and procedures required.5 More recently, microcontact printing (µCP) has attracted increasing interest as a microfabrication method in cell biology.6 µCP offers a way to create complex patterns on surfaces, and it is a simple, fast, and inexpensive technique. µCP relies on the use of a relief stamp to transfer chemicals to designated regions of a surface.7 The stamps are usually fabricated by the curing * Corresponding author. Current address: Grupo de Carbohidratos, Instituto de Investigaciones Quı´micas, CSIC, Isla de La Cartuja, Ame´rico Vespucio s/n, 41092 Sevilla, Spain. Phone: (+34) 954-489568. Fax: (+34) 954-460565. E-mail:
[email protected]. † University of Glasgow. ‡ Danish Polymer Centre. (1) Curtis, A.; Riehle, M. Phys. Med. Biol. 2001, 46, R47-R65. (2) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47-55. (3) Hyun Park, T.; Shuler, M. L. Biotechnol. Prog. 2003, 19, 243-253. (4) (a) Chen, C. S.; Mrksich, M.; Huangm, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (b) Wheeler, B. C.; Corey, J. M.; Brewer, G. J.; Branch, D. W. J. Biomech. Eng. 1999, 121, 73-78. (c) Zhang, S. G.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213-1220. (d) Lauer, L.; Ingebrandt, S.; Scholl, M.; Offenhausser, A. IEEE Trans. Biomed. Eng. 2001, 48, 838-842. (5) (a) Madou M. J. Fundamentals of Microfabrication; CRC Press: Boca Raton, FL, 1996. (b) Geissler, M.; Xia, Y. AdV. Mater. 2004, 16, 1249-1269. (c) Schanze, K. S.; Bergstedt, T. S.; Hauser, B. T.; Cavalaheiro, C. S. P. Langmuir 2000, 16, 795-810. (6) (a) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (b) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75, 1-68. (c) Ito, Y. Biomaterials 1999, 20, 23332342.
of poly(dimethylsiloxane) (PDMS) in contact with a master substrate containing the patterns in relief. The finished stamp is inked with a solution of the chemical to be transferred, rinsed, the solvent allowed to evaporate, and then brought into contact with the surface; the chemistry is only transferred in those regions where the stamp relief allows contact with the surface. To get cells to respond and comply to a desired pattern an appropriate chemical contrast between the pattern and the background has to be chosen. One possibility is to pattern adhesive chemicals, the other is to pattern nonadhesive ones. The selection of the appropriate adhesion-promoting molecule to stamp is therefore very important. During recent years, a number of different methods were published to achieve these cell adhesive surfaces using different synthetic molecules, peptides, or proteins. However, in some cases, complicated chemical syntheses8 or expensive peptides or proteins9 have put this technology out of reach for routine cell biological applications. A further limitation of all surface patterning techniques is the need for sophisticated instrumentation, such as X-ray photoelectron spectroscopy (XPS) or ellipsometry, to locate and analyze the patterned surfaces.10 We describe in this paper an inexpensive, simple, and straightforward protocol to prepare patterned surfaces with the fluorophores fluorescein and rhodamine. This work uses µCP to pattern self-assembled monolayers (SAMs) into regions terminated in carboxylic and methyl groups on glass coverslips modified with an optically transparent film of gold. These terminal carboxylic groups provide a handle for further reactivity. The development of a step-by-step protocol has allowed us to label these patterns with different aromatic fluorescent probes. Surprisingly, the created aromatic platforms on a poly(ethylene glycol) (PEG) background are excellent adhesive platforms for human fibroblasts. The fact that only commercially available linkers, really simple protocols, and optical microscopes have been used for the preparation and analysis of these biomaterials (7) Mrksich, M.; Chen, C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775-10778. (8) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548-6555. (9) (a) Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. J. Cell Sci. 2003, 117, 41-52. (b) Sgarbi, N.; Pisignano, D.; Di Benedetto, F.; Gigli, G.; Cingolani, R.; Rinaldi, R. Biomaterials, 2004, 25, 1349-1353. (c) Brock, A.; Chang, E.; Ho, C.-C.; LeDuc, P.; Juang, X.; Whitesides, G. M.; Ingber, D. E. Langmuir 2003, 19, 1611-1617. (10) Csucs, G.; Michel, R.; Lussi, J. W.; Textor, M.; Danuser, G. Biomaterials 2003, 24, 1713-1720.
