Name: neonatal cardiac scaffolds: novel matrices for regenerative studies
Uploaded: 2016-11-05T14:00:00
Description: Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.

Alle muizen experimenten werden uitgevoerd in overeenstemming met de Amerikaanse Animal Welfare Act en werden aan de Universiteit van Minnesota door de Institutional Animal Care en gebruik Comite goedgekeurd. 1. Werkwijze voor muizenhart Isolation <ol><li> Euthanaseren een neonatale muis door onthoofding met een eenmalig gebruik mes. </li><li> Zwabberen de thorax met 70% ethanol. </li><li> Ontleden de huid van de borst door snijden vanaf de borstwand met standaard schaar en trek de huid lateraal met een paar v...

<ol>
	<li>Yusen, R. 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=Registry+of+the+International+Society+for+Heart+and+Lung+Transplantation:+Thirty-second+official+adult+lung+and+heart-lung+transplantation+report--2015.">Registry of the International Society for Heart and Lung Transplantation: Thirty-second official adult lung and heart-lung transplantation report--2015.</a> J Heart Lung Transplant. 34 (10), 1264-1277 (2015).</li><li>Go, A. 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=Heart+disease+and+stroke+statistics--2014+update:+A+report+from+the+american+heart+association.">Heart disease and stroke statistics--2014 update: A report from the american heart association.</a> Circulation. 129 (3), e28-e292 (2014).</li><li>Kapelios, C. J., Nanas, J. N., Malliaras, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+cardiosphere-derived+cells+for+myocardial+regeneration:+current+progress+and+recent+results.">Allogeneic cardiosphere-derived cells for myocardial regeneration: current progress and recent results.</a> Future Cardiol. 12 (1), 87-100 (2016).</li><li>Ott, H. C., 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=Perfusion+decellularized+matrix:+Using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion decellularized matrix: Using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14 (2), 213-221 (2008).</li><li>Porrello, E. R., Olson, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+blueprint+for+cardiac+regeneration.">A neonatal blueprint for cardiac regeneration.</a> Stem Cell Research. 13 (3 Pt B), 556-570 (2014).</li><li>Porrello, E. R., 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=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331 (6020), 1078-1080 (2011).</li><li>Polizzotti, B. 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=Neuregulin+stimulation+of+cardiomyocyte+regeneration+in+mice+and+human+myocardium+reveals+a+therapeutic+window.">Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window.</a> Sci Transl Med. 7 (281), 281ra45 (2015).</li><li>Jesty, S. 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=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109 (33), 13380-13385 (2012).</li><li>Ambrosy, A. P., 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=The+Global+Health+and+Economic+Burden+of+Hospitalizations+for+Heart+Failure:+Lessons+Learned+From+Hospitalized+Heart+Failure+Registries.">The Global Health and Economic Burden of Hospitalizations for Heart Failure: Lessons Learned From Hospitalized Heart Failure Registries.</a> J Am Coll Cardiol. 63 (12), 1123-1133 (2014).</li><li>Roger, V. L., 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=Executive+Summary:+Heart+Disease+and+Stroke+Statistics-2012+Update+A+Report+From+the+American+Heart+Association.">Executive Summary: Heart Disease and Stroke Statistics-2012 Update A Report From the American Heart Association.</a> Circulation. 125 (22), 188-197 (2012).</li><li>Kennedy-Lydon, T., Rosenthal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration:+epicardial+mediated+repair.">Cardiac regeneration: epicardial mediated repair.</a> Proc R Soc B. 282 (1821), 2147-2172 (2015).</li><li>Williams, C., Sullivan, K., Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partially+Digested+Adult+Cardiac+Extracellular+Matrix+Promotes+Cardiomyocyte+Proliferation+In+Vitro.">Partially Digested Adult Cardiac Extracellular Matrix Promotes Cardiomyocyte Proliferation In Vitro.</a> Adv Healthcare Mat. 4 (10), 1545-1554 (2015).</li><li>Borg, T. 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=Recognition+of+extracellular+matrix+components+by+neonatal+and+adult+cardiac+myocytes.">Recognition of extracellular matrix components by neonatal and adult cardiac myocytes.</a> Dev Biol. 104 (1), 86-96 (1984).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immnol. 111, A3-B1-3 (2015).</li><li>Gilbert, T. W., Freund, J. M., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+DNA+in+biologic+scaffold+materials.">Quantification of DNA in biologic scaffold materials.</a> J Surg Res. 152 (1), 135-139 (2009).</li><li>Akhyari, P., 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=The+quest+for+an+optimized+protocol+for+whole-heart+decellularization:+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities.">The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.</a> Tissue Eng Part C Methods. 17 (9), 915-926 (2011).</li><li>Lu, T. Y., 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=Repopulation+of+decellularized+mouse+heart+with+human+induced+pluripotent+stem+cell-derived+cardiovascular+progenitor+cells.">Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells.</a> Nat Commun. 4 (2307), 1-11 (2013).</li></ol>

