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ケアと、米国国立衛生研究所 (NIH 文書番号 85-23、1996 の改正) によって公開された実験動物の使用の調査ガイドによると。一般に、雄 wistar ラット 3 〜 4 ヶ月の熟成、250-350 g の平均重量はこのプロトコルに使用されます。1 つのラット心は 20 の培養皿には十分 (皿あたり 1 mL; 内径: 35 mm) おおよそセル密度が 1.5 倍の 10 4 セル/1000 mm 2。
 1 です。 メディアの準備および試薬 <ol><li>クレアチン、カルニチン、タウリン培地 (CCT 中) 注: CCT 媒体中 199 クレアチン、カルニチン、タウリンの添加に基づく複雑な媒体であります。
	<ol><li>準備 1 L 中 199: 3.6 g Hepes と 1 h のミックスを追加します。655.5 mg クレアチン (5 mM)、395.4 mg カルニチン (2 mM)、および 625.5 mg タウリン (5 mM) を追加します。カルニチン、タウリンを変更する pH &#60; 7。汚染細胞、例えば 血管内皮細胞や線維芽細胞の増殖を抑制する、するために培地に 10 μ M シトシン β-D-アラビノを追加します。7.4 と滅菌フィルター媒体に (2 mM) を NaOH で pH を調整します。4 CCT 媒体を保存 ° C</li>
	</ol></li> <li>パウエル媒体 <ol><li>1 L パウエル中で溶解する 6.43 g 塩化ナトリウム (110 mM) 0.19 g KCl (2.5 mM)、0.16 g KH 2 PO 4 (1.2 mM)、0.3 g MgSO 4 7 H 2 O (1.2 mM)、5.96 g Hepes (25 mM) と 1.98 g D (+)-アクア滅菌でグルコース一水和物 (10 mM)。7.4 と滅菌フィルター媒体 (2 M) を NaOH で pH を調整します。ストア パウエル培地で 4 ° C</li>
	</ol></li> <li>塩化カルシウム (CaCl 2) <ol><li>100 mM CaCl 2 ソリューション (50 mL) を準備し、500 μ L CaCl 2 を含む因数を準備します。-20 で因数を凍結 ° C</li>
	</ol></li> <li>培地の準備 <ol><li>準備 3 細胞培養媒体: 中古めっき中、めっき中、洗濯中。すべての 3 つの媒体 (表 1) のための基礎として使用 CCT 媒体。CCT 培地 1 ml の培養皿あたりを計算します。したがって、20 の文化料理 20 mL CCT メディアの準備 (内部の直径: 35 mm).</li>
		<li>細胞培養プレート: コートの各細胞培養プレート (内径: 35 mm) 前播種培地 1 ml。使用する前にコーティング プレート 37 ° C 一晩、少なくとも 2 時間を格納します</li>。
	</ol></li></ol> 2。大人の心筋細胞の単離 <ol><li>蛍光準備灌流システム <ol><li>熱メッキ媒体と洗濯中 37 ° C Defreeze 500 μ L CaCl 2 の管およびコラゲナーゼの 25 mg で重量を量る</li>。
		<li>フラッシュ滅菌は、アクアと蛍光灌流システムその後 5 分間システムを循環するパウエル媒体を聞かせてください。</li>
		<li>いずれかの空気泡とガス 95% 酸素媒体なし 80 ml パウエル中蛍光灌流システムを入力します</li>。
		<li>40 ml パウエル中管 (50 mL) を準備、37 ° C に熱するし、95% の酸素とガスします</li>。
		<li>カニューレに削除された心を取り付けるための長さ約 25 cm のスレッドを準備します</li>。
		<li>は、アルコール (70% ボリューム) にかみそりの刃を脱脂してチョッパーに固定します。チョッパーにプラスチック製のディスクをクランプします</li>。
	</ol></li> <li>心の抽出 <ol><li>と 4 ~ 5% イソフルレン雄 wistar ラットを麻酔し、頚部転位と犠牲します。腹部断肋骨アーチの後ろに腹部を開くし、開く胸腔内横隔膜を切ってはさみのペアは同じと</li>。
		<li>は、胸腔内に高度頭蓋胸腺上切断して心臓、肺、胸腺を削除します。すぐに冷たい食塩水に材料を転送します</li>。
		<li>解剖はさみ (大) と心臓から肺、胸腺を削除し、カプセル鉗子で材料を遠ざけているか、によっては新しい食塩に後者転送します</li>。
	</ol></li> <li>分離 <ol><li>カプセル鉗子と解剖はさみ (大小) を使用して心から胸腺、気管、脂肪と結合組織の残基のような余分な組織を削除します。大動脈を明らかにし、第 1 および第 2 鰓建築間解剖はさみ (大小) と断絶</li>
		<li>蛍光灌流システムの滴下を開始します。蛍光灌流システムのカニューレに中心を置き、ワニ クランプと後で準備されたスレッドに最初じっと見つめます。それは血の自由まで心をすすぎします</li>。
		<li>5-6 mL 暖かいパウエル中 25 mg コラゲナーゼを溶かし、12.5 μ L CaCl 2 (30 μ M) を追加しています</li>。
