Name: the isolation and culture of primary epicardial cells derived from human adult and fetal heart specimens
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Description: Watch this Scientific Journal Video about The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens at JoVE.com

この研究はオランダ組織科学的研究 (NWO) (イタリアヴェネツィア 016.146.079)、LUMC 研究フェローシップ AMS と LUMC Bontius スティヒティング (MJG) の両方でサポートされます。

人間の組織標本で実験にはライデン大学医療センター倫理委員会で承認され、ヘルシンキ宣言に準拠しています。細胞文化フロー キャビネットの滅菌装置のすべての手順が実行されます。
1. 準備
<ol><li>ダルベッコ変法イーグル培地 (DMEM 低グルコース) と中 199 (M199) を 1:1 の比率で混合することによって追求メディアを準備します。10% 熱不活化ウシ胎児血清 (FBS、56 ° C で 25 分の不活化熱) を追加 100 U/mL ペニシリンとストレプトマイシン 100 mg/mL を補足します。あらかじめ暖かい EPDC 37 ° C の水浴中。</li>
	<li>あらかじめトリプシンを暖かい 0.25%/EDTA (1:1) 37 ° C の水浴中。</li>
	<li>ゼラチンで井戸をコートします。各ウェルに 0.1% ゼラチン/PBS を追加し、37 ° C で少なくとも 15 分版を孵化させなさい必要な細胞培養プレートのためのガイドラインを表 1にまとめます。細胞をメッキする前にすべての流体を慎重に取り外します。</li>
	<li>ストック溶液 10 mM SB431542 (SB) DMSO (注意) を希釈を準備します。50 μ L の因数は、円錐の底のポリプロピレン製遠心チューブ、エッペン チューブのような-20 ° C で保存SB 因数を一度だけ解凍できることに注意してください。</li>
</ol>2. 検索や成人および胎児心臓標本の記憶
<ol><li>15 mL と 50 mL のチューブで手術中に除去に直接人間の大人心耳を保存 10 %fbs、100 U/mL ペニシリンおよび 4 ° C で 100 mg/mL ストレプトマイシンを含む高グルコース DMEMサンプル サイズで 3-5 cm は、一般的に、最大保存することができます郭清後 48 h。</li>
	<li>分離後 24 h 最大 4 ° C で EPDC 媒体で人工妊娠中絶材料から得られた胎児の心を格納します。このプロトコルの 12-22 週間の間の妊娠期間でサンプルを使用します。心外膜の隔離のため、心臓全体を使用できることを注意してください。</li>
</ol>3. 心外膜層の分離
<ol><li>流キャビネット、PBS の 100 mm 細胞培養皿を準備し、蓋に PBS の独立した液滴 (〜 200 μ L) を配置します。5 mL に予め温めておいた追求中 15 mL チューブを入力します。</li>
	<li>場所細胞培養皿にティッシュ PBS でいっぱい。組織はプロシージャの間によく湿らされたことを確認します。</li>
	<li>顕微鏡を使用して、多くの削除できるだけ組織サンプルから心外膜層鉗子を使用して剥がして。 注: 非常に薄い透明なレイヤー (図 1 a) 心臓の外側に密着、心外膜を認識できます。これは分離が阻害されますから心外膜脂肪組織と血管の混入を避けてください。</li>
	<li>蓋の上の PBS の液滴の心外膜組織の断片を収集します。</li>
	<li>メス (図 1 b) を使用してまたは小さな鉗子の先端が鋭いを使用して小さな断片 (0.5 mm3) に心外膜組織を切断します。作品が、P1 を渡すことに注意してください 000 ピペット先端部 (ステップ 3.6)。</li>
	<li>心外膜組織にトリプシンの 1 つの mL を追加し、作品と P1, 000 ピペット先端部とトリプシンを収集します。1.5 mL の円錐形遠心管 (図 1) に組織を転送します。</li>
	<li>〜 60 rpm で揺れながら 10 分の 37 ° C の水浴のチューブを孵化させなさい。</li>
	<li>風呂の水からチューブを外し、70% エタノールで管の外側を清掃します。</li>
	<li>(図 1) の管の底に沈むし、トリプシンを不活化する 5 mL EPDC 媒体を含んでいる 15 mL チューブに心外膜細胞を含む上清を慎重に転送する組織を許可します。</li>
	<li>1 mL のトリプシン、円錐形遠心分離機管の残りのティッシュを補充して上下に軽くピペッティングで混ぜます。</li>
	<li>3.7 に 3.10 の手順を 2 回繰り返します。最後のステップでトリプシン培養の 30 分の合計後上澄みと残りの心外膜組織に転送 EPDC 媒体を含んでいる管。</li>
