Photoreaction Injection Molding of Biomaterial Microstructures

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Photoreaction Injection Molding of Biomaterial Microstructures Won-Gun Koh and Michael Pishko* Department of Chemical Engineering and the Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802-4420 Received February 14, 2003. In Final Form: May 5, 2003 Reaction injection molding using in situ photoinduced polymer macromer gelation in microfluidic channels was applied to the fabrication of poly(ethylene glycol) (PEG) hydrogel microstructures. These hydrogel microstructures were fabricated using poly(dimethylsiloxane) (PDMS) microchannels as mold inserts alone or in combination with photolithography. These microstructures were formed by flowing a gel precursor solution through the microfluidic network, exposing to light, and finally removing the PDMS mold. Microchannels as narrow as 10 µm wide could be used for molding PEG hydrogels, and the resulting three-dimensional hydrogel microstructures did not delaminate from substrates treated with 3-(trichlorosilyl)propyl methacrylate, a gel adhesion promoter. By exploitation of the laminar flow and poor mixing conditions in a microfluidic channel, single microstructures with heterogeneous chemistries were also created, using peptide-modified structures to promote cell adhesion as a model.

1. Introduction Microfluidic devices have gained much attention over the past several years and have significantly influenced the design and the implementation of modern bioanalytical systems. These devices can handle and manipulate small fluid samples in a much more efficient way with the potential of faster assay response times, the simplification of analysis procedures, and smaller samples required for analysis.1 Microfluidic devices are finding wide applications ranging from synthesis to separations to analysis in applications such as immunoassays, lab-on-a-chip, rapid nucleotide sequencing, and high-throughput screening.2-6 Furthermore, microfluidics may be used to pattern biological materials such as proteins, cells, and planar lipid bilayers on substrates with micrometer-scale resolution.7-10 Patterned polymer microstructures were * To whom correspondence should be addressed. Department of Chemical Engineering, The Pennsylvania State University, 104 Fenske Laboratory, University Park, PA 16802-4420. E-mail: [email protected]. Phone: (814) 863-4810. Fax: (814) 8657846. (1) McCreedy, T. Fabrication techniques and materials commonly used for the production of microreactors and micro total analytical systems. Trends Anal. Chem. 2000, 19, 396-401. (2) Khandurina, J.; Guttman, A. Microchip-based high-throughput screening analysis of combinatorial libraries. Curr. Opin. Chem. Biol. 2002, 6, 359-366. (3) Sanders, G. H. W.; Manz, A. Chip-based microsystems for genomic and proteomic analysis. Trends Anal. Chem. 2000, 19, 364-378. (4) Ismagilov, R. F.; Ng, J. M. K.; Kenis, P. J. A.; Whitesides, G. M. Microfluidic arrays of fluid-fluid diffusional contacts as detection elements and combinatorial tools. Anal. Chem. 2001, 73, 5207-5213. (5) Mao, H.; Yang, T.; Cremer, P. S. Design and characterization of immobilized enzymes in microfluidic systems. Anal. Chem. 2002, 74, 379-385. (6) Krishnan, M.; et al. Microfabricated reaction and separation systems. Curr. Opin. Biotechnol. 2001, 12, 92-98. (7) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Microfluidic networks made of poly(dimethylsiloxane), Si, and Au coated with poly(ethylene glycol) for patterning proteins onto surfaces. Langmuir 2001, 17, 4090-4095. (8) Kane, R. S.; et al. Patterning proteins and cells using soft lithography. Biomaterials 1999, 20, 2363-2376. (9) Folch, A.; et al. Molding of deep poly(dimethylsiloxane) microstructures for microfluidics and biological applications. J. Biomech. Eng. 1999, 121, 28-34.

