Article pubs.acs.org/Langmuir
Micropatterning of Hydrogels on Locally Hydrophilized Regions on PDMS by Stepwise Solution Dipping and in Situ Gelation Sari Sugaya,† Shunta Kakegawa,† Shizuka Fukushima, Masumi Yamada,* and Minoru Seki Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan S Supporting Information *
ABSTRACT: This study presents a simple but highly versatile method of fabricating picoliter-volume hydrogel patterns on poly(dimethylsiloxane) (PDMS) substrates. Hydrophilic regions were prepared on hydrophobic PDMS plates by trapping and melting functional polymer particles and performing subsequent reactions with partially oxidized dextran. Small aliquots of a gelation solution were selectively trapped on the hydrophilic areas by a simple dipping process that was utilized to make thin hydrogel patterns by the in situ gelation of a sol solution. Using this process, we successfully formed calcium alginate, collagen I, and chitosan hydrogels with a thickness of several micrometers and shapes that followed the hydrophilized regions. In addition, alginate and collagen gel patterns were used to capture cells with different adhesion properties selectively on or off the hydrogel structures. The presented strategy could be applicable to the preparation of a variety of hydrogels for the development of functional biosensors, bioreactors, and cell cultivation platforms.
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INTRODUCTION Processes for patterning biological substances onto planar surfaces are attracting attention in many areas related to general biological experiments.1−3 For example, microarray technologies have realized high-density and massively parallel biochemical/pharmacological assays with ultrasmall sample consumption in a single experiment.4−7 Patterned cell culture techniques based on adhesive/nonadhesive microsurfaces have also been utilized for tissue engineering and cellular physiological studies,8 including the spatiotemporal control of cell morphology,9 patterned co-cultures in in vivo mimetic microenvironments,10,11 and cell migration assays.12 Most of the current patterning techniques are based on the selective attachment of thin layers of bioactive/inactive molecules, which are achieved using microcontact printing9 and inkjet printing.13,14 In contrast, micropatterned hydrogel matrices provide 3D supports for immobilizing biological substances,15 which would be suitable for conducting sensitive biochemical assays and protein interaction studies. Furthermore, patterned wettype extracellular matrix (ECM) hydrogels can offer microenvironments resembling in vivo conditions for cell culturing while allowing efficient biomolecule transport. In recent years, various techniques have been proposed to prepare hydrogel micropatterns on planar substrates. The most commonly employed approach is the photopolymerization of hydrogel precursors. Through photo-cross-linking, hydrogel materials such as poly(ethylene glycol) diacrylate (PEGDA),16−18 poly(N-isopropylacrylamide) (pNIPAM),19,20 and acrylate-modified hyaluronic acid21 have been patterned on glass, silicon, and polymer plates with shapes corresponding to © 2012 American Chemical Society
the photomask patterns. In addition, by combining photocross-linking with microfluidics,22−24 embossing,21,25 spotting,26 and direct writing,24,27,28 increasingly complex and functional hydrogel microstructures have been produced. Hydrogel patterns of natural biopolymers have also been reported that benefit from their inherently bioactive and highly biocompatible characteristics. Alginate, chitosan, and agarose hydrogel patterns have been prepared using inkjet devices,29 microchannels,30 or contact printing techniques using a mesh.31 However, most of these patterning techniques are applicable only to a single specific hydrogel material. For example, although collagen is one of the most important ECMs involved in cell adhesion, proliferation, and differentiation32,33 and patterning techniques of dried collagen films or molecules have been well studied,34,35 only a few methods can make patterns of wet-type collagen hydrogels.36,37 Furthermore, in patterning hydrogels of natural biopolymers such as alginate, the highly viscous sol solution makes it difficult to prepare patterns smaller than ∼100 μm. A new technology of hydrogel patterning that can be applied to various types of hydrogel materials and that enables highly precise patterning without necessitating complicated devices and operations is therefore needed. In this study, we propose a simple but highly versatile strategy for constructing picoliter-volume hydrogel patterns on a hydrophobic poly(dimethylsiloxane) (PDMS) plate utilizing Received: April 11, 2012 Revised: September 6, 2012 Published: September 20, 2012 14073
