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Dynamic Micropatterning of Cells on Nanostructured Surfaces Using a Cell-friendly Photoresist SoonHo Kweon,†,§ Kwang Hoon Song,‡,§ HyoungJun Park,‡ Jong-Cheol Choi,‡ and Junsang Doh*,†,‡ †
School of Interdisciplinary Bioscience and Bioengineering (I-Bio) and ‡Department of Mechanical Engineering, Pohang University of Science and Technology, San31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk 790-784, South Korea S Supporting Information *
ABSTRACT: Cellular dynamics under complex topographical microenvironments are important for many biological processes in development and diseases, but systematic investigation has been limited due to the lack of technology. Herein, we developed a new dynamic cell patterning method based on a cell-friendly photoresist polymer that allows in situ control of cell dynamics on nanostructured surfaces. Using this method, we quantitatively compared the spreading dynamics of cells on nanostructured surfaces to those on flat surfaces. Furthermore, we investigated how cells behaved when they simultaneously encountered two topographically distinct surfaces during spreading. This method will allow many exciting opportunities in the fundamental study of cellular dynamics. KEYWORDS: dynamic patterning, cell patterning, nanostructured surface, cell-friendly photoresist, cell migration
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INTRODUCTION Dynamic cellular processes such as adhesion/detachment, spreading, and migration are critical for development, immune responses, and cancer invasion/metastasis.1−5 While live-cell imaging has been widely used to study detailed mechanisms of cellular dynamics, inefficient data acquisition due to heterogeneity in dynamic behaviors of cells within an imaging field-ofview has limited the throughput of experiments. Dynamic cell micropatterning techniques can be a powerful method to enhance the throughput of live-cell imaging by allowing synchronization of cellular morphology and dynamics. In dynamic cell micropatterning techniques, stationary cells surrounded by “cell-repellent” materials such as poly(ethylene glycol) (PEG) or bovine serum albumin (BSA) are triggered to spread or migrate by selectively removing or converting the surrounding cell-repellent materials by various stimuli including light, electric fields, and chemicals.6−14 Using this technique, synchronized cellular dynamics in many imaging fields-of-view can be triggered and nearly simultaneously monitored using a motorized stage; thus, high-throughput live-cell imaging can be performed.12 Nanoscale topography on extracellular matrices (ECMs) is an important biophysical cue for regulating cell behaviors, and nanostructured surfaces have been widely used to investigate the effect of nanotopography on cell function.15−17 Spreading and migration of various types of cells on diverse geometries of nanostructured surfaces have been examined to understand the effect of nanotopography on cellular dynamics, which could be potentially useful to control wound healing, cancer metastasis, and immune responses.18−30 Recently, surfaces containing © XXXX American Chemical Society
multiple types of topographical structures in small areas have been fabricated to investigate how cells migrate in such complex and heterogeneous topographical microenvironments.31−36 Enrichment of cells on certain types of topographical structures has been observed several tens of hours after the initial seeding, indicating that if cells are located at the interfaces between different topographical structures, they preferentially polarize and migrate toward certain topographical structures. However, the dynamics of cells at interfaces between two distinct topographical structures has not yet been systematically examined in detail. Herein, we sought to combine two technologies, namely dynamic micropatterning and nanostructured surfaces, with the aim of significantly improving our understanding of cellular dynamics on nanostructured surfaces. To achieve this goal, we developed dynamic cell micropatterning on nanostructured surfaces using a cell-friendly photoresist poly(2,2-dimethoxy nitro-benzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP, Figure 1A).37,38 Using this technique, we first investigated differences in cell spreading dynamics on nanostructured and flat surfaces. We then further addressed how cells behave if half of the cells contact flat regions and the other half contact nanostructured surfaces during spreading. This technique will be useful in studying dynamic cellular processes under complex topographical microenvironments, particularly for cancer invasion Received: January 10, 2016 Accepted: January 13, 2016
A
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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acrylate) (PUA) nanostructured surfaces (ridges, grooves, and heights of 700, 350, 300 nm, respectively, which represent typical dimensions of fibrillary bundles of ECM molecules)40,41 fabricated on glass coverslips by capillary force lithography (CFL)42 were used (Figure 1B(i)). Nanostructured PUA surfaces were spin-coated with PDMP (Figure 1B(ii)), loaded into a chamber filled with phosphate buffered saline (PBS; 10 mM sodium phosphate, 140 mM sodium chloride, and pH = 7.4), and mounted on a microscope stage. Microscope projection photolithography (MPP)12,37 was then performed by illuminating with UV light (λ = 365 nm) focused through an objective lens, with a transparency photomask inserted in a field diaphragm of the microscope (Figure 1B(iii)). PDMP thin films in UV-illuminated regions were spontaneously dissolved in PBS, resulting in exposure of the underlying nanostructured surfaces. In this process, the thickness of the PDMP thin film is critical; if the PDMP thin film is excessively thin, the nanostructured surface will not be fully covered by the PDMP thin film. If the PDMP thin film is excessively thick, the UV exposure time required for the dissolution of the UVexposed PDMP thin film will become longer, which may potentially damage cells. To optimize the PDMP thin-film thickness, various concentrations of PDMP solutions (3, 6, and 10 w/v % in 1,4-dioxane) were spin-coated on nanostructured surfaces, MPP was performed, and the resulting micropatterned nanostructured surfaces were examined by scanning electron microscopy (SEM). As shown in Figure 1C, PDMP thin films spin-coated from 3 and 6 w/v % PDMP solutions were not sufficiently thick to cover the underlying nanostructured surfaces, and nanoscale topographical structures were visible across the entire surface. In contrast, PDMP thin films spincoated from 10 w/v % PDMP solutions completely covered the nanostructured surface, and only UV-illuminated areas exhibited visible nanostructures. Complete coverage of nanostructures by spin-coating of 10 w/v % of PDMP solution was further confirmed by cross-sectional SEM (Figure 1D). On
Figure 1. Fabrication of micropatterned nanostructured surfaces. (A) Structure and photochemical reaction of PDMP. (B) Schematic procedure of micropatterning on nanostructured surfaces. (C) Representative SEM images of micropatterned nanostructured surfaces fabricated using various concentrations of PDMP solution for spincoating. (D) Cross-sectional SEM image of nanostructured surfaces spin-coated with 10 w/v % PDMP solution.