Figure 1. Schematic diagram of the photoreaction injection molding process for creating hydrogel microstructures. Photoreaction injection molding can be used alone to form structures that conform to the design of the microfluidic network or combined with photolithography to create arrays of structures.

also fabricated using microfluidic systems in combination with injection molding.11-15 For example, Kim and colleagues fabricated polymer microstructures by molding in capillaries for potential applications in electronic, optical, and mechanical devices.12 The formation of micropatterned sol-gel structures containing active proteins with microchannels was also demonstrated, for applications in microfluidic biocatalysis.11 Injection molding relies on the use of mold inserts which contain microstructures produced by micromachining or replica molding. To fabricate polymeric microstructures via reaction injection molding, the mold inserts are filled with monomers which are subsequently polymerized (10) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. Microfluidic networks for chemical patterning of substrates: Design and application to bioassays. J. Am. Chem. Soc. 1998, 120, 500-508.

10.1021/la034257x CCC: $25.00 © 2003 American Chemical Society Published on Web 10/18/2003

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Figure 2. Scanning electron micrograph of hydrogel microstructures replicated from PDMS microchannels.

thermally or photochemically. In thermally initiated reaction injection molding, the relatively slow polymerization process occurs at elevated temperatures (up to 150 °C), whereas light-induced reaction injection molding can achieve very fast polymerization under ambient conditions.13 Since most biomolecules are prone to denaturation by heat or exposure to organic solvents, photopolymerization is a more desirable route for the encapsulation of biological species such as proteins and cells in molded microstructures. In our previous work, we fabricated poly(ethylene glycol) (PEG) hydrogel microstructures by the photoinduced gelation of (meth)acrylateterminated PEG macromers and immobilized active

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enzymes or viable mammalian cells inside these hydrogel microstructures.16-18 In other research, hydrogel microstructures were also photochemically fabricated inside microchannels for use as microactuators, for DNA hybridization systems, and for potential drug screening systems.17,19-21 Here we describe the fabrication of PEG hydrogel microstructures via photoreaction injection molding using microfluidic networks of poly(dimethylsiloxane) (PDMS) as micromolds. Acrylate- or methacrylate-modified macromers were injected into the fluidic system, and light was used to gel the macromers to form a hydrogel. The PDMS replica was then removed, leaving the gelled microstructures on the substrate surface. By the combination of photoreaction injection molding with photolithography, arrays of hydrogel microstructures possessing different chemistries were created. Finally, by exploitation of the slow diffusion-driven mixing that occurs in microfluidic channels, microstructures with heterogeneous chemical structures were created. Microstructures that possessed cell adhesion molecules on one portion of the microstructure and lacked them on another were fabricated as a model system. 2. Materials and Methods Chemicals and Materials. Poly(ethylene glycol) diacrylate (PEG-DA, MW ) 575), anhydrous carbon tetrachloride, and n-heptane were obtained from Aldrich Chemical Co. (Milwaukee, WI). The photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was purchased from Ciba, Tarrytown, NY. PEG-DA with a molecular weight of 4000 was obtained from Polysciences (Warrington, PA). Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic/

Figure 3. FITC-containing cylindrical hydrogel microstructures fabricated from a fluorophore-containing precursor solution: (a) before PDMS microchannels were removed; (b) after PDMS microchannels were removed.

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Figure 4. An array of hydrogel microstructures containing different fluorophores: (a) six microchannels filled with precursor solutions containing FITC and TRITC alternatively; (b) hydrogel microstructures inside microchannels after photopolymerization; (c) the array of hydrogel microstructures obtained after removal of PDMS microchannels; (d) scanning electron microscopy image of the hydrogel array. antimycotic solution, trypsin, ethylenediaminetetraacetate (EDTA), sodium chloride, sodium phosphate, and potassium phosphate monobasic were purchased from Sigma Chemical Co. (St. Louis, MO). 3-(Trichlorosilyl)propyl methacrylate (TPM) was purchased from Fluka Chemicals (Milwaukee, WI). Murine 3T3 fibroblasts were obtained from American Type Culture Collection (Manassas, VA). RGD peptides were purchased from Calbiochem (San Diego, CA). Hydrogen peroxide was purchased from EM Science (Gibbstown, NJ). Sulfuric acid was purchased from Fisher (11) Kim, Y. D.; Park, C. B.; Clark, D. S. Stable sol-gel microstructured and microfluidic networks for protein patterning. Biotechnol. Bioeng. 2001, 73, 331-337. (12) Kim, E.; Xia, Y.; Whitesides, G. M. Polymer microstructures formed by moulding in capillaries. Nature 1995, 376, 581-584. (13) Hanemann, T.; Ruprecht, R.; Hausselt, J. H. Micromolding and photopolymerization. Adv. Mater. 1997, 9, 927-929. (14) Hanemann, T.; et al. Laser micromachining and light induced reaction injection molding as suitable process sequence for the rapid fabrication of microcomponents. Microsyst. Technol. 2002, 7, 209-214. (15) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Microchannel electrophoretic separations of DNA in injection-molded plastic substrates. Anal. Chem. 1997, 69, 26262630.

