Name: neonatal cardiac scaffolds: novel matrices for regenerative studies
Uploaded: 2016-11-05T14:00:00.000+00:00
Duration: 8 min 16 s
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.

<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 authors have nothing to disclose.

اعتماد هذه التقنية على تروية دموية متكررة من القلب يجعل تلافي حدوث الانسداد عنصر حاسم في تحقيق نتائج ناجحة. من القسطرة الأولية للقلب في خطوات 2،2-2،6، إلى التغييرات من حل بين خطوات 2،8-2،14، هناك التلاعب التي يمكن أن تسمح مقدمة من فقاعات الهواء التي حل وسط تدفق سائل الإرو?...

فشل القلب هو شائع وقاتل. وهو مرض التدريجي الذي يؤدي إلى انقباض انخفضت من القلب، الذي يعوق تدفق الدم إلى الأعضاء ويترك مطالب التمثيل الغذائي في الجسم تتم تلبيتها. ويقدر أن 5.7 مليون أميركي يعانون قصور القلب وهذا هو السبب الرئيسي في المستشفى في الولايات المتحدة 9. تكلفة الجماعية لعلاج المرضى في فشل القلب في الولايات المتحدة يتجاوز 300 مليار $ دولار سنويا 9-10. العلاج النهائي الوحيد للفشل نهاية المرحلة القلب هو مثلي زرع القلب. في كل عام، وتشير التقديرات إلى أن هناك حاجة إلى أكثر من 100،000 قلوب المانحة لعمليات زرع القلب في الولايات المتحدة الأمريكية 1-2. ويرجع ذلك إلى عدد محدود من الجهات المانحة، يتم تنفيذ سوى ما يقرب من 2400 عملية زراعة سنويا في الولايات المتحدة (2). بوضوح، يحتاج هذا النقص الجهاز لمعالجتها كما هو مطلوب استراتيجيات أخرى لإنتاج أجهزة إضافية لtransplantaنشوئها، ومن الناحية المثالية، هذه الأجهزة سيكون ذاتي وذلك لتجنب المضاعفات المرتبطة الرفض وكبت المناعة مدى الحياة. العضلية الكبار الثدييات يبرهن على وجود قدرة على التجدد محدودة على الاصابة لكن تشير أدلة حديثة أن قلوب الأطفال حديثي الولادة الثدييات الحفاظ على القدرة على التجدد ملحوظة بعد الاصابة 5-8. على وجه التحديد، وبعد استئصال الجراحي الجزئي، وقد تم اكتشاف نافذة التجديدي بين الولادة وبعد الولادة يوم 7. وتتميز هذه الفترة التجدد بسبب عدم وجود ندبة متليفة، وتشكيل اتساع الأوعية الدموية، وإطلاق سراح من العوامل عائية من النخاب، وcardiomyocyte انتشار 5-8 11. يوفر هذا الإطار التجدد الوقت إمكانية استخدام قلب الأطفال حديثي الولادة كمصدر جديدة من مواد لتطوير قلب bioartificial. ومن المعروف أن المصفوفة خارج الخلية لتقديم العظة هامة لتعزيز cardiomyocytالبريد انتشار والنمو. وقد استكشفت اختلافات واضحة في توافر الجزيئات في المصفوفات حديثي الولادة والبالغين 12 و قدرتها على تشجيع تجديد 13. وقد استخدمت المصفوفات الكبار Decellularized في العديد من الدراسات لتوفير سقالة ECM لإعادة تعمير الخلوي وتوليد قلب bioartificial. في حين أن هذه الدراسات والاكتشافات الجديدة في مجال تكنولوجيا الخلايا الجذعية، تحقق تقدما سريعا، لها عدة عقبات لم تتحقق بعد. على سبيل المثال، القيود في الحفاظ على بنية الأصلي للمصفوفة، والتكامل الخلوي في الجدار مصفوفة، والقدرة على دعم انتشار ونمو كل حد في نجاح هذا النهج. في حين نسبت سمات التجدد متفوقة على قلب الأطفال حديثي الولادة، والجوانب العملية لاستخدام هذه الأنسجة محدودة التنقيب. وبناء على القدرة التجديدية أثبت للقلب الأطفال حديثي الولادة، وقد وضعنا مصفوفات جديدة من خلال تطويرتقنية decellularization للقلب P3 الماوس. وقد تم اختيار قلب P3 لهذه الدراسات كما هو ضمن إطار التجديد القلب كما سبق تحديدها 6 ولكن القلب هو كبير بما يكفي لموسم الحصاد، decellularize وrecellularize. الهدف من هذه الدراسة هو إظهار جدوى إنشاء مصفوفة من قلب فأر حديثي الولادة. توفر دراستنا دليلا على جدوى decellularizing دقيقة واحدة، قلب الأطفال حديثي الولادة مع الحفاظ على السلامة الهيكلية والبروتينية إدارة المحتوى في المؤسسة. علينا أن نبرهن أيضا القدرة على recellularize هذا ECM القلب مع mCherry العضلية التعبير ولقد درست هذه العضلية للتعبير عن مختلف علامات القلب التالية recellularization. ستسمح هذه التقنية لللاختبار تفوق مصفوفة حديثي الولادة لتطوير قلب bioartificial.