	<li>追求中のすべてのセルが収集されると、優しく通過懸濁液 10 mL シリンジ 19 ゲージの針で機械的に細胞 (図 1E) を分離する新しい 15 mL チューブに。 注: を妨げ得ること針を防ぐために針をソリューションを通過する前に上下にピペッティングによる懸濁液を均一に確認します。</li>
	<li>さらに細胞を分離するには、21 g 針付き 3.12 手順を繰り返します。</li>
	<li>50 mL チューブの上に 100 μ m セル ストレーナーを置き、10 mL のピペットを使用してすべての残りの塊 (図 1 f) を削除するストレーナーに心外膜細胞を含む培地を転送します。</li>
	<li>こし器に 5 mL EPDC 媒体をピペッティングして残留細胞を収集するストレーナーを洗浄します。</li>
	<li>15 mL チューブに 50 mL の管から細胞懸濁液をピペットで移しなさい。以来、細胞はチューブの底に沈む、ピペッティングする前にソリューションを軽く振る。 注: この手順は必要ではありません、小さい管は遠心分離後、ペレットの可視化を支援します。</li>
	<li>(図 1) 室温で 5 分間 200 × g で遠心分離機します。</li>
	<li>上澄みを除去し、追求中 (表 1) の必要量で (図 1 H) 細胞ペレットを再懸濁します。 注: 胎児 EPDCs EPDC 培 ALK5 キナーゼ阻害剤 (EPDC + SB) の 10 μ M を直接使用します。大人の細胞は、最初の通過中に SB なしメッキできます。</li>
	<li>ゼラチン コーティング培養皿 (図 1I) に細胞懸濁液をプレートします。井戸のサイズは、心外膜組織サンプル (表 1) のサイズによって決まります。低密度で EMT の発生を誘発することに注意してください。</li>
	<li>37 ° c、5% CO2細胞培養プレートにアタッチするを許可するように、少なくとも 48 時間のためのインキュベーターにセルを配置します。</li>
</ol>4. EPDCs の文化
<ol><li>少なくとも 3 日ごと EPDC + SB 媒体を補充します。</li>
	<li>顕微鏡を使用して、少なくとも 3 日間、細胞を検査します。細胞培養プレートが細胞で完全に覆われているとき密度、すなわちに到達、表面比 1:2 のセルを通路します。Confluency に達する 〜 5-10 日を取ることができますに注意してください。<ol>
<li>例えば、ガラス ピペット、ピペットまたは吸引システムを使用してメディアを吸い出しなさい。</li>
		<li>PBS をセルに追加することによって、細胞を丁寧に洗います。ゆっくり版は渦巻きし、PBS を吸引します。</li>
		<li>プレートにトリプシンを追加します。優しくトリプシン セルはすべてをカバーし、37 ° C で 1 分のプレートをインキュベートするプレートを回転します。 注意: 可能な限り小さなトリプシンとして使用 (表示: 200 μ L/6 ウェル ウェル プレート)</li>
		<li>プレートの底から細胞を機械的に切断するプレートをタップします。顕微鏡を使用して、細胞が切断したかどうかを視覚的に確認します。ない場合は、余分な分インキュベート細胞培養プレート。</li>
		<li>セルに追求 + SB 中の必要量を追加、上下、ピペッティングで再懸濁し、細胞懸濁液を新しいゼラチン コーティング培養プレートに転送します。 注: 一般的には、細胞に保存できます 8 の通路まで石畳形態。</li>
	</ol>
</li>
</ol>5. EPDCs で EMT の誘導
<ol><li>EMT を誘導して、トリプシンと転送新しいゼラチンにセル、4 で説明したように EPDC + SB 中に、表面比 1:2 の井戸をコーティングし、37 ° C、5% CO2で少なくとも 24 時間インキュベート細胞を分離します。</li>
	<li>チェック場合合流は 50-70% と EPDCs ある石畳の形態であるかどうかです。 注: の Confluency EMT を受ける EPDCs の能力に影響します。</li>
	<li>細胞から培地を吸引させ、慎重に PBS のセル (4.2.1 - の手順を参照してください 4.2.2)。</li>
	<li>1 ng/ml TGFβ3 EPDC 培地で細胞を刺激し、37 ° C、5% CO2の 5 日間でインキュベーターで配置します。刺激の中に TGFβ3 を含む追求中が持たない補充するに注意してください。</li>
	<li>セルを毎日監視します。5 日後、EMT を受けた細胞は紡錘形の形態 (図 2 a) によって認識されます。</li>
	<li>文化追求中に SB の TGFβ 添加せず 4 で説明されている方法に従って紡錘形 EPDCs。一般に、紡錘形 EPDCs 注は、20 の通路まで培養することができます。</li>
	<li>免疫蛍光染色と EMT の発生を検証ファロイジン、間葉系のマーカー αSMA またはビメンチンに対する抗体を使用して F-アクチン繊維の応力 (図 2 b) の形成を検出するまたは EMT 関連遺伝子 (の qRT PCR を使用して例えば、WT1, ペリオスチン Col1A1、αSMA、N-カドヘリン、MMP3、カタツムリ、ナメクジ) (図 2および表 2)。</li>
</ol>

