Name: engineering a bilayered hydrogel to control asc differentiation
Uploaded: 2012-05-25T13:00:00
Description: Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

SN blev understøttet af en postdoc Fellowship Grant fra Pittsburgh Tissue Engineering Initiative. DOZ understøttes af et tilskud fra Genève Foundation.

1. Isolering adipøst afledte stamceller (ASC&#39;er) 1, 5 Bemærk: Alle procedurer blev udført ved stuetemperatur, medmindre andet er angivet. <ol><li> Isoler rotte perirenalt og epididymalt fedt-og vaskes med steril Hanks pufrede saltopløsning (HBSS) indeholdende 1% føtalt bovint serum (FBS) som tidligere beskrevet 5. Denne undersøgelse er gennemført i overensstemmelse med dyreværnsloven, gennemførelsesbestemmelserne regler for dyrevelfærd, ...

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	<li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bilayer+construct+controls+adipose-derived+stem+cell+differentiation+into+endothelial+cells+and+pericytes+without+growth+factor+stimulation.">A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.</a> Tissue Eng. Part A. 17, 941-953 (2011).</li><li>Nuttelman, C. R., Tripodi, M. C., 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=Synthetic+hydrogel+niches+that+promote+hMSC+viability.">Synthetic hydrogel niches that promote hMSC viability.</a> Matrix Biol. 24, 208-218 (2005).</li><li>Benoit, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-linked+kinase+production+prevents+anoikis+in+human+mesenchymal+stem+cells.">Integrin-linked kinase production prevents anoikis in human mesenchymal stem cells.</a> J Biomed Mater Res A. 81, 259-268 (2007).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> , (2008).</li><li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell+delivery+into+collagen+gels+using+chitosan+microspheres.">Adipose-derived stem cell delivery into collagen gels using chitosan microspheres.</a> Tissue Eng. Part A. 16, 1369-1384 (2010).</li><li>Bubnis, W. A., Ofner, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+epsilon-amino+groups+in+soluble+and+poorly+soluble+proteinaceous+materials+by+a+spectrophotometric+method+using+trinitrobenzenesulfonic+acid.">The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.</a> Anal. Biochem. 207, 129-133 (1992).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PEGylated+fibrin+patch+for+mesenchymal+stem+cell+delivery.">A PEGylated fibrin patch for mesenchymal stem cell delivery.</a> Tissue Eng. 12, 9-19 (2006).</li><li>Bornstein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+rattail+collagen+used+as+substrate+for+tissue+cultures+on+coverslips+in+Maximow+slides+and+roller+tubes.">Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes.</a> Lab Invest. 7, 134-137 (1958).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+differentiation+of+bone+marrow+stem+cells+is+directed+by+a+tunable+three-dimensional+matrix.">Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix.</a> Acta Biomater. 6, 3395-3403 (2010).</li><li>Rochon, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+epithelial+cells+regulate+the+size+and+morphology+of+tissue-engineered+capillaries.">Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.</a> Tissue Eng. Part A. 16, 1457-1468 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li></ol>

ASC&#39;er er kendt for deres lette isolation og evnen til at differentiere mod forskellige celletyper. Med teknikkerne beskrevet i dette skrift, er vi i stand til at udnytte plasticitet ASC&#39;er ved at eksponere disse celler til flere biomatrices samtidigt. Som celler migrere væk fra deres CSM base og ind i deres omgivende ekstracellulære miljø, cellerne tager udgangspunkt i stilladset og kan enten bibeholde &quot;stemness&quot; (collagen) eller kan induceres til at differentiere mod vaskulære-og vaskulære-støt...

