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.

All mouse experiments were performed in accordance with US Animal Welfare Act and were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.
1. Method for Mouse Heart Isolation
<ol>
	<li>Euthanize a neonatal mouse by decapitation with a single use blade.</li>
	<li>Swab the thorax with 70% ethanol.</li>
	<li>Dissect the skin from the chest by cutting it away from the chest wall with standard scissors while pulling the skin laterally with a pair of #5 forceps.</li>
	<li>Perfor...

<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>

The dependence of this technique on repeated perfusions of the heart makes the avoidance of an embolism a critical component of a successful outcome. From the initial catheterization of the heart in Steps 2.2-2.6, to the changes of solution between Steps 2.8-2.14, there are manipulations that can allow introduction of air bubbles which compromise the flow of perfusate into the myocardium. Due to the diminutive size of the neonatal heart, even minute bubbles in the vasculature can cause a technical infarct, thus rendering...

Heart failure is common and deadly. It is a progressive disease that results in decreased contractility of the heart, which impairs blood flow to organs and leaves the metabolic demands of the body unmet. It is estimated that 5.7 million Americans have heart failure and it is the primary cause of hospitalization in the United States9. The collective cost of treating patients in heart failure in the United States exceeds $300 billion dollars per year 9-10. 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. Clearly, this organ shortage needs to be addressed as other strategies are 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.
Mammalian adult cardiomyocytes demonstrate a limited regenerative capacity upon injury but recent evidence suggests that mammalian neonatal hearts maintain a remarkable regenerative capacity following injury5-8. Specifically, following partial surgical resection, a regenerative window has been discovered between birth and postnatal day 7. This regenerative period is characterized by a lack of fibrotic scar, formation of neovascularization, release of angiogenic factors from the epicardium, and cardiomyocyte proliferation 5-8,11. This regenerative window of time provides the potential for using the neonatal heart as a novel source of material for the development of a bioartificial heart.
The extracellular matrix is known to provide important cues to promote cardiomyocyte proliferation and growth. Distinct differences in the availability of molecules in the neonatal and adult matrices12 and their ability to promote regeneration have been explored13. Decellularized adult matrices have been used in several studies to provide an ECM scaffold for cellular repopulation and the generation of a bioartificial heart. While these studies, and new discoveries in stem cell technologies, are advancing rapidly, several hurdles have yet to be met. For example, limitations in preserving native structure of the matrix, cellular integration into the matrix wall, and ability to support proliferation and growth all limit the success of this approach. While superior regenerative attributes have been ascribed to the neonatal heart, the practical aspects of using such a tissue have limited its exploration.
Based on the demonstrated regenerative capacity of the neonatal heart, we have developed novel matrices by developing a technique of decellularization for the P3 mouse heart. The P3 heart was chosen for these studies as it is within the window of cardiac regeneration as previously determined6 but the heart is large enough to harvest, decellularize and recellularize. The goal of this study is to demonstrate the feasibility of creating a matrix from a neonatal mouse heart. Our studies provide evidence for the feasibility of decellularizing a minute, neonatal heart while maintaining the structural and proteinaceous integrity of the ECM. We also demonstrate the ability to recellularize this cardiac ECM with mCherry expressing cardiomyocytes and we have examined these cardiomyocytes for expression of various cardiac markers following recellularization. This technology will allow for the testing of the superiority of a neonatal matrix for the development of a bioartificial heart.

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	<tbody>
		<tr height="21">
			<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.

Decellularization 
On average, the time to decellularization of a P3 heart using this protocol is approximately 14 hr. given an average heart weight of 23 mg for the P3 neonate.
Acellularity 
Figure 3a demonstrates a fully intact P3 neonatal heart (whole mount). Figure 3b shows the same heart following decellularization. Figures 4a and 4b show the hema...

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 these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in 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

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Bioengineering

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Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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

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