Article pubs.acs.org/ac
Micropatterned Thermoresponsive Surfaces by Polymerization of Monomer Crystals: Modulating Cellular Morphology and Cell− Substrate Interactions Feng Wang,†,§ Hongyan He,†,§ Xinmei Wang,† Zhenqing Li,‡ Daniel Gallego-Perez,† Jianjun Guan,‡ and L. James Lee*,† †
NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, Columbus, Ohio 43212, United States ‡ Department of Materials Science and Engineering, The Ohio State University, Columbus, Ohio 43210, United States ABSTRACT: A novel and facile approach has been developed to create thermoresponsive surfaces with macroscale patterns together with microscale features. The surface patterns were formed by applying macroscale nucleation agent patterns onto saturated N-isopropylacrylamide monomer solution membranes to induce the divergent growth of needlelike monomer crystals; the patterned monomer crystals were then photopolymerized to form patterned thermoresponsive films. A series of analytical tools (i.e., scanning electron microscopy, profilometry, and contact angle measurement) were used to characterize the properties of the patterned films. Cell coculture on this patterned thermoresponsive films enables cell separation and sorting by modulating temperature- and topography-dependent cell−substrate interactions and cell morphology, respectively. This versatile technique allows the formation of various macroscale patterns with microscale features over large areas, and on most solid substrates, within minutes, all of this without the need for expensive equipment and facilities. Such patterned surfaces can act as both in vitro tumor models and separation platforms for cancer studies. This method can also be applied to other cell-based biological studies and clinical applications.
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circular, channel-like pits or island textures with controlled depths and heights from 10 to 100 nm.13,14 The use of carbon nanotubes is another way to produce patterned surfaces. Eliason et al.15 reported the fabrication of polymeric micropatterns with embedded carbon nanotubes by an imprinting process which showed significant alignment of osteoblast-like cells on the surfaces. Electrospinning and plasma deposition can also be used to produce patterned surfaces for controlling cell behavior.2,16,17 Although clean-room facilities are not required, these approaches still need special processing apparatus. Therefore, the development of simpler and lowcost fabrication approaches of polymer micro-/nanopatterns with added functionalities (e.g., topography- and temperaturebased modulation of cell morphology and adhesion, respectively) could have a significant impact in biomaterials and tissue engineering applications. The incorporation of responsive polymers not only allows for controlled cellular behavior based on the pattern properties but also offers the possibility of a smart response to a given stimulus, as in the case of “cell sheet engineering”.18 A popular polymer, poly(N-isopropylacrylamide) (PNIPAAm), which
ith the help of micro-/nanotechnologies, tissuelike multicellular structures and extracellular matrix mimics could be fabricated in various shapes.1,2 Since the topographic features together with mechanical and chemical properties of the microenvironment strongly influence various aspects of cell behavior,3−6 including morphology, proliferation, migration, differentiation, and gene expression,7−9 it is essential to better understand cell−substrate interactions in tissue-engineered constructs. Patterned polymeric systems have been widely applied for cell cultures and tissue-engineered scaffolds because of their biocompatibility, finely tuned mechanical properties, and ease of processing. Typically, polymeric patterned surfaces can be fabricated by soft lithography,2 photolithography,10 and electron-beam lithography.7,11 For instance, human dermal fibroblasts have been aligned on the stripelike micropatterns produced by photolithography with well-controlled orientation.10 These traditional lithography processes require multistep fabrications, including micro-/nanoscale pattern formation, surface treatment, and polymerization, which are expensive and time-consuming. Recently, simple methodologies based on selfassembly of homopolymer blends and diblock copolymers, or electrohydrodynamic instabilities of polymer films, have been proposed to produce nanotopographic substrates with a large area for studying cell responses.12 For example, a polymer demixing technique could produce randomly distributed © 2012 American Chemical Society
Received: August 7, 2012 Accepted: October 2, 2012 Published: October 2, 2012 9439