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Figure 1. Procedure of hydrogel patterning on a PDMS substrate. Functional polymer particles were trapped and fixed in the wells on the PDMS plate to pattern chemically reactive regions with the epoxy group. Dextran was then conjugated to the surface by using ethylenediamine. Next, a certain amount of a gelation solution was trapped on the hydrophilized area simply by dipping the PDMS plate in the gelation solution. Finally, the PDMS plate was dipped in the sol solution to form hydrogels with shapes following the hydrophilized regions. dextran was obtained from Research Organics Inc. PDMS (Silpot 184 W/C) was obtained from Dow Corning Toray Co. Ltd., Tokyo, Japan. Green and red fluorescent polystyrene microbeads with diameters of 0.71 and 0.52 μm, respectively, were obtained from Duke Scientific Corp. Yellow-green fluorescent microbeads with a diameter of 0.1 μm were obtained from Invitrogen Corp. NIH-3T3 (RCB0056) and PC12 (RCB0009) cells were provided by the Riken BRC through the National Bio-Resource Project of the MEXT of Japan. All other chemicals were analytical grade. NIH-3T3 cells were maintained in a minimum essential medium (MEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc.), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich). PC12 cells were maintained in DMEM supplemented with 10% FBS, 10% horse serum (HS, Invitrogen Corp.), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were cultivated in a humidified incubator containing 5% CO2 at 37 °C. Local Hydrophilization of PDMS Plates. Figure 1 shows the procedure for the local hydrophilization of PDMS and subsequent hydrogel patterning. PDMS plates with microwells (depth of ∼20 μm) were fabricated by using standard soft lithography and replica molding techniques.40 PDMS prepolymer, with a mixing ratio of the base and the curing agent of 10:1, was poured onto the SU-8 master and cured at 85 °C for 30 min. Monodisperse poly(stylene-co-glycidyl methacrylate) particles with an average diameter of 6.2 μm (CV value of 11%) and a melting point of ∼130 °C were synthesized via dispersion copolymerization.41,42 The obtained particles, bearing an epoxy group, were suspended in methanol at a density of ∼108 particles/mL. The particle suspension was dropped onto and subsequently removed from the PDMS plate with microwells to accumulate particles inside the wells. Polymer matrices of the particles were melted and fixed by holding the PDMS plate at 180 °C for 3 h. When the particle accumulation and melting processes were repeated three or four times, flat polymeric microdomains were patterned. Next, the PDMS plate was dipped in 10% ethylenediamine in DI water for 6 h. Partially oxidized dextran was prepared by reacting 5% dextran in 12% sodium periodate in DI water for 12 h.39 The PDMS plate was dipped in partially oxidized dextran in DI water for 2 h to conjugate dextran with the amine groups on the polymer surface. Formation of Hydrogel Patterns. To prepare calcium alginate hydrogel patterns, a gelation solution of 1 or 10% w/v CaCl2 was poured over the entire PDMS surface with dextran-conjugated hydrophilic microdomains. The gelation solution was then gently
dextran-conjugated hydrophilic microdomains prepared using functional polymer particles38 and subsequent chemical reactions.39 PDMS is a widely used material for fabricating microstructured experimental platforms and microfluidic devices because of its biocompatibility, optical transparency, gas permeability for cell cultivation, and ease of fabrication/ replication via rapid prototyping and replica molding.40 However, it is not easy to form and stabilize hydrogel constructs on chemically inert, hydrophobic PDMS surfaces, especially when the hydrogel size is small. The approach presented here utilizes the difference in the hydrophilic/ hydrophobic properties of the dextran-conjugated microdomain and the native PDMS surface. The process of hydrogel patterning is shown in Figure 1. First, functional polymer particles with an epoxy group were trapped and melted on the microwells formed on a PDMS plate to fix the polymer matrix inside the microwells. Next, by cross-linking the epoxy group with ethylenediamine and partially oxidized dextran, dextranconjugated microdomains were prepared. The PDMS plate with the patterned hydrophilic regions was then dipped in a gelation solution, leaving a certain amount of the solution trapped on the hydrophilized area. Subsequently, the PDMS plate was dipped in a sol solution in which hydrogel patterns were selectively generated on the patterned domains. In this study, we prepared calcium alginate, collagen I, and chitosan hydrogel patterns by using an aqueous solution of calcium chloride or sodium hydroxide as the gelation solution. In addition, as an application of the constructed hydrogel arrays, we demonstrated selective cell patterning on the PDMS/ alginate and PDMS/collagen surfaces by utilizing the differences in the adhesion abilities of different cell types.