Scientific (Fair Lawn, NJ). PDMS elastomer was purchased as Dow Corning Sylgard 184 (Midland, MI), which is composed of a prepolymer and a curing agent. The chrome sodalime photomask for photolithographically patterning hydrogels was purchased from Advanced Reproductions (Andover, MA). (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) Koh, W. G.; Revzin, A.; Pishko, M. V. Poly(ethylene glycol) hydrogel microstructures encapsulating living cells. Langmuir 2002, 18, 2459-2462. (18) Sirkar, K.; Pishko, M. V. Amperometric biosensors based on oxidoreductases immobilized in photopolymerized poly(ethylene glycol) redox hydrogels. Anal. Chem. 1998, 70, 2888-2894. (19) Beebe, D. J.; et al. Functional hydrogel structures for autonomous flow control inside microfluidic channels. Nature 2000, 404, 588-590. (20) Olsen, K. G.; Ross, D. J.; Tarlov, M. T. Immobilization of DNA hydrogel plugs in microfluidic channels. Anal. Chem. 2002, 74, 14361441. (21) Seong, G. H.; Zhan, W.; Crooks, R. M. Fabrication of microchambers defined by photopolymerized hydrogels and weirs within microfluidic systems: Application to DNA hybridization. Anal. Chem. 2002, 74, 3372-3377.

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Figure 5. FTIR spectra of PEG hydrogels (the blue line is for a hydrogel with RGD peptide, and the red line is for a hydrogel without RGD peptide). The spectra of the RGD-containing gel clearly shows peaks indicative of the amide bond of the peptide. Preparation of Microfluidic Networks. Microfluidic networks were formed from a 10:1 mixture of the PDMS prepolymer and the curing agent. The resulting mixture was poured on the silicon masters and cured at 60 °C for at least 2 h. These 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 treated in an oxygen plasma (Harrick Scientific Co., Ossining, NY) for 1 min to change its hydrophobic surface to hydrophilic. Glass substrates were modified with a TPM monolayer to enhance the adhesion of hydrogel microstructures to glass surfaces. This procedure is described in detail in a previous publication.16 The oxidized microfluidic networks were placed by hand on the TPM-modified glass to form an enclosed channel and pierced from the backside of the network with syringe needles to open paths for incoming fluids. These PDMS microchannel systems were used as mold inserts for photoreaction injection molding. Reaction Injection Molding Using Photopolymerization. Hydrogel microstructures were fabricated using PEG-DA (MW ) 575 or 4000) macromers. The gel precursor solution was composed of 20% w/v of PEG-DA and 0.1% w/v of photoinitiator in cell culture medium or phosphate-buffered saline (PBS). To create these hydrogel microstructures, each independent microchannel was filled with gel precursor solution and then exposed to 365 nm, 300 mW/cm2 UV light (EFOS Ultracure 100ss Plus, UV spot lamp, Mississauga, Ontario, Canada) for 1 s. To make cylindrical hydrogel microstructures within a microfluidic channel, photomasks possessing the desired design were aligned over the microchannels and exposed to light. The precursor solution exposed to UV light underwent free-radical cross-linking and became insoluble in common PEG solvents such as water. Any unreacted PEG-DA prepolymer and photoinitiator inside the gel were assumed to diffuse out of the swollen hydrogel as reported by other investigators using similar hydrogel chemistries.22,23 After the precursor solution gelled, the PDMS microfluidic networks were quickly removed from the glass substrates to (22) Bryant, S. J.; Nuttelman, C. R.; Anseth, K. S. Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro. J. Biomater. Sci., Polym. Ed. 2000, 11, 439-457. (23) Hern, D. L.; Hubbell, J. A. Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing. J. Biomed. Mater. Res. 1998, 39, 266-276.