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nucleation agents. After the solution on the sealing tape reached the saturation level, the nucleation agent was placed onto this thin saturated monomer solution as external stimulation. The tape with crystallized features was then exposed to UV radiation (365 nm, 4 W; the distance between the lamp and the sample was about 3 cm) for 5 min to form the patterned PNIPAAm surface. Smooth PNIPAAm films were prepared via the same method without applying any nucleation agent, and photopolymerization occurred before the monomer solution reached the saturation level. The electrospun fibers were prepared by using the following protocol. NIPAAm and BAC (25:1) were dissolved in dioxane to make a 10% solution. The polymerization was initiated by BPO at 70 °C for 24 h. Polymers were precipitated with acetic ether and dried in a vacuum oven. The aligned electrospun PNIPAAm fiber sheets were fabricated by electrospinning 10% PNIPAAm HFIP solution as previously described.24 Characterization of PNIPAAm Surfaces. A series of analytical tools were used to characterize the surface properties. Optical images were acquired directly on Nikon Eclipse Ti fluorescent microscope in the optical mode. A stylus profilometer (Surfpak-sv-3100, Mitutoyo) was used to measure the surface profiles on the different patterned surfaces. To visually examine the surface morphology, Au/Pd sputter-coated surfaces were imaged using a Hitachi S-2000 SEM. Surface wettability, another major parameter affecting cell adhesion and protein adsorption, was measured using the sessile drop method. DI water (around 3 μL) was micropipetted on various surfaces at different temperatures. A Nikon high-performance camera was then used to capture the water drop profile on the surface. Image J (National Institutes of Health, NIH, Washington, DC) was subsequently used to obtain the contact angle. Cell Culture, Staining, and Morphology Observation. Human breast cancer cell lines, MDA-MB-231 and MCF-7, were grown on tissue culture flasks in DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2 and 95% air. Suspension lymphoblast cells (Jurkat) were maintained in RPMI 1640 supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin. The MDA-MB-231 cells were pretransfected with green fluorescent protein (GFP). MCF-7 and Jurkat cells were stained with CellTracker Orange and CellTracker Blue, respectively. Phenol-free RPMI 1640 medium was used in the staining process. After they were rinsed three times with D-PBS solution, the cells were observed under a fluorescence microscope (Nikon TS100, Japan). Modulation of Cell Detachment and Morphology. The cells were premixed at equal numbers in culture media (DMEM with 10% FBS) and subsequently seeded on various surfaces at a total cell density of 105 cells/mL. The culture dishes with seeded cells were cultured in a CO2 incubator at 37 °C for 20 h and then gently rinsed with PBS twice, followed by adding fresh media. Fluorescence micrographs were taken before and after rinsing to evaluate the removal of the suspension/detached cells and the morphology of the anchorage-dependent cells. Selective cell removal was then carried out by maintaining the samples at room temperature and then at 4 °C for 1 h. Fluorescence microscopy was used to monitor changes in cell morphology and selective cell detachment. Cells cultured on TCPS were used as controls.
undergoes hydrophilic-to-hydrophobic transitions due to inter-/intramolecular hydrogen bonding competition below and above its lower critical solution temperature (LCST) in water, has been widely used for such applications.19 Okano and co-workers20 grafted PNIPAAm on polystyrene substrates to allow the culture of confluent cell monolayers at 37 °C and their recovery as single-cell sheets when the temperature was below the LCST. They also designed the grafting of PNIPAAm on substrates prepatterned with ridges and grooves.21 Selective adhesion of endothelial cells to the ridges resulted in the formation of capillary-like structures after 2−3 weeks. Although their patterning approach is promising, it still requires high expertise and the use of complex fabrication processes. More recently, microcontact printing has been used to micropattern fibronectin onto commercially available thermoresponsive PNIPAAm cell culture dishes. Dang et al.22 used aligned electrospun fibers from thermoresponsive hydroxybutyl chitosan to control and achieve spatial orientation and efficient harvesting (without disruption of the cytoskeletal structures and cell−cell interactions) of mesenchymal stem cell sheets. In addition, patterned thermoresponsive surfaces have also been used in single-cell manipulating and harvesting studies via localized heating/cooling.23 Herein we describe a novel yet facile approach to create patterned PNIPAAm surfaces with large areas and various shapes by photopolymerization of needlelike monomer crystal solutions. Scanning electron microscopy (SEM), profilometry, and contact angle measurement were used to characterize the patterned PNIPAAm surfaces. Since cell behavior can be directed through a precisely designed environment, we also evaluated the ability of such surfaces to selectively modulate cell morphology and cell separation by using a heterogeneous cell population.
