Micropatterning of Nanoengineered Surfaces to Study Neuronal Cell Attachment in Vitro
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1991, 46, 321-327. (8) Decher, G.; Hong, J. D. Buildup of ultrathin multilayer films by a self-assembly process: II. Consecutive adsorption of anionic and cationic bipolar amphiphiles and polyelectrolytes on charged surfaces. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430-1434. (9) Yang, S. Y.; Mendelsohn, J. D.; Rubner, M. F. New Class of Ultrathin, Highly Cell-Adhesion-Resistant Polyelectrolyte Multilayers with Micropatterning capabilities. Biomacromolecules 2003, 4(4), 987-994. (10) Vautier, D.; Karsten, V.; Egles, C.; Chluba, J.; Schaaf, P.; Voegel, J. C.; Ogier, J. Polyelectrolyte multilayer films modulate cytoskeletal organization in chondrosarcoma cells. Journal of Biomaterials Science-Polymer Edition 2002, 13, 713-732. (11) Chluba, J.; Voegel, J.-C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Peptide Hormone Covalently Bound to Polyelectrolytes and Embedded into Multilayer Architectures Conserving Full Biological Activity. Biomacromolecules 2001, 2(3), 800-805. (12) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Selective Deposition of Proteins and Cells in Arrays of Microwells. Langmuir
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(20) Folch, A.; Toner, M. Cellular Micropatterns on Biocompatible Materials. Biotechnol. Prog. 1998, 14(3), 388-392. (21) Jiang, X.; Zheng, H.; Gourdin, S.; Hammond, P. T. Polymer-on- Polymer Stamping: Universal Approaches to Chemically Patterned Surfaces. Langmuir 2002, 18(7), 2607-2615. (22) Jiang, X.-P.; Clark, S. L.; Hammond, P. T. Side-by-Side Directed Multilayer Patterning Using Surface Templates. AdV. Mater. 2001, 13(22), 1669-1673. (23) Lee, C.-S.; Lee, S.-H.; Park, S.-S.; Kim, Y.-K.; Kim, B.-G. Protein patterning on silicon-based surface using background hydrophobic thin film. Biosens. Bioelectron. 2003, 18(4), 437-445. (24) Douvas, A.; Argitis, P.; Diakoumakos, C. D.; Misiakos, K.; Dimo- tikali, D.; Kakabakos, S. E. Photolithographic patterning of proteins with photoresists processable under biocompatible conditions. J. Vac.
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2 nanofilm micropatterns. In all images, green squares are FITC-labeled sPLA 2 nanofilm micropatterns, and cells are at 1 day in vitro. Panels A and B show merged images of phase and FITC-labeled sPLA 2 nanofilm micropatterns. Panel C shows a merged image of cells plated onto TRITC - BSA nanofilms. Original microscope magnification ) 100
× (panel A), 400 × (panel B), and 200 × (panel C). Scale bars indicate 160 microns in panel A, 40 microns in panel B, and 80 microns in panel C. 1754 Biomacromolecules, Vol. 5, No. 5, 2004 Shaikh Mohammed et al.
(26) Douvas, A.; Argitis, P.; Misiakos, K.; Dimotikali, D.; Petrou, P. Biocompatible photolithographic process for the patterning of bio- molecules. Biosens. Bioelectron. 2002, 17(4), 269-278. (27) Havard, J. M.; Vladimirov, N.; Fre’chet, J. M. J.; Yamada, S.; Willson, C. G.; Byers, J. D. Photoresists with Reduced Environmental Impact: Water-Soluble Resists Based on Photo-Cross-Linking of a Sugar-Containing Polymethacrylate. Macromolecules 1999, 32(1), 86-94.
