Molding of Hydrogel Microstructures to Create Multiphenotype Cell

This review with 88 refs. describes the patterning of proteins and cells using a non-photolithog. microfabrication technol., which we call "soft litho...
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Anal. Chem. 2003, 75, 5783-5789

Molding of Hydrogel Microstructures to Create Multiphenotype Cell Microarrays Won-Gun Koh, Laura J. Itle, and Michael V. Pishko*

Department of Chemical Engineering and the Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802-4420

The fabrication of mammalian cell-containing poly(ethylene glycol) (PEG) hydrogel microstructures on glass and silicon substrates is described. Using photoreaction injection molding in poly(dimethylsiloxane) microfluidic channels, three-dimensional hydrogel microstructures encapsulating cells (fibroblasts, hepatocytes, macrophage) were fabricated with cells uniformly distributed to each hydrogel microstructure, and the number of cells in each hydrogel microstructure was controlled by changing the cell density of the precursor solution. PEG hydrogels were modified using an Arg-Gly-Asp (RGD) peptide sequence, with the incorporation of RGD into the hydrogel matrix promoting the spreading of encapsulated fibroblasts over a 24-h period in culture. Cells remained viable encapsulated in these hydrogel microstructures for a period in excess of 1 week in culture. Arrays of hydrogel microstructures encapsulating two or more phenotypes on a single substrate were successfully fabricated using multimicrofluidic channels, creating the potential for multiphenotype cell-based biosensors. Patterned deposition of cells onto surfaces is a prerequisite for the development of cell-based biosensing devices for applications such as high-throughput or high-content drug screening.1 Previously, lithographic techniques and microcontact printing (µCP) have been most widely used to generate patterns of cells on surfaces.2-8 However, these methods are limited to the patterning one phenotype at a time; therefore, it is difficult to deposit different phenotypes of cells or perform simultaneous and different assays on the same substrate. The use of microfluidic networks to pattern cells is one potential method of overcoming * To whom correspondence should be addressed. Department of Chemical Engineering, The Pennsylvania State University, 104 Fenske Laboratory, University Park, PA 16802-4420. Phone: (814) 863-4810. Fax: (814) 865-7846. E-mail: [email protected]. (1) Sundberg, C. J. Curr. Opin. Biotechnol. 2000, 11, 47-53. (2) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (3) Singhvi, R.; Kumer, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698. (4) Amirpour, M. L.; Ghosh, P.; Lackowski, W. M.; Crooks, R. M.; Pishko, M. V. Anal. Chem. 2001, 73, 1560-1566. (5) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Progress 1998, 14, 356-363. (6) Ito, Y. Biomaterials 1999, 20, 2333-2342. (7) Koh, W.; Revzin, A.; Simonian, A.; Reeves, T.; Pishko, M. Biomed. Microdevices 2003, In press. (8) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1995, 29, 749-756. 10.1021/ac034773s CCC: $25.00 Published on Web 09/23/2003

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this limitation. Networks of microfluidic channels have been used to simultaneously pattern different biomolecules onto a surface because microchannels can be effectively isolated from each other. This permits independent solutions of biomolecules to be introduced into each channel. Therefore, many researchers have exploited microfluidic networks for the patterning of proteins, cells, and planar lipid bilayers on substrates at micrometer-scaled resolution.9-13 In addition, patterned polymer microstructures were also fabricated with microfluidic networks via an injection molding technique.14-18 Furthermore, since Beebe et al. fabricated hydrogel structures inside microchannels for use as a microactuator,19 several studies have been performed to incorporate hydrogels into microfluidic networks using in situ photopolymerization for such applications as injection molding, DNA hybridization, or potential drug screening systems.20-23 Photopolymerization has been widely used to encapsulate biomolecules such as proteins and cells inside hydrogels, avoiding denaturation of biomolecules because fast polymerization occurs under ambient condition without toxic solvents.23-31 In our (9) Papra, A.; Bernard, A. J.; Uncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Langmuir 2001, 17, 4090-4095. (10) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (11) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120, 500-508. (12) Folch, A.; Ayon, A.; Hurtado, O.; Schmidt, M. A.; Toner, M. J. Biomech. Eng. Trans. ASME 1999, 121, 28-34. (13) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8-12. (14) Kim, E.; Xia, Y.; Whitesides, G. M. Nature 1995, 376, 581-584. (15) Kim, Y. D.; Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2001, 73, 331337. (16) Hanemann, T.; Pfleging, W.; Hausselt, J.; Zum Gahr, K. H. Microsyst. Technol. 2002, 7, 209-214. (17) Hanemann, T.; Ruprecht, R.; Hausselt, J. H. Adv. Mater. 1997, 9, 927929. (18) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (19) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B. Nature 2000, 404, 588-590. (20) Koh, W.; Pishko, M. Langmuir, submitted. (21) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (22) Seong, G. H.; Zhan, W.; Crooks, R. M. Anal. Chem. 2002, 74, 3372-3377. (23) Koh, W. G.; Revzin, A.; Pishko, M. V. Langmuir 2002, 18, 2459-2462. (24) Bryant, S. J.; Nuttelman, C. R.; Anseth, K. J. Biomater. Sci., Polym. Ed. 2000, 11, 439-457. (25) Liu, V. A.; Bhatia, S. N. Biomed. Microdevices 2002, 4, 257-266. (26) Revzin, A.; Russell, R. J.; Yadavalli, V. K.; Koh, W.; Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Langmuir 2001, 17, 5440-5447. (27) Cruise, G. M.; Hegre, O. D.; Lamberti, F. V.; Hager, S. R.; Hill, R.; Sharp, D. S.; Hubbell, J. A. Cell Transplant. 1999, 8, 293-306. (28) Elisseeff, J.; McIntosh, W.; Anseth, K.; Riley, S.; Ragan, P.; Langer, R. J. Biomed. Mater. Res. 2000, 51, 164-171.

