Article pubs.acs.org/Langmuir
Versatile Gradients of Covalently Bound Proteins on Microstructured Substrates Jordi Comelles,*,†,‡ Verónica Hortigüela,†,‡ Josep Samitier,†,‡,§ and Elena Martínez*,†,‡ †
Institute for Bioengineering of Catalonia (IBEC), C/Baldiri Reixac 11-15, 08028 Barcelona, Spain Centro de Investigación Biomédica en Red, Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain § Department of Electronics, University of Barcelona, c/Martí I Franquès 1, 08028 Barcelona, Spain ‡
S Supporting Information *
ABSTRACT: In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a microfluidic approach to obtain linear gradients with high slope (0.5 pmol·cm−2·mm−1), relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.
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generators,14 T-sensor devices,15 or the so-called premixer gradient generators, which were developed by Jeon and colleagues.11 Among these microfluidic methods, only a few deal with substrate-bound biomolecules, which is a requirement when considering extracellular matrix proteins or some growth factors. Although some examples of proteins immobilized through unspecific adsorption can be found in the literature,16−18 in general more stable binding strategies such as covalent bonding,19 biotin−avidin,20 photopolymerization,21 or photografting22 are preferred. Microfluidic systems allow for fabrication of substrates with complex, well-defined surface-bound biomolecule gradients, but they are still far from capturing the complexity of the in vivo cell microenvironment. In particular, most of these gradients are made on flat polymers or glass, therefore not considering the influence of the physical features of the cellular microenvironment on cell behavior. In fact, during recent years it has become clear that cells are influenced not only by biochemical cues but also by substrate properties such as stiffness and geometry. It has been reported that the geometry of the cellular microenvironment has a major impact on cell fate and function, and it can control cell differentiation23 or guide cell migration,24 ultimately leading to dramatic consequences for tissue function.25 However, it is challenging to combine topographical structures and biomolecular gradients. Wang and co-workers
INTRODUCTION Cells in vivo are exposed, continuously and simultaneously, to a milieu of biological stimuli, which, in turn, trigger cellular responses important to the cells’ biological functions.1 In particular, cues in the shape of biomolecule gradients are signaling mechanisms widely present in nature to direct many biological processes such as cancer metastasis,2,3 immune response,4 or neuronal growth.5 Interest in elucidating these phenomena has led, over the past few decades, to the development of multiple methods for exposing cells to chemical gradients in vitro. Actually, various in vitro assays have been conducted to study the effect of biochemical gradients on cell behavior in classic biology experiments. For example, micropipet, hydrogels, and Boyden and Zigmond chambers have been historically used to generate gradients of concentrations of soluble factors.1,6 However, these gradients form and dissipate during a few hours, thus limiting the cell types and questions that can be addressed.7 In recent years, other methods to create molecular gradients, both on the surface and in solution, have emerged. These include vapor diffusion,8 an immersion technique applied to self-assembled monolayer,9 plasma polymer gradients,10 and microfluidics11 (see Genzer12 for an extensive review). This last technique has the advantage of allowing for accurate control of the gradient length and shape at the micrometer scale,7 which is crucial in the development of graded interfacial zones that mimic transitions in heterogeneous tissues.13 There are several methods for generation of biomolecular gradients using microfluidic technology: steady-state gradient © 2012 American Chemical Society
Received: June 25, 2012 Revised: August 19, 2012 Published: August 22, 2012 13688
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Figure 1. Microfluidic system design and generation of the fibronectin gradients. (A) Scheme of the device: PDMS Y-shaped microfluidics channels were obtained from SU-8 molds. PMMA substrates were spun coated on top of a PMMA sheet. PEN sheet was used as a secondary mold to produce topographical cues on the PMMA surface. Microstructured PMMA substrate and PDMS chip were aligned and reversibly bound. By means of a method relying on the properties of laminar flow, a surface-bound fibronectin gradient is produced on the PMMA surface. (Inset) Schematic representation of a fibronectin gradient superimposed to an array of topographical microstructures. (B) Scheme of the functionalization method used. Carboxylic groups were introduced to the PMMA surface by hydrolysis and then reacted with an NHS linker. Finally, fibronectin molecules attached to these groups, forming a covalent bound.
approach in vitro cell culture methods to in vivo cellular microenvironments.
