Protein functionalized micro hydrogel features for cell ... - Springer Link

Biomed Microdevices (2008) 10:567–571 DOI 10.1007/s10544-008-9167-6

Protein functionalized micro hydrogel features for cell–surface interaction Parijat Bhatnagar & Alan J. Nixon & Il Kim & Jun Kameoka

Published online: 8 February 2008 # Springer Science + Business Media, LLC 2008

Abstract Cross-linked hydrogel features have been patterned using subtractive lift-off of polymerized hydrogel film. Projection lithography and oxygen plasma etch was used to pattern parylene C polymer film. Molecular selfassembly of polymerizable monolayer was obtained in solution-phase and acrylamide based hydrogel was polymerized using free-radical polymerization on this substrate. Parylene C film was mechanically lifted-off to remove the blanket hydrogel film and micro hydrogel features (μhf) were obtained attached to the predefined patterns in the range from 1 to 60 μm. The μhf were functionalized with

aldehyde functional groups, and proteins were coupled to them using Schiff base chemistry followed by reductive amination. Interaction of mesenchymal stem cells with transforming growth factor-beta 1 (TGF-β1) functionalized μhf was studied, and TGF-β1 was found to retain its tumor suppression activity.

Electronic supplementary material The online version of this article (doi:10.1007/s10544-008-9167-6) contains supplementary material, which is available to authorized users.

1 Introduction

P. Bhatnagar (*) Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA e-mail: [email protected] A. J. Nixon Clinial Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA I. Kim Department of Polymer Science & Engineering, Pusan National University, Busan 609-735, South Korea J. Kameoka Department of Electrical Engineering, Texas A&M University, College Station, TX 77843, USA e-mail: [email protected] Present address: P. Bhatnagar Intel Corporation, 5200 NE Elam Young Parkway, MS: RA3-355, Hillsboro, OR 97124, USA

Keywords Integrated microfabrication of hydrogels . Surface immobilization of growth factors . Cell–surface interaction . Biologically friendly microfabrication processes

Hydrogel based materials have proved to be an important biomaterial due to their ability to maintain physiological environment and have been recognized as a preferred interface for protein immobilization (Kiyonaka et al. 2004; Langer and Tirrell 2004). Patterning of proteins on micro hydrogel features (μhf) can be used for high-throughput applications in drug screening (Beske and Goldbard 2002; Bhadriraju and Chen 2002; Sidransky 2002; Koch 2004), early detection biosensors (Giaever and Keese 1993; Cornell et al. 1997; Sidransky 2002; Kaul et al. 2004), and fundamental cell biology studies (Chen et al. 1997; Alberts 1998; Takayama et al. 2001; Orth et al. 2003). Recent advances in the use of microfabrication processes for organ printing and complex tissue engineering motivated us to develop micron size growth factor environment (Hollister et al. 2000; Langer and Tirrell 2004; Hollister 2005; Arcaute et al. 2006; Cunningham et al. 2006; Khademhosseini et al. 2006; Mikos et al. 2006). Earlier, we suggested an e-beam directed patterning of oligonucleotides through dendrimeric interface (Bhatnagar et al. 2006). Here we describe patterning of proteins through hydrogel interface for cell–surface interaction studies. We suggest a

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microfabrication method that produces patterns with 1 μm hydrogel feature aligned to the pre-existing surface topography. A photolithographically patterned polymer film was mechanically lifted-off (Ilic and Craighead 2000) to create cross-linked μhf attached to silicon oxide surface through self-assembled monolayer (SAM). The μhf were chemically modified (Arenkov et al. 2000) to covalently immobilize protein molecules using Schiff base chemistry followed by reductive amination. Further, the interaction of growth factor functionalized μhf with equine mesenchymal stem cells (eMSC) has been demonstrated that resulted into anoikis (apoptosis due to loss of anchorage) and immobilized transforming growth factor-beta 1 (TGF-β1) was found to retain its tumor-suppression activity (Lafon et al. 1996; Hartsough and Mulder 1997; Kim et al. 2003; Luginbuehl et al. 2004; Cao et al. 2006; Reynolds and Kyprianou 2006). Current techniques for μhf patterning include photopolymerization (Arenkov et al. 2000; Rubina et al. 2004; Zourob et al. 2006), and soft lithography (Xia et al. 1999; Suh et al. 2004; Suh et al. 2004) and primarily suffer from alignment and resolution issues. Although e-beam based cross-linking (Hong et al. 2004) does not suffer from these issues, it is not a preferred method for high volume manufacturing. The method we report here produces an array of cross-linked μhf in sub-cellular dimensions capable of delivering localized stimulus to the cells (Chen et al. 1997; Takayama et al. 2001; Orth et al. 2003). Alignment to the pre-existing surface topography was achieved using standard microfabrication tool sets. This technique therefore can also be used to create an interface between microelectronic circuitry and proteins immobilized on μhf (Cornell et al. 1997; Kaul et al. 2004).