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著者が明らかに何もありません。

ここで人間の成人および胎児心に由来する一次心外膜細胞を分離するための詳しいプロトコル文化について述べる。これらの細胞の広範な評価は、以前に公開された17をされています。我々 は両方のセルタイプを ALK5 キナーゼ阻害剤 SB431542 による培養上皮の石畳のような細胞として維持できることを示しています。EMT は開発およびポスト傷害応答中に体内の心外膜?...

心外膜、封筒、心は、極めて重要な心臓の開発と修理 (スミッツらの見直しのための単一細胞上皮層1). 発達、心外膜に起因 proepicardial 器官の開発の中心部の底部にある小さな構造。マウス、および 4 週間後の概念で、人間の発達の日 E9.5、周辺細胞はこのカリフラワー構造からの移行し、開発心筋2をカバーを開始します。単一の上皮細胞層が形成され、一度心外膜細胞の一部は上皮間葉転換 (EMT) に行われます。EMT、中に細胞は細胞接着斑などの上皮性特性を失うし、それらの発展途上の心筋に移行する能力を与える間葉系の表現型を得る。後者の 2 つのセルが形成された心外膜派生セル (EPDCs) が線維芽細胞、平滑筋細胞、および可能性のある心筋細胞、内皮細胞3を含むいくつかの心筋細胞タイプに区別できます。集団のまま (スミッツらの見直し議論の対象と4). さらに、心外膜、その成長と血管新生5,6,7、8を規制する心筋に有益なパラクリン シグナル。複数の研究は、障害の心外膜形成が心筋9,10, 血管11及び伝導システム12、強調、エッセンシャルで発育障害につながることを実証しています。心の形成に心外膜の貢献。
大人の心、心外膜は休眠層として存在が、それは虚血13の時に再開になります。心外膜再ポスト傷害のいくつかを繰り返すプロセス説明にもかかわらず増殖と EMT14などを含む心臓の開発の小さい効率的に。興味深いことに、正確なメカニズムは十分に理解されていないが修復への貢献を心外膜変更 VEGF-A mRNA16心の改善の結果、例えば、サイモシン β415と治療によって改善できるまたは心筋梗塞後の機能。心外膜は、傷ついた心臓の内因性修復を高めるために興味深い細胞ソースとみなされます。
心臓発生のメカニズムは頻繁に締めくくっているけが、中にあまり効率的な方法ではあるが。心外膜の活性化を求めて、それは決定し、胎児および大人の心外膜の全容量を比較できますは重要です。また、治療の観点から、動物実験だけでなく人間の心外膜の応答に関する知識を拡張、重要です。ここでは、分離し、細胞の培養ひと成人および胎児心外膜派生 (EPDCs) 上皮細胞の形態で、EMT を誘導する手法について述べる。このモデルでは、探索し、成人および胎児の心外膜細胞の挙動を比較を目指します。
このプロトコルの主な利点は、徹底的に検討されていない人間の心外膜材料の使用です。重要なは、説明の分離および細胞培養のプロトコルは、これらの 2 つのセルのソース間の直接比較を有効にする両方の胎児および大人の石畳 EPDCs を取得する単一の統一された方法を提供します。さらに、心外膜は分離の場所に基づいて、セルが実際に epicardially 派生17であることが保証されます。
人間の追求の分離メソッドが以前に確立されると、大抵伸長プロトコル細胞培養皿18,19に心臓や心外膜組織の部分をメッキパーツに依存してこれら。このアプローチは、部分的に彼らの上皮性の表現型を失うことが、移行するために、自発的な EMT を受けることになりやすい細胞用により選択します。現在のプロトコルの心外膜は最初の上皮の状態を維持するために分離の EPDCs を可能にする単一細胞液に処理されます。このメソッドは、したがって心外膜 EMT を勉強する固体の in vitroモデルを提供します。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium + GlutaMAX</td><td>Gibco</td><td>21885-025</td><td></td></tr><tr><td>Medium 199&amp;nbsp;</td><td>Gibco</td><td>31150-022</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Trypsin 0.25%</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Penicillin G sodium salt</td><td>Roth</td><td>HP48</td><td></td></tr><tr><td>Streptomycin sulphate</td><td>Roth</td><td>HP66</td><td></td></tr><tr><td>Trypsin 1:250 from bovine