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EXPERIMENTAL SECTION Chemicals and Reagents. N-Isopropylacrylamide (NIPAAm), methanol (≥99% pure), photoinitiator I651 (2,2dimethoxy-2-phenylacetophenone), benzoyl peroxide (BPO), and hexafluoroisopropyl alcohol were purchased from Aldrich Chemicals (Milwaukee, WI). The cross-linker BAC (N,N′bis(acryloyl)cystamine) was purchased from Alfa Aesar (Ward Hill, MA). CellTracker Blue CMF2HC, CellTracker Orange CMTMR, phosphate buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM with 4.5 g/L D-glucose), fetal bovine serum (FBS), penicillin, streptomycin, nonessential amino acids, L-glutamine, and sodium pyruvate were purchased from Invitrogen (Carlsbad, CA). Nunc sealing tape was purchased from Fisher Scientific (Pittsburgh, PA). 1,1,1,3,3,3Hexafluoro-2-propanol (HFIP) was obtained from Oakwood Products (West Columbia, SC). The cell lines, including MDAMB-231, MCF-7, and Jurkat cells, were purchased from ATCC (Manassas, VA). All reagents, unless specified, were used without further purification. Formation of Patterned PNIPAAm Thin Films on Solid Surfaces. NIPAAm, photoinitiator I651, and cross-linker BAC were dissolved in methanol to make a monomer solution with a NIPAAm:I651:BAC:solvent weight ratio of 100:3:3:150. One drop of solution was spread on Nunc sealing tape or a glass substrate and left untouched for 1 min under ambient conditions to allow the solvent to evaporate. A sharp needle or a single-side razor was dipped into another saturated pure NIPAAm solution and blow-dried. Microscale crystals would form at the tip/edge of the needle/razor, and they served as the 9440
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Figure 1. Schematic drawing and SEM micrographs illustrating the formation of patterned PNIPAAm films.
Figure 2. Illustrations and corresponding optical micrographs of the patterned monomer crystals for (a) one-point, (b) two-point, (c) equilateral triangle corner, (d) square corner, and (e) line shape nucleation sites.
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RESULTS AND DISCUSSION Formation of Patterned PNIPAAm Surfaces. The basic concept of the pattern formation by photopolymerization of needlelike monomer crystals from supersaturated solution is illustrated in Figure 1. NIPAAm monomer and a small amount of cross-linker (N,N′-bis(acryloyl)cystamine (BAC)) together with a photoinitiator (2,2-dimethoxy-2-phenylacetophenone (I651)) were dissolved in methanol and coated on a Nunc sealing tape. When the solution reached or passed the saturation point during the solvent evaporation process at room temperature, an external stimulation, such as adding a particle on the thin layer of supersaturated solution as a nucleation site, would trigger fast growth of needlelike monomer crystals in the thin film, resulting in an aligned fiber structure. UV irradiation would then initiate photopolymerization without nitrogen protection and convert the aligned NIPAAm crystals into a stable, thermoresponsive, and patterned PNIPAAm surface. This simple technique is capable of yielding large area (>10 cm2) surfaces on most solid substrates (within minutes), with aligned patterns that could mimic a number of aspects of the in vivo extracellular matrix (ECM),25−27 including white matter tracts, microblood-vessel walls, and the peritumoral microenvironment of some neo-
plasms, all of this without the aid of expensive apparatus, not achievable by existing techniques. Patterned Monomer Crystals by Various Nucleation Sites. Figure 2 shows several surface patterns produced by inducing different external stimulation as the nucleation site on the supersaturated monomer solution. Without any external stimulation, randomly oriented thick monomer crystals with diameters around ∼50 μm would precipitate out of the solution during the solvent evaporation process. By adding nucleation sites, thinner monomer crystals with diameters around 10 μm (perpendicular to the growing direction) would grow quickly from the nucleation center with a crystal growth rate around 250 μm/s. As an example, Figure 2a shows the image of a “star shaped”, spherulite-type crystal pattern formed from a singlepoint nucleation site. In this study, we used a drop of pure NIPAAm particle without any cross-linkers and photoinitiators as the nucleation site. Other nucleation materials such as dust particles could also serve the same purpose. Multiple spherulite patterns were formed by placing two-point (Figure 2b), equilateral triangular, three-point (Figure 2c) and square, four-point (Figure 2d) nucleation sites, while a line shape nucleation site (Figure 2e) led to a “fishbone like” crystal pattern. In each case, the crystals grew divergently from the 9441
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Figure 3. (a) SEM micrographs and (b) surface profiles of the patterned PNIPAAm surface, electrospun PNIPAAm fibers, and a smooth PNIPAAm film.
