This study was mainly supported by grants from the American Heart Association Scientist Development Grant (12SDG12050083 to G.D.), the National Institutes of Health (R21HL102773, R01HL118245 to G.D.) and the National Science Foundation (CBET-1263455 and CBET-1350240 to G.D.).

1. Preparation of Materials for Hydrogel Synthesis
<ol>
	<li>Prepare stock solutions of PEGDA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and fibronectin under sterile conditions and based on calculations (Table 1A).
	<ol>
		<li>Weigh out and dissolve compounds in phosphate-buffered saline (PBS). Typically, maintain PEGDA working solution concentrations between 50 and 200 mg/mL (5-20% weight/volume). Pipette the PEGDA solution through a 0.22-&#181;m syringe filter for sterilization. 
		NOTE:&#160;Keep the PEGDA solutions covered with a foil wrapping to protect them from light. Prepare a fresh solution of PEGDA (recommended) for each experiment.</li>
		<li>Reconstitute fibronectin protein powder based on the manufacturer&#39;s recommendation. Maintain the fibronectin stock solution at 1 mg/mL, aliquot it into 60 to 100 &#181;L aliquots, and store them at -20 &#176;C until use. Defrost the aliquots at 4 &#176;C for several hours or overnight before use. If protein stock is not already provided in sterile conditions, use a 0.22 &#181;m syringe filter for sterilization. 
		NOTE: Fibronectin is used for cell attachment. Other ECM proteins can be substituted.</li>
		<li>Weigh out LAP for the stock solution and dissolve it in PBS; a typical stock concentration is 25 mg/mL. Sonicate if there is any trouble with dissolving. Pass the solution through a 0.22 &#181;m filter for sterilization. Cover the LAP with a foil wrapping and keep it at 4 &#176;C for up to several months. 
		NOTE: LAP is the photoinitiator used for the photochemistry.</li>
	</ol>
	</li>
	<li>Prepare sterile glass slide molds for hydrogel formation.
	<ol>
		<li>Autoclave a polyester sheet; one is needed for each gel mold. Soak two glass slides and three plastic spacers (0.5 mm thick) for each gel mold in 70% ethanol for at least 2 h, but generally allow them to soak overnight. Soak five binder clips for each gel mold in 70% ethanol for 10 min prior to use.</li>
		<li>Place the glass slides, spacers, and binder clips onto a small autoclaved sheet in the cell culture hood to allow them to air dry for several hours.</li>
		<li>Prepare hydrogel molds by placing plastic spacers around the edge of a glass slide. Place the second glass slide on top. Secure the spacers tightly next to each other with binder clips, two on each long side and one on top. 
		NOTE: If this is not properly assembled, the gel solution will leak out.</li>
		<li>Surface-sterilize the mold with cell culture hood UV light for 30 min prior to use. Halfway through, flip the mold to expose both surfaces.</li>
	</ol>
	</li>
</ol>
2. Modifying Proteins with a Free Thiol
<ol>
	<li>Prepare stock solutions using calculations from the spreadsheet for converting amine into thiol on proteins (Table 1A).
	<ol>
		<li>To make the reaction buffer, adjust the PBS to pH 8 and add 5 mM EDTA. Pipette reaction buffer into a 0.22 &#181;m syringe filter for sterilization. 
		NOTE: EDTA is important, as it protects thiols from forming disulfide bonds. Thiol groups must remain in their reduced form for the reaction to occur.</li>
		<li>Weigh out 2-iminothiolane (Traut&#39;s Reagent) and dissolve it in reaction buffer for a stock solution concentration of 2 mg/mL (14 mM). Pass the solution through a 0.22-&#181;m syringe filter for sterilization. Store the stock solution at 4 &#176;C for up to several months. 
		NOTE: 2-iminothiolane is the molecule that reacts with solvent-exposed free amines on the proteins and converts them into free thiols.</li>
		<li>Reconstitute the protein in reaction buffer at a concentration between 0.1 and 1 mg/mL. Unless previously sterile, pipette this solution through a 0.22-&#181;m syringe filter for sterilization. 
		NOTE: This protein is the biochemical signal that will be patterned onto the hydrogel surface. Also, while protein concentrations can vary, higher concentrations are ideal for stronger protein patterns.</li>
	</ol>
	</li>
	<li>React the protein with 2-iminothiolane by mixing both stock solutions together; use Table 1B to calculate the correct volume ratio. Typically, use a molar ratio of 8:1 2-iminothiolane to protein. 
	NOTE: For larger proteins or more dilute concentrations, use a higher molar ratio of 2-iminothiolane. Refer to the manufacturer&#39;s protocol15.</li>
	<li>Incubate the reaction for 1 h at room temperature. Use a desalting spin micro-column to remove the thiolated protein product from the remaining reactants, following the manufacturer&#39;s protocol16. 
	NOTE: The exclusion limit of this resin is 5 KDa.</li>
	<li>Use Ellman&#39;s Assay to quantitatively measure the number of free thiols per protein. Follow the manufacturer&#39;s protocol for the assay17.</li>
</ol>
3. Hydrogel Formation
<ol>
	<li>Create the gel precursor solution based on values calculated from Table 1C. Mix together the PEGDA, fibronectin, and LAP volumes. Pipette the solution vigorously to ensure a homogenous solution, but avoid creating bubbles. Keep the solution protected from light.</li>
	<li>Pipette the gel precursor solution carefully between the two glass slides of the gel mold. Typically, add a volume between 800 and 1,000 &#181;L to the mold. Expose the gel solution in the mold to UV light (wavelength: 365 nm, power: 3-4 mW/cm2) for 1-2 min to form the hydrogel. 
	NOTE: Do not expose the hydrogel to prolonged UV light, as it will limit the surface protein patterning capabilities.</li>