<table border="1"><tbody><tr><td> Navn på reagenset / udstyr </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Hanks balancerede saltopløsning (HBSS) </td><td> Gibco </td><td> 14175 </td><td> Varen </td></tr><tr><td> Føtalt bovint serum </td><td> Hyclone </td><td> SH30071.03 </td><td> Varen </td></tr><tr><td> Collagenase Type II </td><td> Sigma-Aldrich </td><td> C6685 </td><td> Varen </td></tr><tr><td> 70 um nylon mesh filter </td><td> BD Biosciences </td><td> 352350 </td><td> Varen </td></tr><tr><td> 100 um nylon mesh filter </td><td> BD Biosciences </td><td> 352360 </td><td> Varen </td></tr><tr><td> MesenPRO vækstmedium System </td><td> Invitrogen </td><td> 12746-012 </td><td> Varen </td></tr><tr><td> L-glutamin </td&gt; <td> Gibco </td><td> 25030 </td><td> Varen </td></tr><tr><td> CaCl2 0,2 H2O </td><td> Sigma </td><td> C8106 </td><td> Varen </td></tr><tr><td> T75 vævsdyrkningskolbe </td><td> BD Biosciences </td><td> 137787 </td><td> Varen </td></tr><tr><td> Chitosan </td><td> Sigma-Aldrich </td><td> 448869 </td><td> Varen </td></tr><tr><td> Eddikesyre </td><td> Sigma-Aldrich </td><td> 320099 </td><td> Varen </td></tr><tr><td> N-octanol </td><td> Acros Organics </td><td> 150630025 </td><td> Varen </td></tr><tr><td> Sorbitan-mono-oleat </td><td> Sigma-Aldrich </td><td> S6760 </td><td> Varen </td></tr><tr><td> Kaliumhydroxid </td><td> Sigma-Aldrich </td><td> P1767 </td><td> Varen </td></tr><tr><td> Acetone </td><td> Fisher Scientific </td><td> L-4859 </td><td> Varen </td></tr><tr><td> Ethanol </td><td> Sigma-Aldrich </td><td> 270741 </td><td> Varen </td></tr><tr><td> Trinitro benzensulfonsyre </td><td> Sigma-Aldrich </td><td> P2297 </td><td> Varen </td></tr><tr><td> Saltsyre </td><td> Sigma-Aldrich </td><td> 320331 </td><td> Varen </td></tr><tr><td> Ethylether </td><td> Sigma-Aldrich </td><td> 472-484 </td><td> Varen </td></tr><tr><td> 8-um vævskulturplade Inserts </td><td> BD Biosciences </td><td> 353097 </td><td> Varen </td></tr><tr><td> 1,5 ml-mikrocentrifugerør </td><td> Fisher </td><td> 05-408-129 </td><td> Varen </td></tr><tr><td> MTT Reagens </td><td> Invitrogen </td><td> M6494 </td><td> Varen </td></tr><tr><td> Dimethylsulfoxid </td><td> Sigma-Aldrich </td><td> D8779 </td><td> Varen </td></tr><tr><td> Qtracker Cell Labeling Kit (QTracker 655) </td><td> Molekylære prober </td><td> Q2502PMP </td><td> Varen </td></tr><tr><td> Type 1 Collagen </td><td> Travigen </td><td> 3447-020-01 </td><td> Varen </td></tr><tr><td> Natriumhydroxid </td><td> Sigma-Aldrich </td><td> S8045 </td><td> Varen </td></tr><tr><td> 12-brønds vævskulturplader </td><td> BD Biosciences </td><td> 353043 </td><td> Varen </td></tr><tr><td> Fibrinogen </td><td> Sigma </td><td> F3879 </td><td> Varen </td></tr><tr><td> Trombin </td><td> Sigma </td><td> T6884 </td><td> Varen </td></tr><tr><td> Benztriazol Derivat af polyethylen </td><td> Sunbio </td><td> DE-034GS </td><td> Varen </td></tr><tr><td> Tris-buffer Tablet (pH 7,6) </td><td> Sigma </td><td> T5030 </td><td> Varen </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5417R </td><td> Equipment </td></tr><tr><td> Orbital Shaker </td><td> New Brunswick Scienctific </td><td> C24 </td><td> Udstyr </td></tr><tr><td> Befugtet inkubator med luft-5% CO2 </td><td> Thermo Scientific </td><td> Model 370 </td><td> Udstyr </td></tr><tr><td> Omrører </td><td> IKA </td><td> Visc6000 </td><td> Udstyr </td></tr><tr><td> Magnetomrører </td><td> Corning </td><td> PC-210 </td><td> Udstyr </td></tr><tr><td> Vakuumeksikator </td><td> - </td><td> - </td><td> Udstyr </td></tr><tr><td> Particle Size Analyzer </td><td> Malvern </td><td> STP2000 Spraytec </td><td> Udstyr </td></tr><tr><td> Vandbad </td><td> Fisher Scientific </td><td> Isotemp210 </td><td> Udstyr </td></tr><tr><td> Spektrofotometer </td><td> Beckman </td><td> Beckman Coulter DU 800UV/Visible Spektrofotometer </td><td> Udstyr </td></tr><tr><td> Vortex </td><td> Diagger </td><td> 3030a </td><td> Udstyr </td></tr><tr><td> Mikropladelæser </td><td> Molecular Devices </td><td> SpectraMax M2 </td><td> Udstyr </td></tr><tr><td> Lys / fluorescensmikroskop </td><td> Olympus </td><td> IX71 </td><td> Udstyr </td></tr><tr><td> Konfokalt mikroskop </td><td> Olympus </td><td> FV-500 laser konfokal mikroskop </td><td> Udstyr </td></tr><tr><td> Scanning Electron Microscope </td><td> Carl Zeiss MicroImaging </td><td> Leo 435 VP </td><td> Udstyr </td></tr><tr><td> Transmission Electron Microscope </td><td> JEOL </td><td> JEOL 1230 </td><td> Udstyr </td></tr></tbody></table>

