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. 
...

<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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-ene+photopolymerizations+provide+a+facile+method+to+encapsulate+proteins+and+maintain+their+bioactivity.">Thiol-ene photopolymerizations provide a facile method to encapsulate proteins and maintain their bioactivity.</a> Biomacromolecules. 13 (8), 2410-2417 (2012).</li><li>Fairbanks, B. D., Schwartz, M. P., Bowman, 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=Photoinitiated+polymerization+of+PEG-diacrylate+with+lithium+phenyl-2,4,6-trimethylbenzoylphosphinate:+polymerization+rate+and+cytocompatibility.">Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility.</a> Biomaterials. 29 (6), 997-1003 (2009).</li><li>Zuidema, J. M., Rivet, C. 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>

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 width="1237">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">PEG-diacrylate (PEGDA)</td>
			<td style="width:112px;">Laysan Bio</td>
			<td style="width:223px;">ACRL-PEG-ACRL-3400</td>
			<td style="width:665px;">Can also be synthesized or purchased through other venders. Different molecular weights can be used.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:237px;height:60px;">Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
			<td style="width:112px;"></td>
			<td style="width:223px;"></td>
			<td style="width:665px;">Synthesized in lab</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Fibronectin</td>
			<td style="width:112px;">Corning</td>
			<td style="width:223px;">356008</td>
			<td style="width:665px;">Other cell attachment proteins can be used, such as laminin, matrigel</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:223px;">D8537-500ML</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Photomask</td>
			<td style="width:112px;">FineLine Imaging</td>
			<td style="width:223px;">n/a</td>
			<td style="width:665px;">Custom prints on transparent sheets with high resolution DPI.</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Binder Clips</td>
			<td style="width:112px;">Various Vendors</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">Compact UV Light Source (365nm)</td>
			<td style="width:112px;">UVP</td>
			<td style="width:223px;">UVP-21</td>
			<td style="width:665px;">Other UV light sources can be used, calibration of power is required.</td>
		</tr>
		<tr height="57">
			<td height="57" style="width:237px;height:57px;">2-iminothiolane (Pierce Traut&#8217;s Reagent)</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">26101</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="82">
			<td height="82" style="width:237px;height:82px;">Ellman&#8217;s Reagent: DTNB; 5,5-dithio-bis(2-nitrobenzoic acid)</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">22582</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">human umbilical vein endothelial cells (HUVECs)</td>
			<td style="width:112px;">Lonza</td>
			<td style="width:223px;"></td>
			<td style="width:665px;">passage number between 6- 10</td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">EGM-2 Media</td>
			<td style="width:112px;">Lonza</td>
			<td style="width:223px;">CC31-56, CC-3162</td>
			<td style="width:665px;">EGM-2 without growth factors was used in experiments. Full EGM-2 media was used for cell maintainance</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">0.25% Trypsin EDTA</td>
			<td style="width:112px;">Life Tech</td>
			<td style="width:223px;">25200-056</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Trypsin Neutralizer</td>
			<td style="width:112px;">Life Tech</td>
			<td style="width:223px;">R-002-100</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Centrifuge</td>
			<td style="width:112px;">Various Venders</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Hemocytometer</td>
			<td style="width:112px;">Hausser Sci. Bright-line</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="width:237px;height:51px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:223px;">E6758</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">0.22&#181;m filter</td>
			<td style="width:112px;">Cell Treat</td>
			<td style="width:223px;">229743</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">1mL Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Glass Microscope Slides</td>
			<td style="width:112px;">Fisher Sci.</td>
			<td style="width:223px;">12-550C</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Plastic spacers</td>
			<td style="width:112px;">Various Venders</td>
			<td style="width:223px;"></td>
			<td style="width:665px;">0.5mm thickness</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">70% Ethanol</td>
			<td style="width:112px;">BICCA</td>
			<td style="width:223px;">2546.70-1</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Bio-shield</td>
			<td style="width:112px;">Bio-shield</td>
			<td style="width:223px;">19-150-0010</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Bradford Reagent&#160;</td>
			<td style="width:112px;">BIO-RAD</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Desalting Resin - Sephadex G-25</td>
			<td style="width:112px;">GE Healthcare</td>
			<td style="width:223px;">95016-754</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Microspin Columns</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">PI69725</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">AR-G2 rehometer</td>
			<td style="width:112px;">TA Instruments</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></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 ...