used microstructures in a microfluidic channel to overcome the effects caused by shear stress in neuronal growth cones. They developed a gradient generator using the premixer configuration that incorporated 100 μm deep wells onto the observation chambers to reduce both flow velocity and mechanical stress on cells. 26 Other groups proposed orthogonal-gradient systems for high-throughput readout of cell responses. Briefly, orthogonal gradients are characterized by two independent gradients running perpendicular to each other. Therefore, each single (x, y) position on the substrate has unique surface properties arising from the combination of both gradients.27,28 Yang and co-workers combined a topographical gradient (varying grooves width) with a chemical gradient (varying surface hydrophobicity) to determine the range of topography and wettability that produced the highest proliferation rates.27 More recently, another orthogonal gradient was reported,28 where a gradient in the pore size of porous silicon was combined with an orthogonal gradient of adhesive molecules. The short-term adhesion of Mesenchymal Stem Cells was studied, and cells were found to respond more strongly to variations in adhesive molecules than to variations in pore size. In this work, we propose an easy, reliable method to produce highly tunable gradients of covalently bound proteins on topographically modified substrates. We employ poly(methyl methacrylate) (PMMA) as substrate, as it can be both chemically and topographically modified.29,30 Once topographically microstructured and chemically activated by a hydrolysis procedure, the PMMA polymer is reversibly bound to a microfluidic device for fabrication of protein gradients (Figure 1A). These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, long-term stable, and easily tunable. The system was then used to probe the influence of microstructures and such protein gradients on cell adhesion, showing that it was suitable for cell culture experiments. Altogether, this method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to
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MATERIALS AND METHODS
Fabrication of PMMA Microstructured Substrates. Poly(methyl methacrylate) (PMMA) substrates were microstructured by means of the nanoembossing technique performed with a nanoimprint lithography apparatus (Obducat nanoimprinter, Obducat AB, Sweden).30 This technique employs a microstructured hard mold, which is put into contact with the substrate to be structured, and this “sandwich” is heated while applying pressure. Usually silicon-based molds fabricated by expensive and complex microfabrication processes are used. As an alternative, high glass transition temperature polymers such as poly(ethylene naphtalate) (PEN) can be used as molds (Figure 1A).30 First, a SU-8-2015 (Microchem Corp., Newton, MA) positive master with microstructures (15 μm lines or square posts of 15 × 15 μm2) was obtained by standard photolithography.31 The resulting mold (primary mold) was replicated on a PEN sheet 125 μm thick (Goodfellow, U.K.) by nanoembossing to obtain a negative replica (secondary mold). Briefly, the PEN-mold “sandwich” was heated to 200 °C; once this temperature was reached, the “sandwich” was pressed (70 bar) and heated for 30 min. Then, the system was cooled down to 80 °C but maintaining the pressure. When the 80 °C temperature was reached, the pressure was released. This secondary mold was then used again in the nanoimprint lithography instrument to transfer the microstructures to the PMMA substrates. Imprinting parameters used were a 125 °C temperature under 30 bar of pressure. PMMA substrates were prepared by spin coating of PMMA 11% solution in anisole (950PMMA A Resist, MicroChem Corp., Newton, MA) on PMMA sheets 125 μm thick (Goodfellow, U.K.). Morphology and dimensions of the microstructures fabricated on the molds and the PMMA replicas were characterized by optical microscopy (Nikon, Netherlands) and atomic force microscopy (Dimension 3100; Digital Instruments, USA). Fabrication of the Microfluidic Device. A simple microfluidic device was fabricated by reversible sealing of a polydimethylsiloxane (PDMS) cover to the PMMA microstructured substrates (Figure 1A). Y-channel molds of 500 μm in width, 30 mm in length, and 70 μm high were fabricated using SU-8-2015 photoresist following the standard protocol provided by the supplier. PDMS (Sylgard 184 Silicon Elastomer, Dow Corning, Germany) prepolymer was prepared by mixing the base polymer with the curing agent in a 10:1 ratio and degassing the mixture under a vacuum for 1 h. 13689
