This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

该协议遵循动物研究伦理委员会的LUMC准则。 1.准备仪器，培养基和MTCS 注:请在层流罩的所有准备工作。该MTCS灌注腔，泡沫陷阱，站在利伯等人 ，2010 11描述。 <ol><li>消毒镊子和微型剪刀用70％的乙醇。制备一个5毫升注射器用21G针用无菌台氏缓冲液（130毫摩尔NaCl，5.4毫米氯化钾，6.0毫摩尔的Hepes，1.2mM的MgSO 4干燥，1.2mM的磷酸二氢钾 ，20mM的葡萄糖，1.5mM的氯化钙 2·2H 2 O， pH值= 7.2）。制备一个5毫升注射器用21G针用无菌KCl溶液（100毫摩尔）。 </li><li>制备65毫升每心脏培养基:DMEM补充有10％胎牛血清，抗生素/抗真菌剂（A / A; 100单位的青霉素，100微克的链霉素和0.25微克两性霉素B / ml）的胰岛素转铁硒（ITS; 10微克/毫升的胰岛素，5.5微克/毫升运铁蛋白，6.7纳克/毫升亚硒酸钠）和过滤杀菌。 </li><li>通过用70％乙醇和干燥用纸巾喷洒消毒灌注腔和气泡阱。然而组装MTCS（图1D），最初没有灌注腔。放置在支架上的泡沫陷阱。使用蠕动滚子泵具有易于装载泵头。 <ol><li>使用硅管使（见图1D）的贮存器（50毫升管）和泵，泵和气泡阱，所述气泡收集器和储存器之间的连接。使培养箱足够长内的管道，以允许介质达到平衡气体与气体在通过气体可渗透的硅管（1米）和培养箱尽可能短外管培养箱。 </li></ol></li><li>填充50ml管中，用70％的乙醇，开启泵（流速约1毫升/分钟，以允许适当的循环），并让乙醇CIRculate 30分钟进行消毒所有管道。通过清空贮存器和泵出从系统中的乙醇除去乙醇。 </li><li>填充50ml管中，用无菌蒸馏水，开启泵和让水循环30分钟，以除掉剩余的乙醇。通过清空容器和从系统中抽出的水除去水。 </li><li>填充50毫升管45毫升培养基，将支架在孵化器。开启泵（约1毫升/分钟流速）和分发介质填满所有管道，除去所有气泡，并允许介质相适应的气体组合物在孵化器至少1小时。保持培养箱中的标准组织培养条件下（5％CO 2和37℃）。确保没有气泡存在于管中。 </li></ol> 2.隔离小鼠心脏的<ol><li>注入鼠标500单位肝素。这将防止血液凝固和允许移除的Blood从心脏。 10分钟后，麻醉小鼠的感应室使用精密汽化器4％异氟烷。麻醉是足够的，当鼠标不响应脚趾捏。 </li><li>传输鼠标到夹层板和连接到同轴电路面罩来维持麻醉。消毒鼠标用70％酒精的皮毛。用剪刀打开腹腔，以暴露腔静脉和隔膜。 </li><li>暴露心脏，使开始从最后到第一肋外侧切口，反映胸廓过老鼠的头部。停止麻醉鼠标没有呼吸了。观察心脏的跳动。 </li><li>插入21G针连接到一个5毫升注射器含有无菌蒂罗德缓冲到从腹部下腔静脉进入胸腔（通过隔膜）。确保血液可从针头插入腔静脉尾退出。