engineering a bilayered hydrogel to control asc differentiation

Naturlige polymerer i årenes løb har fået større betydning på grund af deres vært biokompatibilitet og evne til at interagere med celler in vitro og in vivo. Et forskningsområde, som holder løfte regenerativ medicin er det kombinatoriske anvendelse af hidtil ukendte biomaterialer og stamceller. En grundlæggende strategi på vævet teknik er anvendelsen af ​​tredimensionale stillads (f.eks decellulariserede ekstracellulær matrix, hydrogeler, mikro / nano-partikler) til at dirigere cellefunktion. Denne teknologi har udviklet sig fra den opdagelse, at celler skal et substrat, hvorpå de kan adhærere, proliferere og udtrykke deres differentierede cellulære fænotype og funktion 2-3. For nylig er det også blevet konstateret, at cellerne ikke kun anvender disse substrater til vedhæftning, men også interagere og tage signaler fra matrixen substratet (f.eks ekstracellulær matrix, ECM) 4. Derfor cellerne og scaffolds har en gensidig forbindelse,tjener til at styre væv udvikling, organisation og ultimative funktion. Adipøst afledte stamceller (ASC&#39;er) er mesenchymal, ikke-hæmatopoietiske stamceller er til stede i fedtvæv, som kan udvise multi-linie differentiering og tjene som en let tilgængelig kilde til celler (dvs. præ-vaskulære endothel og pericytes). Vores hypotese er, at adipøst afledte stamceller kan rettes mod forskellige fænotyper samtidigt ved simpelthen co-dyrkning deraf i dobbeltlagede matricer 1. Vores laboratorium er fokuseret på huden sårheling. Til dette formål har vi skabt en enkelt sammensat matrix fra naturlige biomaterialer, fibrin, collagen og chitosan, der kan efterligne karakteristika og funktioner af en dermal-specifik sårheling ECM miljø.

Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Denne protokol fokuserer på at udnytte den iboende evne stamceller at tage udgangspunkt i det omgivende ekstracellulære matrix og kan induceres til at differentiere til flere fænotyper. Dette fremgangsmåder manuskript strækker vores beskrivelse og karakterisering af en model under anvendelse af en dobbeltlagede hydrogel bestående af PEG-fibrin og collagen, til samtidig co-differentiere adipøst afledte stamceller<sup&gt; 1</sup&gt;.

Bearbejdning af et dobbeltlag Hydrogel at styre ASC differentiering

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Research

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Bioengineering

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