2 Experimental methods Patterning of parylene C film Parylene (1.5 μm thick) C polymer film deposited (chemical vapor deposition) on 100 mm diameter thermally oxidized silicon wafer was patterned using standard projection photolithography. The patterns were transferred into the parylene C film using oxygen plasma in PlasmaTherm 72 reactive ion etcher (electrode area 585 cm2). Microfabrication equipments at Cornell NanoScale Science and Technology Facility (CNF) were employed. Polymerization of hydrogel thin film Parylene C patterns were treated for 2 min with 3% (v/v) solution of 3methacryloxypropyl(trimethoxysilane) (Sigma-Aldrich Corp., St. Louis, MO, USA) in 95% ethanol, 4.7% water and 0.3% acetic acid. Five percent (w/v) monomer (95% acrylamide and 5% methylenebisacrylamide (w/w)) was dissolved in 0.01 M PBS with 40% glycerol (v/v). Free

Biomed Microdevices (2008) 10:567–571

radicals were generated by addition of 4 μl of 25% (w/v) of ammonium persulfate and 4 μl of N,N,N′,N′-tetramethylethylenediamine (Bio-Rad Laboratories, Hercules, CA, USA) per milliliter of gel precursor solution. Pre-polymerized hydrogel solution was dispensed on the wafer and polymerized overnight. Aldehyde functionalization of hydrogel The μhf substrate was treated with aqueous 0.1 M NaIO4 for 20 min followed by 5% glutaraldehyde (in 0.1 M sodium phosphate buffer) for 48 h. Immobilization of proteins on hydrogel TGF-β1 (R&D Systems, Minneapolis, MN, USA) was reconstituted as per manufacturer’s instructions at 0.8 μg/ml in PBS without and with 0.25% Triton X-100 and dispensed on aldehyde functionalized μhf substrate. The surface was washed with PBS after 3 h and treated with 0.1 M NaBH4 for 20 min. Detection protocol included the use of anti-TGF-β1 biotinylated affinity purified polyclonal antibody and streptavidin-Alexa 488 (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA) as the secondary probe. Plating of eMSC cells on the μhf substrate The chips were incubated in 24-well plate in PBS with 0.1% NaN3 for 30 min and copiously washed with PBS in cell culture hood. Equine mesenchymal stem cells (eMSC) were isolated as described elsewhere (Fortier et al. 1998) and passaged five times, washed with Hank’s balanced salt solution, trypsinized, centrifuged at 300×g (4°C for 10 min) and plated at 50,000 cells/well. The cell culture media included low glucose Dulbecco’s modified Eagle’s medium, 1% ITS+ (Cat. # 354352, BD Bioscience, Bedford, MA, USA), 1% penicillin–streptomycin, 1% L-glutamine, and 0.2% amphotericin B (all constituents from Gibco, Invitrogen Corp., Carlsbad, CA, USA). The cells were fixed after 4 days with 3.7% paraformaldehyde solution and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Fluorescence imaging Olympus BX51 upright epifluorescence microscope (Olympus America, Inc., Center Valley, PA, USA) with CoolSnapHQ monochrome CCD camera (Photometrics, Tuscon, AZ, USA) was used with Metamorph V6.1 image analysis software (Universal Imaging Corp., Molecular Devices Corp., Downington, PA, USA).

3 Results and discussion Figure 1(a) illustrates the method for patterning μhf. A photolithographically patterned parylene C (sacrificial)


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Fig. 1 (a) Fabrication scheme for μhf. (b) Relationship between the lateral dimension of μhf and their heights with error bars. Average standard deviation is ±0.002. (c) μhf of different sizes. Larger features are more prominent and higher. (d) SEM image of μhf of 1 μm diameter

polymer film on the silicon oxide surface was etched in oxygen plasma (Ilic, Craighead 2000; Orth et al. 2003). The surface was functionalized with polymerizable SAM and acrylamide based hydrogel was polymerized on the surface using free-radical polymerization. The parylene C film was then mechanically peeled (lift-off) from the surface (Ilic and Craighead 2000; Orth et al. 2003) releasing the μhf covalently attached to SAM on the silicon oxide. The μhf exhibited pyramidal shape with AFM and their heights varied with the planar dimensions (Fig. 1(b)). The observance of area effect is typical of microfabrication processes (Hedlund et al. 1994; Chang 2005). Different sizes of patterned hydrogel features were obtained based on the photolithographically defined patterns in parylene C (Fig. 1(c)). Figure 1(d) shows the SEM image of the μhf with 1 μm feature and space. Through sequential surface treatment, (Arenkov et al. 2000), the μhf on silicon oxide surface was functionalized with aldehyde groups. TGF-β1 was immobilized through primary amines to form Schiff bases with the surface aldehyde groups followed by reductive amination resulting in secondary amide linkage that does not hydrolyze in aqueous environments (Hermanson 1996). Figure 2(a, b) shows the fluorescence detection of TGF-β1 on μhf and effect of non-ionic surfactant (Triton X-100), which was helpful in reducing the non-specific adhesion of the protein on silicon oxide surface (Marsh et al. 2002). The μhf