灌注日Ë台氏缓冲轻轻恒压进入腔静脉，直到心脏部分失去了它的红色。注:心脏跳动仍然允许去除血液从心脏。 </li><li>替换与连接到一个5毫升注射器含有无菌KCl溶液的21G针的针。轻轻灌注KCl溶液放入腔静脉，直到心脏停止跳动，取出针。注意:KCl溶液保留心脏处于松弛舒张阶段，这将允许灌注针更容易地插入冠状动脉系统和灌注。 </li><li>略使用弯钳解除心脏和用剪刀解剖心脏不受周围的组织，但留下的动脉和静脉的近端到心脏的完整和附连到心脏。在心脏转移到15毫升管含冰冷的PBS辅以抗生素/抗真菌剂（A / A）。存储在冰冷的PBS的心脏长达3小时。 </li></ol> 3. Cannulation中的小鼠心脏中灌注腔<ol><li>请在层流罩的所有程序。从15ml试管转移离体心脏到10cm培养皿，并添加PBS辅以A / A。 </li><li>在解剖显微镜下，使用微剪刀和镊子以除去所有非心脏组织，但保留在升主动脉，以至少与头臂动脉和肺静脉，2毫米近端心脏分叉。切断心脏顶点的尖端创造进入左心室腔内（通常为2毫米）。将灌注腔中的解剖显微镜下的流罩。 </li><li>附加一个5毫升注射器用培养基使用硅胶管插入针。填充灌注腔用20ml培养基并把心脏上的旋转台在灌注腔2的钝化针晶（ 图1）之间。调节旋转台的高度，使心脏的定位在前面针。 </li></ol> 4.结扎用于培养的二尖瓣（参见图1） <ol><li>结扎用缝合（丝7.0）主动脉。结扎左心耳与缝合。插入针（NR 1中图1）通过肺静脉进入左心房，并与缝合线近端结扎到其在左心房入口。确保针头不太远进入左心房，因为这会损害二尖瓣。 </li><li>插入针（nr.2 于图1）与直线运动到左心室。从灌注腔转移介质到50ml管。密封针与生物相容性胶心肌。 </li><li>之后胶水已干，小心地连接介质填充注射器针头nr.1，并检查是否有泄漏注入介质插入心脏。确保从针nr.2最后剩余的血液灌注出来的心脏该介质退出。 </li></ol> 5.李gation用于培养的主动脉瓣（见图1） <ol><li>插入在主动脉针（nr.1 在图1中），并结扎用缝合。确保针头不太远进入主动脉，因为这会损害主动脉瓣。插入针（nr.2 于图1）与直线运动到左心室。从灌注腔转移介质到50ml管。密封针与生物相容性胶心肌。 </li><li>之后胶水已干，小心地连接介质填充注射器针头nr.2，并检查是否有泄漏注入介质插入心脏。确保从针nr.1最后剩余的血液灌注出来的心脏该介质退出。 </li></ol> 6.将灌注腔上立场<ol><li>填补了灌注腔与传输介质20毫升将垫片和盖子上腔和拧紧垫圈和螺丝。 </li><li>从incuba取下组装MTCS器。附加灌注腔上的立场。连接管（参见图1D）。将立在培养箱（5％CO 2和37℃）。将管线连接到泵上。打开与约600μl/ min的流速泵。 </li><li>确保在储存和/或气泡阱介质的级别允许流的存在下的可视化由落入传入介质滴。培养后，心脏被从系统中移除，固定和处理以用于组织学检查。 </li></ol>