	<li>Take off the binder clips and gently remove top glass slide by applying opposite pressure to the two side spacers.</li>
	<li>Use an appropriately sized biopsy punch to cut out the hydrogel samples. Punch out multiple hydrogels from the gel rectangle to serve as replicates and control samples.</li>
</ol>
4. Hydrogel Stiffness Measurements
<ol>
	<li>Punch out hydrogels with an 8 mm-diameter biopsy punch for an 8 mm parallel plate rheometer. Load one hydrogel sample into the rheometer (see the Table of Materials).</li>
	<li>Lower the parallel plate geometry that is 8 mm in diameter until making contact with the surface of the gel. Keep a gap distance of 0.5 mm for a 0.5 mm-thick hydrogel sample.</li>
	<li>Perform time sweeps for 5 min at 0.1% strain, 0.1 Hz frequency, and 37 &#176;C. Average the G&#39; values across each time sweep. Run independent hydrogels for replicates of each composition. 
	NOTE: The G&#39; values should be stable across time points; if they are not, percent strain and frequency sweeps should be performed to select the appropriate values14.</li>
</ol>
5. Protein Patterning
<ol>
	<li>Prepare the desired pattern for the photomask using a computer aided design (CAD) program. Print the photomask on a transparent sheet using a high-resolution printer. Soak the photomask in 70% ethanol for 10 min prior to use. Allow the photomask to air dry in a cell culture hood before use.</li>
	<li>Add the thiolated protein solution prepared in step 2 to the surface of the hydrogel for surface patterning. Pipette 1-2 &#181;L/cm2 of thiolated protein solution to the surface of each cut-out gel. 
	NOTE: It is important to minimize the protein volume; only add enough to evenly cover the entire surface of the hydrogel sample.</li>
	<li>Carefully place the photomask on the surface of the hydrogel. Do not allow air bubbles between the photomask and the hydrogel surface. Gently press down on the mask to remove any bubbles that form.</li>
	<li>Expose the hydrogel to a second round of UV light (wavelength: 365 nm, power: 3-4 mW/cm2) for 30-60 s. 
	NOTE: It is important to exposure the pattern to enough UV light to create a strong pattern without causing the loss of protein function.</li>
	<li>Wash the hydrogels with PBS to remove unreacted species and place each hydrogel within the well plate. Be careful when placing the gels into each well; make sure that the patterned surface is face up. Wash the gels in PBS at 4 &#176;C; gels are stable for at least two weeks at 4 &#176;C.</li>
</ol>
6. Preparing Hydrogels for Cell Seeding
<ol>
	<li>Incubate the gels in basal medium for 5-10 min at 37 &#176;C prior to seeding. For human umbilical vein endothelial cells (HUVECs), use EGM-2 medium without growth factors. Minimize the volume of medium added to each well to prevent hydrogel floating. For a 48-well plate, use a 250 &#181;L volume of medium.</li>
	<li>Spin down the plate at 300 x g for 3 min to ensure that the hydrogels are located at the bottom of the well and are not floating. Do this immediately prior to cell seeding.</li>
</ol>
7. Cell Seeding on Hydrogels
<ol>
	<li>Use standard sterile mammalian cell culture procedures for cell seeding and experimental procedures. Remove HUVECs from tissue culture flasks using standard cell detachment protocols. Wash the tissue culture dish with sterile PBS and incubate with trypsin for 3-5 min at 37 &#176;C.</li>
	<li>Quench the trypsinized cell suspension with excess cell medium or trypsin neutralizer solution.</li>
	<li>Spin down the cell suspension in the centrifuge at 300 x g for 5 min. Carefully remove the supernatant and keep the cell pellet.</li>
	<li>Resuspend the cell pellet in basal EGM-2 with no growth factors. Use a hemocytometer for cell counting.</li>
	<li>Add 75,000 cells/cm2 to each hydrogel surface by pipetting slowly into the center of each well so as not to disturb the gels. Place the well plate into a cell culture incubator at 37 &#176;C. For several days, periodically remove the dish for observation.</li>
</ol>
8. Evaluation of Bioactivity
<ol>
	<li>Culture E. coli overnight in suspension in LB broth in an orbital suspension at 37 &#176;C and 200 rpm. Spin down the E. coli culture, decant, and reconstitute the cell pellet in minimal volume of PBS. Weigh out the lysozyme and reconstitute at 1 mg/mL for the stock solution.</li>
	<li>Pipette a small volume (25 &#181;L) of lysozyme stock solution into microcentrifuge tubes. Add varying levels of LAP photoinitiator (0-12 mM) and expose the samples to 1 min of UV light. In a separate group, add the same amount of LAP (2 mM) to lysozyme solution and expose samples to varying UV light times (0-4 min). Include replicates for each treatment.</li>
	<li>Add equal volume of concentrated E. coli solution to each lysozyme sample for a final lysozyme protein concentration of 0.5 mg/mL. Incubate the mixed solutions for 4 h at room temperature. 
	NOTE: This allows time for functional lysozyme to lyse the bacterial cell wall and release proteins into the solution.</li>
	<li>Use untreated lysozyme incubated with E. coli for a positive control; consider this a 100% bioactive measurement. Use lysozyme solution incubated with PBS alone as a negative control.</li>
	<li>Spin down the samples to remove cell debris and keep the supernatant. Run a Bradford assay to quantify the total concentration of protein within the supernatant to measure the amount of bacterial lysate.</li>
	<li>Run a Bradford assay following the manufacturer&#39;s protocol18. Calculate the fold change in protein concentration compared to the negative control.</li>
</ol>