10.1021/la053045s CCC: $33.50 © 2006 American Chemical Society Published on Web 03/31/2006
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Langmuir, Vol. 22, No. 13, 2006 5529 Scheme 1a
a
Synthesis of Fluorescent Micropatterned Surfaces: (a) EDC (2 mM), NHS (5 mM), MES (50 mM, pH 6.5); (b) THF/MeOH 1:1, NaOH, RITC, or FITC.
confirms their flexibility as a routine method for cell patterning experiments. Experimental Section Preparation of the Samples. Molds for the stamps were prepared onto glass slides via photolithographic techniques.5 A negative form of the pattern (stripes from 25 to 50 µm wide or squares from 5 × 5 to 50 × 50 µm) was then created from the topographical surface on the slide by covering the surface with PDMS followed by heatinduced polymerization. The flexible PDMS stamp was peeled back from the glass slide and immersed in solutions (1 mM) of different thiol derivatives (16-mercaptohexadecanoic acid (MHA), L-cysteine, and N-(2-mercaptopropionyl) glycine). The stamp was dried and placed on a metallized glass slide (5-10 nm of titanium and 25100 nm of gold). After that, the slide was immersed immediately in a solution of 1-dodecanethiol in methanol (1 mM) for 5 min. The slide was rinsed and dried. Functionalization of the carboxylated patterned areas were carried on by immersion of the slide into a mixture of (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (2 mM) and N-hydroxysuccinimide (NHS) (5 mM) in 2-(N-morpholino)ethanesulfonic acid (MES) (50 mM, pH 6.5). After 30 min, a PEG-bis-(3aminopropyl) solution was added to a final concentration of 100 mM, and the mixture was stirred an additional 12 h. The samples were immersed in a solution (0.1 mM) of fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RITC) in a solvent mixture of tetrahydrofuran (THF)/methanol (1:1) in basic conditions for 1 h. Samples were analyzed by XPS and fluorescence microscopy (Supporting Information). Cell Results. hTERT-BJ1 fibroblasts were seeded onto patterned glass slides using a serum-containing medium (9% fetal calf serum). After 24 h, the cells were fixed, stained, and observed under scanning electron and fluorescence microscopy (Supporting Information).
Results and Discussion Preparation of the Samples. Micropatterned substrates with chemically modified squares and stripes were created using µCP
techniques.7 Different commercially available thiol derivatives terminating with a carboxylic group were stamped, using a patterned PDMS stamp, on the surface of a thin layer of gold. The thiol derivatives were transferred only to regions of the surface that contacted the raised region of the stamp, providing the sample with carboxylated stripes or squares that corresponded to the structure of the stamps. These thiol derivatives reacted with the gold surface because of the strong Au-S bond.11 The free terminal -CO2H groups on the patterned areas provide a handle for building up these regions and changing their chemical properties.12 A commercially available PEG derivative functionalized with two terminal amine groups was anchored to the carboxylated patterns to repel protein adsorption in these areas (Scheme 1).13 The reaction utilizes EDC and NHS to catalyze reactions between the surface acid groups and the PEG amine groups.14,15 An excess of bis-aminated PEG was used to minimize its double functionalization with the micropatterned surface. The patterns were labeled with fluorescein and rhodamine B dyes for pattern observation under a fluorescence microscope. This reaction was carried out in basic conditions with FITC or RITC.16 To confirm the pattern formation, the samples were observed under a fluorescence microscope. Of the different thiols used, only MHA provided a well-defined pattern surface. L-Cysteine and tiopronin are probably limited with regards to their length and nonhydrophobicity to self-assemble into a dense monolayer. XPS analysis was performed to determine the surface elemental composition for each of the modification steps used in this study. (11) Dubois, L. H.; Nuzzo, R. G. Annu. ReV. Phys. Chem. 1992, 43, 437-463. (12) de la Fuente, J. M.; Fandel, M.; Berry, C. C.; Riehle, M.; Cronin, L.; Aitchison, G.; Curtis, A. S. G. ChemBioChem 2005, 6, 989-991. (13) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (14) Detarm, D. F.; Silverstein, R. J. Am. Chem. Soc. 1996, 88, 1013-1019. (15) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (16) Barrientos, A. G.; de la Fuente, J. M.; Rojas, T. C.; Ferna´ndez, A.; Penade´s, S. Chem.sEur. J. 2003, 9, 1909-1921.