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. Materials for mouse heart isolation&lt;/strong&gt;</td></tr><tr><td>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><td>60 mm Culture dish</td><td>BD Falcon</td><td>353004</td><td>or equivalent</td></tr><tr><td>Phosphate buffered saline pH 7.4 (sterile)</td><td>Hyclone</td><td>SH30256.01</td><td>or equivalent</td></tr><tr><td>Single Use Blade</td><td>Stanley</td><td>28-510</td><td>or equivalent</td></tr><tr><td>Standard Scissors</td><td>Moria Bonn (Fine Science Tools)</td><td>14381-43</td><td>or equivalent</td></tr><tr><td>Spring Scissors 10 cm</td><td>Fine Science Tools</td><td>15024-10</td><td>or equivalent</td></tr><tr><td>Vannas Spring Scissors - 3 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-00</td><td>or equivalent</td></tr><tr><td>#5 Forceps</td><td>Dumnot (Fine Science Tools)</td><td>11295-00</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;2. Materials for decellularization&lt;/strong&gt;</td></tr><tr><td>Inlet adaptor</td><td>Chemglass</td><td>CG-1013</td><td>autoclavable</td></tr><tr><td>Septum</td><td>Chemglass</td><td>CG-3022-99</td><td>autoclavable</td></tr><tr><td>1/8 in. ID x 3/8 OD C-Flex tubing</td><td>Cole-Parmer</td><td>EW-06422-10</td><td>autoclavable</td></tr><tr><td>Male luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K33</td><td>autoclavable</td></tr><tr><td>Female luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K26</td><td>autoclavable</td></tr><tr><td>Prolene 7-0 surgical suture&amp;nbsp;</td><td>Ethicon</td><td>8648G</td><td>or equivalent</td></tr><tr><td>Ring stand</td><td>Fisher Scientific</td><td>S47807</td><td>or equivalent</td></tr><tr><td>Clamp</td><td>Fisher Scientific</td><td>05-769-6Q</td><td>or equivalent</td></tr><tr><td>Clamp regular holder</td><td>Fisher Scientific</td><td>05-754Q</td><td>or equivalent</td></tr><tr><td>60 cc syringe barrel&amp;nbsp;</td><td>Coviden</td><td>1186000777T</td><td>or equivalent</td></tr><tr><td>Beaker</td><td>Kimble</td><td>14000250</td><td>or equivalent</td></tr><tr><td>22 G x 1 Syringe Needle</td><td>BD</td><td>305155</td><td>or equivalent</td></tr><tr><td>12 cc syringe</td><td>Coviden</td><td>8881512878</td><td>or equivalent</td></tr><tr><td>3-way stop cock</td><td>Smith Medical</td><td>MX5311L</td><td>or equivalent</td></tr><tr><td>PE50 tubing</td><td>BD Clay Adams Intramedic</td><td>427411</td><td>Must be formable by heat. Polyethylene recommended.</td></tr><tr><td>1% SDS</td><td>Invitrogen</td><td>15525-017</td><td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.</td></tr><tr><td>1% Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td>Make up fresh solution from a 10% stock and filter sterilize before use.&amp;nbsp;</td></tr><tr><td>Sterile dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Hyclone</td><td>SH30538.02</td><td>Or MilliQ system purified water.</td></tr><tr><td>1x Pen/Strep</td><td>Corning CellGro</td><td>30-001-Cl</td><td>or equivalent&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;3. Materials for DNA quantitation&lt;/strong&gt;</td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700</td><td>&gt;30 U/mg activity</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>or equivalent</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mallinckrodt</td><td>5958-04</td><td>or equivalent</td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1379</td><td>or equivalent</td></tr><tr><td>Tris base/hydrochloride</td><td>Sigma-Aldrich</td><td>T1503/T5941</td><td>or equivalent</td></tr><tr><td>Pico-Green dsDNA assay kit</td><td>Life Technologies&amp;nbsp;</td><td>P7589</td><td>requires fluorimeter to read</td></tr><tr><td