De afhankelijkheid van deze techniek bij herhaalde perfusie van het hart maakt het vermijden van een embolie een essentieel onderdeel van een succesvol resultaat. Vanaf de eerste catheterisatie van het hart in de stappen 2,2-2,6, om de veranderingen van de oplossing tussen Steps 2,8-2,14, zijn er manipulaties dat de invoering van luchtbellen die afbreuk doen aan de stroom van perfusaat in de hartspier kan toestaan. Vanwege de kleine omvang van de neonatale hart, kunnen zelfs minuut bellen in het vaatstelsel een technisc...

Hartfalen komt vaak voor en dodelijk. Het is een progressieve ziekte die resulteert in verlaagde contractiliteit van het hart, die bloedtoevoer aan organen schaadt en laat de metabolische behoeften van het lichaam onvervulde. Men schat dat 5,7 miljoen Amerikanen hebben hartfalen en het is de voornaamste oorzaak van ziekenhuisopname in de Verenigde Staten 9. De gezamenlijke kosten van de behandeling van patiënten bij hartfalen in de Verenigde Staten meer dan $ 300.000.000.000 dollar per jaar 9-10. De enige definitieve therapie voor eindstadium hartfalen is orthotope harttransplantatie. Elk jaar, wordt geschat dat meer dan 100.000 donorharten zijn nodig voor harttransplantatie procedures in de Verenigde Staten 1-2. Vanwege het beperkte aantal donoren, worden slechts bij benadering 2400 transplantaties jaarlijks uitgevoerd in de VS 2. Het is duidelijk dat dit tekort aan organen moet worden aangepakt als andere strategieën nodig zijn om extra organen voor transplanta producerentie en idealiter zou deze organen autoloog zijn om de complicaties van afstoting en levensduur immuunsuppressie voorkomen. Zoogdieren volwassen hartspiercellen aantonen dat er een beperkte regeneratieve capaciteit op schade, maar recent bewijs suggereert dat zoogdieren neonatale harten handhaven van een opmerkelijke regeneratieve capaciteit na letsel 5-8. Specifiek, na gedeeltelijke chirurgische resectie, een regeneratieve venster is ontdekt tussen de geboorte en postnatale dag 7. Deze regeneratieve periode wordt gekenmerkt door een gebrek aan fibrotische littekenvorming, vorming van neovascularisatie, afgifte van angiogene factoren uit het epicard en cardiomyocyt proliferatie 5-8 , 11. Deze regeneratieve tijdvenster biedt het potentieel voor het gebruik van het neonatale hart als een nieuwe bron van materiaal voor de ontwikkeling van een biokunstmatige hart. De extracellulaire matrix is ​​bekend dat belangrijke signalen te verschaffen cardiomyocyt bevorderene proliferatie en groei. Duidelijke verschillen in de beschikbaarheid van moleculen in de neonatale en volwassen matrices 12 en hun vermogen om regeneratie te bevorderen werden onderzocht 13. Volwassen cellen ontdane matrices zijn gebruikt in verscheidene studies een ECM matrix voor cellulaire herbevolking en het genereren van een bioartificiële hart verschaffen. Hoewel deze studies, en nieuwe ontdekkingen in stamcellen technologieën, zijn snel voortschrijdende, verschillende hindernissen moeten nog worden voldaan. Bijvoorbeeld beperkingen bij het behoud natieve structuur van de matrix, cellulaire integratie in de matrix wand, en het vermogen om proliferatie en groei ondersteunen alle grens het succes van deze benadering. Terwijl superieure regeneratieve eigenschappen zijn toegeschreven aan de neonatale hart, zijn de praktische aspecten van het gebruik van een dergelijk weefsel zijn verkenning beperkt. Gebaseerd op de aangetoonde regeneratieve capaciteit van het neonatale hart, hebben we nieuwe matrices ontwikkeld door de ontwikkeling van eentechniek van decellularisatie voor de P3 muis hart. Het P3 hart werd gekozen voor deze studies aangezien het binnen het raam van cardiale regeneratie voorafbepaalde 6 maar het hart is groot genoeg te oogsten, decellularize en recellularize. Het doel van deze studie is om de haalbaarheid van het creëren van een matrix van een neonatale muis hart te tonen. Onze studies leveren het bewijs voor de haalbaarheid van decellularizing een minuut, neonatale hart met behoud van de structurele en eiwithoudende integriteit van de ECM. We demonstreren ook de mogelijkheid om deze cardiale ECM mCherry expressie cardiomyocyten recellularize en wij hebben deze cardiomyocyten onderzocht op expressie van verschillende merkers cardiale volgende hercellularisatie. Deze technologie zal voor het testen van de superioriteit van een neonatale matrix voor de ontwikkeling van een biokunstmatige hart.