and metastasis where the heterogeneity of the topographical structures of ECMs may play an important role.5,39
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RESULTS AND DISCUSSION Micropatterning of PDMP Thin Films Coated on Nanostructured Surfaces. For the dynamic micropatterning of cells on nanostructured surfaces, we first performed micropatterning on nanostructured surfaces by following the procedure schematically shown in Figure 1B. Poly(urethane
Figure 2. Cell spreading dynamics on nanostructured vs flat surfaces. (A) Schematic procedure of single cell spreading experiments on nanostructured surfaces. (B−C) Representative time-lapse differential interference contrast (DIC) images of HeLa cells on nanostructured (B) and flat (C) surfaces. B
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 3. Quantitative analyses of cell spreading dynamics on nanostructured vs flat surfaces. (A) Definition of parameters for cell spreading analysis. (B−E) Kinetics of morphology changes on nanostructured and flat surfaces. X (B), Y (C), elliptical form factors (X/Y) (D), and area (E). n = 40 for each case. (Error bars, SD; Mann−Whitney test, NS, not significant; *p < 0.05; ***p < 0.001.)
cells on nanostructured surfaces, cells on flat surfaces underwent isotropic spreading. To quantitatively analyze the spreading of cells, the direction of the nanogrooves was assigned to the x-axis, and the average length of cells in the x- (X) and y-axes (Y) and the elliptical form factor (X/Y) were measured and plotted (Figures 3A−D). Cells on nanostructured surfaces continuously spread along the nanogroove direction (or x-axis) with a much faster spreading rate than cells on flat surfaces (Figure 3B). In contrast, cell length in the direction perpendicular to the nanogrooves (or yaxis) slightly increased initially and appeared to reach a plateau within 1 h (Figure 3C). Consequently, the average elliptical form factor of cells spreading on nanostructured surfaces continuously increased up to ∼4 while the average elliptical form factor of cells on flat surfaces was ∼1 at all times (Figure 3D). The average area of cells on nanostructured surfaces was larger than that of cells on flat surfaces in the initial stages (t = 1 and 2 h), but the differences were not statistically significant at 3 h (Figure 3E), implying the initial spreading rate of cells was faster on nanostructured surfaces than on flat surfaces. Cytotoxicity of PDMP-based MPP. We have previously demonstrated that PDMP-based MPP had minimal toxicity on HeLa12 and MDCK.43 While relatively thin PDMP films (typically ∼100-nm-thick) that only required 1 s of brief UV exposure for complete dissolution were used in the previous studies,12,43 significantly thicker PDMP films (at least 300-nmthick to cover the entire nanotopography, and typically ∼550 nm measured by cross-sectional SEM images) need to be used for this study, which increases the amount of the polymer dissolved by the photochemical reaction and UV exposure time. Indeed, 3 s of UV exposure was sufficient to completely dissolve 550-nm-thick PDMP thin films immersed in PBS when UV illumination was performed using a Xe lamp (75 W) filtered through a DAPI excitation filter (EX. 365, BS 395). Considering that arc lamps filtered through a DAPI excitation filter are widely used for live cell imaging of cells labeled with Hoechst,44 3 s of UV illumination would be unlikely to harm the cells.45 PDMP thin films exposed to UV undergo a photochemical reaction to generate water-soluble compounds (right-hand side of Figure 1A). To test whether the compounds generated by the photochemical reaction of PDMP (CP-PDMP) are toxic to cells, first CP-PDMP solution in PBS was prepared. A glass vial
the basis of this result, 10 w/v % PDMP solution was used for spin-coating for the rest of the study. Dynamic Micropatterning of Cells on Nanostructured Surfaces. For dynamic micropatterning of cells on nanostructured surfaces, gelatin-coated nanostructured PUA surfaces were spin-coated with PDMP. MPP was performed on PDMP/ gelatin-coated nanostructured surfaces to generate a square array of 12-μm-diameter circles with 120 μm center-to-center distances. Then, HeLa cells in culture medium were applied onto the micropatterned nanostructured surfaces (Figure 2A(i)), incubated for 1 h at 37 °C with 5% of CO2, and washed with fresh culture medium to remove nonadhering cells. Since proteins and cells cannot adhere to PDMP surfaces,37 and gelatin-coated nanostructured areas exposed by the MPP, which promotes cell adhesion, are slightly smaller than the typical diameter of HeLa cells (14.6 ± 1.6 μm),43 single-cell arrays with partially spread cells were formed (Figure 2A(ii)). Typically, 50−80% of gelatin-coated PUA islands were occupied by single HeLa cells. Note that perfect single-cell arrays are not necessary because cells on desired spots can be selected and used for the subsequent experiment by using a programmed motorized stage.12 PDMP thin films surrounding the selected HeLa cells were then removed by UV light illumination without a photomask to trigger the spreading of cells (Figure 2A(iii)). Importantly, brief illumination with UV (typically less than 3 s) that would have negligible effects on cell function and viability12 was sufficient for the dissolution of PDMP thin films surrounding the cells. The dynamics of cell spreading on nanostructured surfaces was monitored by acquiring differential interference contrast (DIC) time-lapse images for 3 h. Using a motorized stage, typically ∼30 cells were monitored in each experiment. Representative time-lapse DIC images and a movie of cells spreading on nanostructured surfaces are shown in Figure 2B and Movie S1 in the Supporting Information (SI), respectively. A rounded cell at the beginning of the experiment became elongated toward the direction of nanogrooves, implying the occurrence of anisotropic spreading. Anisotropic spreading was also observed for cells other than HeLa cells such as HT-1080 and MDCK cells as shown in Figure S1 in the SI. Identical experiments were performed on gelatin-coated flat PUA surfaces, and representative time-lapse DIC images and a movie are shown in Figure 2C and Movie S2 in the SI, respectively. In contrast to C
