Name: engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization
Uploaded: 2013-10-28T21:45:00
Description: Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Denne undersøgelse blev udført i forskningsprogrammet for de biomedicinske Materialer (BMM) anlægge. BMM er medfinansieret af det hollandske økonomiministerium, landbrug og innovation. Det finansielle bidrag fra Nederlandse Hartstichting er taknemmeligt anerkendt.

1.. Kultur for Human Vena Saphena Cells <ol><li> Isoler celler fra vena saphena magna, erhvervet fra en donor i overensstemmelse med retningslinjer for sekundær anvendelse materiale i henhold til protokollen af Schnell et al. 9 og gemme disse i flydende nitrogen. Fra den del af vena saphena magna fra en donor afskårne stykker på 2 x 2 mm til kulturen i en seks-brønds plade. Brug 2 stykker per brønd. Generelt nok celler kan fås til at fylde omkring 3 hætteglas med 0,25 x 10 6 celle...

<ol>
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M., 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=Optimal+cell+source+for+cardiovascular+tissue+engineering:+venous+vs.+aortic+human+myofibroblasts.">Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.</a> Thorac. Cardiovasc. Surg. 49, 221-225 (2001).</li><li>Mol, A., 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=Autologous+human+tissue-engineered+heart+valves:+prospects+for+systemic+application.">Autologous human tissue-engineered heart valves: prospects for systemic application.</a> Circulation. , I152-I158 (2006).</li><li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16, 3261-3270 (2010).</li><li>John, J., Quinlan, A. T., Silvestri, C., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boundary+stiffness+regulates+fibroblast+behavior+in+collagen+gels.">Boundary stiffness regulates fibroblast behavior in collagen gels.</a> Ann. Biomed. Eng. 38, 658-673 (2010).</li><li>Rubbens, M. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41, 763-774 (2013).</li><li>Merryman, W. 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=Correlation+between+heart+valve+interstitial+cell+stiffness+and+transvalvular+pressure:+implications+for+collagen+synthesis.">Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen synthesis.</a> Am. J. Physiol. 290, (2006).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+mechanotransduction+to+biologically+inspired+engineering:+2009+Pritzker+Award+Lecture,+BMES+Annual+Meeting+October+10,+2009.">From cellular mechanotransduction to biologically inspired engineering: 2009 Pritzker Award Lecture, BMES Annual Meeting October 10, 2009.</a> Ann. Biomed. Eng. 38, 1148-1161 (2009).</li><li>Sander, E. A., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39, 714-729 (2010).</li><li>van der Schaft, D. W., 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=Engineering+Skeletal+Muscle+Tissues+from+Murine+Myoblast+Progenitor+Cells+and+Application+of+Electrical+Stimulation.">Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation.</a> J. Vis. Exp. (73), e4267 (2013).</li></ol>

Det beskrevne modelsystem celle befolkede fibrin konstruktioner har et stort potentiale for studiet af cellen og kollagen (re) organisation (de Jonge et al. 15), fx skal anvendes til tissue engineering formål. Ved hjælp fibrin som den oprindelige celle luftfartsselskab, efter fibrin nedbrydning er et væv skabt med celler og endogent matrix alene. På denne måde celler stimuleres til at reagere på stamme enten statisk eller cyklisk karakter, ved at anvende kontraktile kræfter 16,17...

Kardiovaskulære væv har en fremtrædende bærende funktion. Især indhold og tilrettelæggelse af kollagen fibre i den ekstracellulære matrix bidrager til de bærende egenskaber og dominere den samlede væv styrke 1.. I tissue engineering mekanisk konditionering af konstruktionen bliver brugt - typisk bestående af (cyklisk) filterdug regimer - at styrke væv organisation og mekaniske egenskaber 2,3. Fuld forståelse af spændingsinduceret collagen organisation i komplekse væv geometrier at skabe væv med foruddefineret collagen arkitektur er endnu ikke nået. Dette er primært på grund af vores begrænsede viden om collagen remodeling i udviklingslandene væv. Eksisterende modeller hovedsageligt give information om det endelige netto resultat af collagen remodeling med brug af statisk belastning 4-6. Her giver vi et meget tunable model system, der tillader studiet af kollagen (re) organisation i en real-time mode, i 3D, under indflydelsestatisk eller cyklisk belastning. De vævskonstruktioner er fibrin-baserede, sikre, at alle kollagen i konstruktionen er endogent. Celle-og collagen organisation i konstruktionerne visualiseres, og en intern referencesystem tillader os at flytte celler og kollagen strukturer for time-lapse analyse. I denne protokol vil vi beskrive anvendelsen af modellen for humane Vena saphena Cells (HVSCs), eftersom disse celler er kendt for deres forbedrede ekstra cellulær matrix produktion og baseret evne til at omforme matrixen og vores etablerede anvendelse i manipuleret kardiovaskulære væv 7, på arbejdet i de Jonge et al. 8.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Kollagen indhold og organisation i udvikling kollagene væv kan påvirkes af lokale væv stammer og væv tvang. Tissue ingeniører til formål at anvende disse principper for at skabe væv med foruddefinerede kollagen arkitekturer. En fuld forståelse af de nøjagtige underliggende processer af kollagen remodeling at kontrollere den endelige vævsarkitekturen imidlertid mangler. Især er lidt kendt om (re) orientering af collagenfibre i reaktion på ændringer i væv mekaniske belastningsforhold. Vi udviklede et in vitro modelsystem bestående af biaksialt med begrænset myofibroblast-udsåede fibrin konstruktioner, til yderligere at belyse kollagen (gen) orientering i respons på i) vende tilbage biaksial til enaksede statiske belastningsforhold og ii) cyklisk enakset belastning af begrænsede biaksialt konstruktioner før og efter en ændring i lastning retning med brug af Flexcell FX4000T læsseindretningen. Time-lapse konfokal billeddannelse anvendes til VIsualize kollagen (gen) orientering i en destruktiv måde. Celle-og collagen organisation i konstruktionerne kan visualiseres i realtid, og en intern referencesystem tillader os at flytte celler og kollagen strukturer for time-lapse analyse. Forskellige aspekter af modellen systemet kan justeres, ligesom celle kilde eller anvendelse af sunde og syge celler. Tilsætningsstoffer kan anvendes til yderligere at belyse mekanismer underliggende collagen remodeling, ved for eksempel at tilføje MMP&#39;er eller blokering integriner. Form og størrelse af konstruktionen kan let tilpasses særlige behov, hvilket resulterer i en meget afstemmelige modelsystem til at studere celle og kollagen (re) organisation.

Denne model system giver mulighed for dyrkning myofibroblast-podede fibringeler. 1A viser et væv dyrket først under statiske biaksiale begrænsninger. Tissue begrænsninger frigives ved at skære fibringel fra to begrænsninger, for at skabe enaksede statiske begrænsninger og væv komprimerer og remodels bagefter (figur 1A). Til cyklisk belastning, er vævet dyrkes under statiske biaksiale begrænsninger samt. Efter 5 dages cyklisk enakset stamme kan anvendes (figur 1B).</str...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Denne model system starter fra en myofibroblast befolkede fibringel, der kan bruges til at studere endogent collagen (re) organisation realtid på en destruktiv måde. Modelsystemet er meget afstemmelige, da det kan bruges med forskellige cellekilder, mellemstore tilsætningsstoffer, og nemt kan tilpasses til særlige behov.

Engineering Fibrin-baserede vævskonstruktioner fra myofibroblasts og Application of Begrænsninger og stamme at fremkalde Cell og kollagen (Re) Organisation

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

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The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...