この作品は、EU（STREPプロジェクト241577 &quot;大きな心、「第7回欧州フレームワークプログラムは、CP）、メナリーニ国際事業ルクセンブルク（AM）、テレソンGGP07133（CP）とギリアド·サイエンシズ（AM）によってサポートされていました。

（; 2009年5月リニューアル2006/0024713）、ヒトの組織についての実験プロトコルがCareggi大学病院の倫理委員会によって承認された。各患者は、書面によるインフォームドコンセントを与えた。 1。ソリューションと設備の準備溶液は、表1に記載されている。細胞単離手順の簡略化したフローチャートを図1に見出される。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2">ソリューション</td><td> CP </td><td> DB </td><td> KB </td><td> TB </td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23">試薬（MM） </td><td> KH 2 PO 4 </td><td> 50 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 硫酸マグネシウム</td><td> 8 </td><td> 1.2 </td><td> 5 </td><td></td><td></td><td> 1.2 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td><td></td><td></td><td> 10 </td><td> 10 </td><td></td><td></td></tr><tr><td>アデノシン</td><td> 5 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>グルコース</td><td> 140 </td><td> 10 </td><td> 20 </td><td> 10 </td><td></td><td> 10 </td><td> 10 </td></tr><tr><td>マンニトール</td><td> 100 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>タウリン</td><td> 10 </td><td> 20 </td><td> 5 </td><td></td><td></td><td> 20 </td><td> 20 </td></tr><tr><td> NaClを</td><td></td><td> 113 </td><td></td><td> 136 </td><td></td><td> 113 </td><td> 113 </td></tr><tr><td>塩化カリウム</td><td></td><td> 4.7 </td><td> 85 </td><td> 5.4 </td><td> 25 </td><td> 4.7 </td><td> 4.7 </td></tr><tr><td> 塩化マグネシウム</td><td></td><td> &lt;/ TD&gt; <td></td><td> 1.2 </td><td> 5 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td></td><td> 0.6 </td><td> 30 </td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td>のNa 2 HPO 4 </td><td></td><td> 0.6 </td><td></td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> 炭酸水素ナトリウム</td><td></td><td> 12 </td><td></td><td></td><td></td><td> 12 </td><td> 12 </td></tr><tr><td> KHCO 3 </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td>ピルビン酸ナトリウム</td><td></td><td> 4 </td><td></td><td></td><td></td><td> 4 </td><td> 4 </td></tr><tr><td> BDM </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> BHBA </td><td></td><td></td><td> 5 &lt;/ TD&gt; <td></td><td></td><td></td><td></td></tr><tr><td>コハク酸</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> EGTA </td><td></td><td></td><td> 0.5 </td><td></td><td></td><td></td><td></td></tr><tr><td> K 2-ATP </td><td></td><td></td><td> 2 </td><td></td><td></td><td></td><td></td></tr><tr><td>ピルビン酸</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td>クレアチン</td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> KMES </td><td></td><td></td><td></td><td></td><td> 115 </td><td></td><td></td></tr><tr><td rowspan="2">酵素（U / ml）を</td><td>コラゲナーゼタイプV </td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ TD&gt; <td> 250 </td></tr><tr><td>プロテアーゼタイプXXIV </td><td></td><td></td><td></td><td></td><td></td><td> 4 </td><td></td></tr><tr><td> pHは</td><td></td><td> 7.4 KOH </td><td> 7.3のNaOH </td><td> 7.1 KOH </td><td> 7.35のNaOH </td><td> 7.2 KOH </td><td> 7.3のNaOH </td><td> 7.3のNaOH </td></tr></tbody></table> 。表1検体採取、細胞の単離と筋細胞の機能解析 、CP =心筋保護液のために使用される溶液 ; DB =解離緩衝液; KB =クラフト·Bruhe溶液; TB =タイロード緩衝液; PS =ピペット溶液; EB1 =酵素緩衝液1; EB2 =酵素緩衝液2。 <ol><li>心臓麻痺（CP）溶液を調製する。 CP溶液を1週間まで4℃で保存することができる。 </li><li>のCa 2 +を含まない解離緩衝液（DB）を準備します。このソリューションでは、一日の中で使用する必要があります。 </li><li>クラフト·Bruhe（KB）の溶液を調製する。 KB溶液を最大1ほんの4℃で保存することができるK。 </li><li>のCa 2 +を含まないタイロード緩衝液（TB）を準備します。このソリューションでは、一日の中で使用する必要があります。 </li><li>使用前にシリンジフィルターを使用して、すべてのソリューションをフィルタリングします。 </li><li>消化装置（ 図2）、電動モータによって回転一方がシリコーンエラストマーの対向する2ブラシからなる掻き容器を準備する。消化器がカスタムメイドなのです。消化装置の詳細については、 図8であり;デバイスのイメージを図2 Cと2Dである。 70％エタノールと水で組織チャンバーを洗浄します。 </li></ol>心筋のサンプル2。収集および処理<ol><li> 50ミリリットルチューブにcardiplegic（CP）の溶液40ミリリットルを注ぎ、それが細胞単離研究室に手術室から検体の輸送のための氷の中に保管してください。 </li><li>氷冷で洗って、すぐに切除した後に手術室から心室の心筋標本を収集CP液やチューブに保管します。心臓切開手術中に上部の間の心室中隔から切り出し心内膜標本、重み&gt; 100ミリグラムを使用してください。 </li><li>急速に研究室エリアに試料を移す。切除標本から10分以内に検体処理を開始します。 </li><li>氷冷CPバッファ内の試験片を保持しながら、慎重に実体顕微鏡下で微細なハサミを使用して心内膜線維化層を除去する。その後、小片（2-3 mmの長さ）の心筋組織をカット。組織試料の大きさに応じて、それぞれの単離のために100ミリグラムおよび1gの間の心室筋の総量を削減する。 </li><li>組織ミンチが完了すると、きれいな氷冷CP溶液を、消化装置に心筋チャンクを転送する。全組織のわずか1グラムを使用して、心筋のチャンクで2つのシリコンブラシ（3-4 ml）の間の全体のボリュームをいっぱいにならないよう。 </li></ol>心筋チャンクの3。洗浄し、消化<ol><li>アフト小胞体のチャンクを冷性Ca 2 +を含まない解離緩衝液（DB）でチャンバ内のCPバッファを変更し、消化装置の掻き取り室に移す。 </li><li>浴中で加熱された水（ 図1）と接触するようにするためには、チャンバ、恒温槽内の消化装置を配置する。 37.5℃までバスを設定し、徐々に組織室内の温度を上昇させるために、電源を入れます。 1革命/秒に回転速度を設定し、消化器のモーターの電源をオンにします。 </li><li>すべての8分きれいなDBにチャンバー内の溶液を変え、DBで3回の洗浄サイクルを実行します。 DBは、（37℃）まで温め、酸素が心筋の塊と接触して取得する前に飽和している。 </li><li> DB溶液にコラゲナーゼタイプVと4 U / mlのプロテ​​アーゼタイプXXIVの250 U / mlのを追加して、酵素緩衝液1（EB1）を準備します。 DB溶液に250 U / mlのコラゲナーゼタイプVを追加することにより、酵素緩衝液2（EB2）を準備します。ウォームアップ（37℃）とoxygenatEのEB1とEB2。 </li><li> （37℃）100パーセント酸素化EB1と回転の消化器内消化の2 12分のサイクルを実行します。各サイクルで、EB1の〜3ミリリットルを使用しています。ピペット吸引によって溶液を除去し、各サイクルの後に、それを捨てる。 </li><li>細胞回収と〜バッファを溶出させるための冷却（4℃）KB液80ミリリットルのために6 15ミリリットルのチューブを準備します。 </li><li> 37℃で3ミリリットル100パーセントの酸素化EB2で最初の15分の消化サイクルを実行蒸解サイクルの後、15mlチューブ内の最初の解離筋細胞を含む溶液を収集し、12ミリリットルの冷KB溶液と細胞懸濁液を希釈する。室温でフラットチューブに保管してください。 </li><li>次の消化サイクルのコラゲナーゼVの濃度を半分にするために、DB等量の残りEB2溶液を希釈する。 </li><li> 37℃で3ミリリットルEB2と他の5〜12分間消化サイクルを実行する;それらの各した後、15 mlコニカルチューブに緩衝液を含む筋細胞の収集および12ミリリットルキロバイト溶液で希釈する。 30分間室温で6セル​​を含むチューブに保管してください。 </li></ol> 4。細胞再懸濁およびCa 2 +の再適応<ol><li> + -遊離Ca 2 20mlに1 mg / mlウシ血清アルブミン（BSA）を追加するタイロードバッファー（TB）。溶液を濾過。 </li><li>遠心沈降筋細胞を強制的に5分間、100×gでコニカルチューブを含む6筋細胞は上清を除去し、可変量を各チューブ内の細胞を再懸濁（1-3 mlの収量に応じて）室温でのTBを含む、BSAの。 </li><li>徐々に100ミリモル/ LのCaCl 2溶液の少量ずつを追加することにより、緩衝液を含む細胞内のCa 2 +濃度を増加させる。第一及び第二のステップにおけるCa 2 +濃度は、それぞれ、50マイクロモル/ Lおよび100マイクロモル/ Lまで上昇させる。以下のCa 2 +の添加工程は、5分ごとに実行され、濃度が各ステップtで100マイクロモル/ Lで上昇させる0.9ミリモル/ LのOA最終濃度</li><li>単離法の収率を評価する。顕微鏡のガラスボトム室に筋細胞を含む溶液0.5mlを移す。 10倍の客観的な倍率で15顕微鏡視野を評価し、健康的な筋細胞の割合（クリア条線と有意な介在物など棒状の細胞、 図2）を計算します。期待利回りは約20％である。 </li></ol>絶縁型心筋細胞の5。機能評価。 次のプロトコルは、活動電位と細胞内Ca 2 +フラックスの同時録画を含むヒト心筋細胞機能評価の一例です。 <ol><li>穿孔パッチ構成のパッチクランプ実験のためのピペット溶液（PS）を準備します。溶液を最長3ヶ月間-20℃で保存することができる。 </li><li>のCa 2 +を含まないタイロード緩衝液（TB）に1.8モル/ Lの塩化カルシウムを追加します。 USEパッチクランプ/蛍光実験中の心筋細胞の灌流のためにこのソリューションを提供します。 </li><li> 1.5mlチューブへの転送細胞懸濁液の1ミリリットルをし、10マイクロモル/ LのFluoforteと10μlのPowerload濃縮物を追加します。室温で30分間インキュベートする。その後、垂直位置にチューブをセットし、5分間沈降細胞を残す。結核を含む内Ca 2 +の細胞を懸濁します。 </li><li> （37±0.5°Cの温度）小（0.5ml）中に細胞懸濁液0.25mlのを転送し、温度制御された顕微鏡は、0.3ml /分の流速で加熱さmicroperfusorシステムと重力によって灌流記録チャンバを、取り付けられている。 </li><li>マイクロピペットプラーを使用して、3〜5ミクロンの先端径とPSで満たされた3 M 4.5の抵抗でパッチクランプピペットを準備します。 </li><li> PS（250μg/ ml）をバッチにアムホテリシンBを追加し、電極を埋めるために使用します。 </li><li> FO、介在物のない、明確な条線を細胞に棒状を選択GIGAシールをRmとアクセス抵抗が20MΩを下回るまで、5か​​ら10分待ってください。 </li><li>刺激の異なる周波数（0.2 Hzで、0.5と1Hz、各周波数で1分）での短パルス（&lt;3ミリ秒）を使用して、電流クランプモードでの活動電位を誘発する。記録フェーズでは、492±3 nmで明視野照明をオンにして505から520 nmでFluoforteの蛍光を検出する。 Digidata 1440AとPCLAMPの10.0ソフトウェアを使用して蛍光および膜電位信号を取得。必要に応じて記録シーケンスを複数回繰り返します。しかし、各セルの15分以下の記録時間を保つ。 </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+recording+using+the+perforated+patch+clamp+technique.">Whole-cell recording using the perforated patch clamp technique.</a> Methods Mol Biol. 491, 141-149 (2008).</li><li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy.">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127, 575-584 (2013).</li><li>Kuusisto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-grade+inflammation+and+the+phenotypic+expression+of+myocardial+fibrosis+in+hypertrophic+cardiomyopathy.">Low-grade inflammation and the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy.</a> Heart. 98, 1007-1013 (2012).</li><li>Yan, G. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+2+early+afterdepolarization+as+a+trigger+of+polymorphic+ventricular+tachycardia+in+acquired+long-QT+syndrome+:+direct+evidence+from+intracellular+recordings+in+the+intact+left+ventricular+wall.">Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome : direct evidence from intracellular recordings in the intact left ventricular wall.</a> Circulation. 103, 2851-2856 (2001).</li><li>Yue, L., Feng, J., Li, G. R., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+outward+and+delayed+rectifier+currents+in+canine+atrium:+properties+and+role+of+isolation+methods.">Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods.</a> The American Journal of Physiology. 270, 2157-2168 (1996).</li><li>Li, G. R., Feng, J., Yue, L., Carrier, M., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+two+components+of+delayed+rectifier+K++current+in+human+ventricular+myocytes.">Evidence for two components of delayed rectifier K+ current in human ventricular myocytes.</a> Circulation research. 78, 689-696 (1996).</li><li>Viswanathan, P. C., Shaw, R. M., Rudy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence:+a+simulation+study.">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li><li>Volders, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization:+key+role+for+beta-adrenergic+receptor+stimulation.">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li><li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol+antagonizes+prolongation+of+refractory+period+by+the+class+III+antiarrhythmic+agent+E-4031+in+guinea+pig+myocytes.+Mechanism+of+action.">Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li><li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy:+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine.">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