without immobilized proteins did not exhibit any fluorescence when probed with labeled antibodies (negative control, Fig. S1). No non-specific adhesion of probe antibodies was observed on the μhf, but a blocking step with StartingBlock T20 (in phosphate buffered saline (PBS)) Blocking Buffer (Pierce Biotechnology, Inc., Rockland, IL, USA) for 1 h was introduced to prevent the non-specific adhesion of primary antibodies on the silicon oxide surface (Fig. S2). Similar results were obtained with interleukin-4 (IL-4) and interferon-gamma (IFN-γ; Fig. S3), biotin, and protein A. Interaction of cells on functionalized surfaces and celladhesion studies can provide insight into the molecular basis of many diseases and is an area of active research (Lafon et al. 1996; Kim et al. 2003; Cao et al. 2006; Reynolds and Kyprianou 2006). Several applications for interaction of cells in lab-on-a-chip (LOC) systems have been reported (El-Ali et al. 2006), and the above protein functionalized μhf can further be added to the set of available LOC tool-set and utilized for studying cell-signal transduction events. The μhf with immobilized TGF-β1, IL-4 or IFN-γ were seeded with eMSCs and incubated for 4 days. It was observed that cells adhered to the substrate when μhf were functionalized with IL-4 (Fig. 2(c); or IFNγ, not shown). However, when plated on the μhf with TGFβ1, the cells adhered to each other forming a sheet that was observed to barely attach on the corners of the chip but not

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Fig. 2 (a, b) Fluorescent detection of TGF-β1 immobilized on the μhf. Protein was reconstituted in (a) PBS (b) PBS with 0.25% Triton X-100. (c, d) Plated eMSCs (c) cell-sheet adhered to the substrate when μhf were functionalized with IL-4 (d) cells adhered to each other to form a cell-sheet but did not adhere to the substrate when μhf were functionalized with TGFβ1

on its surface. Figure 2(d) is the image of the cell-sheet that consisted of cells adhered to each other but not to the TGF-β1 functionalized μhf substrate. We infer this nonadherent behavior of the cell-sheet was due to the tumorsuppression activity (Lafon et al. 1996; Hartsough and Mulder 1997; Kim et al. 2003; Luginbuehl et al. 2004; Cao et al. 2006; Reynolds and Kyprianou 2006) of TGFβ1, which at high dose has been found to induce anoikis in various cell types. TGF-β1 concentration in the range of 5 to 10 ng/ml has been accepted as normal dose for differentiating MSCs in chondrocytes (Cassiede et al. 1996; Johnstone et al. 1998; Gao et al. 2001; Worster et al. 2001; Luginbuehl et al. 2004; Li et al. 2005). However, we did not characterize the dose effect in this work since this was not our focus. As a control, the cells (Fig. S4) were plated on non-functionalized μhf, and cells were found to adhere similar to Fig. 2(c).

4 Conclusions The intersection of biology and materials has been identified as one of the growing frontiers in materials

science (Stupp et al. 2005; Whitesides and Wong 2006). Significant advancements have been made in the past decade in the field of proteomics. Interaction of cells with protein functionalized surfaces has been an area of considerable interest (Bhadriraju and Chen 2002; Raghavan and Chen 2004). Here we have demonstrated the use of standard microfabrication equipment for fabrication of μhf and its possible application in molecular and cell biology. Micro manipulating techniques such as microcontact printing (Xia and Whitesides 1998; Whitesides et al. 2001), inkjet printing (Lemmo et al. 1998; Heller 2002), dip-pen nanolithography (Piner et al. 1999; Ginger et al. 2004) or photolithography could be employed to immobilize different biomolecules in separate μhf and used for agonist-antagonist type of investigations in a single cell or to emulate extra-cellular matrix for tissue-engineering (Doh and Irvine 2006). Surface patterning of bio-functionalized nanoparticles through biomolecular interaction offers the possibility to construct integrated biosensors (Tokareva et al. 2004) and the use of standard microfabrication equipment allows integration of microelectronic or photonic sensors to be aligned (Tien et al. 2002; Verpoorte 2003) in high-resolution to the biomolecular patterns.

Biomed Microdevices (2008) 10:567–571 Acknowledgements We thank Profs. Harold G. Craighead, and Barbara Baird for their guidance and support; Michael Scimeca, M. Lisa Strassheim, Ryan Young, Sueng-Min Park, Aaron Smith, and Mathieu Foquet for technical help. This material is based upon work supported in part by the Nanobiotechnology Center, an STC Program of the National Science Foundation under Agreement no. ECS-9876771. IK thanks NCRC Program from MOST/KOSEF (R15-2006-022-01001-0).

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