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previous work, we fabricated poly(ethylene glycol) (PEG) hydrogel microstructures and encapsulated viable mammalian cells inside three-dimensional PEG hydrogel microstructures using photopolymerization for biosensor applications.23 Since most mammalian cells exist in a three-dimensional tissue matrix, patterning mammalian cells inside three-dimensional hydrogels provides a more in vivo-like surrounding for cells than immobilizing them onto two-dimensional substrates.32 In this study, we fabricated cell-containing PEG hydrogel microstructures using photoreaction injection molding. The rationale behind using a hydrogel environment in patterning is to mimic the environment where anchorage-dependent cells exist in tissue, that is, a three-dimensional hydrogel composed of proteins and polysaccharides. Peptides promoting cell adhesion were incorporated into PEG hydrogels to optimize the extracellular environment and to encourage cell spreading. Arrays of hydrogel microstructures encapsulating one or more different cell phenotypes were fabricated using microfluidic networks made from poly(dimethylsiloxane) (PDMS). EXPERIMENTAL SECTION Chemicals and Materials. Poly(ethylene glycol) diacrylate (PEG-DA, MW 575), anhydrous carbon tetrachloride, and nheptane were obtained from Aldrich Chemical Co. (Milwaukee, WI), and 2-hydroxy-1[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Ciba, Tarrytown, NY) was used as a photoinitiator. PEGDA (MW 4000) was obtained from Polysciences (Warrington, PA). Acryloyl-PEG-n-hydroxysuccinimide ester (acryloyl-PEG-NHS, MW 3400) was obtained from Shearwater Polymer (Huntsville, AL). 3-(Trichlorosilyl)propyl methacrylate (TPM) was purchased from Fluka Chemicals (Milwaukee, WI). Hydrogen peroxide was purchased from EM Science (Gibbstown, NJ). Sulfuric acid was purchased from Fisher Scientific (Fair Lawn, NJ). Poly(dimethylsiloxane) (PDMS) elastomer was purchased as Dow Corning Sylgard 184 (Midland, MI), which is composed of prepolymer and curing agent. The chrome sodalime photomask for making hydrogel patterns was purchased from Advanced Reproductions (Andover, MA). Murine 3T3 fibroblast, SV-40 transformed murine hepatocyte, and SV-40 transformed murine peritoneal macrophage cell lines were obtained from American Type Culture Collection (Manassas, VA). For cell culture, Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), antibiotic/antimycotic solution, trypsin, ethylenediaminetetraacetate (EDTA), sodium chloride, sodium phosphate, and potassium phosphate monobasic were purchased from Sigma Chemical Co. (St. Louis, MO). The peptide used was Gly- Arg-Gly-Asp-Ser (GRGDS) (Calbiochem., San Diego, CA). Calcein AM; Cell tracker Blue CMAC (7-amino-4-chloromethylcoumarin), Green CMFDA (5-chloromethylfluorescein diacetate); and Orange CMTMR (5(and -6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) were purchased from Molecular Probes (Eugene, OR). (29) Pathak, C. P.; Sawhney, A. S.; Hubbell, J. A. J. Am. Chem. Soc. 1992, 114, 8311-8312. (30) Russell, R. J.; Simonian, A.; Wild, J.; Pishko, M. V. Anal. Chem. 1999, 71, 4909-4912. (31) Mellott, M. B.; Searcy, K.; Pishko, M. V. Biomaterials 2001, 22, 929-941. (32) O’Connor, S.; Andreadis, J.; Shaffer, K.; Ma, W.; Pancrazio, J.; Stenger, D. Biosens. Bioelectron. 2000, 14, 871-881.

