Alessandra d'Azzo(アレッサンドラ・ダッツォ)は、Jewelers for Children(JFC)の遺伝学および遺伝子治療の寄附講座を保持しています。この活動は、NIHの助成金R01GM104981、RO1DK095169、CA021764、メンフィスのアッシジ財団、およびアメリカのレバノンのシリア関連慈善団体によって部分的に支援されました。

マウスにおけるすべての処置は、St. Jude Children's Research Hospital Institutional Animal Care and Use CommitteeおよびNational Institutes of Healthのガイドラインによって承認された動物プロトコルに従って実施された。
1. 溶液と培地の調製
<ol><li>15.4 mL の PBS と 2.5 mL の 20 mg/mL コラゲナーゼ P(最終濃度 5 mg/mL)、2 mL の 11 U/mL ディスパーゼ II (最終濃度 1.2 U/mL)、1.0 μL の 1.0 M CaCl2 (最終濃度 5 mM)を混合して消化溶液を調製します。</li>
	<li>440 mLのDMEMと50 mLのFBS(10%)、5.0 mLのペン/連鎖球菌(それぞれ100 U/mLおよび100 μg/mL)および5.0 mLのグルタミンサプリメント(2 mM)を混合して、500 mLの一次線維芽細胞培地(DMEM完了)を調製します。孔径0.22μmの真空フィルターで培地をろ過滅菌します。</li>
	<li>38.5 mLのポリプロピレンチューブ中で、4°C、100,000 x g のFBSを一晩超遠心分離して、エキソソームフリー血清を調製し、上清を新しいチューブに移します。</li>
	<li>440 mL の DMEM と 50 mL のエキソソームフリー血清(10%)、5 mL のペン/ストレプ菌 (100 U/mL および 100 μg/mL)、5 mL のグルタミンサプリメント (2 mM) を混合して、500 mL のエキソソームフリー培地を調製します。孔径0.22μmの真空フィルターで培地をろ過滅菌します。</li>
	<li>6.057gのトリス塩基を90mLのdH2Oに溶解し、pHをHClで7.4に調整することにより、0.5 M Tris-HCl(pH 7.4)を調製します。</li>
	<li>97.9 mLのdH 2 Oと2mLの0.5 M Tris-HCl(pH = 7.4)および100 μLの1 M Mg(Ac)2を混合して、10 mM Tris-HCl(pH 7.4)/1 mM Mg(Ac)2溶液(スクロースワーキング溶液)を調製します。使用直前にプロテアーゼ阻害剤を溶液に添加し、溶液を氷上に保管してください。</li>
	<li>表1に従って氷上でスクロース密度勾配溶液を調製します。これらの溶液は、2本のスクロースグラジエントチューブを調製するのに十分です。</li>
	<li>100gのTCA粉末に40 mLのdH2Oを加えて100%TCAを調製し、完全に溶解するまで混合します。dH2Oで最終容量を100 mLに調整します。</li>
	<li>20mLのdH2Oと80mLの100%エタノールを混合して80%エタノールを調製する。</li>
	<li>1.8 Lの精製水(dH2O)と200 mLの10倍のランニングバッファーを混合して、ウェスタンブロットのランニングバッファーを調製します。</li>
	<li>1.4 LのdH2Oと200 mLの10x転写バッファーおよび400 mLのメタノールを混合して、ウェスタンブロット転写バッファーを調製します。転写バッファーを4°Cに予冷します。</li>
	<li>122 gのトリス塩基と180 gの塩化ナトリウムを850 mLのdH2O(pH 8.0)に溶解して、20xトリス緩衝生理食塩水(TBS)を調製します。容量をdH1Oで2 Lに調整します。</li>
	<li>5 gの脱脂粉乳を1 mLの10% Tween-20、5 mLの20x TBS、および94 mLのdH2Oに溶解してブロッキングバッファーを調製します。</li>
	<li>3 g の BSA を 1 mL の 10% Tween-20、5 mL の 20x TBS、および 94 mL の dH2O に溶解して抗体バッファーを調製します。</li>
	<li>100 mL の 20x TBS と 20 mL の 10% Tween-20 (10 x) を 1.88 L の dH2O と混合して、洗浄バッファーを調製します。</li>
	<li>1容量のルミノール/エンハンスと1容量の安定な過酸化物緩衝液を混合して現像液を調製します。</li>
</ol>2. マウス腓腹筋(GA)の解剖15,16
<ol><li>氷上で10 mLのPBSを含む50 mLチューブを調製します。</li>