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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During step (iii) in Figure 2A when PDMP films surrounding cells are removed by UV illumination without a photomask, approximately 1.6 × 10−4 mg of CP-PDMP is generated when a 40× objective lens is used (assumed density of PDMP ∼ 1 g/ cm3; 40× objective lens generates a circle pattern with radius ∼300 μm; PDMP film thickness of 550 nm, thus the amount of CP-PDMP generated by the photochemical reaction is π × (300 μm)2 × 550 nm × 1 g/cm3). In each experiment, ∼50 cells in 1 mL of cell culture media are triggered to spread, so the final concentration of CP-PDMP is ∼0.008 mg/mL. Because 0.1 mg/mL of CP-PDMP was not toxic for cells (Figure 4), CP-PDMP generated during dynamic cell micropatterning would not be cytotoxic if CP-PDMP generated by the photochemical reaction diffuses away rapidly. Indeed, the concentration of CP-PDMP estimated by numerical simulation rapidly decreased and reached 0.1 mg/mL within 30 min (detailed simulation methods and results are described in the SI), thus the toxicity of CP-PDMP is likely to be minimal. Cell Spreading Dynamics on Complex Topographical Surfaces. Next, we asked how cells spread when they contact nanostructured and flat surfaces simultaneously. To address this question, we fabricated surfaces containing nanostructured and flat regions adjacent to each other, or nano/flat combined surfaces, by a previously developed method.31 As shown in Figure 5A, two different types of surfaces, “perpendicular” and “parallel” depending on the relationships between nano/flat interfaces and nanogrooves, were fabricated. The geometries shown in Figure 5A are somewhat artificial but can provide insights into fundamental cell behaviors in response to heterogeneous topographical structures. During cancer invasion and metastasis, tumor cells may encounter heterogeneous topographical structures, which may direct migration of tumor cells.5,41 Using these substrates and following procedures shown in Figures 1B and 2A(i,ii), centroids of partially spread HeLa cells were located at the nano/flat interfaces (Figure 5B(i) and Figure S2 in the SI). Then, cell spreading was triggered by illumination without a photomask (Figure 5B(ii)),
containing PDMP immersed in PBS was irradiated by UV to completely dissolve CP-PDMP. Typically, 4 mg/mL of CPPDMP in PBS was used as a stock solution. The viability of cells treated with three different concentrations of CP-PDMP (0.1, 0.01, and 0.001 mg/mL), which is a widely used concentration range to test cytotoxicity of polymers,46,47 was then assessed by performing an MTS assay. Six different types of cells including HeLa (human cervical cancer cell), HaCaT (human keratinocyte cell), HT-1080 (human fibrosarcoma cell), NIH-3T3 (mouse embryonic fibroblast cell), MDA-MB231 (human breast cancer cell), and MDCK (canine kidney epithelial cell) were tested. As shown in Figure 4, the viability of cells treated with CP-PDMP was comparable to that of untreated cells for all the cell types tested, meaning the cytotoxicity of CP-PDMP is minimal.
Figure 4. Cytotoxicity of compounds generated by the photochemical reaction of PDMP (CP-PDMP). MTS assays were performed by adding various concentrations of CP-PDMP to six different types of cells. “Absorbance” represents the viability of cells. Data are averages of three replicates. (Error bars, SD; Mann−Whitney test, NS, not significant).
Figure 5. Dynamics of cells on perpendicular and parallel nano/flat combined surfaces. (A) Representative SEM images of perpendicular and parallel nano/flat combined surfaces. (B) Schematic illustration of cell spreading experiments on nano/flat combined surfaces with centroid located at nano/ flat interfaces. (C−D) Representative still images (DIC) acquired 3 h after spreading with outlines of cells on perpendicular (C) and parallel (D) nano/flat combined surfaces. (E) Elliptical form factor of cells 3 h after the onset of spreading (perpendicular, n = 45; and parallel, n = 32; error bars, SD; Mann−Whitney test, NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). D
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 6. Quantitative analyses of the morphology of cells on perpendicular and parallel nano/flat combined surfaces. (A) Definition of parameters for cell morphology analysis on nano/flat combined surfaces. (B−C) Effect of surface types on morphology of cells on perpendicular (B; n = 45) and parallel (C; n = 32) nano/flat combined surfaces after 3 h of spreading. (Error bars, SD; Mann−Whitney test, NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.)