Figure 4. Comparison of water contact angles on the aligned and electrospun fibers and smooth PNIPAAm surfaces. The values reported for aligned PNIPAAm and spun fibers were obtained in a direction perpendicular to the surface feature.
nucleation site until they reached the edge of the monomer solution film or they impinged against each other. After UVirradiation-induced photopolymerization, the cross-linked PNIPAAm films retained the same macroscale pattern as the monomer crystals. Characteristics of Patterned PNIPAAm Surfaces. It is well-known that cell behavior and protein adsorption onto a substrate are highly affected by the topological features of the substrate (e.g., pattern type, feature dimension, surface roughness) and wettability and chemical nature of the surface. In this study, the SEM images, surface profiles, and water contact angles were used to characterize the surface properties. Figure 3 compares the SEM images and surface profiles on the aligned PNIPAAm, electrospun fibers, and smooth PNIPAAm surfaces. For the aligned PNIPAAm, the striplike patterns from monomer crystals can be clearly seen, and the width of the strips is about several micrometers (shown in Figure 3a). The strips curl along the crystal direction but do not resemble the needle-ike form as in the monomer crystals. According to the cross-section SEM image of aligned PNIPAAm film parallel to the crystal growth direction, the thickness of the film made of aligned PNIPAAm strips is ∼50 μm (data not shown). The surface profile of aligned PNIPAAm perpendicular to the
crystal growth directions reveals that the peak-to-valley distance of the patterned surface can be as high as >8 μm and is typically less than 6 μm (Figure 3b). As a comparison, the electrospun fibers represent a very rough surface and the peak-to-valley distance of the surface can be higher than 24 μm. Flat PNIPAAm films, on the other hand, show smooth surfaces. Changes in topology (i.e., roughness and porosity) also induce changes in the wettability of the surfaces. Figure 4 summarizes the water contact angles on the aligned PNIPAAm, electrospun fibers, and smooth PNIPAAm surfaces. As can be seen, the rough electrospun fibers have the largest water contact angle difference (>60°) between room and body temperatures. The water contact angle on smooth PNIPAAm films only increased 20° when the temperature changed from 20 to 37 °C. These results suggest that the topological differences would affect the hydrophilic-to-hydrophobic transition of PNIPAAm across its lower critical solution temperature (LCST) in water at 32 °C.4,28 It is reported that29 the PNIPAAm electrospun fiber undergoes a transition from the Wenzel state to the metastable Cassie−Baxter state with an increase in temperature: for the hydrophilic nanofibers (T < LCST), the apparent contact angles (θ*) become lower than θY (the ideal Young contact angle) because of the water wicking (Wenzel state); for 9442
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Figure 5. Coculture of Jurkat, MDA-MB-231, and MCF-7 cells on different surfaces (scale bar 100 μm). The images for aligned PNIPAAm are merged images (fluorescence and phase contrast).
Figure 6. Selective detachment of MCF-7 and MDA-MB-231 cells on (a) aligned PNIPAAm and (b) TCPS (scale bar 100 μm).
the hydrophobic nanofibers (T > LCST), θ* becomes greater than θY because of trapped air underneath the liquid inside the rough grooves. Modulation of Cell Detachment. The ability of the patterned PNIPAAm surfaces to selectively modulate cell morphology and cell−substrate interactions was tested using a
heterogeneous cancer cell population. Tissue culture polystyrene (TCPS), smooth PNIPAAm films, and electrospun PNIPAAm fibers were used as controls. A mixture of fluorescently labeled MDA-MB-231 (a highly invasive breast cancer cell line, green), MCF-7 (a mild tumorigenic breast cancer cell line, red), and Jurkat cells (a cancer T-cell line, blue) 9443
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Figure 7. Coculture of MDA-MB-231 and MCF-7 cells on TCPS and aligned PNIPAAm surfaces 48 h after seeding (scale bar 100 μm). The MDAMB-231 cells aligned in parallel with surface features of the aligned PNIPAAm after 48 h.
epithelial to mesenchymal transition. Therefore, our results suggest that selective cell harvesting can be achieved on the patterned PNIPAAm surfaces with minimum perturbations to the cell microenvironment. Controlled Cell Morphology on Pattered Surfaces. The topographic and chemical features of pattered surfaces strongly influence the adhesion and spreading behavior of anchorage-dependent cells. Figure 7 shows that MDA-MB-231 and MCF-7 cells spread randomly and exhibited relatively similar epithelial-like morphologies on the TCPS surface. Cell coculture on the aligned PNIPAAm surfaces, on the other hand, revealed that MDA-MB-231 cells gradually changed to a more elongated shape within 48 h of culture, while MCF-7 cells had a tendency to maintain their original morphology. Similarly, the differences in cell morphology could be attributed to a differential expression of cell adhesion (cell-to-cell and cell-tosubstrate) and cytoskeletal proteins between these two cell types. This, along with the modulation of cell−substrate interactions, may facilitate the identification of specific phenotypes within a heterogeneous cell population (e.g., tissue biopsy).