	<li>CO2チャンバーでマウスを安楽死させ、続いて子宮頸部脱臼させます。</li>
	<li>両足から腓腹筋を取り除き、氷上でPBSを使用してチューブに移します。 注:GA筋をPBSに移す前に、GA筋からヒラメ筋を取り除きます。</li>
</ol>3. マウス初代線維芽細胞の単離と培養
<ol><li>筋肉の重さを量り、バイオセーフティフードの下の10cmの皿に移します。筋肉をメスで細かいペーストになるまで細かく刻みます。</li>
	<li>細かく刻んだマッスルペーストを50 mLチューブに移し、3.5倍の体積/mg組織の消化液をチューブに加え、37°Cで45分間インキュベートします。 懸濁液を10分ごとに5 mLピペットで完全に混合します(これにより、完全な解離に役立ちます)。 注:あるいは、このステップには組織解離剤を使用することができる。</li>
	<li>20 mLのDMEM completeを細胞懸濁液に加え、消化液を不活化します。50 mLチューブに置かれた70 μmのナイロンセルストレーナーに移します。フロースルーを回収し、さらに5 mLのDMEMでセルストレーナーを洗浄します。</li>
	<li>細胞懸濁液を室温(RT)で300 x g で10分間遠心分離し、上清を慎重に除去します。</li>
	<li>ペレットを10 mLのDMEMコンプリートに再懸濁し、細胞を10 cmのディッシュに播種します。</li>
	<li>細胞を37°C、5%CO2、3%O2 (継代0またはP0)で培養する。 注:1)これらの培養物は、生理学的に類似した状態(骨格筋のO2 レベルは約2.5%)を確保するために、低酸素レベルに維持する必要があります17。2)細胞培養の継代数(例:P0)は、培養物が継代培養された回数の記録です。3)P0の初代線維芽細胞は、100%コンフルエントに1日を加えるまでルーチン培養されます(P0のみ)。これは、他の種類の細胞をパージし、培養物に存在する細胞が線維芽細胞のみであり、顕微鏡検査に基づいて筋原性細胞が枯渇していることを保証するためです。生後2ヶ月の成体動物の骨格筋には、約2%の筋原性前駆細胞が含まれています4。さらに、筋原性前駆細胞は通常、コーティングされていない皿に付着しません。したがって、それらは継代培養中に失われます18、19、20。筋原性細胞には、20%FBSと塩基性線維芽細胞増殖因子(bFGF)を添加した別の培地(F10)が必要です18。DMEMコンプリートでは、中程度の線維芽細胞は筋原性細胞よりも増殖率が高く、その結果、最初の継代の前にこれらの汚染細胞が排除されます。</li>
	<li>100%コンフルエントプラス1日でP0細胞をPBSですすぎます。1 mLのトリプシン化溶液を加え、37°C、5%CO2、3% O2 でインキュベートして細胞を剥離します。10 mLのDMEMコンプリートを使用して酵素活性を停止します。</li>
	<li>細胞を300 x g で10分間室温で遠心分離し、ペレットを20 mLのDMEMコンプリートに再懸濁し、15 cmのディッシュ(継代1またはP1)に播種します。細胞は80%〜90%のコンフルエントまで増殖し、継代3まで増殖させることができます。 注:下流の実験に応じて、通路1、通路2(P2)、または通路3(P3)を使用できます。</li>
</ol>4. 細胞の播種と馴化培地の採取
<ol><li>線維芽細胞(P1、P2、またはP3)を10mLのPBSで洗浄し、2.5mLのトリプシン処理溶液を細胞に加え、37°C、5%CO2、3%O2でインキュベートします。10 mLのDMEMコンプリートを使用して酵素活性を停止します。 注:継代4以降、初代細胞は廃棄され、特性が変化するため、それ以上の実験には使用しないでください。</li>
	<li>細胞を回収し、室温で300 x g で10分間遠心分離します。</li>
	<li>細胞ペレットを10 mLのエキソソームフリー培地に再懸濁し、細胞をカウントします。</li>
	<li>1皿あたり1.5–2.0 x 106個の細胞(15 cm)で細胞を播種し、37°C、5%CO 2、3%O2でインキュベートします。</li>
	<li>コンディショニングした培地を氷上の50 mLチューブに16〜24時間集めます。 注:細胞が80%コンフルエントでない場合は、新鮮なエキソソーム遊離培地を再度添加し、さらに16〜24時間後に回収することができます。この 2 つのコレクションは、さらに処理するために組み合わせることができます。</li>
</ol>5. 差動および超遠心分離によるエクソソームの精製
注意: すべてのステップは、4°Cまたは氷上で実行されます。必要に応じて、水で満たされたチューブとチューブのバランスを取ります。
<ol><li>馴化培地を300 x g で10分間遠心分離して生細胞を除去し、上清を新しい50 mLチューブに移します。</li>