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Figure 1. hTERT-BJ1 cells fluorescently stained for F-actin and DNA on (a) a striped FITC pattern, 25 µm wide, with a 25 µm gap (green ) pattern, red ) F-actin, blue ) nucleus); (b) a square FITC pattern, 50 × 50 µm, with 50 µm spacing (green ) pattern, red ) F-actin, blue ) nucleus); (c) a square RITC pattern, 50 × 50 µm, with 50 µm spacing (red ) pattern, green ) F-actin, blue ) nucleus); and (d) a square FITC pattern, 5 × 5 µm, with 10 µm spacing (green ) pattern, red ) F-actin, blue ) nucleus). Scale bars: 50 µm.
The analysis confirmed the coupling of MHA to the gold surface (addition of carbon and oxygen) and the PEG grafting (addition of oxygen and nitrogen) (Supporting Information). Furthermore, from the high-resolution analysis of the carbon 1s peak, an increase in the C-O signal at 286.8 eV was observed (data not shown), confirming the addition of the PEG backbone to the MHA surface. This simple synthetic methodology has allowed us to achieve an inexpensive and fast method of patterning carboxyalkanethiolate SAMs functionalized with long PEG and fluorescent labels. Cell Results. To test the cell behavior on the patterned surface, adhesion experiments were performed using the hTERT-BJ1 cell line. Cells were seeded in a serum-containing medium onto the patterned samples. Following 24 h of incubation, the cells were fixed and stained with rhodamine-conjugated phalloidin (for samples coated with fluorescein on the stamped pattern) or fluorescein-conjugated phalloidin (for samples coated with rhodamine on the stamped pattern) to visualize their actin cytoskeletal response. Typical cell responses are shown in Figures 1 and 2. Results indicate that, in the case of the larger striped and squared structures, cells adhered primarily on the printed regions (aromatic fluorescent platforms) (Figure 1a,b,c). Furthermore, on the smaller patterns, the cells appeared to use the aromatic patterned areas as “stepping stones” to guide adhesion and spreading (Figure 1d). These results show the high affinity that human fibroblasts or serum proteins have for fluorescein or rhodamine surfaces, adopting even the shape of the pattern (Figure 1a,b,c). Cell adhesion was not found on samples with patterns functionalized with PEG but not fluorophores (data not shown), showing a complete functionalization of the carboxylic groups with the PEG derivative.17,18 (17) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305-313.
Scanning electron microscopy (SEM) images of the cells seeded onto the PEG-FITC patterns are shown in Figure 2. Because of the length of the commercial PEG derivative (around 20 nm), the patterns can be observed by SEM but not by light microscopy (Figure 2a,b). Cells are exclusively located in the patterned regions, and cell attachments do not appear to go beyond the pattern (Figure 2c,d,e,f). Both the fluorescence and SEM images clearly show that, while the fibroblasts will adhere and spread on samples without the fluorescence pattern, that is, SAMs of 1-dodecanethiol (data not shown), when presented with the option, the cells have a clear preference for the PEG-FITC or PEG-RITC regions over the alkanethiol areas. Bearing in mind the previously reported affinity of cells to hydrophobic surfaces, in addition to the nonadhesive properties of PEG,19 this result was unexpected. It is assumed that this preferential adhesion is not related to the positive charge of the rhodamine at physiological pH, as the fluorescein has the same influence over cell attachment but does not present such a charge. Instead it is postulated that the aromatic platforms, a common feature of both fluorophores, must be the moiety responsible for this phenomenon. The fact that the cells preferentially adhere to the aromatic platforms could have three possible explanations: First, it could be that adhesive proteins from the culture media are adsorbed only on the aromatic platforms. Second, it could be that the proteins absorbed to the aromatic platforms take up a more adhesive conformation and, for instance, they are more celladhesive than the others attracted to the aliphatic background (18) Scotchford, C. A.; Cooper, E.; Leggett, G. J.; Downes, S. J. Biomed. Mater. Res. 1998, 41, 431-442. (19) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912-6921.