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			<td height="21" style="height:21px;width:451px;">1. Materials For Mouse Heart Isolation</td>
			<td style="width:323px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)</td>
			<td>Jackson Laboratories</td>
			<td>21577</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm Culture dish</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline pH 7.4 (sterile)</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Use Blade</td>
			<td>Stanley</td>
			<td>28-510</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Scissors</td>
			<td>Moria Bonn (Fine Science Tools)</td>
			<td>14381-43</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors 10 cm</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5 Forceps</td>
			<td>Dumnot (Fine Science Tools)</td>
			<td>11295-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Materials For Decellularization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inlet adaptor</td>
			<td>Chemglass</td>
			<td>CG-1013</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Septum</td>
			<td>Chemglass</td>
			<td>CG-3022-99</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/8 in. ID x 3/8 OD C-Flex tubing</td>
			<td>Cole-Parmer&#160;</td>
			<td>EW-06422-10</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K33</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K26</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolene 7-0 surgical suture&#160;</td>
			<td>Ethicon</td>
			<td>8648G</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring stand</td>
			<td>Fisher Scientific</td>
			<td>S47807</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp</td>
			<td>Fisher Scientific</td>
			<td>05-769-6Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp regular holder</td>
			<td>Fisher Scientific</td>
			<td>05-754Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 cc syringe barrel&#160;</td>
			<td>Coviden</td>
			<td>1186000777T</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beaker</td>
			<td>Kimble</td>
			<td>14000250</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22g x 1 Syringe Needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 cc syringe&#160;</td>
			<td>Coviden</td>
			<td>8881512878</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stop cock&#160;&#160;</td>
			<td>Smith Medical&#160;</td>
			<td>MX5311L</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22 x 1 g needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE50 tubing&#160;</td>
			<td>BD Clay Adams Intramedic</td>
			<td>427411</td>
			<td>Must be formable by heat. Polyethylene recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% SDS&#160;</td>
			<td>Invitrogen</td>
			<td>15525-017</td>
			<td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Make up fresh solution from a 10% stock and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile dH2O</td>
			<td>Hyclone</td>
			<td>SH30538.02</td>
			<td>Or MilliQ system purified water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Pen/Strep&#160;</td>
			<td>Corning CellGro</td>
			<td>30-001-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3. Materials For DNA Quantitation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K&#160;</td>
			<td>Fisher</td>
			<td>BP1700</td>
			<td>&#62;30U/mg activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl*6H2O</td>
			<td>Mallinckrodt</td>
			<td>5958-04</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base/hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T1503/T5941</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pico-Green dsDNA assay kit</td>
			<td>Life Technologies&#160;</td>
			<td>P7589</td>
			<td>requires fluorimeter to read</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4. Method for fixation and sectioning of tissue.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin Type A from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td>must be 300 bloom or greater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5. Method for tissue histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomolds 10 x 10 x 5mm</td>
			<td>Tissue-Tek</td>
			<td>4565</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Hacker/Bright</td>
			<td>Model OTF</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides&#160; 25 x 75 x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-550-19</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin 560&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801570</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Define</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3803590</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue buffer&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3802915</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcoholic Eosin Y 515&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801615</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formula 83 Xylene substitute&#160;</td>
			<td>CBG Biotech&#160;</td>
			<td>CH0104B</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount Mounting Medium&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>SP15-500</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Antibody</td>
			<td>Rockland</td>
			<td>600-401-106.1</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Actinin Antibody</td>
			<td>Abcam</td>
			<td>AB9465</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mCherry Antibody</td>
			<td>Abcam</td>
			<td>AB205402</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NKX2.5 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-8697</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-mouse AF488 Antibody</td>
			<td>Life Technology</td>
			<td>&#160;A21202&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-chicken AF594 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>703-585-155&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-goat CY5 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>705-175-147</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>111-587-003&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (Ca, Mg Free)</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Corning CellGro</td>
			<td>25-060-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primeria 100 mm plates</td>
			<td>Corning</td>
			<td>353803</td>
			<td>Primeria surface enhances fibroblast attachment promoting a higher myocyte purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Difco</td>
			<td>215240</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase II</td>
			<td>Sigma-Aldrich</td>
			<td>D8764</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Delbecco&#39;s Minimal Essential Media)</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vitamin B12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V6629</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin coated plates&#160;</td>
			<td>BD Bioscience</td>
			<td>354501</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heart bioreactor glassware</td>
			<td>Radnoti Glass Technology</td>
			<td>120101BEZ</td>
			<td>Must be sterilizable by autoclaving or gas.</td>
		</tr>
	</tbody>
</table>