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of the gradient were computed by averaging the intensities of regions of 500 μm × 200 μm, covering the whole length of the gradient. Microstructured PMMA substrates functionalized with fibronectin were characterized in depth by confocal fluorescent microscopy. In this case, images were obtained using a Leica TCS-SL (Leica Microsystems GmbH, Germany) confocal microscope with a 63× objective. Excitation at 543 nm was provided by a helium−neon gas laser. Confocal microscope images were analyzed using the 3D plugin of ImageJ software. All graphical data are reported as the mean value ± standard error obtained from data sets of three independent experiments. Quantification of Protein Surface Density on the Gradient Sample. Protein coverage was determined by surface plasmon resonance (SPR) measurements as described in Lagunas et al.29 The SPR RT2005 instrument used was purchased from Resonant Technologies (RES-TEC) GmbH (Max-Plank-Institute for Polymer Research, Germany). Sensor chips SPRchip were acquired from GWC Technologies Inc. (Wisconsin). To emulate the experimental conditions in which fibronectin gradients were created on PMMA surfaces, a thin layer of PMMA (50 nm) (950PMMA 2% solution in anisole, MicroChem Corp., Newton, MA) was spun coated onto the gold SPR chips. This polymer thickness was thin enough to still ensure a sensitive detection for the SPR signal. A set of PMMA-covered SPR chips was prepared and activated using NHS as described above. The chips were index matched to the SPR prism and fitted into a 20 μL flow cell connected to the same syringe pump used for gradient preparation. The baseline in the SPR response was established by flowing PBS. Then, a fibronectin solution (20 μg mL−1 in PBS) was injected at the flow rates and incubation times described in Supporting Information Figure 1B. Three different incubation times were analyzed: 39 min 1 s, 17 min 55 s, and 2 min 26 s. After incubation, PBS solution was injected again into the cell to remove any protein adsorbed in a nonspecific way. The reflected intensity measured by the SPR instrument was fitted by means of Fresnel’s equations with the help of the Winspall free software (RES-TEC, Germany). The shift of the SPR peak, obtained before and after fibronectin incubation, was used to calculate the thickness of the fibronectin layer (d) bound to the surface. The fibronectin surface density corresponding to the incubation times assayed, which can be correlated with positions along the gradient, can be then calculated using eq 1
The prepolymer was then poured over the SU-8 mold and cured for 24 h at room temperature. The resulting PDMS replica was peeled off, cut to a microscope slide size, and mechanically bounded to the PMMA substrate. Fluid inlet and outlet holes were perforated using a punch needle. Generation of Protein Gradient. Before being mounted in the microfluidic chips, PMMA substrates were chemically activated to ensure site-selective and stable protein capturing from protein flowing solutions (Figure 1B). For this purpose, PMMA substrates were first immersed into a sodium hydroxide 2 M aqueous solution (Sodium ́ hydroxide pellets, Panreac Quimica S.A.U., Barcelona, Spain) for 2 h at 40 °C to hydrolyze the ester groups of the PMMA, thus obtaining a carboxylate surface.29,32 Subsequently, the substrates were neutralized with hydrochloric acid 0.1 M (hydrochloric acid 37%, Panreac ́ Quimica S.A.U., Barcelona, Spain), rinsed with distilled water, and then dried under a nitrogen stream. Carboxylic acid moieties at the surface were activated by a mixture ́ of N-hydroxysuccinimide (NHS) (Sigma-Aldrich Quimica S.A., Madrid, Spain) (8.9 mg, 0.08 mM) and N-(3-dimethylaminopropyl)́ N′-ethyl carbodiimide (EDC) (Sigma-Aldrich Quimica S.A., Madrid, Spain) (73.4 mg, 0.38 mM) in Milli-Q water (5 mL) at room temperature for 15 min. Finally, the substrates were washed thoroughly with Milli-Q water and blown dried under a nitrogen stream. Both flat and microstructured PMMA substrates were activated following this procedure. Once activated, microfluidic devices were mounted by sealing the PDMS channels onto the PMMA substrates using paperclips. Surface-bound protein gradients were generated along the channel length, in the direction perpendicular to the fluid flow (Figure 1A). To do this, we employed the “step function” method proposed by Georgescu et al., which was adapted to our particular needs.18 Briefly, a protein solution stream was moved along the width of the main channel of the Y-shaped device by varying the flow ratio between the two inlets. One inlet carried a 20 μg mL−1 fibronectin solution ́ (Fibronectin from human foreskin fibroblasts, Sigma-Aldrich Quimica S.A., Spain) and the other one a phosphate saline buffer solution (Dulbecco’s Phosphate Buffer Saline, Invitrogen, Spain). This process controls the exposure time (incubation time) of the activated PMMA surface to the protein solution at