<ol>
	<li>Yao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-effects+of+matrix+low+elasticity+and+aligned+topography+on+stem+cell+neurogenic+differentiation+and+rapid+neurite+outgrowth.">Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth.</a> Nanoscale. 8 (19), 10252-10265 (2016).</li><li>Evans, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+stiffness+affects+early+differentiation+events+in+embryonic+stem+cells.">Substrate stiffness affects early differentiation events in embryonic stem cells.</a> Eur Cells Mater. 18, 1-13 (2009).</li><li>Ye, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Stiffness+and+Nanoscale+Spatial+Organization+of+Cell-Adhesive+Ligands+Direct+Stem+Cell+Fate.">Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate.</a> Nano Lett. 15 (7), 4720-4729 (2015).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310 (5751), 1139-1143 (2005).</li><li>Sridhar, B. V., Doyle, N. R., Randolph, M. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+tethered+TGF-beta1+with+encapsulated+chondrocytes+in+a+PEG+hydrogel+system+enhances+extracellular+matrix+production.">Covalently tethered TGF-beta1 with encapsulated chondrocytes in a PEG hydrogel system enhances extracellular matrix production.</a> J Biomed Mater Res A. 102 (12), 4464-4472 (2014).</li><li>Salinas, C. N., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decorin+moieties+tethered+into+PEG+networks+induce+chondrogenesis+of+human+mesenchymal+stem+cells.">Decorin moieties tethered into PEG networks induce chondrogenesis of human mesenchymal stem cells.</a> J Biomed Mater Res A. 90 (2), 456-464 (2009).</li><li>Joddar, B., Guy, A. T., Kamiguchi, H., Ito, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+gradients+of+chemotropic+factors+from+immobilized+patterns+to+guide+axonal+growth+and+regeneration.">Spatial gradients of chemotropic factors from immobilized patterns to guide axonal growth and regeneration.</a> Biomaterials. 34 (37), 9593-9601 (2013).</li><li>Wylie, R. G., Ahsan, S., Aizawa, Y., Maxwell, K. L., Morshead, C. M., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+controlled+simultaneous+patterning+of+multiple+growth+factors+in+three-dimensional+hydrogels.">Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels.</a> Nat Mater. 10 (10), 799-806 (2011).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103 (4), 655-663 (2009).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nat Biotechnol. 23 (1), 47-55 (2005).</li><li>Saik, J. E., Gould, D. J., Keswani, A. H., Dickinson, M. E., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+hydrogels+with+immobilized+ephrinA1+for+therapeutic+angiogenesis.">Biomimetic hydrogels with immobilized ephrinA1 for therapeutic angiogenesis.</a> Biomacromolecules. 12 (7), 2715-2722 (2011).</li><li>McCall, J. D., Anseth, K. 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J., Gilbert, R. J., Morrison, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+rheological+characterization+of+hydrogels+for+tissue+engineering+strategies.">A protocol for rheological characterization of hydrogels for tissue engineering strategies.</a> J Biomed Mater Res B Appl Biomater. 102 (5), 1063-1073 (2014).</li><li>Truat's Reagent Instructions. Thermo Scientific Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/.../MAN0011238_Trauts_Reag_UG.pdf">https://tools.thermofisher.com/content/sfs/.../MAN0011238_Trauts_Reag_UG.pdf</a> (2017)</li><li>Ellman's Reagent Instructions. Thermo Scientific Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/MAN0011216_Ellmans_Reag_UG.pdf">https://tools.thermofisher.com/content/sfs/manuals/MAN0011216_Ellmans_Reag_UG.pdf</a> (2017)</li><li>Desalting Columns. GE Life Sciences Available from: <a target="_blank" href="https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1478781880316/litdoc52130800_2016111034421.pdf">https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1478781880316/litdoc52130800_2016111034421.pdf</a> (2017)</li></ol>