The overall goal of this methodology is to create immobilized bioactive spatial protein patterns which be used to study cellular responses. This method allows the recapitulating in-vivo signals using biomaterial strategies. This technique allows for accurate mimicry of certain signaling motives that cannot be achieved using standard culturing methods such as, sequential growth factors, cells cells signaling and spatial specific biochemical cues.This protocol is significant to existing methods as it provides a simplistic method for adjusting substrate stiffness and protein patterning. While this method can further tissue engineering and regenerative medicine technologies, it can also be applied to study other systems such as, tissue development and stem cell fate. To begin, prepare stock solutions for the major hydrogel component, polyethylene glycol diacrylate, the photo initiator, lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate and the ECM protein, fibronectin, under sterile conditions as described in the accompanying text protocol.Filter sterilize each solution using a 0.22 micron syringe filter prior to using or storing the individual aliquots. Then, wrap the PEG-DA solution with foil to protect it from light. Wrap the photo initiator stock solution in foil to protect it from light and store the prepared solution at four degree celsius for up to several months.If fibronectin stock solution is frozen, allow sterile aliquot to thaw on ice for several hours or in four degree celsius overnight. Start by autoclaving one polyester sheet for each gel mold. Then, soak two glass slides in three plastic spacers for each gel mold in 70%ethanol for at least two hours.Additionally, soak five binder clips for each gel mold in 70%ethanol for 10 minutes. Place the glass slides, spacers and binder clips onto a small autoclave sheet in the cell culture hood to allow them to air dry for several hours. Next, prepare the hydrogel molds by placing the 0.5 millimeter thick plastic spacers around the edge of a glass slide.Place the second glass slide on top and then secure the spacers tightly next to each other with binder clips. Finally, surface sterilize the mold by placing it in the hood and turning on the UV light for 30 minutes prior to use. Halfway through the sterilization process, flip the mold to be suer to expose both surfaces.Create the gel precursor solutions as described in table one of the accompanying text protocol. Then, mix together the PEG-DA, fibronectin and photo initiator volumes to create the hydrogel. Pipette the solution vigorously to ensure a homogenous solution, but avoid creating bubbles.Pipette the gel precursor solution carefully between the two glass slides of the gel mold. Then, place the mold under a Uv light and expose it for one to two minutes to form the hydrogel. Once gelled, take off the binder clips and gently remove the top glass slide by applying opposite pressure to the two side spacers.Then, using 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. Sterilize the photo mask by soaking it in 70%ethanol for 10 minutes.Then, allow the photo mask to air dry in a cell culture hood. Next, pipette one to two microliters per centimeter square of a thiolated protein solution to the surface of each cut out hydrogel for surface patterning. It is important to spread the protein solution across the surface of the hydrogel evenly to ensure uniform protein immobilization.Carefully place the photo mask on the surface of the hydrogel. Gently press down on the mask to remove any bubbles that form between the mask and the hydrogel surface. Next, place the hydrogel under the UV light and expose it to a second round of UV light for 30 to 60 seconds.Wash the hydrogels with PBS to remove any unreacted species and place each hydrogel within the well plate so that the pattern surface is faced up. Then, wash the gels overnight in PBS at four degrees celsius. Remove PBS from wells, then, add 250 microliters of basal EGM2 into each well and incubate the gels for five to 10 minutes at 37 degree celsius.During incubation, trypsin digest a plate of near-confluent HUVECs into a single cell suspension using standard techniques. Then, spin down the cell suspension and carefully remove the supernatant. Re-suspend the cell pellet and basal EGM2 with no added growth factors and add some of the cell suspension to a hemocytometer.Count the cells to determine the concentration. Next, spin down the plate containing he hydrogels at 300 x G for three minutes, to ensure that the hydrogels are located at the bottom of the well and are not floating. It is critical that hydrogels are not floating within the wells.Floating hydrogels will limit success in cell seeding and cause problems with hydrogel imaging. Slowly pipette 75, 000 cells per centimeters squared onto the center of each hydrogel surface so as to not disturb the gels. Then, place the plate into a cell culture incubator at 37 degrees celsius.For several days, periodically remove the dish to observe the cell migration in response to the protein pattern. Exchange 50%of the media every two to three days. When forming the hydrogels, various precursor PEG-diacrylate concentrations can be used to yield the desired substrate stiffness.Shown here, 20%PEG-DA solution correlates with the stiffness of over 100 kilopascals. It is also important to consider both photo initiator concentration and UV exposure time as an increase in either variable will decrease the bioactivity of attached molecules, represented here by the bioactivity of lysosome. Minimizing the UV exposure during hydrogel formation is critical to maintaining free acrylate functional groups for subsequent protein immobilization reactions.Hydrogels exposed to UV light for longer than two minutes are unable to create immobilized protein patterns shown in red. Additionally, as the UV exposure to the protein pattern increases, more proteins react to the surface. When prepared correctly, cells can be cultured onto these patterned hydrogel substrates to manipulate their behavior.Here, endothelial cells were uniformly seeded onto the surface of VEGF pattern PEG hydrogels. Two days after seeding, the endothelial cells were observed to migrate towards the spatial regions of the hydrogel that contained the immobilized VEGF. Following the development of this protocol, researchers can use it to immobilize any protein or peptide in order to explore new bioactive platforms for in-vitro cell systems.While attempting this procedure, it is important to remember to minimize photo initiator concentration and UV exposure time to maintain bio activity of immobilized molecules. After watching this video, you should have a good understanding of how to pattern bioactive proteins and peptides onto PEG hydrogels, seed cells uniformly onto hydrogels and observe the consequent cell response.

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JoVE Journal

Bioengineering

7.1K visualizações.  Rensselaer Polytechnic Institute. The overall goal of this methodology is to create immobilized bioactive spatial protein patterns which be used to study cellular responses. This method allows the recapitulating in-vivo signals using biomaterial strategies. This technique allows for accurate mimicry of certain signaling motives that cannot be achieved using standard culturing methods such as, sequential growth factors, cells cells signaling and spatial specific biochemical cues.This protocol is significant to existing ...