Diese Forschung wird durch das Basic Science Research Program von der National Research Foundation of Korea (NRF) unterstützt, das vom Bildungsministerium (NRF-2018R1D1A1A02049346) gefördert wird.

Alle Tierforschungsverfahren wurden in Übereinstimmung mit dem Laboratory Animals Welfare Act, dem Guide for the Care and Use of Laboratory Animals und den Richtlinien und Richtlinien für Nagetierexperimente bereitgestellt, die vom Institutional Animal Care and Use Committee (IACUC) in der School of Medicine der Katholischen Universität Korea zur Verfügung gestellt wurden.
1. Herstellung von MSCs und injizierbaren Gelatinehydrogelen
<ol><li>Kultur MSCs in einem 100 mm Kulturgericht bei 37 °C und 5%CO2. Wenn das MSCs-Wachstum 80 % Zusammenfluss erreicht, waschen Sie die Schale zweimal mit DPBS und fügen Sie 1 ml Trypsin-Ersatz bei 37 °C für 3 min hinzu. ANMERKUNG: MSCs wurden nach herkömmlichen Verfahren16aus dem murinen Knochenmark isoliert, in Dulbeccos Modified Eagle es Medium (DMEM) kultiviert, das 10% fetales Rinderserum (FBS) und 1% Antimykotische Lösung enthält, und zwischen Durchgang 7-u20129 für diese Studie verwendet.</li>
	<li>Fügen Sie 9 ml Kulturmedium und Zentrifuge bei 500 x g für 3 min hinzu. Als Nächstes entsorgen Sie den resultierenden Überstand, setzen Sie die Zellen in 1 ml PBS wieder auf und halten Sie die Zellsuspension auf Eis aufrecht.</li>
	<li>Verdünnen Sie 10 l Zellsuspension mit 10 l Trypan-Blau und erhalten Sie die Zellkonzentration mit einem automatisierten Zellzähler.</li>
	<li>MSCs mit einer Dichte von 1 x 107 Zellen/ml auf ein 1 ml-Rohr zurücksetzen und übertragen.</li>
	<li>Bereiten Sie eine 6,25 Gew. GH-Konjugatlösung in PBS vor und trennen Sie sie in 2 Durchstechflaschen. Als nächstes mischen Sie die GH-Lösungen entweder mit 6 g/ml HRP (GH-Lösung A) oder 0,07 Gew. H2O2 (GH-Lösung B). HINWEIS: Bereiten Sie Gelatine-Hydroxyphenylpropionsäure (GH) Konjugate nach den veröffentlichten Protokollen12,15vor.<ol>
<li>Bewahren Sie ein 9:1 Volumetric Ratio der GH-Konjugatlösung zu HRP (GH-Lösung A) bzw. GH-Konjugatlösung zuH2O2 (GH-Lösung B) auf.</li>
	</ol>
</li>
	<li>Vor dem Mischen der MSCs mit GH-Lösung A die Zellsuspension kurz zentrieren bei 1.000 x g und den resultierenden Überstand vorsichtig ansaugen. Anschließend das Pellet, das MSCs enthält, mit GH-Lösung A mischen.</li>
</ol>2. In situ MSC-Lade- und dreidimensionale In-vitro-Kultur
<ol><li>Laden Sie GH-Lösung A (mit MSCs) und GH-Lösung B in beide Seiten einer Doppelspritze. Platte 300 l der kombinierten GH-Lösungen mit MSCs mit einer Enddichte von 5 x 106 Zellen/ml auf einem Acht-Well-Kammerschlitten.</li>
	<li>Nach der In-situ-Hydrogelbildung und der anschließenden MSC-Verkapselung über enzymatische Vernetzung, fügen Sie 700 l DMEM mit 10% FBS und 1% antimykotischer Lösung hinzu.</li>
	<li>Inkubieren Sie die Rutsche bei 37 °C und 5%CO2 und ersetzen Sie das Kulturmedium alle 2-u20123 Tage.</li>
</ol>3. Bestätigung der In-vitro-Proliferation und des Überlebens von MSCs in GH-Hydrogelen
