Article pubs.acs.org/Langmuir
Protein Covalently Conjugated SU‑8 Surface for the Enhancement of Mesenchymal Stem Cell Adhesion and Proliferation Peng Xue,† Jingnan Bao,† Yon Jin Chuah,† Nishanth V. Menon,† Yilei Zhang,‡ and Yuejun Kang*,† †
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459 Singapore School of Mechanical & Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798 Singapore
‡
S Supporting Information *
ABSTRACT: Cell growing behavior is significantly dependent on the surface chemistry of materials. SU-8 as an epoxy-based negative photoresist is commonly used for fabricating patterned layers in lab-on-a-chip devices. As a hydrophobic material, SU-8 substrate is not favorable for cell culture, and cell attachment on native SU-8 is limited attributed to poor surface biocompatibility. Although physical adsorption of proteins could enhance the cell adhesion, the effect is not durable. In this work, SU-8 surface chemistry is modified by immobilizing fibronectin (FN) and collagen type I (COL I) covalently using (3-aminopropyl)triethoxysilane (APTES) and cross-linker glutaraldehyde (GA) to increase surface biofunctionality. The effectiveness of this surface treatment to improve the adhesion and viability of mesenchymal stem cells (MSCs) is investigated. It is found that the wettability of SU-8 surface can be significantly increased by this chemical modification. In addition, the spreading area of MSCs increases on the SU-8 surfaces with covalently conjugated matrix proteins, as compared to other unmodified SU-8 surface or those coated with proteins simply by physical adsorption. Furthermore, cell proliferation is dramatically enhanced on the SU-8 surfaces modified under the proposed scheme. Therefore, SU-8 surface modification with covalently bound matrix proteins assisted by APTES+GA provides a highly biocompatible interface for the enhanced adhesion, spreading, and proliferation of MSCs.
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INTRODUCTION SU-8 is an epoxy-based negative photoresist with excellent mechanical and optical properties, and it has been extensively employed in microfabrication by photoinitiated polymerization.1,2 SU-8 is attractive for manufacturing microstructures due to its capability of high resolution patterning for semiconductor industry. Additionally, SU-8 is able to produce microstructure with high aspect ratio with a wide range of structure thickness.3−7 Moreover, SU-8 has optimal chemical stability and transparent to visible light after cross-linking, which can be used as optical waveguides,8 probes for microscopy,9 MEMS,10−12 and molds for microchip.13 Previous studies have shown that SU-8 could be nontoxic and biocompatible after polymerization,14,15 indicating its potential to serve as the substrate of bioanalytical micro- and nanodevices,16 such as biosensors,17 bioarrays,18 and drug delivery vehicles.19 However, the hydrophobic nature of SU-8 has limited its biological applications in practice, which causes low specific adsorption of probes,20 poor surface wettability,21,22 and limited cell attachment.23 Therefore, strategies to modify the SU-8 surface to improve its biocompatibility could make it a more amenable material for biological applications. Traditional SU-8 surface modification methods are based on physical adsorption and chemical conjugation. It has been reported that biomolecules such as collagen and fibronectin can be physically absorbed on the SU-8 surface to improve cell attachment owing to molecule recognition mechanism.24,25 Chemical modification © 2014 American Chemical Society
of SU-8 can be simply achieved by exposing SU-8 surface to oxygen plasma,26 sulfuric acid,7 or cerium(IV) ammonium nitrate (CAN).23 The surface functionalization is based on generation of functional groups, such as −OH, −COOH, −NH2, or −SH, which can be used for conjugation of biomolecules.27,28 Additionally, epoxide groups on SU-8 surface can directly react with some molecules, such as ethanolamine and amino-functionalized DNA.21,29 These methods were extensively used for diminishing hydrophobicity and enhance the molecule and cell attachment on various surfaces. However, the traditional methods still have typical limitations. Although surface hydrophobicity could be simply reduced by plasma treatment, the hydrophobicity could recover in several hours.26 Moreover, although physically coating matrix proteins on polydimethylsiloxane (PDMS) surface can facilitate initial cell adhesion, cell detachment usually occurs after reaching confluence.30 Obviously, the weak interaction forces, such as electrostatic and van der Waals forces, cannot provide sufficient binding of proteins for cell growth. Therefore, to address these issues, it is desirable to induce much stronger binding force at the substrate−cell interface, such as covalent conjugation of the proteins.31 Among these covalent conjugation methods, surface treatment with 3-aminopropyltriethoxysilane Received: January 5, 2014 Revised: March 4, 2014 Published: March 6, 2014 3110
dx.doi.org/10.1021/la500048z | Langmuir 2014, 30, 3110−3117
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Article