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EXPERIMENTAL SECTION
Materials. Sodium alginate (300−400 mPa·s at 1%, 20 °C), chitosan (10−100 mPa·s at 5 g/L, 20 °C), ethylenediamine, sodium periodate, and trisodium citrate were obtained from Wako Pure Chemical Ind. Ltd., Osaka, Japan. Dextran (Mw = 15 000−20 000) was obtained from Nacalai Tesque Inc., Tokyo, Japan. A collagen I solution from rat tail was obtained from BD Biosciences. FITC-conjugated 14074
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Figure 2. Local hydrophilization of the PDMS plate. (a−c) Micrographs of (a) an empty circular well, (b) particles trapped inside the well, and (c) the hydrophilized surface after conjugation with dextran. (d) FITC-conjugated dextran was employed to confirm selective hydrophilization. The scale bar is 100 μm. removed by pipetting, leaving small aliquots of the CaCl2 solution only on the hydrophilized regions. Next, a 0.9−2.7% sodium alginate solution containing fluorescent microparticles (ϕ = 0.71 or 0.1 μm) was poured on the PDMS plate. After 5 min of incubation at room temperature, the excess solution was gently removed by pipetting and immediately replaced with a 1% CaCl2 solution to prevent hydrogel drying. To pattern collagen I hydrogels, 0.023 M NaOH in phosphatebuffered saline (PBS, Dulbecco’s formula, final pH of 11.9) and 4.62 mg/mL collagen in 0.02 N acetic acid (pH of 3.1) were used as the gelation and sol solutions, respectively. The patterning procedures were the same as those for the calcium alginate hydrogel, except that an additional incubation at 37 °C for 30 min was included after pouring the collagen sol solution. The patterned hydrogels were preserved in the cell culture medium (DMEM with serum). In the case of chitosan hydrogel patterning, 1 M NaOH in DI water and 1.8% chitosan in 0.16 M HCl were used. The patterning procedures were the same as those for the calcium alginate hydrogel. The patterned hydrogels were preserved in 1 M NaOH solution. Cell Patterning on PDMS Plates with Hydrogel Patterns. Confluent cells were harvested by trypsin/EDTA treatment. NIH-3T3 and PC12 cells were suspended in cell culture medium at 7 × 105 and 3 × 106 cells/mL, respectively. Then, 0.5 mL of the cell suspension was dropped onto a 2 × 2.5 cm2 area of the PDMS plate, with cell densities of 7 × 104 and 3 × 105 cells/cm2 for NIH-3T3 and PC12 cells, respectively. After 4 h of incubation in the CO2 incubator, nonadherent cells were removed by aspiration. The patterned cells were cultured for 4 days. So that cell positions could be clearly observed, cell nuclei were stained with Hoechst 33342 (Invitrogen).
the epoxy group was reactive even after the melting of the polymer particles at 180 °C for 3 h. Patterning of Calcium Alginate Hydrogels. Alginate forms hydrogels in the presence of multivalent cations such as Ca2+.44 Previous studies demonstrating the patterning of alginate hydrogels initially formed alginate sol patterns by spotting29 or contact printing,30 followed by cross-linking with the addition of a cation solution. In addition, a positively charged surface was employed to ensure stable adhesion of the negatively charged alginate polymers on the substrate. In contrast, in the present method, we first patterned the Ca2+ solution with a simple dipping process, followed by reaction with the alginate solution to form hydrogels in the patterned regions. Figure 3a,b shows the dextran-modified area before and after trapping the 10% CaCl2 solution. Small aliquots of CaCl2 solution selectively trapped on the dextran-conjugated hydrophilic areas gradually shrunk because of evaporation (Figure 3b). These droplets were not completely evaporated because of the hygroscopic nature of CaCl2 and atmospheric water vapor. Next, by applying a 1.8% sodium alginate solution incorporating green fluorescent particles (0.71 μm) onto the PDMS plate, calcium alginate hydrogels were formed only on the hydrophilized regions (Figure 3c,d). The patterns of calcium alginate hydrogels corresponded well to the shapes of the hydrophilized areas (Figure 3e), although the trapped gelation solution was localized to a specific point in the hydrophilized region. We assumed that the gelation of the alginate sol solution occurred not only within but also beyond the hydrophilic region. However, the gel matrix formed outside of the hydrophilic region was taken off by removing the sol solution as a result of its weak interaction with the hydrophobic PDMS surface. Using this process, we successfully formed hydrogel patterns that were as small as 12.5 μm when smaller hydrophilic microdomains were used (Figure 3f). The formed hydrogels were stable at least for 1 week when stored in 1% CaCl2 solution, distilled water, or 0.9% NaCl solution. The obtained hydrogel patterns were easily dissolved by dipping them in a 0.1 M trisodium citrate solution, a chelating agent for the calcium ion, confirming that the formed patterns were indeed composed of a calcium alginate hydrogel matrix (Figure S1 in Supporting Information). In addition, it is worth noting that local hydrophilization and hydrogel patterning inside PDMS microchannels were possible, although we observed nonspecific hydrogel formation on the PDMS surface and the edges of microchannels (Figure S2 in Supporting Information). We then estimated the volume and thickness of the alginate hydrogels by using confocal laser microscopy (Seika Corp., Tokyo, Japan). Hydrogel patterns incorporating much smaller fluorescent particles (0.1 μm) were prepared, and the z
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RESULTS AND DISCUSSION Formation of Hydrophilic Microdomains on PDMS Plates. Initial experiments were conducted to pattern dextranconjugated hydrophilic regions on a hydrophobic PDMS plate. Figure 2 shows the modification procedure using circular microwell structures. By applying and removing the particle suspension, polymer particles bearing an epoxy group were selectively trapped inside the microwells (Figure 2b) because of the difference in the densities of the particles and the medium and the lateral capillary force exerted at the air−liquid interface.38,43 After the particles were melted once, a modified region with a concave surface was obtained. Therefore, we repeated the trapping and melting process three or four times until we obtained a flat surface (Figure 2c). The epoxy group on the polymeric surface was then reacted with ethylenediamine, followed by conjugation with partially oxidized dextran, to obtain dextran-modified microdomains. By using FITCdextran, the fluorescence of which was not significantly affected by the oxidation process, we were able to observe that the dextran molecules were selectively conjugated to the modified regions even after washing in DI water, indicating that covalent bonding had occurred (Figure 2d). This result suggested that 14075
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Table 1. Heights and Volumes of Calcium Alginate Hydrogels Formed on Square Regions with Different Sizesa conc of sodium alginate (%) 1.8 1.8 1.8 1.8 0.9 2.7 1.8 (conc of CaCl2, 1%)
width of the square region (μm)
average hydrogel height (μm)
estimated hydrogel volume (pL)
12.5 25 50 100 50 50 50
3.8 3.7 4.1 4.4 3.1 4.5 1.9
0.6 2.3 10.2 44.3 7.9 11.3 4.8
a
The hydrogel heights were measured by using confocal laser microscopy. The alginate concentration was changed as indicated. The CaCl2 concentration was 10% except for in the bottom column (1%).