<ol><li>Um die Lebensfähigkeit von 3D-kultivierten MSCs innerhalb von GH-Hydrogelen zu bestimmen, verwenden Sie einen Live/Dead Cell Staining Assay nach der vorgegebenen Inkubationszeit.</li>
	<li>Nach inkubation der gekapselten MSCs in GH-Hydrogelen für 3, 5, 7 oder 14 Tage, das Medium ansaugen und den Brunnen zweimal mit PBS waschen.</li>
	<li>Bereiten Sie eine Färbelösung vor, die 5 l Calcein AM und 20 l Ethidium-Homodimer-1 (EthD-1) in 10 ml DPBS enthält.</li>
	<li>Fügen Sie 200 l der Färbelösung in den Brunnen und inkubieren für 30 min im Dunkeln bei Raumtemperatur.</li>
	<li>Die Färbelösung ansaugen und den Brunnen zweimal mit PBS waschen.</li>
	<li>Trennen Sie die Kammer vorsichtig von der Rutsche und legen Sie einen vollständigen Deckelüberschlag über die GH-Hydrogele. Verwenden Sie eine konfokale Mikroskopie, um den Grad der Proliferation und morphologischen Veränderungen der gekapselten MSCs zu visualisieren. HINWEIS: Fluoreszierende Bilder wurden unter 200-facher Vergrößerung aufgenommen und bei den Anregungs-/Emissionswellenlängen von 470/540 nm für Calcein und 516/607 nm für EthD-1 abgebildet.</li>
</ol>4. Induktion des Myokardinfarkts bei Mäusen
<ol><li>Anästhetisieren Sie 7 Wochen alte männliche C57BL/6-Mäuse (20-u201222 g) mit intraperitonealer Injektion einer Mischung aus Zoletil (30 mg/kg) und Rompun (10 mg/kg) in Saline.</li>
	<li>Vor der Operation, depilate die Brust der Maus mit Haarentfernung Creme und sterilisieren die Haut mit Jod.</li>
	<li>Legen Sie die Maus auf einen Operationstisch und intubieren Sie, indem Sie einen Katheter in die Luftröhre einsetzen, um zusätzlichen Sauerstoff über mechanische Belüftung bereitzustellen.</li>
	<li>Mit einer chirurgischen Schere vorsichtig durch die Haut schneiden und dann mit einer Mikroschere in die Interkostalmuskulatur eindringen. Trennen Sie die zweite und dritte linke Rippe mit einer 5-0 Seidennaht, um eine offene Brusthöhle zu erhalten.</li>
	<li>Vorsichtig ligate die linke vordere absteigende (LAD) Koronararterie mit einem Nadelhalter mit einem 8-0 Polypropylen-Nähte und schneiden Sie die Naht mit Elektrokauterie.</li>
	<li>Beobachten Sie einen sofortigen Farbwechsel in der vorderen linken ventrikulären Wand.</li>
</ol>5. Intramyokardiale Transplantation von MSC-ladenden GH-Hydrogelen
<ol><li>Nach der Induktion des Myokardinfarkts durch LAD-Ligatur, injizieren Sie 10 l MSC-ladende GH-Lösungen in zwei verschiedene Punkte in der Infarktgrenzzone (insgesamt: 2 x 105 MSCs/20 l) mit einer Doppelspritze, die mit einer 26G-Nadel ausgestattet ist.<ol>
<li>Nach dem in Schritt 1 beschriebenen Verfahren können Sie MSC-ladende GH-Lösungen vorbereiten und in eine Doppelspritze übertragen. ANMERKUNG: Zur Beurteilung der Engraftmentierung von MSC-ladenden GH-Hydrogelen im Infarktbereich wurden MSCs und GH-Konjugate mit PHK26 bzw. Fluoresceinisothiocyanat (FITC) voretikettiert.</li>
	</ol>
</li>