Figure 7. Focal adhesion and F-actin distribution of cells on perpendicular and parallel nano/flat combined surfaces. (A,B) DIC, vinculin, and F-actin fluorescence images of cells on perpendicular (A) and parallel (B) nano/flat combined surfaces. Dashed lines: interfaces of flat and nano surfaces. (C,D) Focal adhesion area and F-actin fluorescence intensity of cells on perpendicular (C) and parallel (D) nano/flat combined surfaces (n = 40 for each case; error bars, SD; Mann−Whitney test, NS, not significant).
perpendicular nano/flat combined surfaces (Figure 5E), suggesting that spreading on nanostructured regions dominated the overall spreading of cells; cells contacting nanostructured regions elongated along the nanogroove directions while cells contacting flat regions developed sharp edges perpendicular to nano/flat interfaces. Cell morphology contacting flat vs nanostructured regions was quantitatively analyzed by measuring the longest distance from nano/flat interfaces (d), the length of cells to the direction of nano/flat interfaces (h), and the area of cells contacting each region (Figure 6A). Overall, quantitative analyses of cell morphology (Figure 6B,C) agreed well with trends qualitatively described above. To gain insights into distinct morphologies of cells formed on parallel and perpendicular nano/flat combined surfaces, focal adhesion complexes and filamentous actin (F-actin) were visualized using fluorophore-conjugated anti-vinculin and phalloidin, respectively (Figure 7A,B). Quantitatively, focal adhesion area and F-actin staining intensity (Figure 7C,D) of cells contacting nanostructured regions and flat regions were
and spreading dynamics were observed by time-lapse imaging. Representative time-lapse images and a movie of cells spreading on nano/flat combined surfaces are shown in Figures S3 and S4 and Movies S3 and S4 in the SI, respectively. Over time, distinct morphologies of cells emerged depending on their locations and surface types. Representative still images acquired 3 h after spreading with outlines of cells on perpendicular and parallel nano/flat combined surfaces are shown in Figure 5C and D, respectively. Overall, cells on the perpendicular nano/ flat combined surfaces exhibited rather rounded morphologies with an elliptical form factor of 1.21 ± 0.03 (Figure 5E), close to 1, indicating that the spreading of cells on flat regions dominated the overall spreading of cells. Indeed, cells contacting flat regions appeared to be almost rounded, while cells contacting nanostructured regions were slightly elongated along the nanogroove directions. In contrast, cells on the parallel nano/flat combined surfaces exhibited elongated morphologies with an elliptical form factor of 1.68 ± 0.10 (Figure 5E), which is significantly greater than that of cells on E
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different, but biased migration toward nanostructured surfaces still occurred. It is possible that differences in surface topography directly induced preferential migration toward nanostructured surfaces.49 Alternatively, the distinct morphologies of cells in each region that were developed during spreading induced preferential polarization of cells toward certain directions.13 Recently, it was reported that cells on surfaces containing multiple topographical structures or a gradient of topographical structures tend to preferentially migrate to certain topographical structures.31−34 Such inferences were typically made by observing accumulation of cells on certain topographical structures after several tens of hours from initial seeding because most mesenchymal or epithelial cells migrate with a velocity of ∼10 μm/h. High resolution spatiotemporal dynamics of cells at the interfaces between two distinct topographical structures would provide mechanistic insights into how cells sense complex topographical microenvironments and steer migration directions, but such a study is challenging with randomly located slowly moving cells. In contrast, our method allows us to determine the initial location of cells prior to triggering their spreading. This unique capability enabled us to observe the dynamic behavior of ∼30 cells located at the interfaces between nanostructured and flat surfaces in each experiment and thus systematically compare differences in spreading dynamics of cells on nano/flat combined surfaces with two different configurations. Therefore, the developed method in this study would be particularly useful in assessing detailed dynamics of cells on complex and heterogeneous nanostructured surfaces by increasing experimental throughput.
