L. Rinaldi, S. Schäffer, D. Reitz, H. Thomas 및 A. Weber의 기술 지원은 감사하게 생각합니다. 저자는 광범위한 증거 교정과 원고의 언어 편집을 위해 Dr. E. Martinson에게 감사드립니다. 이 연구는 Giessen Anschubsfinanzierung 대학의 M. Aslam과 D. Gündüz 연구원의 지원을 받았다.

<ol>
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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>

저자는 선언 할 것이 없습니다.

이 기사에서는 심장 근세포, 내피 세포 및 섬유 아세포의 분리 및 배양에 대한 재현 가능한 프로토콜에 대해 설명합니다. 이 프로토콜은 단지 하나의 세포 유형에 반대 cardiomyocytes, 내피 세포, 그리고 섬유 아 세포와 같은 심장 조직의 주요 세포 유형의 동시 고품질 분리를 설명합니다. 격리 절차의 중요한 단계는 심장 조직의 적절한 소화입니다. 소화가 불완전하다면 많은 양의 근육 세포가...

설치류 모델은 오랫동안 건강과 질병에서 심혈 관계 생리학에 대한 우리의 이해를 넓히는 도구로 사용되어 왔습니다. 1 이 동물 모델은 기관 수준에서 질병의 병리 생리학을 이해하고 심혈관 질환 치료에 사용되는 다양한 약리학 적 약물의 약물 동력학 및 약력학을 분석하고 심혈관 질환 발병의 분자 메커니즘 및 특정 세포 유형은 시험 관내 세포 배양 모델의 사용을 필요로한다. 이 목적을 위해 심혈관 시스템으로부터의 상이한 불멸화 된 세포주가 개발되었다. 2 , 3 그러나 새로 분리 된 1 차 세포는 생리 학적으로 기능적으로 살아있는 조직과 유기체와 관련이 있습니다. 심장은 심장 혈관의 모든 주요 유형의 세포를 포함하는 다용도 장기입니다.ystem, 쥐 심장은 여전히 ​​심혈관 생리학의 이해를 위해 일반적으로 사용되는 모델입니다. 지난 수십 년 동안 심장 조직으로부터 개별 세포 유형을 분리하기위한 여러 가지 방법이 설명되었습니다. 4 , 5 , 6 , 7 그러나 이러한 방법은 특정 세포 유형의 분리에만 초점을 맞추어 더 이상 세포 유형을 잃어 버려 세포 분석에 더 이상 사용할 수 없습니다. 여기에는 심장 조직, 즉 심근 세포, 내피 세포 및 섬유 아세포의 주요 세포 유형을 동시에 고품질로 분리 할 수있는 최적화 된 프로토콜이 설명되어 있습니다. 이러한 모든 세포 유형은 다른 실험 설정 8 , 9 , 10 및 동일한 동물의 세포 - 세포 상호 작용 분석에 사용할 수 있습니다.

<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

쥐는 심장 혈관 연구에 사용되는 중요한 동물 모델이며, 랫트 심장 세포는 심근 비대, 섬유증 및 죽상 동맥 경화증과 같은 심혈관 질환 진행의 분자 메커니즘에 대한 생체 외 분석을 위해 일상적으로 사용됩니다. 이러한 세포 메커니즘을 이해하기 위해 심혈관 시스템에서 불후의 세포주를 개발하기 위해 다양한 성공을 거둔 여러 시도가 있었지만, 일차 세포는 이러한 연구를 위해보다 자연스럽고 가까운 생체 내 환경을 제공합니다. 따라서 특정 세포 유형에 종사하는 여러 실험실에서는 관심있는 쥐 심장 세포의 개별 유형을 분리하는 프로토콜을 개발했습니다. 그러나 하나 이상의 셀 유형을 격리 할 수있는 프로토콜이 없습니다. 여기에는 하나의 준비에서 고품질의 주요 심장 세포 유형 (cardiomyocytes, endothelial 세포 및 fibroblasts)의 격리를 허용하고 최적화 된 프로토콜을 설명합니다세포 분석에 사용. 이를 통해 사용 가능한 리소스를 가장 효율적으로 사용할 수 있으므로 시간을 절약하고 연구 비용을 줄일 수 있습니다.

<html> 분리 절차는 계획된 실험에 사용될 수있는 70-80 % 생존 가능하고 막대 모양의 줄무늬 심근 세포 ( 그림 2A 및 2C )의 수율을 초래합니다. 우리의 실험실에서 cardiomyocytes는 일상적으로 칼슘 2 + 신호 분석에 사용됩니다. 그림 5A 는 Fura - AM (5 μm의)와로드 된 cardiomyocytes의 허혈 / reperfusion에 대한 응답으로 세포 내 칼슘 [칼슘 2...

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

쥐 심장에서 다른 심장 세포 유형의 분리를 위해 몇 가지 프로토콜이 개발 및 기술되었습니다. 여기에는 실험 준비 비용을 줄이기 위해 단일 준비에서 고품질 주요 심장 세포 유형 (cardiomyocytes, endothelial cell, fibroblast)을 분리 할 수있는 최적화 된 프로토콜이 설명되어 있습니다.

성인 쥐 심장에서 고품질 Cardiomyocytes, 내피 세포 및 섬유 아세포의 동시 격리

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 조회수.  University Hospital Giessen.  쥐 심장에서 다른 심장 세포 유형의 분리를 위해 몇 가지 프로토콜이 개발 및 기술되었습니다. 여기에는 실험 준비 비용을 줄이기 위해 단일 준비에서 고품질 주요 심장 세포 유형 (cardiomyocytes, endothelial cell, fibroblast)을 분리 할 수있는 최적화 된 프로토콜이 설명되어 있습니다. 

비디오: 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.

성인 쥐 심장 Myocytes의 차 문화

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

연구

생물학

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.

공촛점 이미징에 대한 분리 및 성인 심장 Myocytes의 유전자 조작

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.

쥐 심장의 심실 무료 벽에서 내심 내피 및 관상 내피 세포의 격리

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

발생학

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.

절연 및 성인 쥐 Cardiomyocytes의 재배

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

의학

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.

심 방 Cardiomyocytes 보존된 방출 분수와 함께 대사 증후군 관련 심장 마비의 쥐 모델에서의 격리

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.

분리 및 문화 성인 마우스 세포 신호 전달 용의 Cardiomyocytes과의<em&gt; 시험관</em&gt; 심장 비대

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.

격리, 문화 및 성인 마우스 심근 세포의 형질 도입

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.

마우스 심근 세포를 일관되게 분리하는 간단하고 효과적인 방법

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.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

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.

성인용 마우스 심장에서 심방 심근 세포, 심실 심근 세포 및 비근구의 동시 격리 및 문화

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