As shown in Table 1, the higher alginate concentration resulted in the formation of thicker hydrogels, demonstrating the possibility to control the hydrogel morphology in the z direction. The calcium concentration was also a critical factor in determining the properties of the formed hydrogels. When the CaCl2 concentration was as low as 1% for the circular and square microdomains with sizes of 20−200 μm, hydrogel patterns were still formed but they were thinner than those formed at 10% CaCl2 (Table 1). In contrast, the line patterns shown in Figure 3e were obtained when the CaCl 2 concentration was 1%, but bridges were formed between the hydrogel lines at a higher CaCl2 concentration of 10%. This result suggests that the ratio of the hydrophilized region to the hydrophobic PDMS surface, not the absolute areas of the hydrophilized region, affected the morphology of the formed hydrogel. A lower concentration of the gelation solution was preferable for patterning hydrogels with relatively large areas such as the line array (with a hydrophilic area ratio of 50%). We then examined the effect of the conjugated dextran molecules on the formation and stabilization of hydrogels because conjugation to ethylenediamine introduces the relatively hydrophilic surface property. When the ethylenediamine-conjugated surface was used (i.e., without conjugation to dextran), it was also possible to trap the CaCl2 solution on the modified domain and form calcium alginate hydrogel patterns. However, the hydrogels detached from the patterned surface immediately after dipping the PDMS plate in the preservation solution of 1% CaCl2 (Figure 3g), suggesting that the interactions of the polymeric chains of dextran and alginate were essential to immobilizing the formed thin hydrogel patterns on the surface. In addition, we observed the 3D morphology of the formed hydrogel patterns using confocal laser scanning microscopy (FV1000, Olympus Corp., Tokyo, Japan). Square hydrogels with a width of 100 μm were prepared using 1.8% sodium alginate solution and 10% CaCl2 solution, which contained 0.1 μm fluorescent microbeads. As shown in Figure 4, the hydrogel surface was relatively rough, and bumps with a size of ∼2 μm were observed, which were probably formed at the time of the removal of the sol solution. Techniques for gently removing the sol solution by dilution or aspiration might enable the formation of hydrogels with flatter surfaces. Previous techniques used for alginate hydrogel patterning have limitations, especially in resolution, because dispensing a
Figure 3. Construction of calcium alginate hydrogel micropatterns. Green fluorescent microbeads (0.71 μm) were incorporated to visualize hydrogel morphologies. (a−d) The formation process of hydrogel showing (a) the hydrophilized surface, (b) trapped CaCl2 solution, and (c, d) bright-field and fluorescence images of the obtained hydrogel. (e) Various shapes of calcium alginate hydrogels. (Left) Hydrophilized patterns. (Right) formed hydrogels. Square and line-array patterns were prepared using 10 and 1% CaCl2 solutions, respectively. (f) Bright-field and fluorescent images of small hydrogels with A square shape (width, 12.5 μm). Hydrogel shapes are outlined by dotted lines. (g) Calcium alginate hydrogels formed in the hydrophilic region without conjugated dextran. The detached regions of the calcium alginate hydrogels are indicated by arrows. Scale bars: (a−e, g) 100 μm; (f) 20 μm.
positions of the embedded particles near the surface and the bottom of the hydrogels were determined to calculate the thickness. Table 1 shows the measured average thicknesses and the estimated volumes of the square alginate hydrogels (n = 10) prepared using a 10% CaCl2 solution when the width of the hydrophilic region ranged from 12.5 to 100 μm. The hydrogel thickness slightly increased from 3.7 to 4.4 μm with the increase in the size of the hydrophilic region, although its effect was not significant. The estimated volumes of calcium alginate hydrogels varied from 0.6 to 44.3 pL depending on the area of the hydrophilic domain. In addition, we examined the effects of the alginate concentration on the thickness of the formed hydrogel. 14076
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Figure 4. Three-dimensional morphology of a 100 × 100 μm2 alginate hydrogel and its projection shape onto the x−z plane, which were observed using confocal laser scanning microscopy. The hydrogel was prepared by using a 1.8% sodium alginate solution and a 10% CaCl2 solution.