not significantly different from each other for both perpendicular and parallel nano/flat combined surfaces. Of note, elongated vinculin staining along the nanogrooves was observed in cells contacting nanostructured areas regardless of surface types, similar to cells on nanostructured surfaces (Figure S7B in the SI). In contrast, isotropically oriented vinculin staining was observed in cells contacting flat surfaces, similar to cells on flat surfaces (Figure S6A in the SI). These results indicate that focal adhesion structure is determined by local surface topography. Interestingly, F-actin stress fiber formation along the direction of cell elongation, which is a hallmark of cells aligned on nanogrooved surfaces41,48 (Figure S7B in the SI), was only observed in cells on parallel nano/flat combined surfaces. F-actin distribution of cells on perpendicular nano/flat combined surfaces was rather isotropic and concentrated near cortical regions, similar to cells on flat surfaces (Figure S7A in the SI). These results suggest that Factin stress fiber formation is more influenced by the global configuration of topographical structures determining overall cell morphology. Continuous nanogrooves on parallel nano/flat combined surfaces may promote F-actin stress fiber formation across cells, inducing elongated morphology of cells similar to cells on nanostructured surfaces. In contrast, discontinued nanogrooves on perpendicular nano/flat combined surfaces at nano/flat interfaces may discourage F-actin stress fiber formation in cells, resulting in rounded morphology close to cells on flat surfaces. About 4 h after the onset of spreading, the majority of cells polarized and migrated. Trajectories of migrating cells on two different types of nano/flat combined surfaces are plotted in Figure 8A and B. The majority of cells (40 out of 45 cells) on
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CONCLUSIONS We developed a method for dynamic cell micropatterning on nanostructured surfaces using a cell-friendly photoresist PDMP for the first time, as far as we know. Using this new technique, we first quantitatively studied differences in cell spreading dynamics on nanostructured and flat surfaces. We then further addressed how cells behave when they contact nanostructured and flat surfaces simultaneously during spreading. Importantly, this is the first systematic demonstration of how cells respond when they encounter two topographically distinct surfaces. The detailed mechanisms of the intriguing cellular dynamics observed herein require further investigation, but the unique capability of controlling the initial positions of cells on complex topographical surfaces, and subsequently triggering spreading and migration using light, would allow many exciting opportunities in the fundamental study of cellular dynamics.
Figure 8. Migration of cells on perpendicular and parallel nano/flat combined surfaces. (A,B) Representative trajectories of migrating cells on perpendicular (A) and parallel (B) nano/flat combined surfaces.
perpendicular nano/flat combined surfaces migrated toward the nanostructured surfaces, suggesting that preferential polarization of the molecules/subcellular compartments determining the leading edges occurred toward nanogrooves. Similarly, most of the cells (22 out of 32 cells) on parallel nano/flat combined surfaces migrated along the direction of the nanogrooves with their leading edges biased toward nanostructured surfaces. In the case of cells on parallel nano/flat combined surfaces, biased migration of cells toward nanostructured regions may be due to biased spreading of cells, considering that the average area of cells contacting nanostructured regions is always slightly higher than that of cells contacting flat regions (Figure 6C and Figure S6C in the SI). However, in the case of cells on perpendicular nano/flat combined surfaces, the average areas of cells contacting each region are not significantly different from each other (Figure 6B and Figure S5C in the SI), meaning the spreading rate of cells in each region was not significantly
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MATERIALS AND METHODS
Fabrication of Nanostructured Surfaces. Nanostructured surfaces containing nanoscale ridges/grooves (700 nm ridges, 350 nm grooves, and 300 nm heights) were fabricated by capillary force lithography (CFL) as described previously.42 PDMP Synthesis and Substrate Preparation. Random terpolymer PDMP was synthesized and characterized as described elsewhere.38 Nanostructured or flat PUA surfaces were coated with 0.1% gelatin solution (Sigma) at room temperature for 30 min and rinsed with DI water extensively. Then, 10 wt % PDMP in 1,4-dioxane (Sigma) was spin-coated at 2000 rpm for 2 min and baked at 120 °C for 48 h. Cell Culture. HeLa (human cervical cancer cell), HaCaT (human keratinocyte cell), HT-1080 (human fibrosarcoma cell), NIH-3T3 (mouse embryonic fibroblast cell), MDA-MB-231 (human breast cancer cell), and MDCK (canine kidney epithelial cell) were cultured F