colspan="4">&lt;strong&gt;4. Method for fixation and sectioning of tissue&lt;/strong&gt;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>or equivalent</td></tr><tr><td>Gelatin Type A from porcine skin</td><td>Sigma-Aldrich</td><td>G2500</td><td>must be 300 bloom or greater</td></tr><tr><td colspan="4">&lt;strong&gt;5. Method for tissue histology&lt;/strong&gt;</td></tr><tr><td>Cryomolds 10 x 10 x 5 mm</td><td>Tissue-Tek</td><td>4565</td><td>or equivalent</td></tr><tr><td>Cryostat</td><td>Hacker/Bright</td><td>Model OTF</td><td>or equivalent</td></tr><tr><td>Microscope Slides&amp;nbsp; 25 x 75 x 1 mm</td><td>Fisher Scientific</td><td>12-550-19</td><td>or equivalent</td></tr><tr><td>Hematoxylin 560&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801570</td><td>or equivalent</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2701</td><td>or equivalent</td></tr><tr><td>Define</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3803590</td><td>or equivalent</td></tr><tr><td>Blue buffer&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3802915</td><td>or equivalent</td></tr><tr><td>Alcoholic Eosin Y 515&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801615</td><td>or equivalent</td></tr><tr><td>Formula 83 Xylene substitute&amp;nbsp;</td><td>CBG Biotech&amp;nbsp;</td><td>CH0104B</td><td>or equivalent</td></tr><tr><td>Permount Mounting Medium&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>SP15-500</td><td>or equivalent</td></tr><tr><td>Collagen IV Antibody</td><td>Rockland</td><td>600-401-106.1</td><td>or equivalent</td></tr><tr><td>&amp;alpha;-Actinin Antibody</td><td>Abcam</td><td>AB9465</td><td>or equivalent</td></tr><tr><td>mCherry Antibody</td><td>Abcam</td><td>AB205402</td><td>or equivalent</td></tr><tr><td>NKX2.5 Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-8697</td><td>or equivalent</td></tr><tr><td>Donkey anti-mouse AF488 Antibody</td><td>Life Technology</td><td>A21202</td><td>or equivalent</td></tr><tr><td>Donkey anti-chicken AF594 Antibody</td><td>Jackson Immunoresearch</td><td>703-585-155&amp;nbsp;</td><td>or equivalent</td></tr><tr><td>Donkey anti-goat CY5 Antibody</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>705-175-147</td><td>or equivalent</td></tr><tr><td>Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td><td>Jackson Immunoresearch</td><td>111-587-003</td><td>or equivalent</td></tr><tr><td>Prolong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36930</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;6. Isolation of neonatal ventricular cardiomyocytes using pre-plating&lt;/strong&gt;</td></tr><tr><td>HBSS (Ca, Mg Free)</td><td>Hyclone</td><td>SH30031.02</td><td>or equivalent</td></tr><tr><td>HEPES (1 M)</td><td>Corning CellGro</td><td>25-060-Cl</td><td>or equivalent</td></tr><tr><td>Cell Strainer</td><td>BD Falcon</td><td>352340</td><td>or equivalent</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>or equivalent</td></tr><tr><td>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><td>Trypsin</td><td>Difco</td><td>215240</td><td>or equivalent</td></tr><tr><td>DNase II</td><td>Sigma-Aldrich</td><td>D8764</td><td>or equivalent</td></tr><tr><td>DMEM (Delbecco&#039;s Minimal Essential Media)</td><td>Hyclone</td><td>SH30022.01</td><td>or equivalent</td></tr><tr><td>Vitamin B12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>V6629</td><td>or equivalent</td></tr><tr><td>Fibronectin coated plates&amp;nbsp;</td><td>BD Bioscience</td><td>354501</td><td>or equivalent</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Hyclone</td><td>SH30910.03</td><td>or equivalent</td></tr><tr><td>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.

