著者は、彼女の技術支援と実行組織学のためにクリスティPahrmannに感謝。

<html> 8から12週齢で20gの最小の重量を量るのBalb / Cマウスをチャールズリバー（Sandhoferウェグ7、D - 97633 Sulzfeld）から購入しています。 マウスは、通常の条件、飼育、標準のマウスのペレットと水を自由の下に収容されています。誘導チャンバーを用いて（2％）イソフルランでマウスを麻酔。 <ol><li>首の領域と胸郭の左側にひげをそると80％エタノールを用いて消毒する。 </li><li>その背中にマウスを置き、麻酔を維持するために、その鼻と口にフェイスマスクを置く。 </li><li>マウスが十分anesthesizedであることを確認するために、尾と後ろ足の足をつまんで反射神経をチェックしてください。 </li><li>微視的な視点の下で気管を覆う皮膚、筋肉や組織を分離する正中頸部切開を行います。 </li><li>気管は、気管内チューブ（図1）を挿入するには声門下の2つのカートリッジの環の間の組織に小さな穴をカットさらされている場合。マイクロ手術foreceptsを使用して気管の頭蓋の一部を保持する気管内チューブを挿入します。両方の肺がよく換気されていることを確認するために胸部の動きを確認してください。 </li><li>呼吸速度（RR）は、17〜18センチメートルH 2 Oの吸気圧で、約110分毎にする必要がありますその左側に面して、その右側に横たわって、慎重にマウスを回す。 3番目と4 番目の肋骨の間にleftsided開胸を行い、出血を防ぐために焼灼器を使用して、慎重に組織や筋肉を細かく分析。 </li><li>胸部が開かれると、慎重に胸部を開き、任意のとがったもので肺に触れることなく、心を見つける。今の心を覆っている心膜の一部を削除してください。 </li><li> LADは、肺動脈と左心耳の間に位置しています。一つの縫合（図2）と近位LADを連結する8から0 Proleneの縫合糸（エチコン、ノルダーシュテット、ドイツ）を使用してください。 </li><li> 4 番目と5 番目のリブの間に胸腔チューブを（28G、腐敗したカテーテル）、置きます。 </li><li>肋骨と皮膚を閉じるには、縫合糸（エチコン、ノルダーシュテット、ドイツ）を実行して4から0 Proleneを適応させるために6から0 Prolene実行されている縫合糸（エチコン、ノルダーシュテット、ドイツ）を使用して、レイヤーの胸部切開を閉じます。 </li><li>慎重に2ミリリットル注射器（図3）の助けを借りて胸部を排出します。その背中にマウスを置きます。今すぐ気管内チューブを取り出して、7から0 Prolene縫合を（エチコン、ノルダーシュテット、ドイツ）使用して、1つのシングルステッチで気管カートリッジのリングを適応させる。 </li><li>マウスの顔のマスクを置き、4から0 Prolene実行されている縫合糸（エチコン、ノルダーシュテット、ドイツ）を使用して皮膚を閉じます。 </li></ol>検証 LAD -ライゲーションの成功を確認するために、異なる可能性があります。 トロポニンテストは、動物を安楽死させることなく、唯一の150リットルの血液を使用して、手術後6時間から18時間を行うことができます。血液は、カスタマイズされたトロポニンテストキット（過度にTセンシティブ、ロシュ、マンハイム）（図4）に適用されます。トロポニンが筋細胞のアクチンフィラメントの調節的蛋白質である、筋肉の3つのタイプの異なるアイソタイプがあります。アイソタイプcTnIとcTnTを中心に発見され、組織損傷にリリースされます。トロポニンT（cTnT）のための標準化されたテストは、2つの心臓特異的モノクローナル抗体を、一つのトラップ血液試料中のトロポニンに基づいている、もう一つはマーカーです。 梗塞部位は、3日間（図5）の後に肉眼的に識別できます。 TTC（2,3,5 - 塩化トリフェニルテトラゾリウム）染色対策組織の生存率は梗塞サイズを評価するために使用。エバンスブルー色素（1,5％、1.0mL）中のリン酸緩衝生理食塩水（PBS）は心筋の虚血領域を測定するために左室内腔に注入される。マウスを安楽死であり、心臓はスライスに採取し、切片化されています。組織切片は20分、37 ° Cで1​​％TTC PBS溶液、pH7.4でインキュベートする。組織は2〜8℃で一晩PBS -バッファ、10％ホルマリンで固定されている℃のTTCは、色素エバンスブルー陰性の領域（図6）にex vivoで投与される。 組織学のためにマウスは安楽死されなければならないと心はさらなる処理のために埋め込まれなければなりません。パラフィン切片を実行した後、スライドはH＆E（ヘマトキシリン​​とエオジン）で染色または線維性組織（図7）を可視化するために三色のされています。 <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="図1" /> 図1：気管チューブ。 1A：気管を露出、腹側頸部切開後。 1B：気管は喉頭に尾two軟骨リングを開かれます。 1C：気管チューブを気管に挿入されます。 <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="図2" /> 図2：心臓が左サイド開胸を介して公開され、LADを結紮される。 t&quot;は&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="図3" /> 図3：胸部を閉じた後、胸膜のドレインが行われる。マウスは、麻酔下ではまだです。 <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="図4" /> 図4：トロポニンテストが陽性の場合に2行には、数分後に表示されます。 <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="図5" /> 図5：植心臓を表示します。 5A：色あせた白っぽい色で虚血領域と心のマクロビュー。 5B：梗塞心臓の断面図、虚血領域は、右上隅に表示されます。