each position of the channel width. By decreasing the incubation times we generated a gradient on the surface density of the captured protein. Two independent syringe pumps (Ne-300, New Era Pump Systems Inc., NY) were used to set up the flow rate of the two inlets between 10 and 90 μL min−1 following the step functions represented in Supporting Information, Figure 1. Each of these step functions resulted in a gradient profile with characteristic slope and shape. Once the gradient generation procedure was finished, the PMMA substrates were separated from the PDMS cover and stored at 4 °C in PBS for further processing. Characterization of Protein Gradients. The fibronectin gradients fabricated on flat and microstructured PMMA substrates were characterized by fluorescence microscopy upon sample immunostaining. For this purpose, the PMMA substrates were incubated with a solution containing Anti-Fibronectin primary ́ antibody produced in rabbit (Sigma-Aldrich Quimica S.A., Madrid, Spain) diluted 1:300 in 1% Bovine Serum Albumin (BSA) in PBS ́ (Sigma-Aldrich Quimica S.A., Madrid, Spain) for 1 h. Afterward, PMMA slides were incubated with a secondary antibody Alexa Fluor 568 goat antirabbit IgG (Invitrogen S.A., Barcelona, Spain) diluted 1:1000 in 1% BSA in PBS for 1 h. Samples were then mounted with ́ Fluoromount (Sigma-Aldrich Quimica S.A., Madrid, Spain). The gradient morphology was measured by fluorescence microscopy using an Eclipse E1000 upright microscope (Nikon, Netherlands) equipped with a charge-coupled device (CCD) camera and a G2A long-pass emission filter. Fluorescence intensity was measured 1, 3, and 7 days after gradient formation to check the gradient stability. Fluorescent microscope images were analyzed using ImageJ free software (http://rsb.info.nih.gov/ij, National Institutes of Health, USA). Fluorescence images (0.65 mm2 area) were captured every 5 mm along the length of the main channel (30 mm). Intensity profiles
A=d
(n − n0) ∂n/∂c
(1)
where A is the absorbed amount of protein per area unit, n0 the refractive index of the solution, and ∂n∂c = 0.212 mL g−1.33 Cell Adhesion Experiments. Gradients of surface-bound fibronectin were prepared following the method described above and used in cell adhesion experiments. In this case, a mixture of fluorescently labeled and unlabeled bovine fibronectin was used: 20 μg mL−1 solution of fibronectin in PBS, a mixture (1:39) of rhodamine fibronectin from bovine plasma (Cytoskeleton Inc., CO) and ́ fibronectin from bovine plasma (Sigma-Aldrich Quimica S.A., Madrid, Spain). NIH/3T3 mouse embryonic fibroblast cell lines from passages 6− 15 were expanded for 3 days at 37 °C and 10% CO2 in Dulbecco’s Modified Eagle Medium (D-MEM) (Invitrogen S.A., Barcelona, Spain) supplemented with 10% Fetal Bovine Serum (FBS) (Invitrogen S.A., Barcelona, Spain), 1% L-glutamine (Invitrogen S.A., Barcelona, Spain), 1% penicillin−streptomycin (Invitrogen S.A., Barcelona, Spain), and 1% sodium pyruvate (Invitrogen S.A., Barcelona, Spain) (growth medium). Fibronectin-gradient substrates were preincubated in BSA 1% (w/v) in PBS for 1 h before cell culture. Then, cells were seeded at a density of 2 × 103 cells cm−2 in growth medium without FBS. After 1 h of incubation, nonadherent cells were removed by a gentle wash with PBS. Attached cells were stained for observation of focal contacts (paxillin staining), actin cytoskeleton, and nuclei. For this purpose, cells were fixed with 4% paraformaldehyde (Merk Sharp & Dohme, Madrid, Spain) in PBS for 20 min and washed three times 13690
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Figure 2. Characterization of the fibronectin gradients. (A) Fluorescence microscopy image corresponding to labeling of fibronectin proteins (scale bar = 100 μm). (B) Graph representing the average of the measured intensities of immobilized fibronectin at different points along the gradient (mean value ± standard error (SE) of three independent experiments). Dashed line corresponds to the predicted value of surface coverage estimated from Ilkovic’s model. Position 0 μm is defined by the border of the channel and corresponds to the region of higher exposure time to the fibronectin solution. (C) Fibronectin surface density for different incubation times (corresponding to different positions along the gradient) calculated from SPR measurements (mean value ± SE of three independent experiments). Dashed line corresponds to a linear fitting of the experimental data (y = a·x + b, a = 0.0378, b = 7.6443, R2 = 0.976). with PBS. The remaining free aldehyde groups were then blocked with 50 mM ammonium chloride (20 min at room temperature). After this time, samples were washed twice with PBS and cells permeabilized with a saponin (Fluka, Buchs, Switzerland) solution for 10 min at room temperature (0.1% saponin in PBS containing 1% BSA). Afterward, rabbit anti-Paxillin [Y113] (Abcam, Cambridge, U.K.) diluted 1:200 in 1% BSA in PBS and a solution containing TRITCphalloidin (Fluka, Buchs, Switzerland) diluted 1:500 in 1% BSA in PBS from a stock solution of 1 mg/mL in DMSO were added, and cells were incubated for 1 h at room temperature. Then, cells were washed with PBS and incubated for 1 h at room temperature with the secondary antibody goat antirabbit Alexa 647 (Invitrogen S.A., Barcelona, Spain) and Hoechst (Invitrogen S.A., Barcelona, Spain), both diluted 1:1000 in 1% BSA in PBS. After incubation, cells were washed with PBS and samples mounted with fluoromount. Images were obtained by using a Leica SP2 (Leica Microsystems GmbH, Germany) confocal microscope with objectives of 20× and 63× magnification.
introduce carboxylate groups on the PMMA surface, which were reacted with N-hydroxysuccinimide molecule (NHS). This linker chemistry was used to capture proteins covalently. To produce protein gradients we used microfluidic technology, adapting the “step function” method proposed by Georgescu et al.18 A Y-shaped PDMS channel was used to produce hydrodynamic focusing of the protein solution. The microfluidic device was pressed and fixed onto the activated PMMA surface using mechanical pressure. The main channel was 500 μm in width and 70 μm in height. Two input channels were connected to the main channel. The fibronectin solution (20 μg mL−1) flowed through one channel controlled by one syringe pump, and a buffer solution flowed through the other channel controlled by another pump. By increasing/decreasing the flow rate on the protein solution channel while simultaneously decreasing/increasing the flow rate on the buffer solution channel, the position of the protein stream across the channel can be controlled and thus the gradient steepness (distance between two different protein concentrations). By tuning the time that the protein solution flows over the substrate at each step the gradient slope can be defined (Supporting Information Figure 1). Figure 2A shows an immunofluorescence image of a representative region of a Fn gradient surface obtained on activated flat PMMA after 24 h from its generation. A linear gradient is clearly visible across the channel section, and its
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RESULTS AND DISCUSSION Generation of Surface-Bound Protein Gradients. Fibronectin (Fn) gradients were generated on PMMA surfaces previously activated following the procedure described by Lagunas et al. and Hyun et al. (Figure 1B).29,32 Flexible PMMA sheets were used as support for the spin coating of a PMMA layer. In order to covalently attach proteins to the PMMA surface, a methyl ester alkaline hydrolysis reaction was used to 13691
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concentration and then gently falls to zero following a linear trend. Cosson and colleagues estimated,20 by means of fluorescence calibration using BSA as model protein, a surface density of ∼150 ng cm−2 (1 pmol cm−2). The ‘highest’ protein amount we found by means of SPR is in good agreement with Cosson’s. By fitting the data of Figure 2C by a linear relationship (Supporting Information Figure 5) we estimated the slope of the gradient as Δ = 0.5 pmol·cm−2·mm−1. This gradient slope is 40 times higher than low-slope gradients (Δ = 0.0125 pmol·cm−2·mm−1) reported previously also produced on PMMA.29 Actually, the combination of the surface density within the gradient and its intrinsic length scale will determine the possible applications of the platform.12 Low slope gradients could be used as screening platforms, whereas applications like directed cell migration require higher slopes.38 Therefore, the gradients developed here could be potentially used to study biological problems such as directed cell migration.20,38,39 Stability of the Gradients of Surface-Bound Proteins Generated. One of the main drawbacks of soluble biomolecule gradients is that usually they form and dissipate during a few hours, thus limiting the study of cellular processes that occur over days or weeks.7 Other microfluidic approaches rely on physical adsorption of proteins onto surfaces, leading to unpredictable desorption from surfaces. Moreover, to the best of our knowledge, few works deal with study of the gradient’s behavior along time.18,20,22 Fiddes and colleagues characterized fluorescence intensity up to 4 days after formation, and Cosson et al. and Georgescu et al. measured the gradient profile up to 7 days after formation. Cosson reported a good long-term stability, mainly caused by the strong interaction of the system avidin−biotin. On the other hand, surface gradients generated by physical absorption reported a decrease of fluorescence