obtain the molded hydrogel microstructures. Figure 1 shows a schematic diagram of the photoreaction injection molding process, both using and without using a photomask, for the fabrication of hydrogel microstructures. Characterization of hydrogels was performed using attenuated total reflectance/Fourier transform infrared (ATR/FTIR) spectroscopy (Thermo Nicolet Corp., Madison, WI) and X-ray photoelectron spectroscopy (XPS) (Kratos Analyrical Inc., Chestnut Ridge, NY). Cell Culture. Murine fibroblasts were cultured in DMEM with 4.5 g/L glucose and 10% FBS and were incubated at 37 °C in 5% CO2 and 95% air. Fibroblasts were grown to confluence in 75 cm2 polystyrene tissue culture flasks, and confluent cells were subcultured every 2-3 days by trypsinization with 0.25% (w/v) trypsin and 0.13% (w/v) EDTA.

3. Results and Discussions Formation of PEG Hydrogel Using Photoreaction Injection Molding. The fabrication system for photoreaction injection molding described here consisted of two parts. The first was a microstructured mold insert formed from PDMS, and the second was a TPM-modified glass substrate. These two parts were sealed together to form the complete mold and subsequently filled with hydrogel precursor solution. PDMS microfluidic networks were fabricated by replica molding, which created a PDMS replica possessing three of the four walls necessary for the enclosed microfluidic channels. The angle of the walls was almost 90°, so the microchannel in the PDMS replica was essentially rectangular. The depth of microchannel was fixed about 50 µm, and the width was either 200 or 300 µm. Finally, sealing the replica to a flat glass surface created a complete microchannel network. Here reversible, conformal sealing with TPM-modified glass surfaces was used. Reversible sealing between the PDMS replica and glass occurred due to the softness of PDMS and its ability to conform to minor imperfections in a flat surface, thus making van der Waals contact with these surfaces.24 PDMS microchannels were easily peeled off from the glass

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Figure 6. XPS spectra of (a) PEG hydrogel and (b) peptide-modified PEG hydrogel.

substrate with only moderate force and without leaving significant PDMS residue on the substrate. Therefore, resealing of the replica to the substrate may be performed numerous times with the same PDMS replica. For the photoreaction injection molding of PEG hydrogels, the gel precursor solution must completely fill the microchannels. Since reversible sealing cannot withstand high pressure in the microchannels, the precursor solution should fill the channel by either capillary action or via pressure-driven flow at a low flow rate. For the precursor solutions described here, however, both PDMS and TPMmodified glass surfaces were hydrophobic; therefore, the solution could not flow through the channel by capillary action. To solve this problem, PDMS microchannels were treated with an oxygen plasma to make them hydrophilic. (24) McDonald, J. C.; et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 2000, 21, 27-40.

Oxygen plasma treatment lowered the contact angle of channel surfaces with water to almost zero, allowing channels to be easily filled with the gel precursor solution via capillary action. After the filled channels were exposed to UV light for 1 s, the PDMS replica was removed from the glass substrate. Hydrogel microstructures remained on the TPM-modified substrates after removal of PDMS microchannels and did not detach from the substrate when exposed to an aqueous environment for a week because of the covalent bonding between hydrogel microstructures and surface-tethered methacrylate groups on the substrate. Figure 2 shows that the resultant replicated hydrogel microstructures, which assumed the shape of the microchannels, remained on the glass substrates. Clearly defined three-dimensional hydrogels were fabricated with smooth surfaces without phase separation, and microchannels as narrow as 10 µm wide could be used for the fabrication of hydrogel microstructures.

