Name: isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction
Uploaded: 2018-07-26T14:00:00.000+00:00
Duration: 8 min 31 s
Description: Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

本研究は DZHK (ドイツ語マッシィ心血管研究センター)、によって支えられた (Else Kröner フレゼニウス財団、f. H.) EKFS、BMBF (ドイツ語教育省および研究) と臨床科学者プログラム資金 BIH シャリテによってシャリテ - Universitätsmedizin ベルリン、ベルリン衛生研究所 (f. H.)。

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ここでは、我々 はまずメッツ関連 HFpEF 示す心房改造22をマークのモデルラットから単一セル ACMs の隔離のためのプロトコルを説明します。手順は、過剰な脂肪組織はますます困難な大動脈カニュレーションと同様、手術の準備をすることができます一意に挑戦です。トラブルシューティング ガイドは、表 2アドレス分離のプロシージャの最も一般的な問題です?...

メッツ タイプ 2 糖尿病と心血管疾患のリスク要因のクラスターをについて説明し、増加動脈血圧高脂血症 (トリグリセリドの発生し、高密度リポタンパク質コレステロールを下げる)、増加断食グルコースおよび中心性肥満1。メッツの世界的な有病率は 25-30% になり、絶えず上昇2と推定されます。HFpEF はメッツにしばしば関連付けられている異種臨床症候群です。HFpEF と (すなわち、高血圧性心臓病) その前の段階の間に心筋のリモデリング心房3の改造伴われるも。減らされた収縮機能および左心房の構造的変化は、死亡率の増加、心房細動新規発症の心不全4と関連しています。心房リモデリングはイオン チャネル機能、Ca2 +恒常性、心房の構造、線維芽細胞の活性化と組織の線維化5の変化によって特徴付けられます。左心房改造メッツ関連 HFpEF とその基礎となる病理学的メカニズムはまだよくわかっていないし、さらに詳細な調査が必要です。動物モデルは、貴重なツールになるし、心房の心筋症6,7,8,9の分野で多くの進歩をもたらすと証明しました。
隔離された単一細胞心筋細胞を用いた研究が確証し、生体内で調査結果を補完するためによく使用されます。分離、および潜在的なそれに続く細胞培養、シグナル伝達経路, イオン チャネル電流と興奮収縮連関の調査を可能にします。生理学的な条件の下で心筋細胞は増殖しません。心房性 na 利尿因子の転写制御配列と、シミアン ウイルス 40 large T 抗原遺伝子導入マウスの融合は、AT 110の名前最初の不死化 ACMs の作成をもたらした。AT 1 細胞のそれ以上の開発は HL 1 セルのみ連続継代できませんがまた11を自発的に契約をもたらした。ただし、に比べてあまり組織的微細構造、筋原繊維の11、および過分極側に内側現在12開発の頻度が高いなどの新鮮単離細胞構造および機能に違いを示す彼らは。ラットおよびマウスの様々 なモデルからの心室心筋細胞 (VCM) の分離が確立13,14,15,16,17,18,19します。 一般的に、摘出心臓は蛍光装置に取り付け、Ca2 +による逆行性に灌流-コラーゲンやプロテアーゼなどの消化酵素を含む空きバッファー。カルシウムは生理学的条件を段階的に再導入し。しかし、ACMs の分離に専用プロトコルが利用可能な20,21, 線維の増加と圧力差による心房リモデリングと疾患モデルの有用性は限られています。
この記事で心房改造 (すなわち、特にメッツ関連 HFpEF の ZFS1 ラット モデル)22動物から心房の単一細胞心筋細胞の隔離のためのプロトコルを実装しています。既存の隔離のプロトコル最適化、制御、形態および機能そのまま心筋細胞のより高い収穫につながる心の空洞の内圧を修正する簡単な特注デバイスによって補完されます。次のプロトコルは、研究者のステップ バイ ステップ ガイド、カスタムメイド機器ソリューションのリスト、包括的なトラブルシューティング ガイドの詳細な説明を提供します。