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(29) Hua, F.; Lvov, Y. M.; Cui, T. Spatial Patterning of Colloidal Nanoparticle-based Thin Film by A Combinative Technique of Layer- by-layer Self-assembly and Lithography, J. Nanosci. Nanotechnol. 2002, 2(3), 357-361. (30) Ai, H.; Meng, H.; Ichinose, I.; Jones, S. A.; Mills, D. K.; Lvov, Y. M.; Qiao, X. Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons. J. Neurosci. Methods 2003, 128, 1-8. (31) Li, M.; Kondabatni, K.; Cui, T.; McShane, M. J. Fabrication of 3-D gelatin-patterned glass substrates with layer-by layer and lift-off (LbL- LO) technology. IEEE Trans. Nanotechnol. 2004, 3, 115-123. (32) DeCoster, M. A. Group III secreted phospholipase A 2 causes apoptosis in rat primary cortical neuronal cultures. Brain Res. 2003, 988, 20-28. (33) Yagami, T.; Ueda, K.; Asakura, K.; Hayasaki-Kajiwara, Y.; Nakazato, T.; Sakaeda, T.; Hata, S.; Kuroda, T.; Takasu, N.; Hori, Y. Group IB secretory phospholipase A 2 induces neuronal cell death via apoptosis. J. Neurochem. 2002, 81, 449-461. (34) Yagami, T.; Ueda, K.; Asakura, K.; Hata, S.; Kuroda, T.; Sakaeda, T.; Takasu, N.; Tanaka, K.; Gemba, T.; Hori, Y. Human group IIA secretory phospholipase A 2 induces neuronal cell death via apoptosis. Mol. Pharmacol. 2002, 61, 114-126. (35) Copic, A.; Vucemilo, N.; Gubensek, F.; Krizaj, I. Identification and purification of a novel receptor for secretory phospholipase A 2 in porcine cerebral cortex. J. Biol. Chem. 1999, 274, 26315-26320. (36) Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. Identification and properties of very high affinity brain membrane binding sites for a neurotoxic phospholipase from Taipan venom, J. Biol. Chem. 1989, 264, 11503-11510. (37) Lambeau, G.; Schmid-Alliana, A.; Lazdunski, M.; Barhanin, J. Identification and purification of a very high affinity binding protein for toxic phospholipases A 2 in skeletal muscle. J. Biol. Chem. 1990, 265, 9526-9532. (38) Molecular Probes. Amine-ReactiVe Probes. (39) Sauerbrey, G. Z. Verwendung von Schwingquarzen zur Wgung dnner Schichten und zur Mikrowaage. Z. Phys. Verhandl. 1959, 155, 206- 222. (40) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Assembly of multicomponent protein films by means of electrostatic layer-by- layer adsorption. J. Am. Chem. Soc. 1995, 117, 6117-6123. (41) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Ultrathin multilayer polyelectrolyte films on gold: construction and thickness determination. Langmuir 1997, 13, 3422-3426. (42) Boyan, B. D.; Lossdorfer, S.; Wang, L.; Zhao, G.; Lohmann, C. H.; Cochran, D. L.; Schwartz, Z. Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopog- raphies. Eur. Cell Mater. 2003, 24, 22-27. BM0498631 Study of Neuronal Cell Attachment in Vitro Biomacromolecules, Vol. 5, No. 5, 2004 1755 Fabrication of Interdigitated Micropatterns of Self-Assembled Polymer Nanofilms Containing Cell-Adhesive Materials Javeed Shaikh Mohammed, † Mark A. DeCoster, ‡ and Michael J. McShane* ,†,§
Micropatterns of different biomaterials with micro- and nanoscale features and defined spatial arrangement on a single substrate are useful tools for studying cellular-level interactions, and recent reports have highlighted the strong influence of scaffold compliance in determining cell behavior. In this paper, a simple yet versatile and precise patterning technique for the fabrication of interdigitated micropatterns of nanocomposite multilayer coatings on a single substrate is demonstrated through a combination of lithography and layer-by-layer (LbL) assembly processes, termed polymer surface micromachining (PSM). The first nanofilm pattern is constructed using lithography, followed by LbL multilayer assembly and lift-off, and the process is repeated with optical alignment to obtain interdigitated patterns on the same substrate. Thus, the method is analogous to surface micromachining, except that the deposition materials are polymers and biological materials that are used to produce multilayer nanocomposite structures. A key feature of the multilayers is the capability to tune properties such as stiffness by appropriate selection of materials, deposition conditions, and postdeposition treatments. Two- and four-component systems on glass coverslips are presented to demonstrate the versatility of the approach to construct precisely defined, homogeneous nanofilm patterns. In addition, an example of a complex system used as a testbed for in vitro cell adhesion and growth is provided: micropatterns of poly(sodium 4-styrenesulfonate)/poly-L-lysine hydrobromide (PSS/PLL) and secreted phospholipase A 2 /poly(ethyleneimine) (sPLA 2 / PEI) multilayers. The interdigitated square nanofilm array patterns were obtained on a single coverslip with poly- (diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Cell culture experiments show that cortical neurons respond and bind specifically to the sPLA 2 micropatterns in competition with PLL micropatterns. The fabrication and the initial biological results on the nanofilm micropatterns support the usefulness of this technique for use in studies aimed at elucidating important biological structure-function relationships, but the applicability of the fabrication method is much broader and may impact electronics, photonics, and chemical microsystems.