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Preparation of Peptide-Modified PEG Hydrogel. To incorporate the GRGDS peptide into PEG hydrogels, the peptide was conjugated to PEG by reacting the peptide with acryloyl-PEGNHS, as previously described.33 Briefly, the peptide was dissolved to a final concentration of 1 mg/mL in culture media and then 10% w/v of acryloyl-PEG-NHS was dissolved in peptide solution and reacted at room temperature for at least 2 h. Final precursor solution was prepared by adding 10% w/v of PEG-DA (MW 575 or 4000) and 0.1% v/v 2-hydroxy-1[4-(hydroxyethoxy)phenyl]-2methyl-1-propanone in ethanol as photoinitiator to solution containing acrylated peptide. The solution was sterilized by filtration and added to a suspension of fibroblasts. As described later, this cell-containing precursor solution was exposed to UV light for ∼30 s to convert the precursor solution into a PEG hydrogel. Preparation of Microchannels for Injection Molding. Microfluidic networks were formed from a 10:1 mixture of the PDMS prepolymer and the curing agent. The resulting mixture was poured onto a silicon master and cured at 60 °C for at least 2 h. All masters had a negative pattern of the desired micropattern defined with SU-8 50 negative photoresist (Microlithography Chemical Corp., Newton, MA). After curing, the PDMS replica was removed from the master and pierced from the backside of the network with syringe needles to open paths for incoming liquids. These PDMS microchannels were used as molding insert for reaction injection molding. PDMS replicas were treated with oxygen plasma (Harrick Scientific Co., Ossining, NY) for 1 min to change its hydrophobic surface to hydrophilic. Oxidized microfluidic networks were placed by hand on the glass to form an enclosed channel. Before conformal contacting with PDMS replicas, glass was modified with a 3-(trichlorosilyl)propyl methacrylate (TPM) monolayer as previously described to enhance the adhesion of hydrogel to glass or silicon surfaces.26 Fabrication of Hydrogel Microarray Encapsulating Mammalian Cells. Microarrays of hydrogel microstructures were fabricated with PEG-DA (MW 575 or 4000). Gel precursor solution was composed of 20% w/v of PEG-DA and 0.1% w/v of photoinitiator in cell culture medium or PBS solution. To encapsulate the mammalian cells, a cell suspension was added to the precursor solution. In this study, three different phenotypes of cells, fibroblasts, hepatocytes and macrophages, were used for the fabrication of multiphenotype cell microarrays. Prior to the formulation of the cell-containing precursor solution, each phenotype was incubated with Cell Tracker Green CMFDA (fibroblasts) or Cell Tracker Blue CMAC (hepatocytes) or Cell Tracker Orange CMTMR (macrophage) for later fluorescence imaging. To complete the molding of PEG hydrogels encapsulating mammalian cells, each independent microchannel was filled with cell-containing precursor solution and exposed to 365 nm, 300 mW/cm2 UV light (EFOS Ultracure 100ss Plus, UV spot lamp, Mississauga, Ontario). To make various patterns of hydrogel microstructures, a photomask was aligned over the microchannels before exposure to UV light. After UV photopolymerization, any precursor solution exposed to UV light underwent free-radical cross-linking inside the microchannels and became insoluble in common PEG solvents, such as water. During the UV lightinduced gelation process, cells present in the polymer precursor solution were encapsulated in the resultant hydrogel microstruc(33) Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 39, 266-276.