very viscous alginate sol solution onto small regions is generally difficult and not reproducible. Li et al. reported the formation of circular alginate patterns with diameters of ∼300 μm by solution spotting, but there was a need to use a lowconcentration sol solution (0.65%),29 which might have resulted in the formation of hydrogels of insufficient mechanical strength.45 In addition, alginate hydrogels have normally been patterned on a hydrophilic substrate, which improves hydrogel attachment. In these cases, the patterned aqueous droplets tend to spread on the surface, possibly making the air−liquid boundary shape uncontrollable. Compared to the previous techniques, the present approach achieves high-resolution patterning, even when using an alginate solution of relatively high concentration (1.8−2.7%), with the help of the restricted hydrophilic microdomains. Patterning of Collagen and Chitosan Hydrogels. Collagens form a group of bioactive proteins composed of specific peptide chains and are among the most important ECMs.32,33 Collagen I dissolves in aqueous solution at low pH, but the triplet helices form hydrogels when heated to 37 °C at neutral pH. Various techniques of patterning collagen molecules have been reported, but only a few techniques have achieved the patterning of wet-type hydrogels of pure collagen, including embedding into grooved PDMS plates37 and microchannel-assisted deposition.36 Here, we examined if the present method is applicable to preparing collagen I hydrogel patterns. Figure 5a shows formed collagen hydrogel arrays incorporating red fluorescent microbeads on the hydrophilic domains, using NaOH as the gelation agent. Although a large amount of the collagen sol solution was poured onto small areas of the modified regions with limited amounts of dried NaOH/PBS powder, which were formed after drying the trapped NaOH solution, hydrogel patterns were selectively formed on the dextran-modified microdomain. This suggests the presence of interactions between the dextran and collagen molecules. The hydrogel was stable in the cell cultivation medium containing serum for at least 1 week. The average thickness of the collagen gel was 6.2 μm. Next, we demonstrated the pattern formation of chitosan hydrogels by using a similar procedure. Chitosan is a polysaccharide with high biocompatibility and biodegradability and is used for surgical sutures, DDS carriers, and tissue engineering scaffolds.33,46 Electrochemical methods and meshbased contact printing approaches have been reported for chitosan hydrogel patterning.31,47 However, these techniques do not allow the formation of hydrogels with arbitrary shapes.
Figure 5. Construction of collagen I and chitosan hydrogel micropatterns. Bright-field and fluorescent micrographs of (a) collagen I and (b) chitosan hydrogels. Red fluorescent microbeads (0.52 μm) were incorporated into the hydrogel to observe the hydrogel morphologies clearly. The scale bar is 100 μm.
As shown in Figure 5b, we successfully used our method to pattern chitosan hydrogels in circular shapes, with an average thickness of 9.3 μm. This result clearly demonstrates that the approach presented here achieves the precise patterning of various wet-type and intact hydrogels that cannot be obtained by photopatterning techniques without employing complex operations and experimental setups. Cell Patterning Using Hydrogel Arrays. Patterned cell culture systems are widely utilized for tissue engineering applications, cell-based assays, and drug screening. However, cell-patterning techniques based on wet-type hydrogel microstructures have not been studied well, except for PEGDA hydrogels, mainly because of the lack of proper technologies for preparing hydrogel micropatterns. Here we attempted to culture cells on the patterned hydrogels prepared by using the present technique. It is known that native PDMS surfaces can be used as a cell culture substrate, but specific surface treatment must be employed to promote cell adhesion, depending on the cell type.48 In this study, we used NIH3T3 (mouse fibroblast) and PC12 (rat pheochromocytoma) cells with different adhesive properties. For the highly adherent NIH-3T3 cells, selective attachment was performed on a PDMS surface patterned with nonadherent calcium alginate hydrogels. In contrast, collagen hydrogel patterns were utilized for the PC12 cells, which are less adhesive. The difference in adhesion properties of these cells would be attributed to the different quantities of adhesion ligands/receptors and ECMs expressed on the cell surface.49 Figure 6a shows the patterns of NIH-3T3 cells on the lineshape calcium alginate/PDMS surface. At day 0, cells avoided calcium alginate hydrogels but selectively adhered to the PDMS surface. During cultivation, NIH-3T3 cells proliferated and spread selectively on PDMS because fibroblastic NIH-3T3 cells, which can synthesize the extracellular matrix and modify the neighboring surface, could not adhere to the calcium alginate surface. At day 4, NIH-3T3 cells had formed a confluent monolayer, with alignment along the line direction because of the relatively narrow PDMS patterns. This alignment was consistent with the findings of the previous study that the width of the line-shape adhesive region dominates the cell 14077
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Figure 6. Cell patterning utilizing hydrogel patterns with different adhesion properties. (a) NIH-3T3 cells on a PDMS plate with calcium alginate hydrogel patterns and (b) PC12 cells on a PDMS plate with collagen I hydrogel patterns. Calcium alginate and collagen I hydrogels incorporating green and red fluorescent microbeads, respectively. Cell nuclei were stained with Hoechst 33342 and indicated in blue. Day 0 indicates the micrographs after 4 h of incubation and the removal of nonadherent cells. The cell densities in cell-attached regions were 2.1 × 105 and 2.9 × 105 cells/cm2 for 3T3 cells at days 0 and 4, respectively, and 4.1 × 105 and 8.3 × 105 cells/cm2 for PC12 cells at days 0 and 4, respectively. The scale bar is 100 μm.