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ATR/FTIR spectroscopy was used to monitor the photopolymerization of PEG-DA. Conversion of 93%, consistent with previous results,16,25 was confirmed by measuring the decrease in the terminal CdC stretch of the PEG-DA macromer (RCHdCH2) at 1635 cm-1 before and after UV exposure. Fabrication of Arrays of Hydrogel Microstructures. On the basis of these results, we produced more complicated hydrogel microstructures by combining photolithography with photoreaction injection molding. Examples are shown in Figure 3. Here, after a gel precursor solution containing fluorescein was injected to the microchannels, a photomask with the design of a 100 µm diameter circular array was aligned with the channels and exposed to UV light. As shown in Figure 3a,b, the resulting cylindrical hydrogel microstructures were fabricated inside the microchannel in the UV-illuminated regions while unpolymerized gel precursor solution remained in the microchannel in the unexposed region. When PDMS was removed and the glass slide was rinsed with water, the desired cylindrical elements of PEG hydrogel were obtained as shown in Figure 3c,d. Here, the lateral dimension and height of the hydrogel microstructures could be controlled by the feature size of the mask and the thickness of the photoresist, respectively, with structures as small as 10 µm × 10 µm × 40 µm readily fabricated. To investigate the fidelity of the pattern transfer process when a photomask was used, hydrogel microstructures were fabricated from the photomasks containing circles with diameters of 500, 300, 200, 100, and 50 µm. The diameters of the resulting microstructures were then compared with those of the masks. Because of the gap between the precursor solution and the photomask by the glass substrate, the sizes of the microstructures were larger than the feature size of the mask and the percentage of increase in diameter became severe as the feature size of the mask got smaller. In the case of diameters between 500 and 100 µm, the increase in diameter of the hydrogel microstructures was less than 5%, whereas a diameter difference of 30% was observed for microstructures fabricated from the mask containing circles of 50 µm diameter. In our previous studies, PEG hydrogel microstructures were fabricated on glass or silicon substrates by spincoating a precursor solution onto substrates and exposing to UV light through a photomask.16,17 By using the photoreaction injection molding technique presented here, we discovered clear advantages over previous methods in fabricating hydrogel microstructures. For example, only a small volume of precursor solution was needed to fill a microchannel whereas spin-coating followed by photolithography required much more volume of precursor solution due to the loss of solution during the spin-coating process. Another important advantage was that hydrogel microstructures possessing different chemistries can be easily fabricated on a single substrate (as shown in Figure 4) without the need for multiple spin-coating, alignment, exposure, and developing steps as with conventional photolithography. Because sets of microchannels were fluidically isolated from each other, this permitted the simultaneous introduction of independent gel chemistries into each channel and microstructures could thus be created using only a single photolithographic exposure. To fabricate microstructures in this fashion, a mold insert (25) Mellott, M. B.; Searcy, K.; Pishko, M. V. Release of protein from highly cross-linked hydrogels of poly(ethylene glycol) diacrylate fabricated by UV polymerization. Biomaterials 2001, 22, 929-941.

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Figure 7. A heterogeneous hydrogel microstructure created by laminar flow of precursor solutions inside a microchannel. One gel precursor solution incorporated an RGD cell adhesion peptide, and the other solution contained TRITC: (a) optical transmission microscopy; (b) fluorescence microscopy; (c) optical micrographs of 3T3 fibroblasts attached to the patterned hydrogel microstructure after 10 h.

composed of six channels was first fabricated in PDMS and gel precursor solutions including fluorescein and tetramethylrhodamine were alternatively introduced to each microchannel (Figure 4a). These precursor-solutioncontaining microchannels were then exposed to UV light through a photomask (Figure 4b). Removing the PDMS template and washing away the unreacted precursor