Figure 1. Optical and fluorescence images of fibroblasts encapsulated in (a) PEG hydrogel modified with RGD and (b) unmodified PEG hydrogel.

tures. Finally, PDMS microfluidic networks were quickly removed from the glass substrate to obtain hydrogel microstructures. Cell Culture. All cell lines were incubated at 37 °C in 5% CO2 and 95% air. Murine fibroblasts were cultured in DMEM with 4.5 g/L glucose and 10% FBS. SV-40 transformed murine hepatocytes were cultured in DMEM containing 1.0 g/L glucose, 200 nM dexamethasone, and 4% FBS. Both phenotypes were grown to confluence in 75 cm2 polystyrene tissue culture flasks, and confluent cells were subcultured every 2 to 3 days by trypsinization with 0.25% (w/v) trypsin and 0.13% (w/v) EDTA. SV-40-transformed murine macrophages were cultured in RPMI 1640 medium with 2 mM L-glutamine containing 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% FBS. Confluent cells were subcultured every 2 to 3 days by cell scraping. RESULTS AND DISCUSSION Encapsulation of Fibroblasts inside RGD Peptide-Modified PEG Hydrogels. Previously, we encapsulated mammalian

cells such as fibroblasts and hepatocytes inside PEG hydrogel microstructures.23 Even though encapsulated cells remained viable for over a week, they appeared rounded after 24 h and slowly spread over the course of several days. The slow spreading rate of encapsulated cells was likely caused by the nonadhesive nature of PEG hydrogels toward proteins and, hence, toward cells. With time, encapsulated cells produced their own extracellular matrix on which to adhere. To improve cell adhesion and spreading inside the PEG hydrogel, we incorporated RGD, a cell adhesion peptide, into the hydrogel matrix. RGD, which consists of the short peptide sequence Arg-Gly-Asp, is an integrin binding domain found within the extracellular matrixes (ECM) of many tissues and has been extensively studied in cell adhesion research.33-35 Incorporation of RGD into hydrogels was achieved by functionalizing the amide terminus of the peptide with an acrylate moiety, enabling the adhesion peptide to copolymerize rapidly with (34) Houseman, B. T.; Mrksich, M. Biomaterials 2001, 22, 943-955. (35) Mann, B. K. J. Biomed. Mater. Res. 2000, 60, 86-93.

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Figure 2. Fibroblasts encapsulated in cylindrical hydrogel microstructure modified with RGD.

Figure 3. Hydrogel microstructures containing 3T3 fibroblast fabricated with 300-µm-wide microchannels: (a) microchannels filled with cellcontaining precursor solutions, (b) gelation of the hydrogel inside the microchannel after exposure to UV light, and (c) an array of hydrogel microstructures encapsulating fibroblasts after removing PDMS microchannels.

PEG-DA during photopolymerization. Figure 1a is the optical and fluorescent micrographs of cells encapsulated inside peptidemodified hydrogel after 24 h incubation. To make these hydrogels, 10 µL of cell-containing precursor solution was added to 96-well plates and exposed to UV light. For fluorescent imaging, encapsulated cells were stained with calcein AM, which diffuses through the membrane of living cells and reacts with intracellular esterase to produce a green fluorescence.36 Therefore, these fluorescence images not only showed the morphology of cells but also indicated that cells remained viable after photoencapsulation. Under culture conditions, fibroblasts and macrophages remained viable in these microstructures for over 1 week. Cells were judged to be spread if they were elongated or possessed a polygonal morphology. As shown in Figure 1a, more cells spread inside RGD-modified hydrogel when compared to the mostly rounded cells in the unmodified hydrogel (Figure 1b). Because of the highly (cell) nonadhesive character of PEG (36) Bryant, S. J.; Anseth, K. S. Biomaterials 2001, 22, 619-626.

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Figure 4. Average number of cells in one hydrogel microstructure (300 µm × 300 µm × 50 µm high) as a function of cell density in the precursor solution. Error bars reflect the standard deviation in the number of cells within the microstructure.

Figure 5. Array of hydrogel microstructures encapsulating both fibroblasts (green) and hepatocytes (blue): (a) microchannels filled with fibroblasts and hepatocytes; (b) hydrogel microstructures containing fibroblasts and hepatocytes.

hydrogels, any change in cell morphology inside the hydrogel must be attributed to the incorporation of peptide in the gel backbone. These results demonstrate that RGD peptide sequences can promote cell adhesion and spreading inside PEG hydrogel matrix when chemically incorporated into the hydrogel. We also observed cell spreading inside the hydrogel microstructure modified with peptide after 24 h, as shown in Figure 2. Here, hydrogel microstructures were fabricated using a photomask with an array of 600-µm-diameter circles. Fabrication of Hydrogel Microarray Encapsulating Mammalian Cells. Using photoreaction injection molding, arrays of hydrogel microstructures encapsulating mammalian cells were fabricated. For molding hydrogels, we used a network of microchannels formed from PDMS and placed on TPM-treated glass