were placed on these surfaces. After a 20 h incubation period, PBS buffer was used to rinse the surface gently. As shown in Figure 5, most MDA-MB-231 and MCF-7 cells (94%) remained attached to the TCPS and the aligned PNIPAAm surfaces, while Jurkat cells could be rinsed off easily. Although Jurkat cells could also be easily removed from the smooth PNIPAAm films, the MDA-MB-231 and MCF-7 cells maintained the sphere shape and did not spread on these surfaces. Electrospun fibers, on the other hand, did not support selective and complete removal of anchorage-independent Jurkat cells, because they tended to get trapped within the fiber network. Consequently, TCPS and aligned PNIPAAm showed advantages over the smooth PNIPAAm and the electrospun fibers for separating the anchorage-dependent from anchorage-independent cells. In the following, we further compare TCPS and aligned PNIPAAm. Figure 6 shows the selective cell detachment of adherent cells by using different incubation temperatures. On the aligned PNIPAAm surface, most MDA-MB-231 cells were easily removed after incubation at 20 °C for 1 h, while the MCF-7 cells remained anchored. Subsequent incubation at 4 °C for 1 h led to the release of the MCF-7 cells from the surface. In comparison, most tumor cells remained attached to the TCPS surface after a series of incubation and washing at different low temperatures (shown in Figure 6b). Previous studies with cocultures of epithelial and fibroblast cells reported that fibroblasts could be removed from the surface more selectively and rapidly compared to epithelial cells under controlled trypsinization conditions.30 This is possibly due, in part, to the fact that epithelial cells present more complex cell-to-cell and cell-to-ECM/substrate interactions mediated by several proteins, which presumably stabilize cell anchorage to the surface. In our case, selective detachment of MDA-MB-231 cells could be partially attributed to a more fibroblast-like phenotype (compared to the more epithelial MCF-7 cells) resulting from
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CONCLUSION We have developed a novel and facile method to produce stimuli-responsive surfaces with complex patterns, which can be tailored to meet a specific application (e.g., ECM mimics) by controlled photopolymerization of NIPAAm monomer crystals. The patterns form when an external stimulus is applied to induce the growth of needlelike monomer crystals (from a supersaturated NIPAAm monomer solution). The stimulus acts as a nucleation site and quickly triggers fast crystal growth and propagation. This versatile technique allows the formation of various macroscale patterns with microscale features over large areas and on most solid substrates within minutes, without the 9444
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(11) Wilkinson, C. D. W.; Riehle, M.; Wood, M.; Gallagher, J.; Curtis, A. S. G. Mater. Sci. Eng., C 2002, 19, 263−269. (12) Lim, J. Y.; Dreiss, A. D.; Zhou, Z. Y.; Hansen, J. C.; Siedlecki, C. A.; Hengstebeck, R. W.; Cheng, J.; Winograd, N.; Donahue, H. J. Biomaterials 2007, 28, 1787−1797. (13) Lim, J. Y.; Hansen, J. C.; Siedlecki, C. A.; Hengstebeck, R. W.; Cheng, J.; Winograd, N.; Donahue, H. J. Biomacromolecules 2005, 6, 3319−3327. (14) Curtis, A.; Wilkinson, C. Trends Biotechnol. 2001, 19, 97−101. (15) Eliason, M. T.; Sunden, E. O.; Cannon, A. H.; Graham, S.; Garcia, A. J.; King, W. P. J. Biomed. Mater. Res. A 2008, 86A, 996− 1001. (16) Xu, C. Y.; Inai, R.; Koraki, M.; Ramakrishna, S. Biomaterials 2004, 25, 877−886. (17) Cheng, X.; Wang, Y.; Hanein, Y.; Bohringer, K. F.; Ratner, B. D. J. Biomed. Mater. Res. 2004, 70, 159−168. (18) Matsuda, N.; Shimizu, T.; Yamato, M.; Okano, T. Adv. Mater. 