<table><tbody><tr><td>ZSF-1 Obese rat</td><td>Charles River Laboratories, Inc.</td><td></td><td>21 weeks old</td></tr><tr><td>Fine Iris Scissors</td><td>Fine Science Tools GmbH</td><td>14094-11</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools GmbH</td><td>14001-18</td><td></td></tr><tr><td>Micro Dressing Forceps (curved, serrated)</td><td>Aesculap, Inc.</td><td>BD312R</td><td></td></tr><tr><td>Tissue Forceps (straight, 1 x 2 teeth)</td><td>Aesculap, Inc.</td><td>BD537R</td><td></td></tr><tr><td>Tying Forceps (angled)</td><td>Aesculap, Inc.</td><td>MA624R</td><td></td></tr><tr><td>Rodent and Small Animal Guillotine</td><td>Kent Scientific Corp.</td><td>DCAP</td><td></td></tr><tr><td>Low Cost Induction Chamber 3.0 L</td><td>Kent Scientific Corp.</td><td>SOMNO-0730&amp;nbsp;</td><td></td></tr><tr><td>Butterfly Winged Infusion Set 21 G</td><td>Hospira, Inc.</td><td>181106101</td><td></td></tr><tr><td>Abbocath 16 G</td><td>Hospira, Inc.</td><td>0G7149702</td><td></td></tr><tr><td>Microlance Hypodermic Needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td>modify needle to make cannula</td></tr><tr><td>Braun Original Perfusor Syringe 50 ml</td><td>B. Braun Melsungen AG</td><td>8728810F</td><td></td></tr><tr><td>Braun Inject Solo Syringe 10 ml</td><td>B. Braun Melsungen AG</td><td>2057926</td><td></td></tr><tr><td>Beaker 50ml</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 106 17</td><td></td></tr><tr><td>Duroplan petri dish (100 x 20 mm)</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 755 48</td><td></td></tr><tr><td>Seraflex Suture USP 3/0</td><td>SERAG-WIESSNER GmbH &amp;amp; Co. KG</td><td>IC208000</td><td></td></tr><tr><td>VWR disposable Square Weighin Boats 100ml</td><td>VWR, Inc.</td><td>10803-148</td><td></td></tr><tr><td>Styrofoam surface</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Inc.</td><td>71380</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich, Inc.</td><td>P4504</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich, Inc.</td><td>P5379</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich, Inc.</td><td>S0876</td><td></td></tr><tr><td>Magensium sulfate heptahydrate</td><td>Sigma-Aldrich, Inc.</td><td>230391</td><td></td></tr><tr><td>Magensium chloride</td><td>Sigma-Aldrich, Inc.</td><td>M8266</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich, Inc.</td><td>T0625</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Inc.</td><td>G7528</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich, Inc.</td><td>B0753</td><td></td></tr><tr><td>Calcium chloride solution (1 M)</td><td>Sigma-Aldrich, Inc.</td><td>21115</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich, Inc.</td><td>A9647</td><td></td></tr><tr><td>Liberase</td><td>Roche (Sigma-Aldrich, Inc.)</td><td>LIBTM-RO</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica GmbH</td><td>3862357</td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie Deutschland GmbH &amp;amp; Co. KG</td><td>10182054</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich, Inc.</td><td>L2020</td><td></td></tr><tr><td>WillCo glass-bottom dish 500&amp;micro;l 0.005mm</td><td>WillCo Wells B.V.</td><td>HBST-3522</td><td></td></tr><tr><td>Fluo4 AM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>F14201</td><td>5&amp;micro;M for 20min at RT</td></tr><tr><td>Di-8-ANNEPS</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>D3167</td><td>10&amp;micro;M for 45 min at 37&amp;deg; C&amp;nbsp;</td></tr><tr><td>Mitotracker RED FM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>M22425</td><td>20nM for 30 min at 37&amp;deg; C</td></tr><tr><td>Jacketed reaction vessel 500 ml</td><td>Gebr. Rettberg GmbH</td><td>107024414</td><td></td></tr><tr><td>Jacketed reaction vessel 1000 ml</td><td>Gebr. Rettberg GmbH</td><td>107025414</td><td></td></tr><tr><td>Jacketed bubble trap</td><td>Gebr. Rettberg GmbH</td><td>134720001</td><td></td></tr><tr><td>ED heating immersion circulator</td><td>Julabo GmbH</td><td>9116000</td><td></td></tr><tr><td>Reglo Digital MS-2/6 peristaltic pump</td><td>Ismatec (Cole-Parmer Gmbh)</td><td>ISM 831</td><td></td></tr><tr><td>Voltcraft Thermometer 302 K/J</td><td>Conrad Electronic SE</td><td>030300546</td><td></td></tr><tr><td>Tubing</td><td></td><td></td><td></td></tr><tr><td>LSM 700 microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>ZEN 2.3 imaging software</td><td>Carl Zeiss, Inc.</td><td>410135-1011-240&amp;nbsp;</td><td></td></tr><tr><td>Single channel heater controller TC-324B</td><td>Warner Instruments, LLC</td><td>64-2400</td><td></td></tr><tr><td>8 channel perfusion system</td><td>Warner Instruments, LLC</td><td>64-0185</td><td></td></tr><tr><td>8 channel Multi-Line In-Line Solution Heaters</td><td>Warner Instruments, LLC</td><td>64-0105</td><td></td></tr></tbody></table>

isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction

この記事で述べるメタボリック シンドローム (メッツ) のモデルラットから単一セル心房心筋細胞 (Acm) を分離するため、蛍光ベースの最適化手順-保存された駆出分画 (HFpEF) で心不全の関連。メッツ関連 HFpEF の有病率が上昇していると心房リモデリングと心房細動に関連する心房の心筋症は、心房を改造死亡率の独立した予測因子である関連性の高い臨床的に。隔離された単一細胞心筋細胞を用いた研究が確証し、生体内で調査結果を補完するためによく使用されます。循環容器 rarefication と間質性組織線維化は、この病気の動物モデルからの ACMs の成功単一セル隔離の可能性のある制限要因をもたらします。
我々 は形態および機能そのまま ACMs の収量が大幅に増加手動で分離している最中、心臓腔内圧を調整する装置を採用することによりこの問題を解決しました。取得した細胞はさまざまな細胞培養、機能カルシウム イメージング (すなわち、興奮収縮連関) など、別の実験で使用できます。
順を追ってプロトコル最適化されたソリューションは、包括的なトラブルシューティング ガイドと必要な機器を準備する徹底した指示のリストと研究者を提供します。プロシージャの最初の実装は難しいかもしれないの成功の適応はメッツ関連 HFpEF 実験の広いスペクトルのためのモデルラットの新式の ACM 隔離を実行するリーダーになります。

生後 21 週、実行可能な ACMs (6.1 のステップに従って推定)、カルシウム再適応 (ステップ 5.4-5.7) 後の 60-90% は、このメソッド (図 4 a) によって ZSF 1 肥満ラットから分離できます。ラット、ACMs VCMs24,25と比較してより多くの異種表現型と異なる特徴です。図 4 bは、保存状態の膜およ?...

Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

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

ここでは、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単一セル心房心筋細胞を分離するため、蛍光ベースの最適化手順をについて説明します。心腔の内圧の手動調節、機能そのまま心筋興奮収縮連関研究に適した屈する実装されます。

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

isolation-atrial-cardiomyocytes-from-rat-model-metabolic-syndrome

Research

JoVE Journal

Medicine

10.1K ビュー.  Charité University Medicine. ここでは、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単一セル心房心筋細胞を分離するため、蛍光ベースの最適化手順をについて説明します。心腔の内圧の手動調節、機能そのまま心筋興奮収縮連関研究に適した屈する実装されます。

ビデオ: Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

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 Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

カルシウムの同時測定のための人間の心房筋細胞の分離<sup&gt; 2 +</sup&gt;過渡と膜電流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

生物学

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 ...

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 ...

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 ...

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 ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

ビブラートムカット心筋切れ切り切れからヒト心室心筋細胞を分離

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

心房筋細胞、心室筋細胞、および非筋細胞の同時分離と培養

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

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

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

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