		<li>は滴下心を蛍光灌流システムに接続されているガラス漏斗を移動することによって循環を閉じて解決コラゲナーゼを灌流システムに追加します。毎秒 1 滴のドロップ速度と 25 分間灌流を開始 。
		注: 灌、心が膨らむし、蝋のような外観を取得します</li>。
		<li>は 25 分後に灌流を停止し、中心部を蛍光灌流システムから取り外します。心臓から大動脈、アトリア、および結合組織を削除し、右と左の心室を開きます</li>。
		<li>90 ° の角度に 2 回心臓を切る (切断幅: 0.7 mm; 速度: 0.15 cm/s)。10 の 2 つのメスを使って手動でこのプロセスを繰り返す s 両側</li>。
		<li>は、新しいチューブ (50 mL) に灌流中の 12 mL を転送します。37 で別の 5 分間この媒体およびダイジェストのセルにセル スラリーを注ぐ ° c. ミックス ソリューション毎分</li>。
		<li>新しいチューブ (50 mL) にナイロン メッシュ (200 μ m) を通して消化の心臓を持つソリューションにフィルターを適用します</li>。
		<li>は、上清 29 x g で 3 分間破棄でフィルタ リング ソリューションを遠心し、12.5 μ L CaCl 2 (250 μ M) 細胞ペレットを含む 6 mL 暖かいパウエル培地を追加します。滑らかな揺れ動きを通じてペレットを再懸濁します。再び 2 分破棄上清の 29 x g で遠心し、6 mL 暖かいパウエル媒体 25 μ L CaCl 2 (500 μ M) で置換を追加します。穏やかな揺れ動きを通じて細胞ペレットを溶解し、12 mL 暖かいパウエル媒体含む 120 μ L CaCl 2 (1 mM) を追加します。3 回目 16 × g で 1 分間遠心します。再度、上清を削除します</li>。
		<li>細胞ペレットを混ぜてあらかじめ加温めっき中です</li>。
		<li>は、培養皿からプレめっき媒体を削除します。メッキ各培養プレートに、分離心筋細胞を含む培地 1 mL を転送します。インキュベート, 37 ° C で新鮮な分離心筋</li>
		<li>は、培養皿からメッキ媒体を削除します。洗浄媒体各培養皿に 1 mL を追加し、媒体を変更することがなく 6 日に 37 ° C でプレートを蓄える</li>。
		<li>の異なった化学薬品と ARVC 治療の影響を調査するためまず洗浄媒体と、その後別の化学物質を追加することによってメッキ媒体を更新します 。
		注: 光学顕微鏡レベルで評価: 各セルの準備と 150 に 300 心筋細胞が光顕で一日監視必要があります。外見によるとグループにすべてカウント心筋細胞を分割 (例えば、 &#34; 棒状 &#34;、&#34; ダウン ラウンド &#34;、&#34; 拡散 &#34; と &#34; 異常な外観 &#34;)。カテゴリ &#34; 拡散 &#34; 偽足のような構造を持つすべての心筋細胞が含まれています。&#34; 異常な外観 &#34; 不規則な表面を持つすべて ARVC しない検出のまま細胞膜が含まれています</li>。
	</ol></li></ol> 3。例実験 <ol><li><ol><li>共焦点レーザー顕微鏡による培養中に ARVC の分析の形態と構造変換を 大人の心筋細胞の免疫蛍光/蛍光染色 をします。ファロイジン TRITC を使用して F-アクチンの構造を調査する &#34; ロッド形 &#34;、&#34; ダウン ラウンド &#34; と &#34; 拡散 &#34; ARVC。メーカーによると染色を実行 &#39; s プロトコル。ファロイジン TRITC 染色のための例は参照 ا ら に指定します。 11. 蛍光/蛍光染色と違い実験的治療 (例えば の間培われた大人の心筋収縮装置のデ- と再 differentiation でSwiprosin-1、イオノマイシン) 調べることができます</li>。
	</ol></li> <li>リアルタイム定量的 RT-PCR (qRT PCR) <ol><li>異なる遺伝子の mRNA 発現の変化を調査する qRT PCR を行う (例えば、 OSM Swiprosin 1, β-MHC) ARVC の栽培中。拡大培養皿を使用して十分なサンプル サイズ (内径: 60 mm) 2 mL の量。ARVC 5 培養皿の 1 つのサンプルが得られます。MRNA の単離と製造元によると cDNA の変換 &#39; s プロトコル</li>。
	</ol></li> <li>イムノブロット法 <ol><li>ARVC の培養中に (例えば Swiprosin 1) 蛋白質発現の変化を調査するため西部のしみを実行します。サンプルあたり 1 つの培養皿 (1 mL) を使用します</li>。
	</ol>
	</li>
</ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+evaluation+of+the+contractile+apparatus+in+primary+cultures+of+rabbit+cardiac+myocytes.">Morphometric evaluation of the contractile apparatus in primary cultures of rabbit cardiac myocytes.</a> Circ. Res. 69 (1), 86-94 (1991).</li></ol>