<html> Decellularization في المتوسط، والوقت لdecellularization من قلب P3 باستخدام هذا البروتوكول ما يقرب من 14 ساعة. يعطى متوسط ​​وزن القلب من 23 ملغ لحديثي الولادة P3. Acellularity يوضح الشكل...

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

في هذه الدراسات، ونحن نقدم منهجية للرواية، حديثي الولادة، السقالات القلب الفئران للاستخدام في الدراسات التجدد.

حديثي الولادة القلب السقالات: مصفوفات رواية للدراسات التجدد

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

neonatal-cardiac-scaffolds-novel-matrices-for-regenerative-studies

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.7K Views.  University of Minnesota.  في هذه الدراسات، ونحن نقدم منهجية للرواية، حديثي الولادة، السقالات القلب الفئران للاستخدام في الدراسات التجدد. 

Video: Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

الشعرية قوة الطباعة الحجرية لهندسة الأنسجة القلبية

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

هندسة الأنسجة: بناء ومتعددة الخلايا 3D سقالة لتسليم الطبقات صفائح خلية

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

تلفيق عضلي يبني الأنسجة المهندسة

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Biology

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

ديسيلولاريزيشن مماثلة لانسجة القلب الجنين والكبار Explants كمثل 3D منهاجي في المختبر الدراسات

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

أسلوب صافي المستندة إلى القالب لإنشاء أنسجة القلب خالية من السقالة ثلاثي الأبعاد

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

تصنيع السقالات مصفوفة ديسيلولاريزيد المستمدة من الغضروف

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

إعداد الأنسجة القلبية المهندسة على شكل شبكة مشتقة من خلايا iPS البشرية لإصلاح عضلة القلب في فيفو

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

توليد ، فحص عالي الإنتاجية ، وبنوك حيوية للكرويات القلبية المشتقة من الخلايا الجذعية متعددة القدرات التي يسببها الإنسان

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

3D توليد أنسجة عضلة القلب البشرية باستخدام الكتابة الكهربائي الذائب لسقالات بولي كابرولاكتون وخلايا القلب المشتقة من hiPSC

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

زراعة القلب في الجسم الحي لدراسة الانتقال البطاني إلى اللحمة المتوسطة

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

تسليط الضوء على المؤلف: نموذج زرع قلب الفئران غير المتجانسة لحديثي الولادة لدراسة الانتقال البطاني إلى اللحمة المتوسطة  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

نموذج زراعة قلب الفئران غير المتجانسة لحديثي الولادة لدراسة الانتقال البطاني إلى اللحمة المتوسطة

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...