私たちは、説明され、人間の心室の心筋の外科サンプルから実行可能な筋細胞を単離するための方法を検証しています。首尾よく心房外科試料から単離した細胞に使用されていた以前に記載されたプロトコルから始めて、病気の心室の心筋から単一の実行可能な筋細胞の分離を可能にする技術が開発され、微調整した。初期の報告では、冠灌流を介してバッファを含む酵素の配信が遅延整流?...

心筋の電気生理学的特性の解剖は、単一の心筋細胞を単離するための技術の開発の後に顕著に進んでいる。心臓の興奮収縮連関（ECカップリング）の理解における最近の進歩はまた、完全な組織の全ての生理学的特性を保持している実行可能な単一の心筋細胞を単離する能力によって可能になってきた。パッチクランプ法は日常的機能および心臓筋細胞膜のイオン電流の薬理学的調節を研究するために使用される。のCa 2 +感受性色素と細胞内カルシウム動態の記録はまた、定期的に、ECカップリングの生理学上だけでなく、細胞内Ca 2 +の病理学的変化に重要なデータを提供し、健康で病気の様々なモデルから単一の心筋細胞上で実行される機械的な障害や心疾患増加催不整脈の負担につながる恒常性。情これらの研究からのTiONは、臨床現場における薬物の電気生理学的および機械的な影響を理解するために重要です。しかし、貫通電流および心臓の活動電位と心臓力学の特定の機能を占め、ECカップリングタンパク質における種特異的な違いがあります。非ヒト哺乳動物から単離した筋細胞の研究は、生物物理学的特性および特定の膜貫通イオンチャネルおよびEC結合タンパク質の生理的役割を解明しつつ、それらは必ずしも人間の心筋細胞の関連のモデルを提供していません。人間の心筋からの実行可能な筋細胞の単離は、完全に心疾患の病態生理を理解し、新たな治療アプローチを検証することが不可欠である。 心耳は、多くの場合、外科的手技中に破棄されたように、ヒト心房組織は、容易に入手可能である。成人の心臓の活動電位とイオンのCuの初期定量的研究rrentsを酵素的に孤立性心房細胞の1-4を採用。活動電位または単離された成人の心室細胞からの電流の記録は、その後3,5-10を報告されている。これらの研究のほとんどは、外植された心臓から得られた細胞を使用し、単離された細胞を得るためにコラゲナーゼ冠動脈セグメントまたは切除組織の比較的大量の暴露のコラゲナーゼ灌流のいずれかを利用している。これらの研究は、健康な心からの人間の心室心筋細胞からターミナル心不全患者からの膜貫通イオン電流の数の詳細な特性を可能にした。 L型の録音は、Ca 2 +電流（I CA-L）を 5-7、一過性外向きカリウム電流（I） は 8は 、整流カリウム内向き電流（私はκ1）8、遅延整流カリウム電流のさまざまなコンポーネントは、（私はκ 9）が報告されている。の進歩と精製単離手順10は 、脱分極12期および拡張期脱分極および早期のビートにつながる面白い、現在13に増加した後に遅れて活動電位延長11を含む、ターミナル心不全で増加催不整脈性のイオン根拠の明確な特性評価を可能にした。 アダルト心筋細胞は通常、様々な酵素混合物で心臓全体のCa 2 +トレラント細胞14を高収率で生産する技術の逆行性灌流によって小さな動物から単離される。組織の断片からの心筋細胞の単離は、冠状動脈の灌流によって達成されるものと比較しているため、個々の筋細胞への酵素の限られたアクセス、おそらく本質的にあまり成功しています。なぜなら、未使用のドナー心臓の非常に限られた入手可能性、定期的に正常なヒト心室細胞を得るための唯一の実用的な方法は、酵素digestioである待機的外科手術中に切除しばしば非常に小さな組織フラグメントのN。徹底的に細胞レベルで特徴付けられている唯一のヒト疾患モデルでは、移植された心臓へのアクセスのために、端末心不全である。しかし、端末の心不全は、少数の患者が発生し、多くの場合、根本的な原因15は比較的独立している心筋細胞の深刻な改造の共通の経路を伴う。疾患の初期非失敗の段階で患者からの単一心筋細胞の機能を評価する能力は、異なる遺伝性または後天条件の具体的な病態生理を理解することが重要です。肥大型心筋症（HCM）は語っ例です。 HCMは、流出路閉塞および拡張機能障害16による催不整脈リスクと収縮の変化を一般的な（1/500個体）心肥大が特徴継承心臓状態、増加している。 HCMの心臓から心筋細胞Uセル構造の変化（肥大、筋原線維混乱）とEC-カップリング17を含む複雑なリモデリング過程をndergo。しかし、HCMでの筋細胞機能障害のほとんどの情報は、トランスジェニック動物モデルから来ている。 HCM患者のごく少数が端末心不全に向かって進化し、心臓移植を必要とするため、HCMの心は非常にまれに、標準的な方法を用いた細胞単離のために使用できません。しかし、HCM患者の少なくとも30％が、収縮期（HCM）18中に流出路の血流を変化させる大規模な中隔肥大に起因する閉塞症状を発症する。 HCMにおける障害物の救済のための最も効果的な利用可能な治療の選択肢は、手術中隔の心筋切除術である：この外科手術中、上側の中隔の可変サイズの部分は、トランス大動脈アプローチによって除去される。肥大した中隔のこの部分は、新鮮な組織からの細胞を単離するための、したがって提供されています。 人間ventriculaの単離のための方法Rシングル、小型の経静脈心内膜心筋生検標本からの筋細胞は、以前に開発され、19を公表されている。私たちは、中隔心筋切除術および弁置換処置を受けた患者を受けHCMの患者を含む心臓手術を、受けている患者からの心室の心筋サンプルから単中隔の筋細胞を単離するための方法を実施しました。単離プロトコールの詳細な説明に加えて、代表的な電気生理学的およびCa 2 +の蛍光測定は、単離されたヒト心室筋細胞の生存率およびパッチクランプ及び細胞内Ca 2 +の研究の実現可能性を実証し、提示される。