Discoveries in the areas of cell-biomaterial and cell-cell interactions are highly significant 1 because of the great insight they can provide into fundamental cell biology 2 and their potential value for medicine and biotechnology. It is generally understood that the ability to engineer and control cellular behavior is highly dependent on the presentation of physical and chemical cues. 1-3
Therefore, the development of complex functional biointerfaces, where the positioning of and interaction between cells of different types can be precisely controlled, requires the placement of biomaterials with differing functionality in specific configurations on the same substrate. 2 The capability to capture and maintain a permissive environ- ment for multiple cell types is particularly important for cases where coculture is desired, such as patterned neuronal networks, where the incorporation of neuron-supporting glial cells is required, 4 and hepatocyte cultures, where the presence of fibroblasts is critical for preserving characteristics of native liver tissue.
5 While randomly oriented cocultures of multiple cell types have been used to better mimic in vivo systems, the type and degree of cell-cell interactions in such systems are not typically controllable at a desirable level. Therefore, recent efforts have targeted the development of approaches to obtain cell cultures employing adhesive patterns to enhance microenvironmental control through the spatial localization of multiple cell types relative to each other. 4,6,7
In addition to engineering systems that achieve patterning of cells on surfaces, controlling the compliance of the underlying substrate is also important. While mechanical forces have long been known to play a critical role in cellular interactions with the extracellular matrix, particularly for adherent cells, 8-12
an appreciation of substrate stiffness as a significant factor in * Corresponding author. Mailing address: P.O. Box 10137, 911 Hergot Avenue, Ruston, LA 71272. Tel.: 318-257-5100. Fax: 318-257-5104. E-mail: *mcshane@coes.latech.edu. † Institute for Micromanufacturing, Louisiana Tech University. ‡ Louisiana State University Health Sciences Center. § Biomedical Engineering Program, Louisiana Tech University. (1) Magnani, A.; Priamo, A.; Pasqui, D.; Barbucci, R. Mater. Sci. Eng., C 2003, 23, 315-328. (2) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (3) Dowell-Mesfin, N. M.; Abdul-Karim, M. A.; Turner, A. M. P.; Schanz, S.; Craighead, H. G.; Roysam, B.; Turner, J. N.; Shain, W. J. Neural Eng. 2004,
(4) Yang, I. H.; Co, C. C.; Ho, C. C. J. Biomed. Mater. Res. 2005, 75, 976- 984. (5) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. Biotechnol. Prog. 1998, 14, 378-387. (6) Khademhosseini, A.; Suh, K. Y.; Yang, J. M.; Eng, G.; Yeh, J.; Levenberg, S.; Langer, R. Biomaterials 2004, 25, 3583-3592. (7) Fukuda, J.; Khademhosseini, A.; Yeh, J.; Eng, G.; Cheng, J.; Farokhzad, O. C.; Langer, R. Biomaterials 2006, 27, 1479-1486. (8) Tan, J. L.; Tien, J.; Pirone, D. M.; Gray, D. S.; Bhadriraju, K.; Chen, C. S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1484-1489. (9) Parker, K. K.; Brock, A. L.; Brangwynne, C.; Mannix, R. J.; Wang, N.; Ostuni, E.; Geisse, N. A.; Adams, J. C.; Whitesides, G. M.; Ingber, D. E. FASEB
(10) Brock, A.; Chang, E.; Ho, C.-C.; LeDuc, P.; Jiang, X.; Whitesides, G. M.; Ingber, D. E. Langmuir 2003, 19, 1611-1617. (11) Galbraith, C. G.; Yamada, K. M.; Sheetz, M. P. J. Cell Biol. 2002, 159, 695-705. (12) Balaban, N. Q.; Schwarz, U. S.; Riveline, D.; Goichberg, P.; Tzur, G.; Sabanay, I.; Mahalu, D.; Safran, S.; Bershadsky, A.; Addadi, L.; Geiger, B. Nat.
