Name: culturing mouse cardiac valves in the miniature tissue culture system
Uploaded: 2015-10-19T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

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

このプロトコルは、動物実験倫理委員会のLUMCのガイドラインに従っています。 楽器の調製、培養培地、及びMTCS 注：層流フード内のすべての準備を行います。 MTCS灌流チャンバー、気泡トラップとスタンドリーバーら 、2010 11に記載されています。 <ol><li> 70％エタノールで鉗子とマイクロはさみを消毒します。無菌タイロード緩衝液（130mMの塩化ナトリウム、5.4のKCl、6.0 mMのヘペス、1.2 mMの硫酸マグネシウム、1.2 mMのKH 2 PO 4、20mMのグルコース、1.5mMの塩化カルシウム2・2H 2 Oで21G針で5 mlシリンジを準備pH値= 7.2）。無菌KCl溶液（100 mM）ので21G針で5 mlシリンジを準備します。 </li><li>心あたりのメディアの65ミリリットルを準備し、10％ウシ胎児血清を補充したDMEM、抗生物質/抗真菌剤（A / A、ペニシリン100単位、ストレプトマイシン100μgの、およびアムホテリシンB / mlを0.25 UG）、インスリントランスフェリンセレン（ITS;を10μg/ mlのインスリン、5.5 / mlのトランスフェリン、6.7 ngの/ mlの亜セレン酸ナトリウム）、フィルター滅菌します。 </li><li> 70％エタノール、紙タオルで乾燥して噴霧することにより、灌流チャンバー及び気泡トラップを滅菌します。最初に灌流チャンバーなしに、しかし、MTCS（ 図1D）を組み立てます。スタンドに気泡トラップを配置します。簡単に負荷のポンプヘッドと蠕動ローラーポンプを使用してください。 <ol><li>シリコンチューブを使用すると、リザーバ（50mlチューブ）、ポンプ、ポンプおよび気泡トラップ、気泡トラップとリザーバ（ 図1Dを参照）との間の接続を行います。媒体が気体透過性のシリコンチューブ（1メートル）とできるだけ短くインキュベーター外管を通してインキュベーターでガスでガス平衡に到達することを可能にする十分な長さインキュベーター内部の配管をしてください。 </li></ol></li><li> 、70％エタノールで50mlのチューブを埋める（適切な循環を可能にするために、1ml /分の周りの流速）は、ポンプをオンにし、エタノールのCIRを聞かせてすべてのチューブを滅菌するために30分間culate。貯蔵器を空にし、システムからエタノールをポンピングすることにより、エタノールを除去します。 </li><li> 、滅菌蒸留水で50mlのチューブを埋めるポンプをオンにして、水が残っているエタノールを取り除くために30分間循環させて。貯蔵器を空にし、システムから水をポンプで水を除去します。 </li><li>メディアの45ミリリットルと50ミリリットルチューブを埋める、インキュベーター中にスタンドを置きます。ポンプ（1 ml /分の周りの流速）をオンにして、すべてのチューブを埋めるための媒体を循環させ、すべての気泡を除去し、培地は、少なくとも1時間インキュベーター内ガス組成に適応することができます。標準的な組織培養条件（5％CO 2、37℃）でインキュベーターを維持します。気泡がチューブ内に存在していないことを確認してください。 </li></ol>マウスの心臓の2の単離<ol><li>ヘパリン500単位でマウスを注入します。これは、凝固血液を防止し、Bの除去を可能にするであろう心の底からlood。 10分後、精密気化器を用いて、4％イソフルランで誘導チャンバ内でマウスを麻酔。マウスはつま先のピンチに応答しない時の麻酔で十分です。 </li><li>解剖ボードにマウスを移し、同軸回路に接続されたフェイスマスクを用いて麻酔を維持します。 70％のアルコールでマウスの毛を消毒します。はさみは、腹腔を開いて使用すると、大静脈及びダイヤフラムを露出させます。 </li><li>心臓を露出するために、最後から最初の肋骨に起動横切開を行い、マウスの頭の上に胸郭を反映しています。マウスはもう呼吸していないされているように麻酔を停止します。心臓の鼓動を観察します。 </li><li> （ダイアフラムを介して）胸腔内に腹部から下大静脈に無菌タイロード緩衝液を含む5 mlシリンジに取り付けた21G針を挿入します。血液が針の挿入から大静脈の尾側から出ることができることを確認してください。目を灌流電子タイロード緩衝液を静かにと大静脈への一定の圧力で心臓の一部が、その赤い色を失うまで。注意：心臓は、心臓からの血液の除去を可能鼓動のまま。 </li><li>無菌のKCl溶液を含む5 mlシリンジに取り付けた21G針で針を交換してください。静かに心臓が鼓動を停止するまで大静脈にKCl溶液を灌流し、針を取り除きます。注意：KCl溶液を灌流針と冠動脈系の灌流を容易に挿入できるようになりますリラックスした拡張期に心を保持します。 </li><li>わずかに湾曲した鉗子を用いて心臓を持ち上げ、ハサミを使用して、周辺組織から自由に心を分析無傷と心臓に接続されている心臓の近位に動脈と静脈を残します。氷冷PBSを含む15mlチューブに心を移し抗生物質/抗真菌剤（A / A）を補いました。最大3時間、氷冷PBS中で心を保管してください。 </li></ol> 3. Cannulatio灌流チャンバー内にマウスハーツのN <ol><li>層流フード内のすべての手順を実行します。 10センチメートルシャーレに15mlチューブから分離された心を移し、PBSを追加し、A / Aを補いました。 </li><li>解剖顕微鏡下では、すべての非心臓組織を除去するために、マイクロはさみとピンセットを使用していますが、上行大動脈を維持する心臓に腕頭動脈と肺静脈、2mmの近位との少なくとも分岐します。左心室の内腔（通常は2ミリメートル）へのアクセスを作成するために、心尖の先端をカット。解剖顕微鏡下流フードで灌流チャンバーを配置。 </li><li>シリコンチューブを使用して、挿入針に培地で5 mlシリンジを取り付けます。培地20mlで灌流チャンバーを記入し、灌流チャンバー（ 図1）で2平滑化した針との間で回転ステージに心を置きます。心臓は、目の前に配置されるように、回転ステージの高さを調整します針。 </li></ol>僧帽弁を培養するための4ライゲーション（図1参照） <ol><li>縫合糸（絹7.0）で大動脈を連結。縫合糸で左心耳を結紮。左心房への肺静脈を通って（図1に1をNR）針を挿入し、左心房内のエントリに縫合糸の近位で連結します。これは、僧帽弁に損傷を与えるよう針が左心房にすぎないことを確認します。 </li><li>左心室に直線運動で針（ 図1の nr.2）を挿入します。 50mlのチューブに灌流チャンバーからメディアを転送します。生体適合性の接着剤で心筋に針をシール。 </li><li>接着剤が乾燥した後、慎重にnr.1針や漏れがあるかどうかを確認するための媒体で満たされた注射器を接続することにより、心に培地を注入します。針nr.2と最後に残った血液からの媒体出口は心の外に灌流されていることを確認します。 </li></ol> 5.リー大動脈弁を培養するためのゲーション（図1を参照） <ol><li>大動脈に注射針（ 図1の nr.1）を挿入し、縫合糸で連結します。これは大動脈弁を損傷するよう針が大動脈にすぎないことを確認します。左心室に直線運動で針（ 図1の nr.2）を挿入します。 50mlのチューブに灌流チャンバーからメディアを転送します。生体適合性の接着剤で心筋に針をシール。 </li><li>接着剤が乾燥した後、慎重にnr.2針や漏れがあるかどうかを確認するための媒体で満たされた注射器を接続することにより、心に培地を注入します。針nr.1と最後に残った血液からの媒体出口は心の外に灌流されていることを確認します。 </li></ol>スタンドに6.灌流チャンバー<ol><li>メディア転送20mlで灌流チャンバーを埋めます。室にガスケットと蓋を置き、ワッシャー、ネジで締めます。 </li><li> incubaから組み立てMTCSを削除TOR。スタンドに灌流チャンバーを取り付けます。チューブを接続します（図1Dを参照してください ）。インキュベーター（5％CO 2、37℃）にスタンドを置きます。ポンプにチューブを接続します。 600μL/分の周りの流速でポンプの電源を入れます。 </li><li>貯留および/または気泡トラップ媒体のレベルが入ってくるメディア滴の落下によって流れの存在の視覚化を可能にすることを確認します。培養後、心臓は、システムから除去固定し、組織学的検査のために処理されます。 </li></ol>

