Name: simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart
Uploaded: 2017-05-19T14:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

Die technische Unterstützung von L. Rinaldi, S. Schäffer, D. Reitz, H. Thomas und A. Weber ist dankbar anerkannt. Die Autoren danken auch Dr. E. Martinson für umfangreiche Korrekturlesen und Sprachbearbeitung des Manuskripts. Die Studie wurde von der Universität Giessen Anschubsfinanzierung für M. Aslam und D. Gündüz unterstützt.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+metabolism+in+cultured+adult+rat+cardiac+fibroblasts:+response+to+angiotensin+II+and+aldosterone.">Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone.</a> J Mol Cell Cardiol. 26 (7), 809-820 (1994).</li><li>Shahzad, T., 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=Mechanisms+involved+in+postconditioning+protection+of+cardiomyocytes+against+acute+reperfusion+injury.">Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury.</a> J Mol Cell Cardiol. 58, 209-216 (2013).</li><li>G&#252;nd&#252;z, 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=Insulin+Stabilizes+Microvascular+Endothelial+Barrier+Function+via+Phosphatidylinositol+3-Kinase/Akt-Mediated+Rac1+Activation.">Insulin Stabilizes Microvascular Endothelial Barrier Function via Phosphatidylinositol 3-Kinase/Akt-Mediated Rac1 Activation.</a> Arterioscler.Thromb.Vasc.Biol. 30, 1237-1245 (2010).</li><li>Lipps, 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=N-terminal+fragment+of+cardiac+myosin+binding+protein-C+triggers+pro-inflammatory+responses+in+vitro.">N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.</a> J Mol Cell Cardiol. 99, 47-56 (2016).</li><li>Abdallah, 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=Interplay+between+Ca2++cycling+and+mitochondrial+permeability+transition+pores+promotes+reperfusion-induced+injury+of+cardiac+myocytes.">Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.</a> J Cell Mol Med. 15 (11), 2478-2485 (2011).</li><li>G&#252;nd&#252;z, 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=Effect+of+ticagrelor+on+endothelial+calcium+signalling+and+barrier+function.">Effect of ticagrelor on endothelial calcium signalling and barrier function.</a> Thromb Haemost. , (2016).</li><li>G&#252;nd&#252;z, 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=Opposing+effects+of+ATP+and+adenosine+on+barrier+function+of+rat+coronary+microvasculature.">Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.</a> J Mol.Cell Cardiol. , (2012).</li></ol>

Autoren haben nichts zu erklären.

In diesem Artikel wird ein reproduzierbares Protokoll für die Isolierung und Kultur von Herz-Myozyten, Endothelzellen und Fibroblasten beschrieben. Dieses Protokoll beschreibt die gleichzeitige und qualitativ hochwertige Isolierung der wichtigsten Zelltypen von Herzgewebe, dh Kardiomyozyten, Endothelzellen und Fibroblasten im Gegensatz zu nur einem Zelltyp. Ein kritischer Schritt in der Isolation ist die richtige Verdauung des Herzgewebes. Wenn die Verdauung unvollständig ist, kann noch eine große Anzahl von Myozyten erhalten werden, aber ihre Qualität ist sehr schlecht und sie sind meist hyperkontrahiert und runde (Abbildung 2B ) und von keinem Wert in Experimenten. Darüber hinaus wird auch die Ausbeute an Endothelzellen und Fibroblasten reduziert. Dieses Problem kann durch die Steuerung der Verdauungszeit gelöst werden. Vor dem Entfernen des Herzens aus dem Perfusionssystem kann die Weichheit des Herzens mit Hilfe von Pinzetten und Fingerspitzen überprüft werden. Ist das Gewebe noch nicht spürbar sOft kann das Herz für weitere 5-10 min perfundiert werden. Ein zweiter kritischer Schritt bei der gesunden Kardiomyozytenisolation ist die inkrementelle Wiederherstellung von Ca 2+ in den Isolationspuffern. Eine rasche Wiederherstellung hoher Konzentrationen von Ca 2+ in den Medien führt zu gerundeten Kardiomyozyten. Die Qualität des Wassers, das für die Herstellung von Puffern verwendet wird, ist ein weiterer wichtiger Faktor, der die Qualität der Kardiomyozyten beeinflusst. Daher wird die Verwendung von gutem Wasser empfohlen. Die Reinheit der Endothelzellen hängt stark von den Waschschritten ab. EDTA muss in dem Waschpuffer vorhanden sein, um eine unspezifische Bindung von nicht-endothelialen Zellen an die magnetischen Kügelchen zu verhindern. Die Anwesenheit von Blutzellen in der Zellsuspension wird die Ausbeute an Endothelzellen stark reduzieren; Daher ist das richtige Waschen des Herzens sehr kritisch nach der Montage des Herzens im Perfusionssystem und vor der Zugabe von Kollagenase. Restliche Blutkomponenten reduzieren auch tDie Aktivität der Kollagenase und damit die Verdauung des Herzens. Die häufigste Kontamination in der Fibroblastenzellkultur ist das Auftreten von Satellitenkolonien von Epithelzellen (Abbildung 4C ). Diese Verunreinigung kann durch zweistufige Kultivierung von Fibroblasten vermieden werden. Im ersten Schritt wird die Konzentration des FCS im Fibroblastenzellkulturmedium auf 5% reduziert und die Zellsuspension wird an der Zellkulturschale für 30 min im Inkubator haften gelassen. Epithelzellen haften schnell, aber die Fibroblastenzell-Adhäsionszeit wird in Gegenwart einer niedrigen Serumkonzentration erhöht. Nach 30 min Inkubation wird die Zellsuspension, die nicht adhärierende Fibroblasten enthält, sorgfältig zu einer neuen Zellkulturschale entfernt. Auf diese Weise kann die Ausbeute an Fibroblasten reduziert werden, aber ihre Reinheit wird stark erhöht.