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Langmuir 2006, 22, 2738-2746 10.1021/la0525473 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/18/2006 modulating cell behavior has only recently developed. 13 For example, recent work has demonstrated that myocytes sense differences between surfaces of varied elasticity, and express native phenotype only when exposed to substrates of stiffness typical of normal muscle. 14 These findings support the concept that mechanical factors influence different cell types in funda- mentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands. 15 The above points may be summarized by stating two key general requirements for in vitro systems aimed at providing scaffolding for multiple cell types: (1) the patterns must possess the appropriate physical-chemical properties to support the cells of interest and (2) the patterns must be oriented properly relative to one another. 6 The former factor involves adhesive moieties that will selectively capture cells, nontoxicity, stability, and appropriate stiffness for the cells of interest. The second point requires a strategy for constructing the patterns that has desirable features with regard to materials that may be deposited and the relative alignment of multiple patterns. The most commonly used biomaterial patterning techniques are based on soft lithography (i.e., microcontact printing ( µCP),
patterning using microfluidic networks ( µFNs), elastomeric membranes, and laminar flow patterning), 16-20
and employ elastomeric micromolds made of poly(dimethylsiloxane) (PDMS), as well as conventional photolithography. 21 The soft lithography techniques have advantages that include rapid prototyping, low cost, and the ability to pattern on nonplanar substrates. Of these, microstamping is one that has found widespread use in biological studies; however, alignment of separate elastomeric stamps across large areas is difficult to achieve, and therefore deposition of patterns of different types with desired relative alignment is not practiced. A handful of examples of multicomponent patterning of adjacent biomaterial patterns on a single substrate have been reported, including multilevel stamps, 22 two-dimensional (2D) and three-dimensional (3D) µFNs,
23-25 photolithography, 21 thermobiolithography, 26 and photochemical reactions 27-30 com-
bined with lithography. These approaches may potentially be adapted to pattern multilayer films; however, each has its own advantages and disadvantages. For example, because of the elastomeric nature of PDMS, large-area patterning with 2D or 3D microfluidics is problematic. Thermobiolithography suffers from low resolution and difficulty in the alignment of multiple stamps during pattern transfer. In addition to the disadvantages noted, none of the techniques described above can provide significant control over the elasticity of the materials deposited. In contrast, the ionic layer-by-layer (LbL) self-assembly technique, 31,32
an elegant method with the ability to deposit multilayer films that are highly interpenetrated, precise, and uniform in nature with minimal constraints on the size, geometry, or materials, 33 can be applied to obtain multilayer structures with varying composition, thickness (from a few nanometers to microns), roughness, elasticity, porosity, and so forth. The compliance of multilayer nanofilms is highly tunable, as the effective modulus depends not only on the constituent molecules, but also on the assembly conditions and the number of adsorbed layers. Multilayer films have been shown to exhibit thickness-dependent stiffness when deposited on solid substrates, and the effective modulus was found to increase 10-fold after cross-linking (e.g., ∼50 kPa up to ∼800 kPa for native and cross-linked poly-L-lysine/hyaluranon multilayers, respec- tively).
34 Much higher moduli have been reported for poly- (allylamine hydrochloride)/poly(sodium 4-styrenesulfonate) (PAH/ PSS) nanofilms, ranging from hundreds of megapascals in the hydrated state to gigapascals when dried. 35 These nanofilms can be constructed from functionalized macromolecules, enzymes, and proteins embedded at different depths to obtain complex biomimetic nanoarchitectures, 36-42
and a number of recent studies have described the use of multilayers of biomaterials for cell- related applications. 43-46
However, several of these studies share a common limitation in that the multilayer films are homogeneous across the plane. Thus, while LbL can enable the tuning of substrate physical-chemical properties, including stiffness, a suitable micropatterning technique is required for the spatial definition of multicomponent micropatterns of these multilayers. Two-component multilayer films have been patterned using a technique based on µCP. Chemically patterned surfaces were utilized to construct polymer-on-polymer 47 structures and side- (13) Discher, D. E.; Janmey, P.; Wang, Y. L. Science 2005, 310, 1139-1143. (14) Engler, A. J.; Griffin, M. A.; Sen, S.; Bo¨nnemann, C. G.; Sweeney, H. L.; Discher, D. E. J. Cell Biol. 2004, 166, 877-887. (15) Yeung, T.; Georges, P. C.; Flanagan, L. A.; Marg, B.; Ortiz, M.; Funaki, M.; Zahir, N.; Ming, W.; Weaver, V.; Janmey, P. A. Cell Motil. Cytoskeleton
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