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">The mold is custom made from standard PTFE 2.5 cm diameter rods</td>
		</tr>
	</tbody>
</table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

疾患のある心臓の心筋細胞は、細胞構造、励起収縮連関および膜イオン電流の変化を伴う複雑なリモデリングプロセスに供される。これらの変更は増加催不整脈リスクと心臓病患者における収縮期および拡張機能障害につながる収縮の変化の原因であると思われる。しかし、心疾患における心筋細胞機能の変化は上のほとんどの情報は、動物モデルから来ている。 ここでは、説明し、心臓手術操作を受けている患者からの心室の心筋の小さな外科的サンプルから実行可能な筋細胞を単離するためのプロトコルを検証する。プロトコルが詳細に記載されている。電気生理学的および細胞内カルシウムの測定は、この方法で得られたヒト心室心筋細胞における単一細胞の測定数の実現可能性を実証することが報告されている。 プロトコルは、彼を報告した再様々な心臓疾患の存在下で、人間の心臓の機能的変化の細胞および分子基盤の将来の調査のために役立ちます。また、この方法は、細胞レベルで新規な治療標的を同定するために、ダイレクト並進値と、ヒト心筋細胞に新規化合物の有効性を試験するために使用することができる。

非障害の非肥大手術患者21と比較して、上述の方法は、心筋切除術の手術を受けた肥大型心筋症（HCM）を有する患者の心室中隔から単離された心筋細胞の機能異常を特徴づけるために用いた。このセクションに含まれている結果は、ワーク21から誘導され、この技術は、心臓の疾患状態における心筋細胞機能の変化を特徴付けるために使用することができる方法の例として示?...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

心疾患の細胞に基づく現在の知識は、ほとんどの動物モデルの研究に依存しています。ここでは、説明し、人間の心室心筋の小さな外科的サンプルから単実行可能な心筋細胞を得るための新しい方法を検証する。ヒト心室筋細胞は、電気生理学的研究および薬物試験のために使用することができる。

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 ...

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

17.1K ビュー.  University of Florence. 心疾患の細胞に基づく現在の知識は、ほとんどの動物モデルの研究に依存しています。ここでは、説明し、人間の心室心筋の小さな外科的サンプルから単実行可能な心筋細胞を得るための新しい方法を検証する。ヒト心室筋細胞は、電気生理学的研究および薬物試験のために使用することができる。 

ビデオ: Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

人間頸管サンプルの採取、単離、およびフローサイトメトリー分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

慢性血栓塞栓性肺高血圧症とピグレットにおける右室機能の評価

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

分離の心室心筋の筋小胞体カルシウム拡張予備能と細胞内のカルシウム除去の評価

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

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 ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

開発とマウス免疫寛容樹状細胞の機能解析

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

カルシウム処理特性評価のための心室様HiPSC由来心筋細胞と高品質細胞製剤の生成

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内在的に発現したヒトRYR1変異体の機能特性評価

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

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 ...