	<li>2,000 x g で10分間遠心分離して死細胞を除去し、上清を38.5 mLのポリプロピレンチューブに移します。</li>
	<li>上清を10,000 x g で30分間遠心分離し、細胞小器官、アポトーシス小体、および膜断片を除去します。上清を38.5 mLの超透明チューブに移します。</li>
	<li>上清を100,000 x g で1.5時間超遠心分離し、エクソソームをペレット化します。</li>
	<li>上清を慎重に廃棄し、約1 mLの馴化培地を残し、エキソソームペレットを総量30 mLの氷冷PBSで洗浄します。</li>
	<li>100,000 x g で1.5時間遠心分離し、エキソソームペレットを乱さないように注意しながら、ピペッティングにより上清を慎重に廃棄します。</li>
	<li>ペレットを氷冷PBS(15 cmディッシュあたり~25 μL)に再懸濁し、BCAタンパク質アッセイキットとマイクロプレートリーダーを使用して562 nmでタンパク質濃度を測定します。 注:タンパク質濃度は、0.1% Triton-X100 in dH 2 Oで1:2希釈して測定します。80%のコンフルエントさでの一次線維芽細胞の15cmディッシュ(20mLのコンディショニング培地)からのエキソソームの収量は、3~4μgです。</li>
</ol>6. ショ糖密度勾配によるエクソソームのキャラクタリゼーション
<ol><li>1.5 mL の 2.0 M、2.5 mL の 1.3 M、2.5 mL の 1.16 M、2.0 mL の 0.8 M、2.0 mL の 0.5 M スクロース溶液をピペッティングして、13 mL の超透明チューブにスクロースグラジエントをロードします。</li>
	<li>30〜100 μgのエキソソームを0.25 Mショ糖溶液と混合し、容量を1 mLに調整します。</li>
	<li>グラジエントの上にエキソソームを慎重にロードし、サンプルを 100,000 x g および 4 °C で 2.5 時間超遠心分離します。</li>
	<li>超遠心分離機からチューブを取り出し、氷上に置き、グラジエントの上部から 1.0 mL のフラクションを回収します。氷上で1.7 mLのチューブに移します。</li>
	<li>110 μL の 100% TCA を各チューブに添加し、よく混合し、室温で 10 分間インキュベートします。</li>
	<li>10,000 x g およびRTで10分間遠心分離し、上清を慎重に廃棄します。</li>
	<li>ペレットを予冷した80%エタノール500μLに再懸濁し、サンプルを-20°Cで10分間洗浄します。</li>
	<li>10,000 x g 、4°Cで10分間遠心分離し、上清を廃棄します。</li>
	<li>ペレットを室温で10〜15分間乾燥させ、in vitro実験のためにペレットをPBSに再懸濁します。BCAタンパク質アッセイキットを使用してタンパク質濃度を測定するか、ウェスタンブロット分析のために14.5 μLのdH2O、5 μLの4x Laemmliバッファー、および0.5 μLの1 M DTTにペレットを直接再懸濁します。</li>
</ol>7. ウェスタンブロット解析によるエクソソーム検出
<ol><li>ステップ5.7のエキソソーム5〜10 μgを5 μLの4x Laemmliバッファー、および0.5 μLの1 M DTTと混合し、最終容量をdH2Oで20 μLに調整します。 ステップ6.9のサンプルを含むすべてのサンプルを98°Cで5分間加熱します。</li>
	<li>サンプルを10%TGX染色フリーゲルにロードし、メーカーの推奨に従ってタンパク質ラダーをロードします。 注:目的のタンパク質の分子量に応じてゲルの割合を選択してください。</li>
	<li>ローディング色素がゲルの底部に達するまで、ゲルを100 Vで流速バッファー中で~1時間泳動させます。 注:実行時間は、使用する機器、またはゲルの種類と割合によって異なる場合があります。</li>
	<li>ゲルを画像化します。染色されていないゲルの画像は、イムノブロットのローディングコントロールとして使用されます。</li>
	<li>PVDFメンブレンを使用して、80 Vで1.5時間、または30 V、4°Cで一晩ゲルを転写します。</li>
	<li>メンブレンをブロッキングバッファーで室温で1時間ブロッキングします。</li>
	<li>特異的抗体のメンブレン(表2)を抗体バッファー中で、4°Cで一晩撹拌しながらインキュベートします。</li>
	<li>メンブレンを洗浄バッファーで洗浄し、メンブレンを適切な二次抗体(表2)とともに室温で1時間、攪拌しながらインキュベートします。</li>
	<li>メンブレンを洗浄バッファーで洗浄し、現像液とカメラベースのイメージャーを使用してメンブレンをイメージングします。または、X線フィルムを使用してメンブレンを現像します。</li>
</ol>