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The authors declare no conflicts of interest.

マウス心臓弁の培養における重要なステップは、流れの適切な向きを確保するために、マウスから心臓の切除および弁に対して垂直に針の生存率および連結を確実にするためにできるだけ短く灌流チャンバー内ライゲーションの間の時間を作る挙げ。さらに、メディアなし灌流チャンバー内ライゲーション後の流れをチェックする適切な挿入や針の連結を確実にします。これは、無菌培養を?...

心臓弁疾患は、西洋世界における罹患率および死亡率の主要な原因です。その有病率は年齢とともに増加し、それは人口75年の10％以上と1古い影響を与えます。ハートの全身一部、大動脈弁と僧帽弁の弁は、ほとんど影響を受けています。心臓弁疾患は、機械的特性2の変化をもたらすバルブの高度に組織構造の喪失によって特徴づけられます。構造的完全性は、弁の機能のために極めて重要です。 弁のリーフレットは、非常に層状のパターン3,4で構成されて弁膜間質細胞（VIC）、弁膜内皮細胞（VEC）、および細胞外マトリックス、から構成されています。 VICには、ECM合成、分解と組織を担当しています。要因としては、血流から発せられる電気部品やその機能を組織のVICにECMの行為に常駐しています。加えて、機械的な力は、圧縮または引張応力がVICの5の動作に影響を与える、層状または振動せん断応力が生じる心周期中のリーフレットに作用します。 弁の構造が制御されている方法を理解するためには、最初のVICは、心周期の間に経験した刺激の多様なセットにどのように反応するかを理解する必要があります。in vitro試験は、心臓弁膜細胞の特性や能力について非常に有益でした。 in vitroでのこれらの細胞の応答は、しかし、常に正確にin vivoでの応答6を模倣しないことがあります。例えば、刺激にVICの応答は、ECおよびECM組成5の存在に依存します。また、刺激に対する弁膜細胞の応答は、リーフレット7に、その特定の場所に依存します。生化学的な刺激に加えて、弁の細胞の挙動は、Oに作用する機械的な力によって決定されますn個のバルブ8。バルブの各領域は、血行力学的応力の自身の特定のセットに供されます。現在のex vivoでのモデルは、機械的な力が弁膜構造5の重要な決定因子であることを示してきましたが、関連するメカニズムはまだ不明です。 in vivoモデルは、弁膜開発9,10の根底にある分子メカニズムへの洞察を提供してきたが、大人弁膜生物学への洞察はまだとらえどころのないです。 したがって、ex vivoでのフロー・モデルとは、心臓弁は、時間11の長期間の心に彼らの自然な位置で培養することができる開発されました。これは、弁がその天然のコンフィギュレーションに残りおよびVICは、VICの応答が可能な限り自然な刺激になって、 インビボのと同じ環境を経験するという利点を有します。また、心の中で彼らの自然な位置にある弁の文化はそれぞれを施す容易関連する血行動態ストレスに弁膜地域。このex vivoでのモデル、 すなわち、小型組織培養系（MTCS）において、弁は、心臓弁のリモデリングにおけるそれらの役割の研究を可能にする種々の生化学的及び血行力学的な刺激を受けることができます。

<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.

大動脈弁（ 図2）または僧帽弁11には、少なくとも 3日間培養することができます。 （大動脈弁と僧帽弁用拡張期位置のための収縮期位置を表す）開位置に培養することにより、弁膜細胞が生存したまま。 TUNEL陽性細胞（ 図2H、I）、または切断されたカスパーゼ3の発現の欠如によって決定されるように何らの細胞死が観察されない（図示していないリーバー<...

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.

ミニチュア組織培養系でマウス心臓弁の培養

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

Research

JoVE Journal

Bioengineering

8.8K ビュー.  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.

ビデオ: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

マウス胚心臓から心外膜細胞の体外培養で

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

工学

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.

異心臓移植のマウスモデルを用いた肺動脈弁の移植

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

医学

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.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

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

生物学

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.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

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.

冠動脈血管新生のインビトロモデル

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.

ネズミ弁内皮細胞の単離

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.

成体マウスの心筋細胞の単離、培養および形質導入

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.

成人マウスおよびラット心筋細胞の分離、トランスフェクション、長期培養

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.

マウス間質弁細胞の分離を用いて大動脈弁の石灰化を 研究

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://app.jove.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.

大動脈弁間質細胞のカルシウム濃度を測定するための石灰化アッセイ

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. ...

ミニチュア組織培養系でマウス心臓弁の培養

要約

さらに動画を探す

この動画の章

マウス胚心臓から心外膜細胞の体外培養で

異心臓移植のマウスモデルを用いた肺動脈弁の移植

小説

細胞シグナリングのための成体マウスの心筋細胞の単離と文化

冠動脈血管新生のインビトロモデル

ネズミ弁内皮細胞の単離

成体マウスの心筋細胞の単離、培養および形質導入

成人マウスおよびラット心筋細胞の分離、トランスフェクション、長期培養

マウス間質弁細胞の分離を用いて大動脈弁の石灰化を研究

大動脈弁間質細胞のカルシウム濃度を測定するための石灰化アッセイ

ミニチュア組織培養系でマウス心臓弁の培養

要約

さらに動画を探す

この動画の章

マウス胚心臓から心外膜細胞の体外培養で

異心臓移植のマウスモデルを用いた肺動脈弁の移植

小説

細胞シグナリングのための成体マウスの心筋細胞の単離と文化

冠動脈血管新生のインビトロモデル

ネズミ弁内皮細胞の単離

成体マウスの心筋細胞の単離、培養および形質導入

成人マウスおよびラット心筋細胞の分離、トランスフェクション、長期培養

マウス間質弁細胞の分離を用いて大動脈弁の石灰化を 研究

大動脈弁間質細胞のカルシウム濃度を測定するための石灰化アッセイ

マウス間質弁細胞の分離を用いて大動脈弁の石灰化を研究