<ol>
	<li>Atkins, B. Z., Hueman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+diastolic+properties+of+injured+Heart.">Cellular cardiomyoplasty improves diastolic properties of injured Heart.</a> J Surg Res. 85 (2), 234-242 (1999).</li><li>Jaquet, K., KT, K. r. a. u. s. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+myocardial+scar+size+after+implantation+of+mesenchymal+stem+cells+in+rats:+what+is+the+mechanism.">Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism.</a> Stem Cells Dev. 14 (3), 299-309 (2005).</li><li>Joung, B., Kim, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mononuclear+stem+cells+transplanted+in+rat+infarct+myocardium+improved+the+electrical+conduction+without+evidence+of+proarrhythmic+effects.">Bone marrow mononuclear stem cells transplanted in rat infarct myocardium improved the electrical conduction without evidence of proarrhythmic effects.</a> Yonsei Med J. 48 (5), 754-764 (2007).</li></ol>

資金本研究は、大学病院ハンブルク（MK、女性ハビリテーショングラント）、DFG（SS、SCHR992/3-1）、ISHLT（TD、研究賞2009年）によってサポートされていました。 文すべての動物は、動物実験は医学研究とによって公開実験動物の管理と利用のためのガイドのための国立協会が定めるガイドラインおよび規制に準拠して行われた、実験動物のケアの原則に準拠して人間のケアを受け国立衛生研究所（厚生出版物85から23の国立研究所、1985年改訂）。 すべての動物は、チャールズリバーラボラトリーズ（Sulzfeld、ドイツ）から入手し、大学病院ハンブルクエッペンドルフの動物医療施設で維持した。動物は、標準の固形飼料と水を自由に摂取を受けた。

要約すると、このビデオでは、LAD -ライゲーションモデルのステップバイステップを視覚化し、容易に実現可能な外科的処置として、マウスのLAD -ライゲーションを識別します。

lad-ligation: a murine model of myocardial infarction

心筋梗塞や心筋虚血の研究のモデルは、心筋虚血の急性および慢性のpathobiologicalと病態生理学的プロセスを調査し、将来の治療法の開発と最適化するために不可欠です。 心筋虚血を作成するための二つの異なる方法が実験用げっ歯類で実行されます。最初の方法は、低温ペンが心臓（1-3）の表面に適用されているクライオ心筋梗塞、高速だが不正確なテクニックを、作成することです。科学者は、クライオ傷跡が虚血につながることを保証できない場合、このメソッドを使用して、また広大な心筋障害は、心筋梗塞に関連していない病態生理学的な副作用を示すことが作成されます。第二の方法は、左前下行枝（LAD）の恒久的なライゲーションです。ここでLADはほぼ即座に見ることができる虚血を形成し、単一のステッチと連結する。周囲の心筋組織がほとんど影響されないままLADを閉じることで、それ以上の血流は、その領域で許可されていません。この手術は、心筋梗塞に関連する心筋虚血の発生pathobiologicalと病態生理学的側面を模倣する。 このビデオで紹介した方法では、LADを結紮することにより、マウスの心筋梗塞モデルの外科的手順を示しています。このモデルでは、心筋梗塞でpathobiologicalおよび病態生理学的ならびに免疫生物学的研究のために便利です。示した手法は、高い精度を提供し、組織切片とよく相関している。

Watch this Scientific Journal Video about LAD-Ligation: A Murine Model of Myocardial Infarction at JoVE.com

LAD-Ligation: A Murine Model of Myocardial Infarction

このビデオでは、心筋虚血のpathobiologicalと病態生理学的プロセスを研究するために高速で信頼性の高いモデルを使用する方法を示します。

LAD -ライゲーション：心筋梗塞のマウスモデル

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

lad-ligation-a-murine-model-of-myocardial-infarction

Nanyang Technological University

Research

JoVE Journal

Biology

44.4K ビュー.  University Heart Center Hamburg. このビデオでは、心筋虚血のpathobiologicalと病態生理学的プロセスを研究するために高速で信頼性の高いモデルを使用する方法を示します。

ビデオ: LAD-Ligation: A Murine Model of Myocardial Infarction

Murine Pancreatic Islet Isolation

マウス膵島単離

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

後脚虚血症のマウスモデル

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

マウス コリン欠乏エチオニン (CDE) ダイエット慢性肝障害モデル

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

垂直方向のスリーブ胃切除術のマウスモデル

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

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 ...