Figure 1. Schematic illustration of SU-8 surface modification. Fibronectin fragment is conjugated shown as an example. (3-Aminopropyl)triethoxysilane (APTES), glutaraldehyde (GA), Tween 20, sulfuric acid, Triton X-100, bovine serum albumin (BSA), and formalin were obtained from Sigma-Aldrich, Singapore. 1×PBS was purchased from First Base, Singapore. TRITC-conjugated phalloidin and DAPI were purchased from Millipore, Singapore. Collagen type I (COL I), fibronectin (FN), DMEM, fetal bovine serum (FBS), penicillin, PrestoBlue cell viability reagent, micro-BCA Protein Assay Kit, and CyQUANT Cell Proliferation Assay Kit were obtained from Life Technologies, Singapore. Surface Modification of SU-8. SU-8 3010 was added into a 48-well cell culture plate with 100 μL per well. The SU-8 was then transferred into a vacuum oven and degassed for 30 min to remove all the bubbles. The excess solvent was evaporated by a prebake for 5 min at 65 °C and 15 min at 95 °C. Subsequently, SU-8 was polymerized by exposure to UV light (wavelength 365 nm, radiation intensity 17 mW cm−2) for 15 min to ensure complete cross-linking and postbaked for 2 min at 65 °C and 5 min at 95 °C. The cured SU-8 samples were divided into three groups (group I: protein-free; group II: protein; and group III: APTES + GA + protein), and their surfaces were treated as follows. For the group III (APTES + GA + protein), the surface was incubated with 95% sulfuric acid at 80 °C for 10 s followed by washing with DI water thoroughly.7 Then the substrate was immersed in 10% APTES at 50 °C for 2 h, followed by washed thrice with DI water. Subsequently, the substrate was incubated with 2.5% GA at room temperature for 1 h. Excessive GA was removed, and the specimen was washed thrice with DI water. Both group II (protein) and group III (APTES + GA + protein) were then incubated with 0.1 mg mL−1 of collagen type I (COL I) and fibronectin (FN) at 4 °C overnight. Eventually, the samples were washed thrice with DI water followed by removing the excessive proteins. All the samples were sterilized by irradiation under UV light for 60 min prior to the following experiments. Tissue culture plates (TCP) (Nunc, Singapore) were used as control. Atomic Force Microscopy (AFM) Analysis. Surface roughness measurements were performed with an atomic force microscope (MFP-3D, Asylum Research) in intermittent tapping mode. Silicon cantilevers with a nominal resonance frequency of 75 kHz, and its phase was corrected to zero. Images were processed using Igor.Pro.622A (WaveMetrics). After a second-order polynomial plane correction and subsequent line-wise leveling, the root-mean-square (RMS) surface roughness was calculated. All images were recorded with a scanning rate of 500 × 500 nm2 at a resolution of 512 × 512 pixels. Picture quality was maximized using proportional and integral gains. The error bars were calculated after measurement of five different images obtained. Characterization of Surface Wettability. The wettability of SU-8 surfaces was characterized by measurements of contact angle using a theta optical tensiometer (Attension, Finland). Briefly, 5 μL of DI water droplet was dripped to contact with the surface of specimen substrate. Subsequently, the contact angle formed between the substrate surface and the tangent of the static sessile droplet surface
(APTES) and glutaraldehyde (GA) has exhibited great potential for protein immobilization, which is similar to the well-known organosilane based on silicon derivatization.32 APTES acts as a short molecular spacer to avoid direct surface−protein interactions (i.e., the weak interaction) and to conquer the steric hindrance from the vicinity of the support. The proteins are then covalently coupled to the APTES molecules activated by GA.33,34 It was reported that SU-8 microwell could be functionalized by the above-mentioned method as a biosensor to detect C-reactive proteins by fluorescent sandwich immunoassay.33 However, such chemically modified SU-8 surface has not yet been applied to stabilize cell attachment and improve cell proliferation. Considering the recent development of lab-on-a-chip technology in tissue engineering and regenerative medicine, the demand for highly biocompatible chip devices is ever increasing in order to create a cell-friendly microenvironment. Therefore, this paper presents a novel surface chemical modification scheme to enhance the cell affinity and proliferation on the surface of SU-8, which serves as a backbone material for patterning most of the current lab-on-a-chip devices. Mesenchymal stem cells (MSCs) are selected as a model for cellular analysis on the modified SU-8 surfaces. MSCs are multipotent stromal cells that are able to self-renew and differentiate into multiple connective tissue cell lineages and have become a promising cell source for tissue engineering and regenerative medicine, which are stem cell based applications and require highly biocompatible substrate or scaffold to support long-term cell adhesion and proliferation. To improve the surface biocompatibility, collagen type I (COL I) and fibronectin (FN) are immobilized on the surface of SU-8, since they are two critical extracellular matrix (ECM) proteins affecting the MSC morphology, migration, proliferation, and differentiation.35−37 In this report, biocompatible SU-8 surface is activated by APTES+GA and covalently conjugated with COL I and FN to investigate the enhancement of MSC adhesion and proliferation. Briefly, APTES is first bound to the SU-8 surface functionalized by hydroxyl group followed by activation with GA. COL I and FN are covalently conjugated to the surface modified with APTES+GA (Figure 1). The properties of the modified SU-8 surfaces are characterized, and the adhesion, spreading, and proliferation of MSCs are investigated quantitatively.
