Name: patterning bioactive proteins or peptides on hydrogel using photochemistry for biological applications
Uploaded: 2017-09-15T14:00:00
Description: Watch this Scientific Journal Video about Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications at JoVE.com

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. 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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. 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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.

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	<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 ...

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