	<li>Stellen Sie die geöffnete Brusthöhle wieder her und schließen Sie die Muskeln und die Haut mit 5-0 Nähten. HINWEIS: Entfernen Sie vor dem Brustverschluss die Luft mit einer Katheterspritze.</li>
	<li>Entfernen Sie das Luftröhrenrohr und legen Sie die Maus während der Bergung in einen Käfig unter einer Infrarotlampe.</li>
	<li>Bei postoperativer Analgesie subkutane Ketoprofen-Injektionen (5 mg/kg pro Tag) mindestens 72 h verabreichen. Alle Mäuse sollten für einen angemessenen Zeitpunkt genau überwacht werden, um eine ordnungsgemäße Genesung nach chirurgischen Eingriffen sowie eine angemessene Schmerzbehandlung zu gewährleisten.</li>
</ol>6. Echokardiographie
<ol><li>Vier Wochen nach der Transplantation die Maus zunächst mit 5% Isofluran befeuchten und dann die Isoflurankonzentration auf 1% anpassen.</li>
	<li>Depilate die Brust mit Haarentfernung Creme und legen Sie die Maus auf einem Heizkissen. Ultraschall-Wandlergel auf die Brust auftragen.</li>
	<li>Erfassen Sie zweidimensionale parasternale Kurzachsenansichten und zeichnen Sie M-Modus-Tracings auf der Ebene des Papillenmuskels auf. ANMERKUNG: Platzieren Sie einen linearen Array-Wandler (7-u201215 MHz) in der linken parasternalen Linie und zeigen Sie die anatomischen Strukturen an.</li>
	<li>Messen Sie entsprechende Linien für LVAW, LVID und LVPW, um Herzwanddicke, Kammerabmessung und fraktionierte Verkürzung zu erhalten. HINWEIS: Vergleichen Sie die Herzfunktion einschließlich der Auswurffraktion (EF), der fraktionierten Verkürzung (FS) und des endsystolischen Volumens (ESV) auf der Ebene des Papillenmuskels, um eine ordnungsgemäße Bewertung an derselben anatomischen Stelle zu gewährleisten.</li>
</ol>7. Histologische Bewertung
<ol><li>Zur vorgegebenen Zeit nach der Transplantation von MSC-ladenden GH-Hydrogelen in das Infarktherz, euthanisieren Sie die Maus in einerCO2-Kammer und sammeln Sie das Herz für die histologische Analyse15.</li>
	<li>Für Hämatoxylin und Eosin (H&E) und Massons Trichromfärbung (MT) fixieren Sie das sezierte Herzgewebe in 4% Paraformaldehyd (PFA) und betten Sie es in Paraffin ein. Als nächstes schneiden Sie paraffin-eingebettete Herzblöcke mit einem Mikrotom in 4 m serielle Abschnitte und färben die Abschnitte mit MT-Färbung nach Standardprotokollen17.</li>
	<li>Erfassen Sie Bilder auf einem Diascanner mit 20-facher Vergrößerung und berechnen Sie die Infarktgröße der Behandlungsgruppen. Infarktgröße (%) = Gesamtinfarktumfang / Gesamtumfang LV x 100</li>
	<li>Berechnen Sie beide Umfangswerte nach Messung der Mittellinielänge. Messen Sie bei LV-Mittellinienumfangdien die Mittellinienlängen zwischen den Endokard- und Epikardialoberflächen. Bei Midline-Infarktumfangen die Infarktlängen messen, einschließlich mehr als 50 % der Gesamtdicke von Myokard18. HINWEIS: Alle Bildanalysen wurden mit ImageJ-Software durchgeführt.</li>
	<li>Messen Sie die Wanddicke der Narbe auf den Papillenmuskelebenen.</li>
	<li>Berechnen Sie den Bruchteil der Kollagenfläche. Kollagenfläche (%) = Gesamtfläche der interstitiellen Fibrose/Myozytenfläche x 100</li>
</ol>