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EXPERIMENTAL SECTION
Reagents. SU-8 3010 was purchased from Microchem. Deionized water was collected from Millipore Synthesis A10 (Molsheim, France). 3111
dx.doi.org/10.1021/la500048z | Langmuir 2014, 30, 3110−3117
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was determined through the Drop Shape Analysis software. Each data point was determined based on six measurements at six different positions on the specimen. Surface Protein Quantification. The amount of protein retained on different SU-8 substrates was determined by a micro-BCA (bicinchoninic acid) Protein Assay Kit. After overnight incubation in 0.1 mg mL−1 protein solution, the substrate specimen was incubated with 0.05% Tween 20 (Sigma-Aldrich, Singapore) for 30 min followed by washing thrice with DI water. The absorbance of specimens was measured at wavelength of 562 nm with Multiskan Spectrum microplate reader (Thermal Scientific, Singapore) to quantitate the relative protein concentration retained on native and chemically modified SU-8 substrates. Cell Culture. MSCs were obtained from porcine bone marrow based on the method reported previously.38 The cells were cultured in DMEM (containing GlutaMAX-1, 1 g/L D-glucose, and 110 mg/L sodium pyruvate) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL) mixture in a humidified atmosphere at 37 °C and 5% CO2. Suspended cells were washed away after 3 days, and adhered MSCs were further cultured upon reaching confluence. A stable cellular morphology and differentiation capability of MSCs could be maintained in their earlier passages under the protocol established above.39 MSCs undergoing passages 2−4 were used for this experimental study. Characterization of Cell Adhesion. For evaluation of cell adhesion ability on different modified SU-8 surfaces, MSCs were seeded into a 48-well plate at a density of 1 × 104 cells per well (12 500 per cm2) and incubated for 90 min. Cells with similar density were seeded on a TCP as control. Unbound cells were washed out with 1 × PBS and the attached cells were frozen at −80 °C. A CyQUANT Cell Proliferation Assay Kit was used to determine the cell adhesion capacity. Briefly, after curing by 1×CyQUANT GR dye for 5 min, fluorescence was measured by a plate reader (Infinite M200 series, Tecan Asia, Singapore) with an excitation wavelength at 485 nm and emission wavelength at 535 nm. The reading was calculated as a fold value relative to the fluorescence intensity of the TCP. Since the number of cells adhered on the surface is correlated to the DNA content (reflected by fluorescence intensity) after cells lysis, this method could offer an accurate cell number quantification under various conditions of surface modifications. Cell spreading area was determined by fluorescence imaging after cell staining as described in the following. MSCs were seeded into a 6-well plate at a density of 1.5 × 104 per well (1560 per cm2) and incubated for 4 h. Subsequently, 10% formalin was introduced to fix the adhered cells overnight. After permeating with Trition X-100 for 5 min and blocking with 1% BSA for 30 min, the staining was conducted by incubating with TRITC-conjugated phalloidin for 45 min and DAPI for 5 min. The images were captured under an inverted fluorescence microscope (IX71, Olympus, Singapore) and were processed to determine the area of individual cell spreading by Image-Pro Plus (Media Cybernetics, Rockville, MD). Cell Proliferation Assay. The proliferation activity of MSCs cultured on different modified SU-8 surfaces was investigated by PrestoBlue cell viability reagent. Briefly, MSCs were seeded in a 48-well plate at a density of 1.5 × 103 cells per well (1875 per cm2) and incubated in a humidified atmosphere of 5% CO2 at 37 °C. After incubation at predetermined time duration (i.e., 2, 4, and 7 days), the culture medium was removed and the cells were washed with 1×PBS. Subsequently, the cells were incubated with DMEM containing 10% PrestoBlue reagent for 1 h. DMEM with 10% PrestoBlue reagent incubated in the blank wells served as control. The absorbance of PrestoBlue reagent reduction was measured at 570 and 600 nm with a Multiskan Spectrum microplate reader (Thermo Scientific, Singapore), respectively. Since the viable cell number correlated with the PrestoBlue reduction rate, the absorbance readings were translated to the percentage reduction of the PrestoBlue reagent based on the provided protocol. Statistical Analysis. Statistical significance study was conducted by using Microcal Origin 8.5.1. The results were analyzed based on one-way analysis of variance (ANOVA). A p-value of 0.05 or less was considered to be statistically significant.
Article
RESULTS Surface Wettability. The surface wettability of the tissue culture plate, native SU-8, and the protein-bound SU-8 (modified and unmodified) was investigated based on measurement of the water droplet contact angle formed on the substrate (Table 1). The tissue culture plate and native SU-8 exhibited
Table 1. Contact Angle on Culture Plate and Different SU-8 Surfacesa substrate type tissue culture plate native SU-8 APTES + GA collagen type 1*
contact angle (deg) 92.6 ± 2.9 105.4 ± 1.1 67.3 ± 6.5 94.2 ± 7.0
substrate type APTES + GA + collagen type 1# fibronectin* APTES + GA + fibronectin
contact angle (deg) 16.0 ± 1.7 87.0 ± 0.6 11.9 ± 2.2
Data shown as means ± SD (n = 6, #p value