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The authors declare no conflicts of interest.

在培养该心脏小鼠阀关键步骤包括使来自鼠标切除心脏的和结扎在灌注腔尽可能短，以确保在垂直于阀的可行性的针和结扎之间的时间，以确保流动的正确方向。此外，检查在灌注腔结扎后的流量不中确保适当的插入和针结扎。关键是要保持无菌培养和防止气泡在油管，这有可能被困在心脏阻碍的流动。 用于心脏的灌注介质的总体积是45毫升（注意，即通过心脏灌注介质是不...

心脏瓣膜疾病是发病率和死亡率在西方世界的一个主要原因;其患病率随着年龄的增加，它会影响人口75年的10％以上和更老1。心脏的全身性部分的阀门，主动脉和二尖瓣，大多是受影响的。心脏瓣膜病的特征是所述阀的高度组织的结构，这导致的机械性能2变更的损失。结构完整性，因此对阀的功能是至关重要的。 所述瓣膜的瓣叶被包括瓣膜间质细胞（VIC），瓣膜内皮细胞（VEC），和细胞外基质，这是高度组织在一个分层图案3,4的。所述的VIC负责对ECM合成，降解和组织。因素从血液，内皮细胞发出或居住在编排上其功能的VIC的ECM行为。此外，机械力造成层流或振荡剪切应力，影响的VIC 5的行为压缩或拉伸应力在心动周期中作用于单张。 为了理解如何在阀的结构被调整，首先必须理解的VIC到心脏周期期间所经历的刺激的多样化如何作出反应。 在体外研究已经非常翔实有关瓣膜细胞的特征和能力。这些细胞在体外的反应，但是，可能不总是精确地模仿体内响应6;例如，VIC的对刺激的响应是依赖于内皮的存在和ECM 组合物5。此外，瓣膜细胞对刺激的响应取决于其特定的位置，在小叶7。除了生化刺激，瓣膜细胞的行为是通过作用Ø机械力确定n个阀 8。阀的各区域进行其自己的特定的一组血液动力学应力。尽管目前的体外模型表明，机械力的作用是瓣膜结构 5的重要决定因素，相关的机制仍不清楚。虽然体内模型提供了深入了解潜在的心脏瓣膜发展9,10的分子机制，见解成人瓣膜生物学仍然是难以捉摸的。 因此，开发了离体的流动的模型，其中该心脏瓣膜可以在其在心脏中自然位置的时间11长时间进行培养。这具有的优点是，阀保持在其天然构型和的VIC经历相同的环境作为体内，使得的VIC响应的刺激尽可能自然。此外，阀门在其自然位置在心脏的文化有利于使各瓣膜区域相关的血液动力学应力。在此体外模型，也就是说，微型组织培养系统（MTCS），所述阀可进行不同的生物化学和血液动力学刺激允许其在心脏瓣膜重塑作用的调查。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400-045</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-06</td><td></td></tr><tr><td>Silk 7-0</td><td>Ethicon</td><td>768G</td><td></td></tr><tr><td>100 mm culture dish</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>50 ml tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>5 ml syringe</td><td>BD</td><td>309649</td><td></td></tr><tr><td>21 G needle</td><td>BD</td><td>304432</td><td></td></tr><tr><td>Heparin</td><td>LEO</td><td>012866-08</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>Tedpella</td><td>1346</td><td></td></tr><tr><td>Silicon tubing</td><td>Thermo Scientific</td><td>8060-0020</td><td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td></tr><tr><td>Silicon tubing for pump</td><td>Masterflex</td><td>96400-13</td><td>I.D. x O.D. x Wall: 0.8 x1.59 x 0.40 mm</td></tr><tr><td>Biocompatible glue (Histoacryl)</td><td>B. Braun</td><td>1050071</td><td></td></tr><tr><td>precision vaporizer</td><td>Dr&amp;auml;ger</td><td>Vapor 200</td><td></td></tr><tr><td>peristaltic roller pump</td><td>Masterflex</td><td>7521-35</td><td></td></tr><tr><td>Easy-load pump head</td><td>Masterflex</td><td>7518-00</td><td></td></tr><tr><td>Flow chamber</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr><tr><td>Bubble trap</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr></tbody></table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

主动脉瓣（图2）或二尖瓣11可以是培养至少3天。通过在打开位置（其代表主动脉瓣和二尖瓣舒张位置收缩位置）培养，瓣膜细胞保持存活。无细胞死亡被观察为由于缺少TUNEL阳性细胞 （图2H，I）或裂解的caspase-3表达的测定（未示出和Lieber等人，2010 11）。胶原分布（如可视化通过马森三色染色）类似于天然条件时在1％血清?...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

在小型组织培养体系培养老鼠心脏瓣膜

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.8K 次观看.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

视频: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

科研

医学

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

生物学

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

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

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠状血管生成体模型

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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.

分离培养和成年小鼠心肌细胞转导

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

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

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

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

小鼠间质瓣细胞分离研究主动脉瓣体外钙化

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Source: Bouchareb, R. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62419">Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.</a>&#160; J. Vis. Exp. &#160;(2021).
This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.

钙化测定法测量主动脉瓣间质细胞中的钙浓度

Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.&#160; J. Vis. Exp. ...