pancreas</td><td>Serva</td><td>37289</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E4884</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Culture plates 6 well</td><td>Greiner bio-one</td><td>657160</td><td></td></tr><tr><td>Culture plates 12 well</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>Culture plates 24 well</td><td>Greiner bio-one</td><td>662160</td><td></td></tr><tr><td>SB 431542</td><td>Tocris</td><td>1614</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Merck</td><td>102931</td><td></td></tr><tr><td>100-1000&amp;micro;L Filtered Pipet Tips</td><td>Corning</td><td>4809</td><td></td></tr><tr><td>10-ml pipet</td><td>Greiner bio-one</td><td>607180</td><td></td></tr><tr><td>5-ml pipet</td><td>Greiner bio-one</td><td>606180</td><td></td></tr><tr><td>Cell culture dish 100/20 mm</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010056</td><td>Or home-made and sterilized</td></tr><tr><td>Eppendorf tubes 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>15-ml centrifuge tubes</td><td>Greiner bio-one</td><td>188271</td><td></td></tr><tr><td>50-ml centrifuge tubes</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>10 mL Syringe</td><td>Becton Dickinson</td><td>305959</td><td></td></tr><tr><td>Needles 19 Gauge</td><td>Becton Dickinson</td><td>301700</td><td></td></tr><tr><td>Needles 21 Gauge</td><td>Becton Dickinson</td><td>304432</td><td></td></tr><tr><td>EASYstrainer Cell Sieves, 100 &amp;micro;m</td><td>Greiner bio-one</td><td>542000</td><td></td></tr><tr><td>TGF&amp;beta;3&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>243-B3</td><td></td></tr><tr><td>Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma</td><td>A2547&amp;nbsp;</td><td></td></tr><tr><td>Anti-Mouse Alexa Fluor 555</td><td>Invitrogen</td><td>A31570</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Pipet P1,000</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Pipet controller</td><td>Integra</td><td>155 015</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Inverted Light Microscope</td><td>Olympus</td><td>CK2</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Waterbath</td><td>GFL</td><td>1083</td><td></td></tr></tbody></table>