Nagetiermodelle werden seit langem als Werkzeuge verwendet, um unser Verständnis der Herz-Kreislauf-Physiologie in Gesundheit und Krankheit zu erweitern. 1 Obwohl diese Tiermodelle es uns erlauben, die Pathophysiologie einer Erkrankung auf organischer Ebene zu verstehen und die Pharmakokinetik und Pharmakodynamik verschiedener pharmakologischer Mittel zur Behandlung von Herz-Kreislauf-Erkrankungen, des Verständnisses der molekularen Mechanismen der Entwicklung von Herz-Kreislauf-Erkrankungen und des Beitrags eines bestimmten zu analysieren Zelltyp erfordert die Verwendung von in vitro Zellkulturmodellen. Zu diesem Zweck wurden verschiedene immortalisierte Zelllinien aus dem Herz-Kreislauf-System entwickelt; 2 , 3 aber frisch isolierte primäre Zellen sind physiologisch und funktionell relevanter für lebende Gewebe und Organismen. Das Herz ist ein vielseitiges Organ, das alle gängigen Zelltypen des Herz-Kreislaufs enthältYstem, und das Rattenherz ist immer noch ein häufig verwendetes Modell für das Verständnis der Herz-Kreislauf-Physiologie. In den letzten Jahrzehnten wurden verschiedene Methoden zur Isolierung einzelner Zelltypen aus Herzgewebe beschrieben; 4 , 5 , 6 , 7 Diese Methoden konzentrieren sich jedoch nur auf die Isolierung eines spezifischen Zelltyps, was zum Verlust von anderen Zelltypen führt, die für die zelluläre Analyse nicht mehr verwendet werden können. Hier wird ein optimiertes Protokoll beschrieben, das die gleichzeitige und qualitativ hochwertige Isolierung der wichtigsten Zelltypen von Herzgewebe, dh Kardiomyozyten, Endothelzellen und Fibroblasten, ermöglicht. Alle diese Zelltypen können in verschiedenen Versuchsaufbauten 8 , 9 , 10 und zur Analyse von Zell-Zell-Wechselwirkungen aus demselben Tier verwendet werden.

<table><tbody><tr><td>anti-vWF</td><td>Santa Cruz Biotech.</td><td>SC-14014</td><td></td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102378</td><td></td></tr><tr><td>Carnitin</td><td>Sigma-Aldrich</td><td>C0283</td><td></td></tr><tr><td>Collagenase type II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Creatin</td><td>Sigma-Aldrich</td><td>C0780</td><td></td></tr><tr><td>D-Glucose</td><td>Merck</td><td>108342</td><td></td></tr><tr><td>Dil-Ac-LDL</td><td>Thermo Scientific</td><td>L3484</td><td></td></tr><tr><td>EDTA Solution (0.2 M)</td><td>Biochrome AG</td><td>L2113</td><td></td></tr><tr><td>Embeding solution</td><td>Citiflour</td><td>AF1-25</td><td></td></tr><tr><td>Endothelial cell medium MV2</td><td>PromoCell</td><td>C-22022</td><td></td></tr><tr><td>Foetal calf serum (FCS)</td><td>Biochrome AG</td><td>S0615</td><td></td></tr><tr><td>Gentamicin</td><td>Serva Chemicals</td><td>47991</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>TU 061219</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Roche/Sigma</td><td>11243217001</td><td></td></tr><tr><td>M199 medium</td><td>Thermo Scientific</td><td>11150059</td><td></td></tr><tr><td>M199 medium (Powder)</td><td>Biochrome AG</td><td>T061</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sigma-Aldrich</td><td>63138</td><td></td></tr><tr><td>Mouse anti-rat CD31 antibody (TLD-3A12)</td><td>Thermo Scientific</td><td>MA1-81051</td><td></td></tr><tr><td>NaCl solution (0.9%), Sterile</td><td>B. Braun</td><td>30820080</td><td></td></tr><tr><td>Pan mouse IgG beads (Dynabeads)</td><td>Thermo Scientific</td><td>11041</td><td></td></tr><tr><td>Paraformaldehyde (PFA) 4% Solution</td><td>Santa Cruz Biotech.</td><td>sc-281692</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) 1x&amp;nbsp;</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Plastic consumables</td><td>Greiner Bio-One</td><td></td><td></td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>4933</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>7873</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>6404</td><td></td></tr><tr><td>Sodium Hydroxide Solution (2 N)</td><td>Merck</td><td>109136</td><td></td></tr><tr><td>Sterile filtration system</td><td>Thermo Scientific</td><td>5660020</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T8691</td><td></td></tr><tr><td>TO-PRO</td><td>Thermo Scientific</td><td>T3605</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Water, Sterile</td><td>B. Braun</td><td></td><td></td></tr></tbody></table>

simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart

Die Ratte ist ein wichtiges Tiermodell, das in der Herz-Kreislauf-Forschung verwendet wird, und Ratten-Herzzellen werden routinemäßig zur in-vitro- Analyse der molekularen Mechanismen der kardiovaskulären Erkrankung wie Herzhypertrophie, Fibrose und Atherosklerose verwendet. Obwohl mehrere Versuche mit variablem Erfolg gemacht wurden, um immortalisierte Zelllinien aus dem kardiovaskulären System zu entwickeln, um diese zellulären Mechanismen zu verstehen, bieten primäre Zellen eine natürliche und nahe an in vivo Umgebung für solche Studien. Daher haben verschiedene Laboratorien, die an einem bestimmten Zelltyp arbeiten, Protokolle entwickelt, um einzelne Arten von Ratten-Herzzellen von Interesse zu isolieren. Ein Protokoll, das die Isolierung von mehr als einem Zelltyp erlaubt, fehlt jedoch. Hier wird ein optimiertes Protokoll beschrieben, das die Isolierung von hochwertigen Hauptkardiometertypen (Kardiomyozyten, Endothelzellen und Fibroblasten) aus einer einzigen Präparation ermöglicht und es ermöglichtEinsatz für zelluläre Analysen. Dies ermöglicht eine effizienteste Nutzung der verfügbaren Ressourcen, die Zeit sparen und die Forschungskosten senken können.

<html> Das Isolationsverfahren führt zu einer Ausbeute von 70 - 80% lebensfähigen, stabförmigen, gestreiften Kardiomyozyten ( Abb. 2A und 2C ), die für geplante Experimente verwendet werden können. In unserem Labor werden Kardiomyozyten routinemäßig zur Analyse der Ca 2+ -Signalisierung verwendet. 5A zeigt intrazelluläre Calcium [Ca 2+ ] i- Oszillationen als Reaktion auf Ischämie / Reperfusion in Kardiomyozyten, die mit Fura-AM (5 &amp; mgr; M) beladen sind. Abbildung 5B zeigt Veränderungen in [Ca 2+ ] i in Endothelzellen und Fibroblasten nach Zugabe von ATP (100 μM). Die Veränderungen in [Ca 2+ ] i in verschiedenen Herzzelltypen wurden wie zuvor beschrieben analysiert. 6 , 11 , 12 Re 1 &quot;src =&quot; / files / ftp_upload / 55601 / 55601fig1.jpg &quot;/&gt; Abbildung 1: Modifiziertes Langendorff Perfusionssystem. ( A ) Das gesamte Langendorff-Perfusionssystem zur Isolierung von Herzzellen ( B ) Vergrößerte Ansicht des hängenden Herzens in der &quot;Herzkammer&quot;, die auch für die Sammlung von Perfusionspuffer für die Rezirkulation verwendet wird. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/55601/55601fig2.jpg" /> Abbildung 2: Erwachsene Ratten-Kardiomyozyten. ( A ) Typische stabförmige, gesunde Kardiomyozyten 2 h nach dem Plattieren in lamininbeschichteten Zellkulturschalen ( B ) Ein typisches Beispiel für eine schlechte Vorbereitung durch unvollständige Gewebeverdauung. 2 h nach dem Plattieren in laminin-beschichteten Zellkulturschalen, moSt der Zellen sind hyperkontrahiert und schauen abgerundet, und es gibt wenige stabförmige Kardiomyozyten. Maßstab = 10 μm ( C ) Konfokale mikroskopische Aufnahme von Einzelkardiomyozyten, gefärbt für die Aktinvisualisierung, wie zuvor beschrieben. 13 Maßstab = 50 μm. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 3" src="/files/ftp_upload/55601/55601fig3.jpg" /> Abbildung 3: Rattenkardiale Endothelzellen. ( A ) Phasenkontrastmikroskopische Aufnahme der nichtkonfluenten Endothelzellmonoschicht 3 Tage nach der Isolierung. Dunkle Flecken sind die magnetischen Perlen, die noch an den Elternendothelzellen befestigt sind. Maßstab = 10 μm ( B ) Phasenkontrastmikroskopische Aufnahme der konfluenten Endothelzellmonoschicht 7 Tage nach der Isolierung ( C) Immunfärbung von Endothelzellen mit Dil-Ac-LDL-Aufnahme (Rot) und Nuklearfärbung (Blau) ( D ) Immunfärbung von Endothelzellen mit Färbung von vWF (Rot) und Nuklearfärbung (Blau) ( E ) Zellen, die nur mit Sekundärantikörper inkubiert wurden Und Kernfleck (blau) (Maßstab = 50 μm) <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 4" src="/files/ftp_upload/55601/55601fig4.jpg" /> Abbildung 4: Ratten-Herz-Fibroblasten. ( A ) Typische spindelförmige Herzfibroblasten am zweiten Tag der Isolation. ( B ) Konfluente, reine Fibroblasten nach 7 Tagen Kultur. ( C ) Ein typisches Beispiel für eine schlechte Zubereitung, die mit Epithelzellen kontaminiert ist. Zellen, die durch Pfeilspitzen angegeben sind, sind die Herzepithelzellen<html> . Maßstab = 10 μm. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55601/55601fig4large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 5" src="/files/ftp_upload/55601/55601fig5.jpg" /> Abbildung 5: Änderungen in [Ca 2+ ] i in Herzzellen. ( A ) Repräsentative Verfolgungen von Veränderungen des intrazellulären Calciums [Ca 2+ ] i, gemessen durch Fura-2-Verhältnis in Ratten-Kardiomyozyten, die einer chemischen Ischämie ausgesetzt sind (Na-Cyanat, 2 mM), gefolgt von Reperfusion ( B ) Repräsentative Spuren von Veränderungen in intrazellulärem Calcium [Ca 2+ ] i, gemessen durch Fura-2-Verhältnis in Ratten-Herzendothelzellen und Fibroblasten, behandelt mit ATP (100 &amp; mgr; M), wie angegeben. &gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.

Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

Mehrere Protokolle wurden entwickelt und beschrieben für die Isolierung von verschiedenen Herzzelltypen von einem Rattenherz. Hier wird ein optimiertes Protokoll beschrieben, das die Isolierung von hochwertigen Hauptkardiometertypen (Kardiomyozyten, Endothelzellen und Fibroblasten) aus einer einzigen Präparation ermöglicht, wodurch die experimentellen Kosten reduziert werden.

Gleichzeitige Isolierung von hochwertigen Kardiomyozyten, Endothelzellen und Fibroblasten aus einem erwachsenen Rattenherz

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

simultaneous-isolation-high-quality-cardiomyocytes-endothelial-cells

Universidad San Sebastian

Research

JoVE Journal

Biology

13.3K Aufrufe.  University Hospital Giessen.  Mehrere Protokolle wurden entwickelt und beschrieben für die Isolierung von verschiedenen Herzzelltypen von einem Rattenherz. Hier wird ein optimiertes Protokoll beschrieben, das die Isolierung von hochwertigen Hauptkardiometertypen (Kardiomyozyten, Endothelzellen und Fibroblasten) aus einer einzigen Präparation ermöglicht, wodurch die experimentellen Kosten reduziert werden. 

Serie: Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Primäre Kultur der erwachsenen Ratte Herz Myozyten

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Forschung

Biologie

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolation und genetische Manipulation von Adult Herzmuskelzellen für konfokale Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

Isolierung von Endokardialen und koronaren Endothelzellen aus der ventrikulären freien Wand des Rattenherzes

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Entwicklungsbiologie

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Isolierung und Kultivierung von Erwachsenen Ratte Kardiomyozyten

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Medizin

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

Isolierung von Vorhofflimmern Herzzellen aus einem Rattenmodell der metabolischen Syndrom im Zusammenhang mit Herzinsuffizienz mit erhaltener Auswurffraktion

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation und Kultur der erwachsenen Maus Kardiomyozyten für Cell Signaling und<em&gt; In-vitro-</em&gt; Herzhypertrophie

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolierung, Kultur und Transduction von erwachsenen Maus Kardiomyozyten

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

Eine einfache und effektive Methode zur konsistenten Isolierung von Maus-Kardiomyozyten

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolation, Transfektion und Langzeitkultur von Erwachsenenmaus und Rattenkardiomyozyten

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Gleichzeitige Isolierung und Kultur von Atrial Myozyten, ventrikulären Myozyten und Nicht-Myozyten aus einem erwachsenen Mausherz

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...