intensity,22 in contrast to covalently linked gradients prepared with the same method, where major stability is reported.18,22 Our gradient formation strategy involves hydrolysis of PMMA and use of the NHS linker chemistry in order to obtain covalently attached surface protein gradients, which results in highly stable links, as it was seen when comparing gradients on nonhydrolyzed and hydrolyzed PMMA (Figure 3A), 24 h after gradient formation (stored in PBS at 4 °C). It can be seen that the gradient slope is well preserved in hydrolyzed PMMA, whereas it is almost lost in nonhydrolyzed surfaces. In nonhydrolyzed surfaces the maximum intensity value is a 40% higher than the minimum intensity value, whereas 150% increment is observed in hydrolyzed PMMA. Moreover, according to Figure 3A, the signal intensity observed in nonhydrolyzed gradients is similar to the background level in hydrolyzed surfaces. Therefore, the hydrolysis step and further chemical modification increase the quality of the gradient considerably. To assess their stability over time, the intensity profiles of fibronectin gradients were measured after 3 and 7 days of their fabrication while stored in PBS at 4 °C. The intensity profiles of the long-term stored gradients in comparison with the initial profiles (measured after 24 h of gradient formation) are shown in Figure 3B. It was observed that the shape of the gradient was preserved along the time frame chosen. Protein Surface-Bound Gradients on Topographical Microstructured Substrates. As stated before, use of a thermoplastic polymer as substrate allows introduction of a microstructured surface topography at the gradient region, thus combining chemical and topographical cues.27 For this purpose,
morphology is maintained along the 75% of the total channel length (25 mm). Experimental data points previously obtained were fitted using Ilkovic’s model to determine the linear-shaped gradient (Supporting Information Figures 2 and 3).18,34 As reported by Georgescu et al., Ilkovic’s equation (eq 2) is valid for low-to-intermediate surface coverage,34 which we believe corresponds to our experimental conditions, as the experimental data points do not show any saturation plateau.
θ=
2·cb Γmax
D·t π
(2)
In this equation, cb corresponds to the bulk protein concentration (the concentration of the protein in the solution), D is the fibronectin diffusion coefficient (8.5 × 10−8 cm2 s−1),35 and Γmax is a normalization parameter representing the maximum surface coverage (expressed in surface density values, molecules deposited per area unit). By using these values the experimental data set could be successfully fitted to Ilkovic’s equation and a value of maximum surface coverage of Γmax = 6.99 × 1011 molecules·cm−2 was obtained (Supporting Information Figure 3). This value, corresponding to a densely packed fibronectin monolayer, gives a diameter of 13 nm for one fibronectin molecule, in good agreement with the values found in the literature.36,37 Figure 2B shows the intensity values obtained at different positions across the width of the microfluidic channel. Black dots correspond to experimental data points, which represent the mean value and standard deviation of three independent experiments. The dashed line corresponds to the theoretical profile estimated from modeling of the adsorption kinetics performed. Note that the experimental results are in an excellent agreement with the shape predicted by Ilkovic’s model, thus demonstrating the predictability of the experimental approach employed. The Y-shaped method used relies on two assumptions related to the laminar flow regime: (a) a clear interface between the two solutions is generated, as mixing by lateral diffusion is limited by the laminar flow regime, thus allowing control of the region exposed to the protein stream, and (b) the assumption that the flow velocity nearby the walls is close to zero, thus allowing the proteins to react. Moreover, the laminar flow regime is demonstrated by the fact that the gradient shape was almost constant during the first 15 mm of the channel and then decreased slightly (Supporting Information Figure 4). This is in good agreement with the work of Georgescu et al.,18 who observed that the gradient profile was maintained along the entire length of the channel because of the high flow rates, which limit lateral diffusion along the channel. Gradient Surface Density Quantification. There is little information in the literature about the absolute values of protein’s surface density, beyond ‘arbitrary units’ quantification. To provide a quantitative estimation of the fibronectin surface gradient, surface plasmon resonance (SPR) was used to measure the surface density as a function of incubation time. SPR reflection curves were obtained before and after protein binding. Then, the average thickness of the protein layer was estimated using a multilayer model and Fresnel’s equations.29 Figure 2C shows the fibronectin surface density for different incubation times. These incubation times can be related to their corresponding positions along the width of the gradient (top x axis). The fibronectin surface density starts with a value of around 100 ng cm−2 (0.23 pmol cm−2) at regions of maximum 13692