orientation.50 However, PC12 cells did not adhere to either the PDMS or the alginate surfaces. We then attempted to perform patterned culturing of PC12 cells on the PDMS/collagen I-patterned surface. After PC12 cells were seeded and incubated for 4 h, PC12 cells adhered mainly to the collagen I hydrogel region, with few on the PDMS surface, because collagen molecules support the adhesion of PC12 cells (Figure 6b).51 PC12 cells proliferated on the collagen hydrogels during cultivation, whereas the number of cells on the PDMS surface gradually decreased. The pattern of PC12 cells was stably maintained for at least 7 days. It is worth noting that NIH-3T3 cells spread over the entire surface of the PDMS-collagen I and PDMS-chitosan patterns. These results clearly demonstrate that the selective patterning of cells was possible by employing hydrogel patterns with proper adhesiveness, with the help of PDMS surfaces with moderate cell adhesion characteristics. These patterned hydrogels could be applicable to highly functional cell cultivation systems mimicking in vivo microenvironments, cell-based pharmacological assays, and studies of cell−surface interactions.
chitosan) were successfully patterned. The size, shape, and alignment of the hydrogels were finely controlled by the hydrophilized areas that were defined by the microwell structures on the PDMS surface. As an application of the hydrogel arrays, patterned cell cultures were successfully demonstrated. The presented method for hydrogel patterning is highly versatile and will be useful in developing micrometersized biosensors, bioreactors, and cell cultivation platforms because it has the potential to be incorporated into microfluidic systems and does not necessitate complicated devices and operations.
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ASSOCIATED CONTENT
S Supporting Information *
Fluorescence micrographs showing calcium-alginate hydrogel patterns before and after treating with a solution of a chelating agent. Local hydrophilization and hydrogel patterning inside a PDMS microchannel. This material is available free of charge via the Internet at http://pubs.acs.org.
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CONCLUSIONS In this study, we present a new process to fabricate picolitervolume, micrometer-order-thick hydrogel patterns precisely on PDMS plates by utilizing dextran-conjugated hydrophilic microdomains. This was possible by employing simple operation procedures including stepwise dipping, the dispensing of a gelation solution, and in situ gelation on the restricted hydrophilized regions. Three types of hydrogels composed of pure hydrogel molecules (calcium alginate, collagen I, and
AUTHOR INFORMATION
Corresponding Author
*Tel and Fax: +81-43-290-3398. E-mail: m-yamada@faculty. chiba-u.jp. Author Contributions †
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest. 14078
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(20) Kuckling, D.; Hoffman, J.; Plöntner, M.; Ferse, D.; Kretschmer, K.; Adler, H. J. P.; Arndt, K. F.; Reichelt, R. Photo Cross-linkable Poly(N-isopropylacrylamide) Copolymer III: Micro-Fabricated Temperture Responsible Hydrogels. Polymer 2003, 44, 4455−4462. (21) Khademhosseini, A.; Eng, G.; Yeh, J.; Fukuda, J.; Blumling, J., 3rd; Langer, R.; Burdick, J. A. Micromolding of Photocrosslinkable Hyaluronic Acid for Cell Encapsulation and Entrapment. J. Biomed. Mater. Res., Part A 2006, 79, 522−532. (22) Koh, W. G.; Itle, L. J.; Pishko, M. V. Molding of Hydrogel Microstructures to Create Multiphenotype