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

Denne undersøgelse blev hovedsagelig støttet af tilskud fra American Heart Association videnskabsmand udvikling Grant (12SDG12050083 til G.D.), National Institutes of Health (R21HL102773, R01HL118245 til G.D.) og National Science Foundation (CBET-1263455 og CBET-1350240 til G.D.).

1. forberedelse af materialer til Hydrogel syntese <ol><li>Forbered stamopløsninger af PEGDA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) og fibronektin under sterile forhold og baseret på beregninger (tabel 1A).
	<ol><li>Afvejes og opløses forbindelser i fosfatbufferet saltopløsning (PBS). Typisk, opretholde PEGDA arbejder løsning koncentrationer mellem 50 og 200 mg/mL (5-20% vægt/volumen). Afpipetteres PEGDA løsning gennem et 0,22 µm sprøjte filter for sterilisation. 
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<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. 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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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Denne protokol indeholder en metode til at skabe bioaktive protein mønstre for biologiske applikationer. Der er flere ændringer, der kan gøres til at tilpasse denne protokol for forskellige eksperimenter. Første varierer celle tilslutningskrav for de forskellige celletyper. Hvis fattige celle tilknytning til geler er i første omgang observeret, anbefales øger koncentrationen af ECM proteiner inden for forløber løsningen. Andre ECM proteiner kan bruges i stedet for fibronektin, herunder forskellige typer af kollag...

Der er mange stimuli, der påvirker celle adfærd. Generelt, typisk celle dyrkningsbaserede teknikker stole på opløselige faktorer at fremkalde cellulære svar; der er dog begrænsninger for denne tilgang. Disse metoder er ikke præcist vise alle signaling motiver almindeligt forekommende in vivo. Sådanne signaling mekanismer omfatter afsondret vækstfaktorer, celle-celle signalering og rumligt-specifikke biokemiske stikord. Derudover substrat stivhed kan spille en vigtig rolle i adfærd og stamceller Celledifferentiering og er ikke let manipuleres ved hjælp af fælles celle dyrkningsbaserede metoder1,2. Biomateriale tilgange tilbyder en ny platform for at begynde at udforske disse signaling mekanismer. Især er hydrogels gode kandidater til tuning substrat stivhed3,4, blokeret proteiner og peptider5,6, og skabe rumligt bestemte mønstre7, 8.
Hydrogels er almindeligt anvendt som stilladser i vævsmanipulering på grund af deres biofysiske og biokemiske fællestræk med ekstracellulære matrix (ECM)9,10. Naturlige polymerer er fælles valg til stilladser, da de er biokompatible og findes i mange væv i kroppen. Begrænsning af brug af naturlige polymerer som substrater er, at de ikke lette at manipulere kemiske fraspaltning for bioconjugation. På den anden side er syntetiske hydrogels, som sådan som PIND, fremragende platforme for målrettede kemi11,12. Derudover PIND hydrogels fremkalde ikke en cellulære reaktion og derfor bruges som inert backbones til oprettelse af bioaktive stilladser.
Både photopolymerization og thiol-en Klik for at oprette bioaktive hydrogels, kemi reaktioner er ansat. Disse photoreactions kræver en photoinitiator og en UV-lyskilde. Når photoinitiators bliver introduceret til UV-lys, bryde obligationer til at danne radikaler. Teser radikaler er nødvendige for at indlede reaktionen, men kan indvirke negativt på protein bioactivity12,13. Derfor er det afgørende at optimere photoinitiator og UV eksponering gange at opretholde protein bioactivity.
I denne metode, er hydrogels syntetiseret gennem acrylat-acrylat kæde vækst photopolymerization. PIND-diacrylate (PEGDA) monomerer reagere med hinanden om at danner forgrenede polymer netværk ansvarlig for strukturen af hydrogel. Koncentrationen af PEGDA monomerer i forløber gelopløsning vil kontrollere substrat stivhed. På grund af lille pore størrelse af hydrogel, ECM proteiner som fibronektin nemt kan indarbejdes i hydrogel med henblik på celle vedhæftet fil. Endelig, disse hydrogels kan være overflade-mønstret med bioaktive peptider eller proteiner via thiol-en klik kemi reaktioner. Her, vil ureageret gratis akrylater under ordningen hydrogel for reagere med frie dithioler beliggende på protein eller peptid når den udsættes for UV-lys. Efter proteiner eller peptider er immobiliseret på hydrogel overflade, kan hydrogel opbevares ved 4 ° C i flere uger uden at miste bioactivity. Dette giver bekvemmelighed, fleksibel eksperimentelle planlægning og muligheden for samarbejde mellem labs. Samlet set denne platform giver mulighed for biomekaniske og rumlige biokemiske kontrol, uafhængigt af hinanden, mulighed at påvirke cellulære adfærd.

<table width="1237">
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		<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

Der er mange biologiske stimuli, der kan påvirke adfærd og stamceller Celledifferentiering. Generelle celle kultur tilgange er afhængige af opløselige faktorer inden for mellemlang til kontrol celle adfærd. Men opløseligt tilføjelser kan ikke efterligne visse signalering motiver, såsom matrix-bundet vækstfaktorer, celle-celle signalering, og rumlige biokemiske stikord, som er fælles indflydelse på celler. Derudover spiller biofysiske egenskaber af matricen, som substrat stivhed, en vigtig rolle i celle skæbne, der ikke er nemt manipuleres ved hjælp af konventionelle celle dyrkning praksis. I denne metode beskriver vi en enkel protokol for at give mønstrede bioaktive proteiner på syntetisk polyethylenglycol (PEG) hydrogels ved hjælp af fotokemi. Denne platform giver mulighed for den uafhængige kontrol af substrat stivhed og rumlige biokemiske stikord. Disse hydrogels kan opnå en lang række fysiologisk relevante stivhed værdier. Derudover overflader af disse hydrogels kan være photopatterned med bioaktive peptider eller proteiner via thiol-en klik kemi reaktioner. Disse metoder er blevet optimeret for at bevare protein funktion efter overflade immobilisering. Dette er en alsidig protokol, der kan anvendes til protein eller peptid af interesse at skabe en række mønstre. Endelig kan celler seedede på overflader af disse bioaktive hydrogels følges over tid som de reagerer på rumligt specifikke signaler.

Protokol til at oprette bioaktive mønstre på overfladen af PIND hydrogels er illustreret i figur 1. Et regneark blev udviklet for at beregne mængden og koncentrationen for hver stamopløsning (tabel 1A). Proteiner til at blive immobiliseret på overfladen af hydrogel er modificeret med 2-iminothiolane (figur 1B). Denne reaktion er udført ved hjælp af diskenheder fra tabel 1B...

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

I denne metode, vi bruger photopolymerization og klik kemi teknikker til at skabe protein eller peptid mønstre på overfladen af polyethylenglycol (PEG) hydrogels, at give immobiliseret bioaktive signaler sammen med studiet af cellulære svar in vitro- .

Mønster bioaktive proteiner eller peptider på Hydrogel ved hjælp af fotokemi for biologiske applikationer

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Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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

Light-medieret dannelse og mønsterdannelse af hydrogeler til celledyrkningsapplikationer

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

A Versatile Method of Patterning Proteins and Cells

Et alsidigt Metode til mønstring Proteiner og celler

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Hurtig og skalerbar forsamling og læsning af bioaktive proteiner og immunostimulanter i forskellige syntetiske Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Biologiske forenelighed profil på biomaterialer for knogle regenerering

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Klargøring og karakterisering af Nanoliposomer til Indstødelse af bioaktive hydrofile Gloformede proteiner

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...