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Die Autoren haben keine Interessenkonflikte, um mit diesem Werk zu erklären.

Injizierbare GH-Hydrogele haben aufgrund ihrer Fähigkeit, verschiedene therapeutische Wirkstoffe in situ homogen zu integrieren, ein großes Potenzial für In-vivo-Anwendungen. Darüber hinaus können ihre physikalischen und biochemischen Eigenschaften aufgrund krankheitsabhängiger Anforderungen leicht manipuliert werden. In dieser Hinsicht wurden injizierbare Hydrogele vorgeschlagen, um die großen Einschränkungen in der aktuellen Herzstammzelltherapie anzugehen, die durch schlechtes Überleben und Zellretention (d. ...

Die Herzstammzelltherapie hat sich als möglicher Ansatz für die Myokardreparatur und -regeneration1,2herauskristallisiert. Trotz der jüngsten positiven Ergebnisse in Tiermodellen und klinischen Studien ist die Anwendung einer Stammzelltherapie zur Myokardreparatur aufgrund der geringen Retention und des schlechten Überlebens der injizierten Zellen an den infarktierten Herzgeweben3,4begrenzt. Als Ergebnis wurde der Einsatz von zellbasierter Gewebetechnik, einschließlich injizierbarer Biomaterialien5, Herzpflaster6und Zellblätter7, intensiv untersucht, um die Zellretention und -integration innerhalb des Wirtsmyokards zu verbessern.
Unter den verschiedenen möglichen Ansätzen zur biotechnischen Herzgewebereparatur sind injizierbare Hydrogele in Kombination mit geeigneten Zelltypen, wie mesenchymale Stammzellen (MSCs), embryonale Stammzellen (ESCs) und induzierte pluripotente Stammzellen (iPSCs), eine attraktive Option, um Zellen effektiv in die Myokardregionen8,9zu liefern. Gelatine, ein bekanntes natürliches Polymer, kann aufgrund seiner großen Biokompatibilität, seiner beträchtlichen biologischen Abbaubarkeit und der reduzierten Immunogenität im Vergleich zu einer Vielzahl von Biomaterialien, die in biomedizinischen Anwendungen verwendet werden, als injizierbare Matrix verwendet werden. Obwohl gelatinebasierte injizierbare Plattformen ein großes Potenzial haben, bleibt ihre Anwendbarkeit in vivo aufgrund ihrer geringen mechanischen Steifigkeit und leichten Abbaubarkeit in der physiologischen Umgebung begrenzt.
Um diese Einschränkungen zu überwinden, wurde ein neuartiges und einfaches Design von gelatinebasierten Hydrogelen, die aus Hydroxyphenylpropionsäure bestehen, für In-vivo-Anwendungen vorgeschlagen. Gelatine-Hydroxyphenylpropionsäure (GH) Konjugate können in Gegenwart eines Enzyms, Der Meerrettichperoxidase (HRP), in situ vernetzt werden und anschließend verschiedene Medikamente, Biomoleküle oder Zellen innerhalb des Hydrogels verkapseln, was auf ein großes Potenzial in gewebetechnischen Anwendungen10,11,12,13,14hindeutet. Darüber hinaus haben wir vor kurzem die therapeutische Wirkung von GH-Hydrogelen untersucht, die gekapselte MSCs enthalten, und ihre Verwendung bei erfolgreicher Herzreparatur und -regeneration nach MI in einem murinen Modell15nachgewiesen. In diesem Protokoll beschreiben wir eine einfache Technik für die Verkapselung und in vitro dreidimensionale (3D) Proliferation von MSCs in GH-Hydrogelen. Wir führen auch ein chirurgisches Verfahren ein, das entwickelt wurde, um ein murines MI-Modell über koronare Arterienligation und intramyokardiale Transplantation von MSC-ladenden GH-Hydrogelen in das infektiierte Herz zu erzeugen.

<table>
	<tbody>
		<tr>
			<td>4 % paraformaldehyde (PFA)</td>
			<td>Intron</td>
			<td>IBS-BP031-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>AILEE</td>
			<td>SK534</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>ETHICON</td>
			<td>M8732H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Nunc LAB-TEK</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocath Plus (22GA) catheter</td>
			<td>BD Angiocath Plus</td>
			<td>REF382423</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimyocotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>GYROGEN</td>
			<td>1582MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slipe</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Deluxe High Temperature Cautery kit</td>
			<td>Bovie</td>
			<td>QTY1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EOSIN</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>K49350783 739</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fechtner conjunctiva forceps titanium</td>
			<td>WORLD PRECISISON INSTRUMENTS</td>
			<td>WP1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>HEBU</td>
			<td>HB0458</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Ildong Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>50300</td>
			<td>Homeothermic Blanket System</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>50301</td>
			<td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>SIGMA-ALDRICH</td>
			<td>HHS80</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxide (HRP; 250-330 U/mg)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2; 30 wt % in H2O)</td>
			<td>SIGMA-ALDRICH</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Green Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD cell staining kit</td>
			<td>Thermo Fisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>HANIL</td>
			<td>Micro 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>KASCO</td>
			<td>37-1452</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>HEBU</td>
			<td>HB7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>MT staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1079-1SET</td>
			<td>Weigert&#8217;s iron hematoxylin solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HT15-1KT</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>LK LABKOREA</td>
			<td>H06-660-107</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PHK26 staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>HEBU</td>
			<td>HB7454</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M micopore</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue cassette</td>
			<td>Scilab Korea</td>
			<td>Cas3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer gel</td>
			<td>SUNGHEUNG</td>
			<td>SH102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trout-Barraquer needle holder curved</td>
			<td>KASCO</td>
			<td>50-3710c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips</td>
			<td>Affiniti 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>JUNSEI</td>
			<td>25175-0430</td>
			<td></td>
		</tr>
	</tbody>
</table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