Cell Microarrays. Anal. Chem. 2003, 75, 5783−5789. (23) Heo, J.; Crooks, R. M. Microfluidic Biosensor Based on an Array of Hydrogel-Entrapped Enzymes. Anal. Chem. 2005, 77, 6843−6851. (24) Cheung, Y. K.; Gillette, B. M.; Zhong, M.; Ramcharan, S.; Sia, S. K. Direct Patterning of Composite Biocompatible Microstructures Using Microfluidics. Lab Chip 2007, 7, 574−579. (25) Chan-Park, M. B.; Yan, Y.; Neo, W. K.; Zhou, W.; Zhang, J.; Yue, C. Y. Fabrication of High Aspect Ratio Poly(ethylene glycol)Containing Microstructures by UV Embossing. Langmuir 2003, 19, 4371−4380. (26) Yadavalli, V. K.; Koh, W.-G.; Lazur, G. J.; Pishko, M. V. Microfabricated Protein-Containing Poly(ethylene glycol) Hydrogel Arrays for Biosensing. Sens. Actuators, B 2004, 97, 290−297. (27) Krsko, P.; Sukhishivili, S.; Mansfield, M.; Clancy, R.; Libera, M. Electron-Beam Surface-Patterned Poly(ethylene glycol) Microhydrogels. Langmuir 2003, 19, 5618−5623. (28) Hong, Y.; Krsko, P.; Libera, M. Protein Surface Patterning Using Nanoscale PEG Hydrogels. Langmuir 2004, 20, 11123−11126. (29) Li, H.; Leulmi, R. F.; Juncker, D. Hydrogel Droplet Microarrays with Trapped Antibody-Functionalized Beads for Multiplexed Protein Analysis. Lab Chip 2011, 11, 528−534. (30) Nelson, C. M.; Raghavan, S.; Tan, J. L.; Chen, C. S. Degradation of Micropatterned Surfaces by Cell-Dependent and -Independent Processes. Langmuir 2003, 19, 1493−1499. (31) Zawko, S. A.; Schmidt, C. E. Simple Benchtop Patterning of Hydrogel Grids for Living Cell Microarrays. Lab Chip 2010, 10, 379− 383. (32) Prockop, D. J.; Kivirikko, K. I. Collagens: Molecular Biology, Diseases, and Potentials for Therapy. Annu. Rev. Biochem. 1995, 64, 403−434. (33) Sionkowska, A. Current Research on the Blends of Natural and Synthetic Polymers as New Biomaterials: Review. Prog. Polym. Sci. 2011, 36, 1254−1276. (34) Folch, A.; Toner, M. Cellular Micropatterns on Biocompatible Materials. Biotechnol. Prog. 1998, 14, 388−392. (35) Roth, E. A.; Xu, T.; Das, M.; Gregory, C.; Hickman, J. J.; Boland, T. Inkjet Printing for High-Throughput Cell Patterning. Biomaterials 2004, 25, 3707−3715. (36) Tan, W.; Desai, T. A. Microfluidic Patterning of Cells in Extracellular Matrix Biopolymers: Effects of Channel Size, Cell Type, and Matrix Composition on Pattern Integrity. Tissue Eng. 2003, 9, 255−267. (37) Raghavan, S.; Nelson, C. M.; Baranski, J. D.; Lim, E.; Chen, C. S. Geometrically Controlled Endothelial Tubulogenesis in Micropatterned Gels. Tissue Eng. A 2010, 16, 2255−2263. (38) Yamamoto, M.; Yamada, M.; Nonaka, N.; Fukushima, S.; Yasuda, M.; Seki, M. Patterning Reactive Microdomains inside Polydimethylsiloxane Microchannels by Trapping and Melting Functional Polymer Particles. J. Am. Chem. Soc. 2008, 130, 14044−14045. (39) Yu, L.; Li, C. M.; Liu, Y.; Gao, J.; Wang, W.; Gan, Y. FlowThrough Functionalized PDMS Microfluidic Channels with Dextran Derivative for ELISAs. Lab Chip 2009, 9, 1243−1247. (40) Duffy, D. C.; McDonald, J. C.; Schueller, O. J.; Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). Anal. Chem. 1998, 70, 4974−4984. (41) Yang, W.; Hu, J.; Tao, Z.; Li, L.; Wang, C.; Fu, S. Dispersion Copolymerization of Styrene and Glycidyl Methacrylate in Polar Solvents. Colloid Polym. Sci. 1999, 277, 446−451.
ACKNOWLEDGMENTS This research was supported in part by grants in aid (23106007 and 23700554) and by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We thank the National Bio-Resource Project (NBRP) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan for kindly providing NIH-3T3 and PC12 cells.