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solution with water resulted in an array of hydrogel microstructures which contain both fluorescein and tetramethylrhodamine as shown in Figure 4c. We anticipate that by use of this technique, multiple cell phenotypes and proteins may be created in an array of hydrogel microstructures with a lower probability of chemical crosscontamination between structures than one would see with multiple spin-coating procedures. Patterning Heterogeneous Hydrogel Microstructures inside Microchannels. An important characteristic of flow inside microfluidic channels is that the flow has a low Reynolds number and is laminar. When two or more streams with a low Reynolds number are introduced to a single microchannel simultaneously, the combined streams flow parallel to each other with mixing between the streams occurring only by diffusion. Using this flow property inside a microchannel, two precursor solutions with different chemistries were introduced to a Y-shaped microchannel using a syringe pump (Harvard Apparatus, Holliston, MA). One precursor solution containing PEGDA, initiator, and tetramethylrhodamine was introduced on one branch of the Y, while the other precursor solution containing RGD peptides in addition to PEG-DA and initiator was introduced in the other branch. Here, the peptides were conjugated to the hydrogel network by reacting the peptides with acryloyl-PEG-N-hydroxysuccinimide (acryloyl-PEG-NHS, 3400 Da; Shearwater Polymers, Huntsville, AL).23 As the two solutions were united in the microfluidic system, they remained distinct and did not visibly mix. Photogelation of the two precursor solutions was performed, and then the PDMS microfluidic mold was removed to obtain the final hydrogel microstructures. Structural characterization of these heterogeneous hydrogels was performed using ATR-FTIR and XPS. For the RGD-peptide-containing hydrogels, analysis of IR spectra showed peaks at 1653 and 1540 cm-1 characteristic of the CdO stretch and the N-H bend in the peptide amide (Figure 5). Peptide incorporation was further investigated with XPS. As shown in Figure 6, an N1s signal was not observed for the PEG hydrogel without peptide, whereas an N1s signal was observed for the peptide-modified hydrogel. Spectrum B also indicates the presence of Na, likely from the sodium salt of aspartic acid (D) in the RGD peptide. Both FTIR and XPS results suggest that RGD peptide was successfully incorporated to the PEG hydrogel. Figure 7a,b shows the resultant heterogeneous hydrogel microstructure visualized with bright field and fluorescence microscopy. As shown in these images, a hydrogel microstructure having a polarized chemistry was fabricated inside the microchannel, as is clear from the interface between the two regions shown in Figure 5a and the

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fluorescence image in Figure 7b. To demonstrate that the two regions of the hydrogel microstructure were functionally distinct, 3T3 murine fibroblasts were seeded on the patterned substrate and attached cells were observed after 10 h of incubation. Because of the extremely hydrophilic nature of PEG, cells were unable to adhere to the region of the microstructure that did not have the RGD adhesion peptide whereas cell adhesion improved dramatically on the surface of the region that incorporated RGD as shown in Figure 7c. For a more quantitative evaluation of cell adhesion and spreading on the peptide-modified hydrogel, cells were seeded onto both hydrogels with and without RGD peptide at 106 cells/mL and photomicrographs of adhered cells were taken after 24 h of incubation to obtain the number of adhered cells, percentage of spread cells, and average area of cells. Compared to the control hydrogel (i.e., no RGD peptide), there was a dramatic improvement in cell adhesion and spreading on the peptide-modified hydrogel as incubation time increased. The number of adherent cells per square centimeter increased 115% on the peptide-modified hydrogel surface. RGD peptide in PEG hydrogel resulted in approximately 45% of adherent cells beginning to spread and increased the average area of adhered cells by 155% compared to the “rounded” state. The creation of hydrogel microstructures that show such differences in cell adhesion and spreading may allow one to create novel biomaterial microstructures to promote the development of microstructured tissue. 4. Conclusion Here we described the rapid fabrication of PEG hydrogel microstructures using photoreaction injection molding with potential applications in multianalyte biosensing or tissue engineering. Microchannels in PDMS as narrow as 10 µm wide were used as mold inserts to create hydrogel microstructures. Three-dimensional PEG hydrogel microstructures were successfully fabricated by combining this photoreaction injection molding process with photolithography, and the use of multiple fluidic microchannels enables the fabrication of an array of hydrogel microstructures containing different chemistries. A hydrogel microstructure having a heterogeneous, spatial defined chemistry for cell adhesion was also fabricated inside a single microchannel, thus demonstrating the potential of this technique to create substrates for the micropatterned growth of tissue. Acknowledgment. We gratefully acknowledge financial support from the National Institutes of Health (5R01 EB000684-02). LA034257X