Eines der Hauptprobleme, mit denen aktuelle Herzstammzelltherapien zur Vorbeugung von Herzinsuffizienz nach dem Infarkt konfrontiert sind, ist die niedrige Retentions- und Überlebensrate transplantierter Zellen innerhalb des verletzten Myokards, wodurch ihre therapeutische Wirksamkeit eingeschränkt wird. In jüngster Zeit hat der Einsatz von Gerüstbiomaterialien Aufmerksamkeit für die Verbesserung und Maximierung der Stammzelltherapie gewonnen. Ziel dieses Protokolls ist es, eine einfache und einfache Technik zur Transplantation von mesenchymalen Stammzellen (MSCs) aus Knochenmark unter Verwendung injizierbarer Hydroxyphenylpropionsäure (GH)-Hydrogele einzuführen; Die Hydrogele sind als Zellabgabeplattform für Herzgewebe-Engineering-Anwendungen aufgrund ihrer Fähigkeit, in situ vernetzt zu sein, und hoher Biokompatibilität günstig. Wir präsentieren eine einfache Methode zur Herstellung von MSC-ladenden GH-Hydrogelen (MSC/Hydrogels) und bewerten deren Überleben und Proliferation in dreidimensionaler (3D) In-vitro-Kultur. Darüber hinaus zeigen wir eine Technik zur intramyokardialen Transplantation von MSC/Hydrogelen bei Mäusen, die einen chirurgischen Eingriff beschreibt, um einen Myokardinfarkt (MI) über links anterior absteigende (LAD) koronare Arterienligation und anschließende MSC/Hydrogel-Transplantation zu induzieren.

Um MSCs effektiv an das infarktierte Myokard zu liefern, wurden in diesem Protokoll mSC-ladebare in situ-vernetzte Hydrogele verwendet, die in Abbildung 1 beschrieben sind. Vor der In-vivo-Transplantation wurden die Proliferation und das Überleben von MSCs in GH-Hydrogelen durch einen 3D-In-vitro-Färbe-Assay bestätigt (live: grün; tot: rot). Wie in Abbildung 2dargestellt, zeigten repräsentative Bilder eine ausreichende Verbreitung von MSCs, die verzweigte N...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Die Stammzelltherapie hat sich als effiziente Strategie zur Reparatur von verletztem Herzgewebe nach einem Myokardinfarkt herauskristallisiert. Wir bieten eine optimale In-vivo-Anwendung für die Stammzelltransplantation mit gelatinenhydrogelen, die enzymatisch vernetzt werden können.

Intramyokardiale Transplantation von MSC-Loading injizierbaren Hydrogelen nach Myokardinfarkt in einem Murine-Modell

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Research

JoVE Journal

Medicine

4.3K Aufrufe.  The Catholic University of Korea. Die Stammzelltherapie hat sich als effiziente Strategie zur Reparatur von verletztem Herzgewebe nach einem Myokardinfarkt herauskristallisiert. Wir bieten eine optimale In-vivo-Anwendung für die Stammzelltransplantation mit gelatinenhydrogelen, die enzymatisch vernetzt werden können.

Serie: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Akuter Myokardinfarkt bei Ratten

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Forschung

Medizin

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Koronare Ligation und intramyokardialen Injection in einem Mausmodell der Infarkt

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Myokardinfarkt und Functional Outcome Assessment in Schweine

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Ein Hydrogel Konstruieren und Fibrin-basierte Kleber Umgangs Therapeutics in einem murinen Myokardinfarkt Modell liefern.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Primärer Ergebnisbeurteilung in einem Schwein Modell eines akuten Myokardinfarkts

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myokardinfarkts bei neugeborenen Mäusen, ein Modell von Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Murine linken vorderen absteigenden (LAD) Koronararterienligatur: ein verbessertes und vereinfachtes Modell für Myokardinfarkts

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Murine Myokardinfarkt Modell mit permanenten Ligation der linken Anterior absteigende Koronararterie

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Ein Cryoinjury-Modell zur Untersuchung des Myokardinfarkts in der Maus

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Verzögerte intramyokardiale Lieferung von Stammzellen nach Ischämie-Reperfusionsverletzung in einem Murine-Modell

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...