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Invitrogen) at 37 °C with 5% CO2. Microscope Projection Photolithography (MPP). A modified Zeiss Axio Observer Z1 epifluorescence microscope with a 40× (PlanNeofluar, NA = 1.30) and 100× (Plan-Neofluar, NA = 1.30) objective lens and a Roper scientific Coolsnap HQ CCD camera was used for imaging and micropatterning. The microscope was automatically controlled by Axiovision 4.6 (Carl Zeiss). A PDMP-coated nanostructured or flat surface was mounted in a magnetic chamber (Chamlide, Live Cell Instrument, Korea) filled with PBS, and the chamber was loaded onto the microscope stage. A transparency filmbased photomask printed by a high-resolution image setter with 40 000 dpi resolution (Microtech., Korea) was inserted at the field diaphragm of the microscope, and UV light was illuminated with a xenon lamp (75 W, Osram) through a DAPI filter (EX 365, BS 395). For large area pattern generation, a motorized microscope stage was used as described previously.12 Scanning Electron Microscopy (SEM). Samples were coated by platinum-sputtering, and SEM was performed using a Philips XL30S. Cytotoxicity Assay of Compounds Generated by Photochemical Reaction of PDMP. The compounds generated by the photochemical reaction of PDMP (CP-PDMP) were dissolved in PBS. A glass vial containing 4 mg of PDMP and 1 mg of PBS was irradiated by UV to generate CP-PDMP and completely dissolve CP-PDMP in PBS. Then, cells were treated with three different concentrations of CP-PDMP (0.1, 0.01, and 0.001 mg/mL), and the viability of cells was assessed by performing MTS assays (CellTiter96 AQueous One Solution Cell Proliferation Assay, Promega). Six different types of cells including HeLa (human cervical cancer cell), HaCaT (human keratinocyte cell), HT-1080 (human fibrosarcoma cell), NIH-3T3 (mouse embryonic fibroblast cell), MDA-MB-231 (human breast cancer cell), and MDCK (canine kidney epithelial cell) were plated into a 96-well plate at a density of 104 cells in 100 μL of cell culture media for 24 h. Then, three different concentrations of CP-PDMP (0.1, 0.01, and 0.001 mg/mL) in cell culture media were added. After 36 h of incubation, 20 μL of CellTiter96 AQueous One Solution reagent was added to each well of the 96 well plate. Then, the plate was incubated for 2 h, and the absorbance of each well at 490 nm was measured using a 96-well plate reader (Infinite M200 PRO NanoQuant, TECAN). Dynamic Micropatterning of Cells. Dynamic micropatterning of cells was performed as described previously.12 A photomask printed with an array of circles (180-μm-diameter) was used for the first MPP to generate ∼12 μm gelatin-coated islands surrounded by PDMP thin films. Then, HeLa cells were adhered onto the gelatin-micropatterned substrates by applying cell suspensions (1 mL with density of 1 million cells/mL) to the gelatin-island containing substrates, incubating in an incubator equipped with 37 °C and 5% CO2 for 1 h, and gently washing to remove unbound cells. Then, the HeLa cell-micropatterned substrates were mounted on the microscope stage equipped with a Chamlide TC incubator system (Live Cell Instrument, Korea) to maintain constant culture conditions (37 °C, 5% CO2). Substrates were then illuminated with UV light for 3 s without a photomask using a 40× objective lens to remove the PDMP thin film surrounding cells and trigger spreading. Subsequently, time-lapse images were acquired every 20 min up to 13 h. Typically, the spreading of 30 cells was simultaneously monitored in a single experiment using a motorized stage. Acquired images were analyzed and processed with ImageJ (NIH). Fabrication of Nano/Flat Combined Surfaces. PDMP surfaces containing flat regions adjacent to nanostructured regions were fabricated by sequentially applying CFL and MPP on PDMP films (prepared by spin-coating 10 wt % PDMP in 1,4-dioxane at 2000 rpm for 60 s) as previously described.31 Then, nano/flat structures on PDMP were replicated twice by PUA-based CFL as described above to obtain PUA surfaces containing structures identical to nano/flat structures on PDMP. Fluorescence Imaging of Vinculin and F-actin. Cells on substrates were fixed for 10 min using 4% paraformaldehyde at 4 °C and permeabilized using 0.1% Triton X-100 for 5 min. Alexa-fluor 647
conjugated phalloidin (Invitrogen) and rabbit anti-vinculin antibody (Invitrogen) were added and incubated overnight. Then, biotinylated anti-rabbit antibody (Invitrogen) and Alexa-fluor 555 streptavidin (Invitrogen) were added and incubated for 40 min at 4 °C in order. A modified Zeiss Axio Observer.Z1 epi-fluorescence microscope with 40× (Plan-Neofluar, NA = 1.30) objective lenses and a Roper Scientific CoolSnap HQ CCD camera was used for imaging. An XBO 75W/2 xenon lamp (75W,Osram) and Cy3 (EX BP 550/24, BS 570, EMBP 605/70) and Cy5 (EX BP 620/60, BS 660, EMBP 770/75) filter sets were used for fluorescence imaging. The microscope was automatically controlled by Axiovision 4.6 (Carl Zeiss), and acquired images were processed with ImageJ (NIH).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00318. Computer simulation of the diffusion of CP-PDMP and supporting figures (PDF) Representative movie of a spreading cell on a nanostructured surface. Elapsed time: mm:ss (AVI) Representative movie of a spreading cell on a flat surface. Elapsed time: mm:ss (AVI) Representative movie of a spreading cell on a perpendicular nano/flat combined surface. Elapsed time: mm:ss (AVI) Representative movie of a spreading cell on a parallel nano/flat combined surface. Elapsed time: mm:ss (AVI) Representative movie of a migrating cell on a perpendicular nano/flat combined surface after spreading. Elapsed time: mm:ss (AVI) Representative movie of a migrating cell on a parallel nano/flat combined surface after spreading. Elapsed time: mm:ss (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions §
These authors contributed equally to the work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (J.D., Grant No. A121177/ HI12C1079) and the Industrial Technology Innovation Program (No. 10048358) funded by the Ministry Of Trade, Industry & Energy (MI, Korea), and the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (Grant No. 2011-0030075).
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REFERENCES
(1) Gumbiner, B. M. Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis. Cell 1996, 84 (3), 345−357. (2) Luster, A. D.; Alon, R.; von Andrian, U. H. Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 2005, 6 (12), 1182−1190. (3) Ley, K.; Laudanna, C.; Cybulsky, M. I.; Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 2007, 7 (9), 678−689.