the isolation and culture of primary epicardial cells derived from human adult and fetal heart specimens

心外膜, 心筋を覆う上皮細胞層は、虚血性損傷後、心臓の開発中に、心の修復反応での重要な役割にいます。アクティブになったとき、心外膜細胞は再生心筋細胞を提供するでは上皮間葉転換 (EMT) 知られているプロセスを経る。さらに、必須のパラクライン因子の分泌を介して、心外膜が貢献しています。心外膜の再生の可能性を完全に鑑賞するには、ひと細胞モデルが必要です。ここでは、人間の成人および胎児の心臓組織から一次派生心外膜細胞 (EPDCs) を導出する新規細胞培養モデルを概説します。EPDCs を分離するには、心外膜が心臓標本の外から解剖、単一細胞懸濁液に処理します。次に、EPDCs はメッキ、ALK 5-キナーゼ阻害剤の上皮性の表現型を維持するために SB431542 を含む追求培地で培養しました。EMT は TGFβ 刺激によって誘導されます。このメソッドは、制御された設定で人間の心外膜 EMT の過程の研究を有効にした最初の時間のため、心臓再生を助けることができる EPDCs の secretome でより多くの洞察を得ることが容易になります。さらに、この統一的なアプローチは、人間の成人および胎児心外膜挙動の直接比較のためことができます。

ここでは、我々 は人間成人および胎児心臓組織 (図 1) から EPDCs を分離するための単純なプロトコルを概説します。(図 1 a) 心臓の外側の心外膜の容易にアクセス可能な場所のこのプロトコルを活用します。心耳垂染色後郭清基になる下の細胞外マトリックスと心筋組織のままそのまま (図 1 j) WT1 + 心?...

Watch this Scientific Journal Video about The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens at JoVE.com

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

心外膜細胞と心筋壁に成長因子を提供することによって開発し、中心部の修復に重要な役割を果たしています。ここでは、研究と彼らの発達と大人の特性の比較を可能にする培養ひと心外膜細胞に手法について述べる。

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

the-isolation-culture-primary-epicardial-cells-derived-from-human

Research

JoVE Journal

Developmental Biology

7.7K ビュー.  Leiden University Medical Center. 心外膜細胞と心筋壁に成長因子を提供することによって開発し、中心部の修復に重要な役割を果たしています。ここでは、研究と彼らの発達と大人の特性の比較を可能にする培養ひと心外膜細胞に手法について述べる。

ビデオ: The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜の伸長文化検定と<em&gt; ex vivoで</em&gt;心外膜由来の細胞移動の評価

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

発生生物学

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

ヒト心臓組織からの血管周囲多能前駆細胞集団の単離

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

マウス胚心臓から心外膜細胞の体外培養で

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

工学

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

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

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

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

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

医学

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

ラット心臓の心室自由壁からの心内膜細胞と冠状動脈内皮細胞の分離

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

マウスの心臓から初代メラノサイト様細胞を単離する

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

生物学

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

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

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

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

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

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

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

免疫細胞化学とプロイド分析のための固定心からの心筋細胞の分離

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...

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 ...

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

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心外膜の伸長文化検定と