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K., 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=Comparative+analysis+of+exosome+isolation+methods+using+culture+supernatant+for+optimum+yield,+purity+and+downstream+applications.">Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.</a> Science Reports. 9 (1), 5335 (2019).</li><li>Ayala-Mar, S., Donoso-Quezada, J., Gallo-Villanueva, R. C., Perez-Gonzalez, V. H., Gonz&#225;lez-Valdez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+challenges+in+the+recovery+and+purification+of+cellular+exosomes.">Recent advances and challenges in the recovery and purification of cellular exosomes.</a> Electrophoresis. 40 (23-24), 3036-3049 (2019).</li><li>Boutagy, N. E., 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=Isolation+of+Mitochondria+from+Minimal+Quantities+of+Mouse+Skeletal+Muscle+for+High+Throughput+Microplate+Respiratory+Measurements.">Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements.</a> Journal of Visualized Experiments. (105), e53217 (2015).</li><li>. Muscle Dissection in mouse Available from: <a target="_blank" href="https://www.youtube.com/watch?v=Wh0gXfrHaH8">https://www.youtube.com/watch?v=Wh0gXfrHaH8</a> (2016)</li><li>Mas-Bargues, C., 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=Relevance+of+oxygen+concentration+in+stem+cell+culture+for+regenerative+medicine.">Relevance of oxygen concentration in stem cell culture for regenerative medicine.</a> International Journal of Molecular Sciences. 20 (5), 1195 (2019).</li><li>Bois, P. R., Grosveld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKHR+(FOX01a)+is+required+for+myotube+fusion+of+primary+mouse+myoblasts.">FKHR (FOX01a) is required for myotube fusion of primary mouse myoblasts.</a> The EMBO Journal. 22 (5), 1147-1157 (2003).</li><li>Machida, S., Spangenburg, E. E., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+rat+muscle+progenitor+cells+have+decreased+proliferation+and+myotube+formation+during+passages.">Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.</a> Cell Proliferation. 37, 267-277 (2004).</li><li>Syverud, B. C., Lee, J. D., VanDusen, K. W., Larkin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+satellite+cells+for+skeletal+muscle+tissue+engineering.">Isolation and purification of satellite cells for skeletal muscle tissue engineering.</a> Journal of Regenerative Medicine. 3 (2), 117 (2014).</li></ol>