slides. The depth of the microchannels was fixed at ∼50 µm, and the width was either 200 or 300 µm. Prior to sealing the microchannels to the glass slide, the PDMS microchannels were treated in an oxygen plasma to render their surfaces hydrophilic. Microchannels were filled with polymer precursor solution by capillary action. Figure 3 shows a microfluidic channel containing fibroblasts at various stages in the photoreaction injection modeling process. Microchannels were filled with a gel precursor solution containing fibroblasts in culture media (Figure 3a) and then exposed to UV light through a photomask. Only the exposed regions underwent photopolymerization and gelled inside the microchannel, encapsulating cells present in the precursor solution, as shown in Figure 3b. After removing the PDMS template and rinsing with culture Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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Figure 6. Fabrication of 6 × 6 array of hydrogel microstructure containing three phenotypes of cells: fibroblasts (green), macrophage (red), hepatocytes (blue).

media, the desired array of cell-containing hydrogel microstructures was obtained (Figure 3c). Since reversible, conformal sealing with glass surfaces was used here, PDMS microchannels were easily peeled from glass substrate with only moderate force and without leaving significant residue. Therefore, resealing can be done numerous times with the same PDMS microchannel template. Resultant cell-containing hydrogel microstructures did not detach from TPM-modified glass substrates in an aqueous environment for a week because of covalent bonding between the hydrogel microstructures and surface-tethered methacrylate groups. The number of cells in one hydrogel microstructure depends on the cell density of the precursor solution. To investigate the relationship between cell density and the number of cells in a hydrogel microstructure, precursor solutions with different cell densities were injected into each microchannel. After hydrogel microstructures (300 × 300 × 50 µm) were fabricated, the number of cells in each microstructure was counted to obtain the average cell density. As shown in Figure 4, the average number of cells in one hydrogel microstructure increased almost linearly with cell density, whereas the standard deviation was low and relatively unchanged as cell density changed. In our previous work, we fabricated hydrogel microstructures encapsulating mammalian cells using spin-coating and photolithography.23 Because of the nature of the spin-coating process, it was difficult to obtain a uniform distribution of cells in each hydrogel microstructure, especially when low cell density was used. However, when using photoreaction injection molding in combination with photolithography, a relatively constant number of cells per microstructure could be obtained. 5788

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Fabrication of Multiphenotype Cell Microarrays. Photoreaction injection molding offers several advantages over previously described methods of encapsulating mammalian cells in hydrogel microstructures. For example, a small volume of cellcontaining precursor solution was sufficient to fill and be photopolymerized inside the microchannel, whereas cell patterning techniques based on spin-coating required a much larger volume of precursor solution because of solution loss during the spincoating procedure. Another important advantage of photoreaction injection molding is the possibility to encapsulate different phenotypes on the same array. For future applications to multianalyte cell-based biosensors, we fabricated an array of hydrogel microstructures containing different phenotypes. Figure 5 shows the array of hydrogel microstructures containing murine fibroblasts and hepatocytes. Before cells were introduced to the microchannel, fibroblasts were stained with Cell Tracker Green and hepatocytes with Cell Tracker Blue for fluorescence imaging. Two microchannels were fabricated with PDMS, and precursor solutions containing each cell line were introduced to each channel (Figure 5a). These precursor solution-containing microchannels were exposed to UV light through the photomask. After photopolymerization, the PDMS channels were removed, and any unreacted precursor solution was washed away with culture media or PBS solution. The resulting array of hydrogel microstructures containing both fibroblasts and hepatocytes is shown in Figure 5b. We also fabricated an array of hydrogel microstructures containing three different cell phenotypes (fibroblasts, hepatocytes, and macrophages) in spatially defined regions using procedures described earlier (Figure 6).

CONCLUSION We fabricated PEG hydrogel microstructures encapsulating mammalian cells using photoreaction injection molding. Molding was performed using a network of microchannels formed with PDMS, with photolithography performed through the mold to define hydrogel microstructures. Cells were uniformly distributed to each hydrogel microstructure, and the number of cells in one hydrogel microstructure was easily controlled by changing cell density in the precursor solution. Incorporation of the RGD peptide into the hydrogel matrix enhanced cell adhesion and spreading inside hydrogel. Furthermore, different cell phenotypes were immobilized in one array of hydrogel microstructures, demonstrating the potential to create multiphenotype cell-based biosen-

sors for applications in high-throughput drug screening or pathogen detection. ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Aeronautics and Space Administration (NAG 9 1277), the Pennsylvania State University Materials Research Institute, the Center for Optical Technologies, and the Pennsylvania Department of Community and Economic Development. Received for review July 9, 2003. Accepted August 20, 2003. AC034773S

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