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REFERENCES
(1) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface Engineering Approaches to Micropattern Surfaces for Cell-Based Assays. Biomaterials 2006, 27, 3044−3063. (2) Quist, A. P.; Oscarsson, S. Micropatterned Surfaces: Techniques and Applications in Cell Biology. Expert Opin. Drug Discovery 2010, 5, 569−581. (3) Colpo, P.; Ruiz, A.; Ceriotti, L.; Rossi, F. Surface Functionalization for Protein and Cell Patterning. Adv. Biochem. Eng. Biotechnol. 2010, 117, 109−130. (4) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray. Science 1995, 270, 467−470. (5) Schena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier, E.; Davis, R. W. Microarrays: Biotechnology’s Discovery Platform for Functional Genomics. Trends Biotechnol. 1998, 16, 301− 306. (6) Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.; Takeuchi, S.; Noji, H. Microfabricated Arrays of Femtoliter Chambers Allow Single Molecule Enzymology. Nat. Biotechnol. 2005, 23, 361− 365. (7) Yua, L.; Liua, Y. S.; Gan, Y.; Li, C. M. High-Performance UVCurable Epoxy Resin-Based Microarray and Microfluidic Immunoassay Devices. Biosens. Bioelectron. 2009, 24, 2997−3002. (8) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Microscale Technologies for Tissue Engineering and Biology. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2480−2487. (9) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric Control of Cell Life and Death. Science 1997, 276, 1425−1428. (10) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. Microfabrication of Hepatocyte/Fibroblast Co-cultures: Role of Homotypic Cell Interactions. Biotechnol. Prog. 1998, 14, 378−387. (11) Khetani, S. R.; Bhatia, S. N. Microscale Culture of Human Liver Cells for Drug Development. Nat. Biotechnol. 2008, 26, 120−126. (12) Nie, F. Q.; Yamada, M.; Kobayashi, J.; Yamato, M.; Kikuchi, A.; Okano, T. On-Chip Cell Migration Assay Using Microfluidic Channels. Biomaterials 2007, 28, 4017−4022. (13) Calvert, P. Inkjet Printing for Materials and Devices. Chem. Mater. 2001, 13, 3299−3305. (14) Khan, M. S.; Fon, D.; Li, X.; Tian, J.; Forsythe, J.; Garnier, G.; Shen, W. Biosurface Engineering through Ink Jet Printing. Colloids Surf., B 2010, 75, 441−447. (15) Rubina, A. Y.; Kolchinsky, A.; Makarov, A. A.; Zasedatelev, A. S. Why 3-D? Gel-Based Microarrays in Proteomics. Proteomics 2008, 8, 817−831. (16) Revzin, A.; Russell, R. J.; Yadavalli, V. K.; Koh, W. G.; Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Fabrication of Poly(ethylene glycol) Hydrogel Microstructures Using Photolithography. Langmuir 2001, 17, 5440−5447. (17) Liu, V. A.; Bhatia, S. N. Three-Dimensional Photopatterning of Hydrogels Containing Living Cells. Biomed. Microdev. 2002, 4, 257− 266. (18) Revzin, A.; Tompkins, R. G.; Toner, M. Surface Engineering with Poly(ethylene glycol) Photolithography to Create High-Density Cell Arrays on Glass. Langmuir 2003, 19, 9855−9862. (19) Hoffmann, J.; Plö ntner, M.; Kuckling, D.; Fischer, W. Photopatterning of Thermally Sensitive Hydrogels Useful for Microactuators. Sens. Actuators, B 1999, 77, 139−144. 14079
dx.doi.org/10.1021/la3014706 | Langmuir 2012, 28, 14073−14080
Langmuir
Article
(42) Horak, D.; Shapoval, P. Reactive Poly(glycidyl methacrylate) Microspheres Prepared by Dispersion Polymerization. J. Polym. Sci., Polym. Chem. 2000, 38, 3855−3863. (43) Tanaka, M.; Shimamoto, N.; Tanii, T.; Ohdomari, I.; Nishide, H. Packing of Submicrometer-Sized Polystyrene Particles within the Micrometer-Sized Recessed Patterns on Silicon Substrate. Sci. Technol. Adv. Mater. 2006, 7, 451−455. (44) A. Maerinesen, G. Alginate as Immobilization Material. Biotechnol. Bioeng. 1989, 33, 79−89. (45) LeRoux, M. A.; Guilak, F.; Setton, L. A. Compressive and Shear Properties of Alginate Gel: Effects of Sodium Ions and Alginate Concentration. J. Biomed. Mater. Res. 1999, 47, 46−53. (46) Rinaudo, M. Chitin and Chitosan: Properties and Applications. Prog. Polym. Sci. 2006, 31, 603−632. (47) Fernandes, R.; Wu, L.-Q.; Chen, T.; Yi, H.; Rubloff, G. W.; Ghodssi, R.; Bentley, W. E.; Payne, G. F. Electrochemically Induced Deposition of a Polysaccharide Hydrogel onto a Patterned Surface. Langmuir 2003, 19, 4058−4062. (48) Lee, J. N.; Jiang, X.; Ryan, D.; Whitesides, G. M. Compatibility of Mammalian Cells on Surfaces of Poly(dimethylsiloxane). Langmuir 2004, 20, 11684−11691. (49) Hynes, R. O. Cell Adhesion: Old and New Questions. Trends Genet. 1999, 15, M33−M37. (50) Shen, J. Y.; Chan-Park, M. B.; He, B.; Zhu, A. P.; Zhu, X.; Beuerman, R. W.; Yang, E. B.; Chen, W.; Chan, V. Three-Dimentional Microchannel in Biodegradable Polymeric Films for Orientation and Phenotype of Vascular Smooth Muscle Cells. Tissue Eng. 2006, 12, 2229−2240. (51) Greene, L. A.; Farinelli, S. E.; Cunningham, M. E.; Park, D. S. Culture and Experimental Use of PC12 Rat Pheochromocytoma Cell Line. In Culturing Nerve Cells, 2nd ed.; Banker, G., Goslin, K., Ed.; MIT Press: Cambridge, MA, 1998; pp 161−188.
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