2007, 19, 3089−3099. (19) Lin, S. Y.; Chen, K. S.; Liang, R. C. Polymer 1999, 40, 2619− 2624. (20) Tsuda, Y.; Shimizu, T.; Yarnato, M.; Kikuchi, A.; Sasagawa, T.; Sekiya, S.; Kobayashi, J.; Chen, G.; Okano, T. Biomaterials 2007, 28, 4939−4946. (21) Tsuda, Y.; Yamato, M.; Kikuchi, A.; Watanabe, M.; Chen, G. P.; Takahashi, Y.; Okano, T. Adv. Mater. 2007, 19, 3633−3636. (22) Dang, J. M.; Leong, K. W. Adv. Mater. 2007, 19, 2775−2779. (23) Ichikawa, A.; Arai, F.; Yoshikawa, K.; Uchida, T.; Fukuda, T. Appl. Phys. Lett. 2005, 87, 191108−1−191108−3. (24) Guan, J.; Wang, F.; Li, Z.; Chen, J.; Guo, X.; Liao, J.; Moldovan, N. I. Biomaterials 2011, 32, 5568−5580. (25) Petrie, R. J.; Doyle, A. D.; Yamada, K. M. Nat. Rev. Mol. Cell Biol. 2009, 10, 538−549. (26) Johnson, J.; Nowicki, M. O.; Lee, C. H.; Chiocca, A.; Viapiano, M. S.; Lawler, S. E.; Lannutti, J. J. Tissue Eng. Part C 2009, 15, 531− 540. (27) Giese, A.; Westphal, M. Neurosurgery 1996, 39, 235−250. (28) Zhang, T.; Wang, J.; Chen, L.; Zhai, J.; Song, Y.; Jiang, L. Angew. Chem., Int. Ed. 2011, 50, 5311−5314. (29) Konosu, Y; Matsumoto, H; Tsuboi, K.; Minagawa, M; Tanioka, A. Langmuir 2011, 27, 14716−14720. (30) Owens, R. B. J. Natl. Cancer Inst. 1974, 52, 1375−1378. (31) Wang, J. W. J.; Sutti, A.; Wang, X. G.; Lin, T. Soft Matter 2011, 7, 4364−4368. (32) Karni, T. C.; Casanova, D.; Cahoon, J.; Qing, Q.; Bell, D.; Lieber, C. M. Nano Lett. 2012, 12, 2639−2644. (33) Sun, M.; McGowan, M.; Kingham, P. J.; Terenghi, G.; Downes, S. J. Mater. Sci. Mater. Med. 2010, 21, 2765−2774. (34) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Chen, G. P.; Okano, T. Biochem. Biophys. Res. Commun. 2006, 348, 937−944. (35) Hartman, O.; Zhang, C.; Adams, E. L.; Farach-Carson, M. C.; Petrelli, N. J.; Chase, B. D.; Rabolt, J. F. Biomacromolecules 2009, 10 (8), 2019−2032.
need for expensive equipments and facilities. The incorporation of thermoresponsive PNIPAAm enables cell separation and sorting by modulating temperature- and topography-dependent cell−substrate interactions and cell morphology, respectively. These functionalities are demonstrated by coculturing cells with different characteristics. When the temperature is lowered to or below the LCST (∼32 °C), the PNIPAAm patterns undergo a hydrophobic-to-hydrophilic transition, thus resulting in selective detachment of the cells, which is determined by the temperature and the cell phenotype. In contrast to normal cells and mild tumorigenic cancer cells, highly invasive cancer cells align better with, and migrate along, fiberlike or tubelike microstructures present in the parenchyma and stroma to facilitate tumor cell dissemination and metastasis.31 The aligned PNIPAAm patterns could presumably be used to identify more invasive tumor cell phenotypes in a heterogeneous cell population, including circulating tumor cells (CTCs) in patient blood, which could potentially be sorted on the basis of their adhesion and morphological properties and then isolated (by lowering the temperature) for further biomolecular analysis.23 Such patterned surfaces can act as both an in vitro tumor model and a separation platform for cancer studies. This method can also be applied to other cellbased biological studies and clinical applications. Examples include neuronal networks,32,33 cell sheet based tissue engineering,9,34 and ECM mimics for drug testing and discovery.35
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AUTHOR INFORMATION
Corresponding Author
*Tel: (614) 292-2408. Fax: (614) 292-3769. E-mail: leelj@ chbmeng.ohio-state.edu. Author Contributions §
These authors contributed equally to this paper.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the National Science Foundation sponsored Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices (NSEC-CANPBD) at The Ohio State University.
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