表示される結果は、フランツィスカ ا の博士論文の一部です。

大人の心筋細胞生体内での挙動は他の細胞 (例えば、神経細胞、内皮細胞、線維芽細胞、炎症細胞) と電気シンシチウム彼らを形作る多くの相互作用を受けます1。したがって、分離と培養 ARVC の専ら大人の心筋細胞のストレス適応の勉強が必要です。隔離・培養 ARVC の主な効果は、: 1) 細胞外マトリックスと細胞間の連絡先から切断します。2) 収縮刺激から切断...

生体内で大人の心筋細胞間の細胞間接触に基づく電気シンシチウムとして働きます。さらに、心臓線維芽細胞、内皮細胞、神経細胞、炎症細胞1のような隣接する細胞に影響を与えています。心筋細胞の細胞内組織を適応能力を研究するために心臓肥大、心不全、分離ラット心室の栽培が主要な最初のステップみたように負荷条件を変更心筋 (ARVC) は必要な2,3,4です。歴史的には、心筋細胞最初から分離された鶏胚心5,6.数年後、終末分化した心筋細胞の最初の分離は、カルシウム枯渇7を使用して記述されていた。ただし、これらの大人の心筋細胞はカルシウム耐性をなかったし、したがって、機能アッセイには使用できませんでした。最後に、1976 年に新しいプロトコルと有効にパウエル ツイスト生理的条件8下大人の心室心筋細胞を調査します。最初のステップとして彼らは低カルシウム濃度とステップワイズ法で生理学的濃度 ca がその後増加の下で成人の心筋細胞を分離しました。今日、分離と大人の心筋細胞の培養のためのほとんどのプロトコルはこのカルシウム プロトコルで動作し、高密度細胞接触1の酵素の消化力のコラゲナーゼを使用します。
育成に成功した、牛胎児血清 (FCS) またはオンコスタチン M (OSM) が必要です。ARVC 実行、デ- と再-differentiation 筋分解と改革9,10、11,12を含む広汎な構造変更をします。このプロセスは再発現胎児型遺伝子、β-ミオシン重鎖 (β-MHC) のような伴われる拡散4,11、とも呼ばれる、偽足のような構造体の形成と肥大から知られています。13。 さらに、swiprosin-1 (EFHD2)、新たに同定されたタンパク質は栽培 ARVC11の再分化の過程で大きな役割を果たしています。その結果、文化 ARVC 文化2,4,14の 2 〜 3 週間後の収縮を自発的に表示する、広範囲にわたる多セルに変換します。
最近の発見は、心臓再脱 differentiation 模倣文化の条件下で発生すると機能を生体内で見られる心臓改造10,15時に明らかにしました。心筋リモデリングは、心疾患16の間にキーのプロセスです。心疾患は、工業化社会で死の主な原因で、まだ、大人の心筋細胞の生物学の理解を深めることが重要です (人; 2015)。ARVC の分離と培養は新たな戦略と心臓疾患の治療のための薬を開発することができます。この原稿分離 ARVC の耕作のためのプロトコルが提供されます。さらに、このメソッドのいくつかの重要な部分は、ディスカッション セクションでハイライトされます。

