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 studien var hovedsakelig støttes av tilskudd fra American Heart Association forsker Development 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 av materialer for Hydrogel syntese <ol><li>forberede lager løsninger PEGDA, litium fenyl-2,4,6-trimethylbenzoylphosphinate (LAP) og fibronectin under sterile forhold og basert på beregninger (tabell 1A).
	<ol><li>Veie og oppløse forbindelser i fosfat-bufret saltvann (PBS). Vanligvis opprettholde PEGDA arbeider løsning konsentrasjoner mellom 50 og 200 mg/mL (5-20% vekt/volum). Pipetter PEGDA løsningen gjennom 0.22-µm sprøyte filter for sterilisering. 
		Merk: Holde PEGDA lø...

<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. 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Denne protokollen inneholder en metode for å lage bioaktive protein mønstre for biologiske applikasjoner. Det er flere endringer som kan gjøres til å tilpasse denne protokollen for forskjellige eksperimenter. Først varierer celle vedlegg kravene for ulike celletyper. Hvis dårlig celle vedlegg til geléer er først observert, anbefales øker konsentrasjonen av ECM protein i forløperen løsningen. Andre ECM proteiner kan brukes i stedet for fibronectin, inkludert ulike typer kollagen, laminin eller en kombinasjon av...

Det er mange stimuli som påvirker celle atferd. Generelt, typisk celle dyrking teknikker stole på løselig faktorer å lokke fram mobilnettet svar; Det er imidlertid begrensninger på denne tilnærmingen. Disse metodene er ikke nøyaktig vise alle signalnettverk motiver vanligvis grunnlegge i vivo. Slike signalnettverk mekanismene inkluderer sequestered vekstfaktorer, celle-celle signalisering og romlig-spesifikke biokjemiske signaler. Videre substrat stivhet kan spille en viktig rolle i celle atferd og stamcelleforskningen differensiering og er ikke lett manipulert bruker vanlig celle dyrking praksis1,2. Biomateriale tilnærminger tilbyr en ny plattform for å begynne å utforske disse signalnettverk mekanismer. Spesielt er hydrogels gode kandidater for tuning substrat stivhet3,4, immobilizing proteiner og peptider5,6, og skape romlig bestemte mønstre7, 8.
Hydrogels er ofte brukt som stillaser i vev engineering på grunn av deres Biofysiske og biokjemiske fellestrekk med ekstracellulær matrix (EFM)9,10. Naturlig polymerer er vanlig valg for stillaser, som de er biokompatible og finnes i mange vev i kroppen. Begrensning av bruk av naturlige polymerer som underlag er at de mangler lett manipulert kjemiske moieties for bioconjugation. På den annen side, er syntetiske hydrogels, slik som PEG, gode plattformer for målrettet kjemikalier11,12. Dessuten PEG hydrogels framprovosere ikke en cellulær respons og derfor brukes som inert infrastruktur for oppretting av bioaktive stillaser.
Klikk kjemi reaksjoner er ansatt for å opprette bioaktive hydrogels, både photopolymerization og thiol-ene. Disse photoreactions krever en photoinitiator og en UV lyskilde. Når photoinitiators er introdusert til UV lys, bryte obligasjoner til skjemaet radikaler. Avhandlinger radikaler er nødvendig for å starte reaksjonen, men kan negativt påvirke protein bioactivity12,13. Derfor er det avgjørende å optimalisere photoinitiator og UV eksponeringstider å opprettholde protein bioactivity.
I denne metoden er hydrogels syntetisert gjennom acrylate-acrylate kjeden vekst photopolymerization. PEG-diacrylate (PEGDA) monomerer reagere med hverandre for å danne forgrenet polymer nettverk ansvaret for oppbygning av hydrogel. Konsentrasjonen av PEGDA monomerer i gel forløper løsningen kontrollerer substrat stivhet. På grunn av lille pore størrelse av hydrogel, ECM proteiner som fibronectin lett kan innlemmes i hydrogel for cellen vedlegg. Endelig disse hydrogels kan overflaten-mønstret med bioaktive peptider eller proteiner via thiol-ene Klikk kjemi reaksjoner. Her reagere Ureagert gratis acrylates innenfor hydrogel systemet med gratis thiols plassert på protein eller peptid utsettes for UV-lyset. Når proteiner eller peptider er immobilized på hydrogel overflaten, kan hydrogel lagres på 4 ° C i flere uker uten å miste bioactivity. Dette gir praktiske, fleksible eksperimentelle planlegging og mulighet for samarbeid mellom laboratorier. Samlet tillater denne plattformen biomekaniske og romlig biokjemiske kontroll, uavhengig av hverandre, for sjansen å påvirker mobilnettet atferd.

<table width="1237">
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	</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

Det er mange biologiske stimuli som kan påvirke celle atferd og stamcelleforskningen differensiering. De generelle celle kultur tilnærminger er avhengige av løselig faktorer innen mediet til kontroll cellen atferd. Løselig tillegg kan imidlertid etterligne visse signalering motiver, som matrix-bundet vekstfaktorer, celle-celle signalisering og romlig biokjemiske bunker, som er vanlig påvirkninger på celler. Videre spille Biofysiske egenskaper av matrix, som substrat stivhet, en viktig rolle i celle skjebne, som ikke er lett manipulert med konvensjonelle cellen dyrking praksis. I denne metoden beskriver vi en enkel protokoll for å gi mønstret bioaktive proteiner på syntetiske polyetylenglykol (PEG) hydrogels med fotokjemi. Denne plattformen gir uavhengig kontroll av underlaget stivhet og romlig biokjemiske signaler. Disse hydrogels kan oppnå en rekke fysiologisk relevante stivhet verdier. I tillegg overflater av disse hydrogels kan være photopatterned med bioaktive peptider eller proteiner via thiol-ene Klikk kjemi reaksjoner. Disse metodene har blitt optimalisert for å beholde protein funksjon etter overflate immobilisering. Dette er en allsidig protokoll som kan brukes til protein eller peptid rundt å lage en rekke mønstre. Til slutt, celler seeded på overflater av disse bioaktive hydrogels kan overvåkes over tid som de reagerer romlig sender.

Protokollen for å lage bioaktive mønstre på overflaten av PEG hydrogels er illustrert i figur 1. Et regneark ble utviklet for å beregne volumet og konsentrasjon for hvert lager løsning (tabell 1A). Proteiner til bli immobilisert på overflaten av hydrogel endres med 2-iminothiolane (figur 1B). Denne reaksjonen utføres med volumene fra tabellen 1B. Forløperen hydrogel løsn...

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 metoden vi bruker photopolymerization og klikk kjemi teknikker for å lage protein eller peptid mønstre på overflaten av polyetylenglykol (PEG) hydrogels, gir immobilisert bioaktive signaler for studier av mobilnettet svar i vitro .

Mønstre bioaktive proteiner eller peptider på Hydrogel bruker fotokjemi for biologiske programmer

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A Versatile Method of Patterning Proteins and Cells

En allsidig Måte mønster 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

Rask, skalerbar montering og lasting av bioaktive proteiner og Immunostimulants i ulike syntetisk 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 kompatibilitet profil på biologisk materiale for bein gjenfødelse

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

Utarbeidelse og karakterisering av Nanoliposomes for å felle av bioaktive hydrofile globular proteiner

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