著者の誰一人として、宣言すべき利益相反はありません。

このプロトコルで概説されているように、培地からのエキソソームの単離を成功させるための重要なステップは、成体骨格筋からの初代マウス線維芽細胞培養の適切な確立と維持です。これらの培養物は、生理学的に類似した状態(骨格筋のO2レベルは~2.5 %)を確保するために、低酸素レベルに維持する必要があります15。初代線維芽細胞は、培養に何度も通すと特性?...

エクソソームは、30〜150nmのサイズの不均一な細胞外小胞です。それらは、組織や器官に遍在的に分布していることから、生理学的および病理学的プロセスにおける確立された主要なプレーヤーです1,2。エクソソームは、タンパク質、脂質、DNAタイプ、RNAタイプの複雑なカーゴを運び、それらはそれらが由来する細胞の種類によって異なります1,2,3。エクソソームは、異なる機能を持つタンパク質(すなわち、CD9およびCD63を含むテトラスパニン)が豊富であり、融合イベントの原因となります。例えば、熱ショックタンパク質であるHSP70やHSP90は、抗原の結合や提示に関与しています。さらに、アリックス、Tsg101、およびフロチリンは、エクソソームの生合成と放出に関与し、これらのナノベシクルのマーカーとして広く使用されています2,3,4。
また、エクソソームにはさまざまなRNA(マイクロRNA、長鎖ノンコーディングRNA、リボソームRNA)が含まれており、これらはレシピエント細胞に伝達され、下流のシグナル伝達に影響を与えます3。単一の単位膜で囲まれているため、エクソソームの生理活性は、タンパク質および核酸のカーゴだけでなく、制限膜1の脂質成分にも依存する。エキソソーム膜には、ホスファチジルセリン、ホスファチジン酸、コレステロール、スフィンゴミエリン、アラキドン酸、その他の脂肪酸が豊富に含まれており、これらはすべてエクソソームの安定性と膜トポロジーに影響を与える可能性があります2,3。カーゴと脂質の配置の結果として、エクソソームは受容細胞のシグナル伝達経路を開始し、正常組織生理学の維持に関与します1,2,4,5。特定の病理学的条件(すなわち、神経変性、線維症、および癌)の下で、それらは病理学的刺激を誘発し、伝播することが示されています4,6,7,8,9,10,11。
エクソソームは、近隣または遠隔の部位にシグナルを伝播する能力があるため、疾患の診断や予後のための貴重なバイオマーカーとなっています。さらに、エクソソームは治療用化合物のビヒクルとして実験的に使用されている2,12。これらのナノベシクルを臨床に応用する可能性は、最大の収量、純度、および再現性を達成するために、単離法をますます重要にしています。エクソソームを単離するためのさまざまな技術が開発され、実施されています。一般に、エキソソームは、示差遠心分離、サイズ排除クロマトグラフィー、および免疫捕捉(市販のキットを使用)によって、馴化細胞培養培地または体液から単離することができる。各アプローチには、前に説明した独自の長所と短所があります1、2、13、14。
概説されたプロトコルは、1)成体マウス腓腹筋からの一次線維芽細胞の単離と培養、および2)これらの細胞によって培地に放出されたエクソソームの精製と特性評価に焦点を当てています。機能研究のための初代線維芽細胞からのエキソソームの単離のための十分に確立されたプロトコルは、現在不足しています。初代線維芽細胞は大量のエクソソームを分泌しないため、単離と精製のプロセスが困難になります。このプロトコルは形態学的な完全性および機能活動を維持している間大きい培養容積からのたくさんの純粋なexosomesの浄化を記述する。馴化培地から得られた精製エキソソームは、レシピエント細胞に特異的なシグナル伝達経路を誘導するために、in vitroでの取り込み実験で成功裏に使用されています。また、複数の生体試料から採取したエキソソームカーゴの比較プロテオミクス解析にも使用されています4。

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

エクソソームは、ほぼすべての細胞から放出され、すべての体液中に分泌される小さな細胞外小胞です。これらの小胞を単離するために、超遠心分離、限外ろ過、サイズ排除クロマトグラフィーなど、多くの方法が開発されています。しかし、すべてが大規模なエクソソームの精製や特性評価に適しているわけではありません。ここでは、成体マウスの骨格筋から単離された初代線維芽細胞の培養を確立し、その後、これらの細胞の培養培地からエキソソームを精製および特性評価するためのプロトコルを概説します。この方法は、連続した遠心分離ステップとそれに続くスクロース密度勾配の使用に基づいています。次に、エキソソーム調製の純度を、一連の標準マーカー(Alix、CD9、CD81など)を用いたウェスタンブロット解析によって検証します。プロトコルは機能調査のための電子顕微鏡検査、質量分析および通風管の実験のためのbioactiveexosomesを隔離し、集中する方法を記述する。スケールアップやスケールダウンが容易で、さまざまな細胞タイプ、組織、体液からのエクソソーム単離に適応させることができます。

このプロトコルは、費用対効果の高い方法で大量の調整培地からエキソソームを精製するのに適しています。この手順は再現性が高く、一貫性があります。 図1 は、マウス初代線維芽細胞の培養液から精製したエクソソームの透過型電子顕微鏡(TEM)画像です。 図2 は、標準的なエキソソームマーカーのタンパク質発現パターンと、細胞質(LDH)および...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

このプロトコルは1)大人マウス腓腹筋からの第一次線維芽細胞の隔離そして文化、また2)西部のしみの分析に先行しているスクロース密度勾配と結合される差動ultrarifjugeation方法を使用してexosomesの浄化そして性格描写を示す。

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

isolation-characterization-exosomes-from-skeletal-muscle

Research

JoVE Journal

Biology

3.2K ビュー.  St. Jude Children's Research Hospital. このプロトコルは1)大人マウス腓腹筋からの第一次線維芽細胞の隔離そして文化、また2)西部のしみの分析に先行しているスクロース密度勾配と結合される差動ultrarifjugeation方法を使用してexosomesの浄化そして性格描写を示す。

ビデオ: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

生物学

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨格筋からのミトコンドリア単離

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含有エキソソームの単離と解析

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

ヒト骨格筋からの血管由来の多能前駆体の単離

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

ハイスループットマイクロプレート呼吸測定のためのマウスの骨格筋の最小量からミトコンドリアの単離

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

高解像度の呼吸計測測定のための差動遠心分離することにより骨格筋から無傷ミトコンドリアの単離

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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 ...