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(24) Kwon, K. W.; Park, H.; Doh, J. Migration of T Cells on Surfaces Containing Complex Nanotopography. PLoS One 2013, 8 (9), e73960. (25) Kwon, K. W.; Park, H.; Song, K. H.; Choi, J. C.; Ahn, H.; Park, M. J.; Suh, K. Y.; Doh, J. Nanotopography-guided migration of T cells. J. Immunol. 2012, 189 (5), 2266−2273. (26) Lamers, E.; te Riet, J.; Domanski, M.; Luttge, R.; Figdor, C. G.; Gardeniers, J. G. E.; Walboomers, X. F.; Jansen, J. A. Dynamic cell adhesion and migration on nanoscale grooved substrates. European Cells and Materials 2012, 23, 182−194. (27) Lamers, E.; van Horssen, R.; te Riet, J.; van Delft, F. C. M. J. M.; Luttge, R.; Walboomers, X. F.; Jansen, J. A. The influence of nanoscale topographical cues on initial osteoblast morphology and migration. European Cells and Materials 2010, 20, 329−343. (28) Liliensiek, S. J.; Wood, J. A.; Yong, J.; Auerbach, R.; Nealey, P. F.; Murphy, C. J. Modulation of human vascular endothelial cell behaviors by nanotopographic cues. Biomaterials 2010, 31 (20), 5418− 5426. (29) Sun, X.; Driscoll, M. K.; Guven, C.; Das, S.; Parent, C. A.; Fourkas, J. T.; Losert, W. Asymmetric nanotopography biases cytoskeletal dynamics and promotes unidirectional cell guidance. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (41), 12557−12562. (30) Yim, E. K. F.; Reano, R. M.; Pang, S. W.; Yee, A. F.; Chen, C. S.; Leong, K. W. Nanopattern-induced changes in morphology and motility of smooth muscle cells. Biomaterials 2005, 26 (26), 5405− 5413. (31) Kwon, K. W.; Choi, J.-C.; Suh, K.-Y.; Doh, J. Multiscale Fabrication of Multiple Proteins and Topographical Structures by Combining Capillary Force Lithography and Microscope Projection Photolithography. Langmuir 2011, 27 (7), 3238−3243. (32) Jeon, H.; Koo, S.; Reese, W. M.; Loskill, P.; Grigoropoulos, C. P.; Healy, K. E. Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces. Nat. Mater. 2015, 14 (9), 918−923. (33) Kim, D. H.; Han, K.; Gupta, K.; Kwon, K. W.; Suh, K. Y.; Levchenko, A. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients. Biomaterials 2009, 30 (29), 5433−5444. (34) Kim, D. H.; Seo, C. H.; Han, K.; Kwon, K. W.; Levchenko, A.; Suh, K. Y. Guided cell migration on microtextured substrates with variable local density and anisotropy. Adv. Funct. Mater. 2009, 19 (10), 1579−1586. (35) Londono, C.; Loureiro, M. J.; Slater, B.; Lücker, P. B.; Soleas, J.; Sathananthan, S.; Aitchison, J. S.; Kabla, A. J.; McGuigan, A. P. Nonautonomous contact guidance signaling during collective cell migration. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (5), 1807−1812. (36) Tsai, W. B.; Lin, J. H. Modulation of morphology and functions of human hepatoblastoma cells by nano-grooved substrata. Acta Biomater. 2009, 5 (5), 1442−1454. (37) Kim, M.; Choi, J.-C.; Jung, H.-R.; Katz, J. S.; Kim, M.-G.; Doh, J. Addressable Micropatterning of Multiple Proteins and Cells by Microscope Projection Photolithography Based on a Protein Friendly Photoresist. Langmuir 2010, 26 (14), 12112−12118. (38) Kang, J.; Choi, J.-C.; Kim, M.; Jung, H.-R.; Doh, J. Chapter 4 Photopatterning with a Printed Transparency Mask and a ProteinFriendly Photoresist. In Methods in Cell Biology, Matthieu, P., Manuel, T., Eds.; Academic Press: 2014; pp 55−72. (39) Bordeleau, F.; Tang, L. N.; Reinhart-King, C. A. Topographical guidance of 3D tumor cell migration at an interface of collagen densities. Phys. Biol. 2013, 10 (6), 065004. (40) Sniadecki, N.; Desai, R.; Ruiz, S.; Chen, C. Nanotechnology for Cell−Substrate Interactions. Ann. Biomed. Eng. 2006, 34 (1), 59−74. (41) Teixeira, A. I.; Abrams, G. A.; Bertics, P. J.; Murphy, C. J.; Nealey, P. F. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J. Cell Sci. 2003, 116 (10), 1881−1892. (42) Suh, K.-Y.; Park, M. C.; Kim, P. Capillary Force Lithography: A Versatile Tool for Structured Biomaterials Interface Towards Cell and Tissue Engineering. Adv. Funct. Mater. 2009, 19 (17), 2699−2712.