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Figure 6C, PMMA microstructured replicas were successfully obtained using the secondary PEN molds. In order to check the stability of the PMMA microstructures after the hydrolysis process employed for surface functionalization, atomic force microscopy (AFM) measurements of the morphology of the structures were performed before and after the hydrolysis step. Supporting Information Figure 6D and 6E corresponds to AFM topography images of 10 μm wide lines before and after the hydrolysis procedure, respectively. Comparison of sectional profiles of the structures (Figure 4B) shows no significant differences between the structures before and after hydrolysis, implying that the topographical structures created are stable against the chemical functionalization procedure. Taking into account this information, microstructured PMMA surfaces were used to fabricate substrates combining topography and surface-bound protein gradients. The microfluidic system was then pressed to a microstructured PMMA layer (previously functionalized with NHS). A pillar array (square posts 15 μm in width and 1 μm in height in Figure 4A), which has been previously reported to affect cell morphology40 and migration,41 was aligned by eye with the PDMS channel. After the setup was prepared, a fibronectin surface gradient was produced using the protocol described above to obtain a gradient with a linear shape. Figure 4C shows a fluorescence microscopy image of the variation in fibronectin fluorescence intensity along the width of the channel. A question regarding the protein distribution on the topographical microstructures arises. To find out the allocation of fibronectin, confocal microscopy analysis was carried out. Figure 4D shows the z projection of the fluorescence intensity at different heights. It can be seen that the intensity signal is higher at the outline of the microstructures. This effect could be attributed to the presence of protein at the lateral walls of the microstructures, thus indicating that protein is covering completely the three dimensions of the surface. In order to clarify this observation, Figure 4E shows a Z profile of the fluorescence intensity where the presence of fibronectin is covering the entire shape of the microstructures. It can be noted that the fluorescence signal follows the silhouette of the posts, so proteins are linked to the top, bottom, and walls of the microstructures. Ghibaudo also reported a homogeneous adsorption of fibronectin molecules on micropillar topography, although they incubated the protein solution in static conditions.41 Therefore, the microfluidic method used here can reproduce similar results as the traditional procedure and has the advantage of producing gradient-shaped coverage. Proof of Concept: Cell Adhesion Experiments. Once generated, the gradients of surface-bound fibronectin on the microstructured substrates were recovered by removing the PDMS channel. This out-chip option for cell culture experiments has multiple advantages. First, it avoids undesired effects on cells produced by the shear stress caused by the laminar flow.1,26 Second, it makes comparisons with traditional wellplate cell culture experiments easier. Thus, the system developed is suitable for standard cell culture procedures. The fibronectin molecule is a protein of the extracellular matrix involved in cell adhesion processes. Thus, if the density range of the fibronectin gradient generated is relevant for cell adhesion, we should find differences in cell morphology and spreading characteristics depending on their location in the gradient. Moreover, cells should also be influenced by the imposed microstructure.40 To probe it, we performed an
Figure 3. Characterization of gradient stability. (A) Average of the measured intensities of immobilized fibronectin at different positions along the gradient (mean value ± SE of three independent experiments) for covalently linked fibronectin (black diamonds) and adsorbed fibronectin (open squares) 24 h after gradient formation. (B) From top to bottom, average intensity of the fibronectin signal along the gradient length at 24 h, 3 days, and 7 days after gradient formation (mean value ± SE of three independent experiments). Samples were stored at 4 °C in PBS during the prescribed time before immunostaining.