The authors have nothing to disclose.

This protocol provides a method for creating bioactive protein patterns for biological applications. There are several modifications that can be made to adapt this protocol for different experiments. First, cell attachment requirements will vary for different cell types. If poor cell attachment to the gels is initially observed, increasing the concentration of the ECM protein within the precursor solution is advised. Other ECM proteins can be used instead of fibronectin, including different types of collagen, laminin, or...

There are many stimuli that influence cell behavior. Generally, typical cell culturing techniques rely on soluble factors to elicit cellular responses; however, there are limitations to this approach. These methods are unable to accurately display all signaling motifs commonly found in vivo. Such signaling mechanisms include sequestered growth factors, cell-cell signaling, and spatially-specific biochemical cues.Furthermore, substrate stiffness can play an important role in cell behavior and stem cell differentiation and is not easily manipulated using common cell culturing practices1,2. Biomaterial approaches offer a new platform to begin exploring these signaling mechanisms. In particular, hydrogels are excellent candidates for tuning substrate stiffness3,4, immobilizing proteins and peptides5,6, and creating spatially specific patterns7,8.
Hydrogels are commonly used as scaffolds in tissue engineering due to their biophysical and biochemical commonalities with the extracellular matrix (ECM)9,10. Natural polymers are common choices for scaffolds, as they are biocompatible and are found in many tissues of the body. The limitation of using natural polymers as substrates is that they lack easily manipulated chemical moieties for bioconjugation. On the other hand, synthetic hydrogels, as such as PEG, are excellent platforms for targeted chemistries11,12. Additionally, PEG hydrogels do not elicit a cellular response and therefore are used as inert backbones for creating bioactive scaffolds.
To create bioactive hydrogels, both photopolymerization and thiol-ene click chemistry reactions are employed. These photoreactions require a photoinitiator and a UV light source. When photoinitiators are introduced to UV light, bonds break to form radicals. Theses radicals are necessary for initiating the reaction but can negatively affect protein bioactivity12,13. Therefore, it is crucial to optimize photoinitiator and UV exposure times to maintain protein bioactivity.
In this method, hydrogels are synthesized through acrylate-acrylate chain growth photopolymerization. PEG-diacrylate (PEGDA) monomers react with each other to form branched polymer networks responsible for the structure of the hydrogel. The concentration of PEGDA monomers within the gel precursor solution will control the substrate stiffness. Due to the small pore size of the hydrogel, ECM proteins such as fibronectin can be easily incorporated within the hydrogel for the purpose of cell attachment. Finally, these hydrogels can be surface-patterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. Here, unreacted free acrylates within the hydrogel system will react with free thiols located on the protein or peptide when exposed to UV light. After the proteins or peptides are immobilized on the hydrogel surface, the hydrogel can be stored at 4 &#176;C for several weeks without losing bioactivity. This offers convenience, flexible experimental planning, and the possibility for collaboration between labs. Overall, this platform allows for biomechanical and spatial biochemical control, independent of each other, for the opportunity to influence cellular behavior.