(4) Wiseman, B. S.; Werb, Z. Stromal Effects on Mammary Gland Development and Breast Cancer. Science 2002, 296 (5570), 1046− 1049. (5) Friedl, P.; Alexander, S. Cancer Invasion and the Microenvironment: Plasticity and Reciprocity. Cell 2011, 147 (5), 992−1009. (6) Auernheimer, J.; Dahmen, C.; Hersel, U.; Bausch, A.; Kessler, H. Photoswitched Cell Adhesion on Surfaces with RGD Peptides. J. Am. Chem. Soc. 2005, 127 (46), 16107−16110. (7) Li, Y.; Yuan, B.; Ji, H.; Han, D.; Chen, S.; Tian, F.; Jiang, X. A Method for Patterning Multiple Types of Cells by Using Electrochemical Desorption of Self-Assembled Monolayers within Microfluidic Channels. Angew. Chem., Int. Ed. 2007, 46 (7), 1094−1096. (8) Nakanishi, J.; Kikuchi, Y.; Inoue, S.; Yamaguchi, K.; Takarada, T.; Maeda, M. Spatiotemporal Control of Migration of Single Cells on a Photoactivatable Cell Microarray. J. Am. Chem. Soc. 2007, 129 (21), 6694−6695. (9) van Dongen, S. F. M.; Maiuri, P.; Marie, E.; Tribet, C.; Piel, M. Triggering Cell Adhesion, Migration or Shape Change with a Dynamic Surface Coating. Adv. Mater. 2013, 25 (12), 1687−1691. (10) Vignaud, T.; Galland, R.; Tseng, Q.; Blanchoin, L.; Colombelli, J.; Théry, M. Reprogramming cell shape with laser nano-patterning. J. Cell Sci. 2012, 125 (9), 2134−2140. (11) Yamaguchi, S.; Yamahira, S.; Kikuchi, K.; Sumaru, K.; Kanamori, T.; Nagamune, T. Photocontrollable Dynamic Micropatterning of Non-adherent Mammalian Cells Using a Photocleavable Poly(ethylene glycol) Lipid. Angew. Chem., Int. Ed. 2012, 51 (1), 128−131. (12) Choi, J.-C.; Doh, J. High-throughput quantitative imaging of cell spreading dynamics by multi-step microscopy projection photolithography based on a cell-friendly photoresist. Lab Chip 2012, 12 (23), 4964−4967. (13) Jiang, X.; Bruzewicz, D. A.; Wong, A. P.; Piel, M.; Whitesides, G. M. Directing cell migration with asymmetric micropatterns. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (4), 975−978. (14) Rolli, C. G.; Nakayama, H.; Yamaguchi, K.; Spatz, J. P.; Kemkemer, R.; Nakanishi, J. Switchable adhesive substrates: Revealing geometry dependence in collective cell behavior. Biomaterials 2012, 33 (8), 2409−2418. (15) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Engineering Substrate Topography at the Micro- and Nanoscale to Control Cell Function. Angew. Chem., Int. Ed. 2009, 48 (30), 5406−5415. (16) Kim, D.-H.; Provenzano, P. P.; Smith, C. L.; Levchenko, A. Matrix nanotopography as a regulator of cell function. J. Cell Biol. 2012, 197 (3), 351−360. (17) Lord, M. S.; Foss, M.; Besenbacher, F. Influence of nanoscale surface topography on protein adsorption and cellular response. Nano Today 2010, 5 (1), 66−78. (18) Diehl, K. A.; Foley, J. D.; Nealey, P. F.; Murphy, C. J. Nanoscale topography modulates corneal epithelial cell migration. J. Biomed. Mater. Res., Part A 2005, 75 (3), 603−611. (19) Biggs, M. J. P.; Richards, R. G.; Gadegaard, N.; Wilkinson, C. D. W.; Oreffo, R. O. C.; Dalby, M. J. The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells. Biomaterials 2009, 30 (28), 5094−5103. (20) Biggs, M. J. P.; Richards, R. G.; McFarlane, S.; Wilkinson, C. D. W.; Oreffo, R. O. C.; Dalby, M. J. Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves. J. R. Soc., Interface 2008, 5 (27), 1231−1242. (21) Driscoll, M. K.; Sun, X.; Guven, C.; Fourkas, J. T.; Losert, W. Cellular contact guidance through dynamic sensing of nanotopography. ACS Nano 2014, 8 (4), 3546−3555. (22) Fujita, S.; Ohshima, M.; Iwata, H. Time-lapse observation of cell alignment on nanogrooved patterns. J. R. Soc., Interface 2009, 6 (Suppl. 3), S269−S277. (23) Hamilton, D. W.; Oates, C. J.; Hasanzadeh, A.; Mittler, S. Migration of periodontal ligament fibroblasts on nanometric topographical patterns: Influence of filopodia and focal adhesions on contact guidance. PLoS One 2010, 5 (12), e15129. H
DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (43) Choi, J.-C.; Jung, H.-R.; Doh, J. Dynamic Modulation of SmallSized Multicellular Clusters Using a Cell-Friendly Photoresist. ACS Appl. Mater. Interfaces 2013, 5 (23), 12757−12763. (44) Martin, R. M.; Leonhardt, H.; Cardoso, M. C. DNA labeling in living cells. Cytometry, Part A 2005, 67 (1), 45−52. (45) Purschke, M.; Rubio, N.; Held, K. D.; Redmond, R. W. Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochemical and Photobiological Sciences 2010, 9 (12), 1634− 1639. (46) Taylor, M. S.; Daniels, A. U.; Andriano, K. P.; Heller, J. Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J. Appl. Biomater. 1994, 5 (2), 151−7. (47) Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 2003, 24 (7), 1121−1131. (48) Uttayarat, P.; Toworfe, G. K.; Dietrich, F.; Lelkes, P. I.; Composto, R. J. Topographic guidance of endothelial cells on silicone surfaces with micro- to nanogrooves: Orientation of actin filaments and focal adhesions. J. Biomed. Mater. Res., Part A 2005, 75A (3), 668− 680. (49) Kim, D.-H.; Seo, C.-H.; Han, K.; Kwon, K. W.; Levchenko, A.; Suh, K.-Y. Guided Cell Migration on Microtextured Substrates with Variable Local Density and Anisotropy. Adv. Funct. Mater. 2009, 19 (10), 1579−1586.
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DOI: 10.1021/acsami.6b00318 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