several designs of different topographical microstructures of 15 μm of smallest lateral size were produced in SU-8 photoresist (Supporting Information Figure 6A) following the procedure introduced by Greener and colleagues.31 This mold was then replicated in a poly(ethylene naphtalate) (PEN) sheet (Supporting Information Figure 6B), thus obtaining a negative replica of the designed structures. PEN has a high glass transition temperature (around 150 °C), which is higher than the glass transition temperature of PMMA (105 °C), so it can be used as a mold in an imprint process.30 Moreover, PEN freestanding molds have a surface energy and a flexible behavior that avoid fracture during imprinting and simplify the peel-off step. PEN also has a good chemical resistance, allowing use of different solvents during the cleaning steps, making it reusable. As it can be seen in Figure 4A and Supporting Information 13693
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Figure 4. Fibronectin gradient on microstructured PMMA surface. (A) Array of posts on a PMMA surface (scale bar 250 μm). (B) Profiles of micrometric grooves on PMMA before (black) and after (red) hydrolysis. (C) Fluorescence microscopy image corresponding to labeling of fibronectin proteins on a micropost array (scale bar 100 μm). (D) Z projection of a confocal image of the same fibronectin gradient. Dashed line corresponds to the cross section plotted in C. (E) Z profile of the fluorescence signal of the fibronectin molecule. Shape of the microstructures is visible. Height of the cross section is 4 μm.
better on regions with ‘high’ concentration of fibronectin (∼100 ng cm−2) and were able to form large focal contacts in cell periphery, whereas this behavior was not observed at ‘low’ fibronectin (∼20 ng cm−2) regions. Thus, this indicates that cells are able to sense differences within the fibronectin concentration range of our gradient. Apart from biochemical signals, cells cultured on our platform are also sensing topographical signals. Micrometric topographic features are reported to induce several effects in cell behavior: rearrangement of cell cytoskeleton, which in turn alters organelle morphology;40,44 deformation of the cancer cell’s nucleus;45 or effects in migration.41,44 Cells on our topographical motifs (Figure 5B and 5D) showed a higher branched morphology than cells on flat PMMA (Figure 5C and 5E), both on ‘high’ and on ‘low’ fibronectin densities. The differences in cytoskeleton morphology attributed to the surface topography are in agreement with previous observations.44 It can be seen that cells’ protrusions are sensing the topographical posts, adhering at the walls and at the top of the structures (arrows in Figure 5D), which can be related with higher stabilization of focal adhesion on pillars.41,44 Altogether, cell assays combining fibronectin gradients and microstructures at the same time can be performed, thus allowing the study of the combined effects of both chemical and topographical signals in cellular process such as cell adhesion or cell migration.
adhesion assay culturing NIH 3T3 mouse embryonic fibroblasts on our platform (Figure 5A). After 1 h of incubation, cells adhered on ‘high’ fibronectin density regions were more spread (Figure 5D and 5E) than those adhered on ‘low’ fibronectin density regions (Figure 5B and 5C). Cells on ‘high’ fibronectin density also show a higher dendritic phenotype than cells on ‘low’ density, both on flat and on structured PMMA. This means that, within the range of surface densities assayed, increasing ligand density increases the cell spreading. This is in agreement with Lagunas et al.,42,43 where a relation between cell spreading area and the density of adhesive molecules is established. It has been reported that an increase in the surface density of adhesive molecules leads to an increase of cell area, favoring cell spreading. Moreover, the fibronectin gradient imposed produced differences in cell focal contact formation (insets in Figure 5B−E). Large focal contacts can be seen at the cell periphery in ‘high’-density regions (insets in Figure 5D and 5E), but no visible focal contacts were present at ‘low’-density regions (insets in Figure 5B and 5C). The interaction between cells and the extracellular matrix proteins is mediated by focal contacts.43 These adhesion sites are formed upon protein clustering, and it has been reported that focal contact formation depends on the extracellular matrix density on the substrate. Typically, formation of large focal contacts starts happening around 60 min after cell seeding on high extracellular matrix densities, but there is little clustering at low densities, even at 24 h.43 This is in good agreement with the observations of the 1 h adhesion experiment performed here. Our results show that cells spread 13694
dx.doi.org/10.1021/la3025638 | Langmuir 2012, 28, 13688−13697
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Article
Figure 5. Cell adhesion experiments. (A) Fluorescence microscopy image of NIH 3T3 fibroblast cells plated on a fibronectin gradient on a microstructured PMMA surface. Fibronectin is labeled in red, cell nuclei is in blue, and actin cytoskeleton is stained in green (scale bar = 35 μm). (B) High-magnification confocal image of 3T3 cells at a “low” fibronectin density region on a microstructured surface (