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Figure 2. SEM images of hTERT-BJ1 cells on (a,c) square FITC patterns (50 × 50 µm, 50 µm spacing) and (b,d,e,f) striped FITC patterns (75 µm wide, 75 µm gap). The pattern is easily visible due to the height of the PEG chain. The cells follow the pattern closely with their outline and seem to “explore” the edge with filopodia adhering to the transitional area.
because of the soft yielding hydrated nature of the PEG. Third, it could be that the cells are attracted by the aromatic platforms directly. It has been shown that cells continuously synthesize and deposit their own extracellular matrix (ECM) proteins to migrate,20 and that they are able to deposit new ECM proteins.9c The interaction of the ECM proteins and the fluorescent aromatic platforms could be due to the π-π stacking interaction21 between both π-systems, the aromatic rings from the patterns, and the aromatic amino acids such as phenylalanine and tyrosine from the ECM proteins. (20) (a) Madri, J. A.; Stenn, K. S. Am. J. Pathol. 1982, 106, 180-186. (b) Wicha, M. S.; Liotta, L. A.; Vonderhaar, B. K.; Kidwell, W. R. DeV. Biol. 1980, 80, 253-266. (21) This is the average of three independent reports on the inelastic mean free path of Au 4f photoelectrons, which vary from 31 to 34 Å, as cited in (a) Zharnikov, M.; Frey, S.; Heister, K.; Grunze, M. J. Electron Spectrosc. Relat. Phenom. 2002, 124, 15-24. (b) Laibins, P. E.; Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017-7021. (c) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibins, P. E. J. Phys. Chem. B 1998, 102, 426-436. (22) Burley, S. K.; Petsko, G. A. AdV. Protein Chem. 1998, 39, 125-192 and references therein.
Further investigations to understand this unexpected cell behavior and how ECM proteins from the medium or from the cells intervene are actually under study.
Conclusions SAMs have played a major role in addressing the mechanisms of cell adhesion in response to chemical patterns. The combination of SAMs and µCP with the use of different fluorescent markers provides a remarkably convenient system to control the attachment of cells to the substrate. Attachment of the cells on patterned SAMs has previously been observed only after the substrates were precoated with fibronectin and adhesion peptides. In this paper, no adhesive proteins were employed, and yet the cells responded strongly to the patterns, clearly preferring the patterned areas with the fluorescent markers as opposed to the alkanethiolate-coated areas. In conclusion, the methods presented here introduce a fast, cheap, and simple technique for fabricating chemically patterned surfaces. The synthetic strategy used to generate these SAMs
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produced a surface that was well-defined and stable on a molecular scale. The chemical patterns were created step-by-step, with each individual molecule, and thus the patterns, being built up on the substrate within a few hours. This methodology will prove to be very useful to cell biologists for the design of micropatterned cellular structures. Acknowledgment. This work was supported by MEC and the University of Glasgow. J.M.F. thanks the MEC for a postdoctoral fellowship. N.G. is grateful for financial support from the Royal Society of Edinburgh. We thank A. S. G. Curtis
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and L. Casaderova for fruitful discussions, M. Robertson for preparing gold slides, A. McIntosh for technical support, and L. Cronin for the use of his laboratory facilities. Supporting Information Available: Preparation of SAMs and patterning using µCP, functionalization of the surface, XPS analysis of the surface, cell culture preparation, immunolabeling, and SEM analysis. This material is available free of charge via the Internet at http://pubs.acs.org. LA053045S