<table><tbody><tr><td>PEG-diacrylate (PEGDA)</td><td>Laysan Bio</td><td>ACRL-PEG-ACRL-3400</td><td>Can also be synthesized or purchased through other venders. Different molecular weights can be used.</td></tr><tr><td>Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td><td></td><td></td><td>Synthesized in lab</td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td>Other cell attachment proteins can be used, such as laminin, matrigel</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>D8537-500ML</td><td></td></tr><tr><td>Photomask</td><td>FineLine Imaging</td><td>n/a</td><td>Custom prints on transparent sheets with high resolution DPI.</td></tr><tr><td>Binder Clips</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Compact UV Light Source (365nm)</td><td>UVP</td><td>UVP-21</td><td>Other UV light sources can be used, calibration of power is required.</td></tr><tr><td>2-iminothiolane (Pierce Traut&amp;rsquo;s Reagent)</td><td>Thermo Sci.</td><td>26101</td><td></td></tr><tr><td>Ellman&amp;rsquo;s Reagent: DTNB; 5,5-dithio-bis(2-nitrobenzoic acid)</td><td>Thermo Sci.</td><td>22582</td><td></td></tr><tr><td>human umbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td></td><td>passage number between 6- 10</td></tr><tr><td>EGM-2 Media</td><td>Lonza</td><td>CC31-56, CC-3162</td><td>EGM-2 without growth factors was used in experiments. Full EGM-2 media was used for cell maintainance</td></tr><tr><td>0.25% Trypsin EDTA</td><td>Life Tech</td><td>25200-056</td><td></td></tr><tr><td>Trypsin Neutralizer</td><td>Life Tech</td><td>R-002-100</td><td></td></tr><tr><td>Centrifuge</td><td>Various Venders</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Sci. Bright-line</td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>E6758</td><td></td></tr><tr><td>0.22&amp;micro;m filter</td><td>Cell Treat</td><td>229743</td><td></td></tr><tr><td>1mL Syringe</td><td></td><td></td><td></td></tr><tr><td>Glass Microscope Slides</td><td>Fisher Sci.</td><td>12-550C</td><td></td></tr><tr><td>Plastic spacers</td><td>Various Venders</td><td></td><td>0.5mm thickness</td></tr><tr><td>70% Ethanol</td><td>BICCA</td><td>2546.70-1</td><td></td></tr><tr><td>Bio-shield</td><td>Bio-shield</td><td>19-150-0010</td><td></td></tr><tr><td>Bradford Reagent&amp;nbsp;</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>Desalting Resin - Sephadex G-25</td><td>GE Healthcare</td><td>95016-754</td><td></td></tr><tr><td>Microspin Columns</td><td>Thermo Sci.</td><td>PI69725</td><td></td></tr><tr><td>AR-G2 rehometer</td><td>TA Instruments</td><td></td><td></td></tr></tbody></table>

patterning bioactive proteins or peptides on hydrogel using photochemistry for biological applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

The protocol to create bioactive patterns on the surface of PEG hydrogels is illustrated in Figure 1. A spreadsheet was developed to calculate the volume and concentration for each stock solution (Table 1A). Proteins to be immobilized onto the surface of the hydrogel are modified with 2-iminothiolane (Figure 1B). This reaction is performed using the volumes from Table 1B. The pre...

Watch this Scientific Journal Video about Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications at JoVE.com

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.1K Views.  Rensselaer Polytechnic Institute. In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Video: Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Developmental Biology

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

Biochemistry

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...