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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 
			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 &#956;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

15.4K 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 Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

医学

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 ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

成体マウスからの心房筋細胞の単離

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超音波増強原発性成人線維芽細胞分離

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

皮膚心筋細胞を用いた心機能のインビトロ評価

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

ラットにおける骨髄間葉系幹細胞の単離と培養

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

蛍光タグ付きサルコメアタンパク質を用いた多能性幹細胞由来心筋細胞のサルコメア短縮

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

成体マウスからの心房と心室筋細胞の同時分離のためのランゲンドルフ法の改変

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...