<table><tbody><tr><td>Langendorff perfusion system</td><td>inhouse construction</td><td></td><td>double-walled with a water&lt;br/&gt; based heating system</td></tr><tr><td>Tissue chopper Mc Ilwain</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Aortic Cannula, OD 1,8 mm</td><td>inhouse construction</td><td></td><td></td></tr><tr><td>Abdominal shears</td><td>Aeskulap</td><td>BC772R</td><td></td></tr><tr><td>Capsule forceps</td><td>Eickemeyer</td><td>171307</td><td></td></tr><tr><td>Dissecting scissor large</td><td>Aeskulap</td><td>BC562R</td><td></td></tr><tr><td>Dissecting scissor small</td><td>Aeskulap</td><td>BC163R</td><td></td></tr><tr><td>Mash with Polyamid</td><td>Neolab</td><td>4-1413</td><td>mash size 200 &amp;mu;m</td></tr><tr><td>plastic disc</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Collagenase Typ II</td><td>Worthington</td><td>LS004177</td><td></td></tr></tbody></table>

isolation and cultivation of adult rat cardiomyocytes

そのままの心で隣接するセル大人の心筋細胞に影響を与えます。分離法と成人の心筋細胞の培養は、特定の治療法や環境下でこれらの細胞の動作の正確な調査が可能です。この原稿は、ラットの心室心筋細胞 (ARVC) の培養と分離を成功させるためのプロトコルを示します。
頚部転位深麻酔下でラットが犠牲に。中心部を抽出し、大動脈がカバーされていません。その後、蛍光灌流システム カルシウム枯渇とコラゲナーゼ処理の灌流が実行されます。その後、心室の組織を取得しますみじん切り、再循環、フィルター、CaCl2の段階的な追加で 3 つの遠心分離手順に続いて生理カルシウム濃度に到達するまで。ARVC 細胞培養皿にメッキパーツを多数。細胞培養液を更新した後 ARVC できます栽培される 6 日間の血清を含む培養液を変更せず。ARVC の分離は、カルシウム敏感なプロセスです。細胞内カルシウム濃度のわずかな変化は、質と隔離されたセルの実行可能性の減少を引き起こします。
新鮮な分離 ARVC は棒状のものです。栽培の最初の日以内に、彼らは、棒状形態とフォーム偽足のような構造 (拡散) を失います。この形態の形成の間に ARVC は当初改革アクチン繊維の応力およびde novo sarcomerogenesis によって続いて収縮要素を低下します。栽培の 1 週間後は、ほとんど ARVC は明確に検出可能な十字紋と広範な外観を表示します。このプロセスは細胞内カルシウム濃度に敏感イオノマイシンによる治療は拡散減衰です。Β-ミオシン重鎖 (β-MHC)、オンコスタチン M (OSM) swiprosin 1 (EFHD2)、このプロセスでのデ- と再-differentiation キー マーカー。最近の研究はことを心臓改造中に見られる生体機能模倣心臓再脱 differentiation 発生培養条件下で示唆しています。したがって、ARVC の分離と培養心筋細胞の生物学を理解する上で重要な役割を再生します。

アダルト文化における心筋細胞:図 1は、最後の洗浄後、新鮮単離アダルト心筋 2 h の概要を示します。すべての心筋細胞の約 75% は、棒状の形態を持っていた。残りの 25% は、円形の形態とない検出そのまま細胞膜 (図 1) 異常な外観を示した。まで栽培 (6 日目) の最後にすべての心筋細胞の 15% を示した広がり、...

Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

Isolation and Cultivation of Adult Rat Cardiomyocytes

ここでは、分離と培養ラット心室心筋細胞 (ARVC) のためのプロトコルを提案する.分離 ARVC は、短期および長期の栽培に使用できます。分離と ARVC の栽培は、心疾患の新しい治療法の開発に重要な役割を再生できます。

ラット心筋細胞の分離と培養

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

isolation-and-cultivation-of-adult-rat-cardiomyocytes

Research

JoVE Journal

Medicine

15.5K ビュー.  Justus Liebig University Giessen. ここでは、分離と培養ラット心室心筋細胞 (ARVC) のためのプロトコルを提案する.分離 ARVC は、短期および長期の栽培に使用できます。分離と ARVC の栽培は、心疾患の新しい治療法の開発に重要な役割を再生できます。

ビデオ: Isolation and Cultivation of Adult Rat Cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

発生生物学

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.

成体ラットの心筋細胞の初代培養

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

生物学

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.

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

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 and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

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

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.

保存された駆出率とメタボリック症候群関連心不全ラットモデルから心房の心筋細胞の単離

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

医学

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 ...

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 ...

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.

マウス心筋細胞を一貫して単離するための簡単で効果的な方法

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

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

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 mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

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 ...