neonatal cardiac scaffolds: novel matrices for regenerative studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

decellularisatie Gemiddeld is de tijd decellularisatie P3 van een hart met dit protocol is ongeveer 14 uur. een gemiddelde hart gewicht van 23 mg voor de P3 pasgeborene. Acellularity Figuur 3a toont een volledig intacte P3 neonatale hart (hele mount). Figuur 3b toont dezelfde hart volgende cellen ontdoen. De figuren 4a en 4b de hematoxyline en eo...

Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

In deze studies geven we nieuwe methodologie voor neonatale muizen cardiale scaffolds voor gebruik in regeneratieve studies.

Neonatale Cardiac Steigers: Novel Matrices voor Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Elastomeer PGS Steigers in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Dichtheidsgradiënt Meerlaagse polymerisatie (dGMP): een nieuwe techniek voor het maken van meerdere compartimenten, Aanpasbare Steigers for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

A Novel Driedimensionale Flow Kamer Device aan chemokine-gerichte extravasatie van Cellen doorgeven Studie onder Fysiologische stroomtoestanden

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Harmonic Nanoparticles for Regenerative Research

Harmonische Nanodeeltjes voor Regeneratieve Onderzoek

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Electrospun Nanovezel Steigers met gradaties in Fiber Organisatie

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Imaging Cell Viability on Non-transparent Scaffolds ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â Using the Example of a Novel Knitted Titanium Implant

Imaging levensvatbaarheid van de cellen op niet-transparante Steigers - Met behulp van het voorbeeld van een Novel Gebreide Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Novel Process for 3D Printing Decellularized Matrices

Nieuwe proces voor afdrukken in 3D Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

Onderling verbonden macroporeuze 3D-steigers van Microgel-staven

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

A Novel Cell Injection Method with Minimum Invasion (Video) | JoVE 

Een nieuwe celinjectiemethode met minimale invasie

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Evaluation of Hydrodynamic Properties in Mounted Porcine Aortic Valves

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

Advancing Cardiac Procedure Testing Prior to Embarking on Large Animal Studies (Video) | JoVE

Een ex vivo varkensmodel voor hydrodynamisch testen van experimentele aortaklepprocedures en nieuwe medische hulpmiddelen

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...