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

Il supporto tecnico di L. Rinaldi, S. Schäffer, D. Reitz, H. Thomas e A. Weber è riconosciuto con gratitudine. Gli autori inoltre desiderano ringraziare il dott. E. Martinson per un&#39;ampia lettura delle prove e la modifica del linguaggio del manoscritto. Lo studio è stato sostenuto dalla sovvenzione dell&#39;Università di Giessen Anschubsfinanzierung a M. Aslam e D. Gündüz.

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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>

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In questo articolo viene descritto un protocollo riproducibile per l&#39;isolamento e la coltura di miociti cardiaci, cellule endoteliali e fibroblasti. Questo protocollo descrive l&#39;isolamento simultaneo e di alta qualità dei principali tipi di cellule del tessuto cardiaco, vale a dire cardiomiociti, cellule endoteliali e fibroblasti, a differenza di un solo tipo di cellula. Un passo critico nella procedura di isolamento è la corretta digestione del tessuto cardiaco. Se la digestione è incompleta, può a...

I modelli di roditori sono da tempo usati come strumenti per ampliare la nostra comprensione della fisiologia cardiovascolare in salute e malattie. 1 Anche se questi modelli animali ci permettono di comprendere la fisiopatologia di una malattia a livello di organo e di analizzare la farmacocinetica e la farmacodinamica di vari agenti farmacologici utilizzati per il trattamento delle malattie cardiovascolari, la comprensione dei meccanismi molecolari dello sviluppo delle malattie cardiovascolari e il contributo di un particolare Tipo di cellule richiede l&#39;uso di modelli di coltura cellulare in vitro . A tal fine sono state sviluppate diverse linee cellulari immortalate del sistema cardiovascolare; 2 , 3 tuttavia, le cellule primarie appena isolate sono fisiologicamente e funzionalmente più pertinenti ai tessuti e agli organismi viventi. Il cuore è un organo versatile che contiene tutti i principali tipi di cellule del sistema cardiovascolareYstem, e il cuore del ratto è ancora un modello comunemente utilizzato per la comprensione della fisiologia cardiovascolare. Negli ultimi decenni sono stati descritti diversi metodi per l&#39;isolamento di singoli tipi di cellule dal tessuto cardiaco; 4 , 5 , 6 , 7 tuttavia, questi metodi si concentrano solo sull&#39;isolamento di un determinato tipo di cellule che determina la perdita di altri tipi di cellule che non possono più essere utilizzati per l&#39;analisi cellulare. Qui viene descritto un protocollo ottimizzato che consente l&#39;isolamento simultaneo e di alta qualità dei principali tipi cellulari di tessuto cardiaco, cioè cardiomiociti, cellule endoteliali e fibroblasti. Tutti questi tipi di cellule possono essere utilizzati in diverse configurazioni sperimentali 8 , 9 , 10 e per l&#39;analisi delle interazioni di cellule cellule dallo stesso animale.

<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

Il ratto è un importante modello di animali utilizzato nella ricerca cardiovascolare e le cellule cardiache del ratto vengono usate in maniera routinaria per l&#39;analisi in vitro dei meccanismi molecolari della progressione della malattia cardiovascolare come l&#39;ipertrofia cardiaca, la fibrosi e l&#39;aterosclerosi. Sebbene siano stati fatti diversi tentativi con successo variabile per sviluppare linee cellulari immortalizzate dal sistema cardiovascolare per comprendere questi meccanismi cellulari, le cellule primarie offrono un ambiente più naturale e vicino all&#39;ambiente in vivo per tali studi. Pertanto, diversi laboratori che lavorano su un particolare tipo di cellule hanno sviluppato protocolli per isolare singoli tipi di cellule cardiache di ratto di interesse. Manca un protocollo che consente l&#39;isolamento di più di un tipo di cella. Qui viene descritto un protocollo ottimizzato che consente l&#39;isolamento di tipi di cellule cardiache di elevata qualità (cardiomiociti, cellule endoteliali e fibroblasti) da una singola preparazione e consente diR per l&#39;analisi cellulare. Ciò consente l&#39;uso più efficiente delle risorse disponibili, che possono risparmiare tempo e ridurre i costi di ricerca.

<html> La procedura di isolamento comporta una resa del 70-80% di cardiomiociti vivi, a forma di stelo e striato ( figure 2A e 2C ) che possono essere utilizzati per esperimenti pianificati. Nel nostro laboratorio i cardiomiociti vengono utilizzati abitualmente per l&#39;analisi della segnalazione Ca 2+ . La Figura 5A mostra oscillazioni intracellulari di calcio [Ca 2+ ] i in risposta all&#39;isch...

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

Sono stati sviluppati e descritti diversi protocolli per l&#39;isolamento di diversi tipi di cellule cardiache da un cuore del topo. Qui viene descritto un protocollo ottimizzato che consente l&#39;isolamento di tipi di cellule cardiache di elevata qualità (cardiomiociti, cellule endoteliali e fibroblasti) da una singola preparazione, riducendo i costi sperimentali.

Isolamento simultaneo di cardiomiociti di alta qualità, cellule endoteliali e fibroblasti da un cuore di ratto adulto

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

Research

JoVE Journal

Biology

13.3K Views.  University Hospital Giessen.  Sono stati sviluppati e descritti diversi protocolli per l'isolamento di diversi tipi di cellule cardiache da un cuore del topo. Qui viene descritto un protocollo ottimizzato che consente l'isolamento di tipi di cellule cardiache di elevata qualità (cardiomiociti, cellule endoteliali e fibroblasti) da una singola preparazione, riducendo i costi sperimentali. 

Video: 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.

Cultura primario di miociti adulti Cuore Rat

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

Ricerca

Biologia

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.

L&#39;isolamento e la manipolazione genetica di adulti miociti cardiaci per confocale 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.

Isolamento delle cellule endocartali e coronarie dal muro libero ventricolare del cuore di ratto

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

Biologia dello sviluppo

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.

Isolamento e coltura dei cardiomiociti di ratto adulto.

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

Medicina

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.

Isolamento dei cardiomiociti atriali da un modello del ratto di metabolico sindrome-relativo infarto con la frazione conservata di espulsione

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.

Isolamento e Cultura del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

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.

Isolamento, cultura e trasduzione di topo adulto cardiomiociti

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.

Un metodo semplice ed efficace per isolare in modo coerente i cardiomiociti di topo

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.

Isolamento, trasfezione e cultura a lungo termine di cardiomiociti per topi e ratti adulti

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.

Isolamento simultaneo e cultura di miociti atriali, miociti ventricolari e non miociti da un cuore di topo adulto

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

Isolamento simultaneo di cardiomiociti di alta qualità, cellule endoteliali e fibroblasti da un cuore di ratto adulto

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