手術のためのパートI.準備<ol><li>ラットの手術の前日は、すべてのソリューションが行われていることを確認して、まだどの酵素を追加しないでください。バッファ、Bおよび洗浄緩衝液は、すべてのストレージの前に無菌のためにフィルタリングする必要があります。バッファはすべて4℃で保存してください。 </li><li> 6ウェル組織培養プレートを10〜20μgの/ 2mLの1XSFM mLのラミニンと同様前日でメッキされている必要がありますし、37 CO 2インキュベーター内で° Cで保存してください。 </li><li>手術の日に、70％エタノールでクランプ、はさみ、そして鉗子を滅菌により、手術器具を準備し、それらをペーパータオルの上に乾燥させます。 </li><li> 0.5mLのヘパリン溶液（90 U / ml）で満たされた1 mLの注射器が準備されています。 </li><li>今では逆に蠕動ポンプを用いて、70％エタノールを通して実行することにより、灌流装置を洗浄することをお勧めします。一度再蒸留水で装置を洗浄、エタノールで洗浄が終了し、最後に流れるが、37℃の水浴中に置かれたビーカーに収集される緩衝液Aですすいでください。 </li><li>洗浄灌流装置を準備するには、緩衝液Bの40mLでバッファと左シリンジの管の40mLで右シリンジのチューブを埋める</li><li>首相装置を流れるようにバッファを許可することでカテーテルの先端に灌流チューブ。バッファーを40 mLのレベルの注射器に詰め替え。このステップの後にチューブの中に閉じ込めに気泡がないことを確認します。 </li><li>今、バッファの注射器のコックのバルブを閉じ、灌流チューブが今緩衝液Bでプライミングされるように緩衝液Bで処理を繰り返します。 </li><li>最後に、灌流装置は、緩衝液Bストップコックバルブを開いた状態で残っていますが、フローとIVラインにレギュレータを使用して停止されます。 </li><li>収集ビーカー内の処理のフローもバッファーを捨てる。 </li><li>手術前に行う最後のことは、他のソリューションは、前日フィルター処理されたのと同様に酵素溶液をお使い原液に必要な酵素を追加し、フィルタすることです。一度フィルタリング、この溶液を室温で放置することができます。 </li></ol>パートII。ラットの心臓の解剖<ol><li>以下の標準プロトコルは、ガス室におけるイソフルランでラットを安楽死させる。 </li><li> 70％エタノールで胸部の切開の領域を滅菌する。 </li><li>はさみを使って胸を開いて、心臓を公開。 </li><li> 0.5mLのヘパリン溶液（90 U / mL）で左心室を注入する。 </li><li>氷冷緩衝液Bで大動脈そのまま、場所の大部分で心を取り除く</li></ol>パートIII。心筋細胞の分離<ol><li>次の手順が適切に付着されている場合典型的なラットの心臓では、棒状の心筋細胞の高い割合を（デッド丸い細胞とは対照的に）生成する必要があります。 </li><li>開始するには、カテーテルに大動脈をクランプ。カテーテルの先端は、冠状動脈を通って心臓の良好な血流を確保するために心臓に遠くプッシュされるべきではない。 </li><li>一度クランプ、縫合糸でカテーテルに大動脈を結ぶ。 </li><li>次に、高速の滴下速度を（〜60滴/分）を可能と心を洗い流すためにバッファーBを可能にするためにIVラインの​​レギュレータを開きます。 </li><li>バッファBの洗浄時には、心房と心にしがみついて、任意の脂肪や肺組織を除去するために小さなハサミとピンセットを使用してください。 </li><li>バッファBが終了した後、緩衝液Aに流れを切り替える</li><li>バッファーを5分間流すことが許されている間、いくつかの小さなタスクを迅速に実行する必要があります。 </li><li>最初に、37℃の水浴中で酵素液を温める。 </li><li>最後に、バッファーをシリンジ（まだチューブ内に存在）から枯渇しているときに、注射器で酵素溶液50mlをロード灌流装置の</li><li>それは、酵素溶液のperfusesの心までは、現在すべてのフロースルー水浴（ピペット）でコレクションのビーカーからを破棄する必要があります。 </li><li>とすぐに酵素液は心臓をperfusesとして、シリンジAを補給するために収集ビーカーから酵素溶液を転送するように設定されている蠕動ポンプを活性化する</li><li> 10分間心臓を流れるように酵素溶液を許可。心臓ダイジェストとして、それは傲慢な表情を開始します。 </li><li> 0.1mMのCa 2 +の効果的な濃度になるように注射器で酵素溶液に0.1 M CaCl 2の37.5μLを加える。 </li><li>後10分はシリンジに0.1 M CaCl 2をさらに50μLを添加することにより0.2mMのためにカルシウムの濃度を増加させ、血流がさらに10分間進行させる。 </li><li>心室をカットオフし、20mLの酵素液を含む小さな滅菌ビーカーに移す。 </li><li>ビーカーに0.1 M CaCl 2をさらに40μLを添加することにより0.4mMのためにカルシウム濃度を増加させる。 </li><li>そっと小さな滅菌ハサミで10以上の作品に心を飾らない。 </li><li> 37℃で5分間ビーカーをインキュベート° C緩やかに振盪した。 </li><li>に0.1 M CaCl 2を40mLのを追加します。0.6mmのCa 2 +の有効濃度を与え、穏やかにプラスチック製のトランスファーピペット3〜5回でハートの部分を砕いて粉にする。 </li><li> 37℃でさらに5分間インキュベート° Cをした後、0.1 M CaCl 2の40 mLを加え、ゆっくりと前と同じように砕いて粉末にする。 </li><li>滅菌500μmのメッシュで濾過を使用して、非消化結合組織から消化単一心筋細胞を分離する。 </li><li>細胞は室温で10分間、50 mlチューブに定住することができます。 </li><li>転送上清をピペットで捨てます。 </li><li>優しく洗浄バッファー＃1で細胞を再懸濁し、細胞は、室温で10-20分のために解決することができます。 </li><li>細胞がセトリングしている間、倒立顕微鏡下で懸濁液中の細胞の質と生存率を評価する機会として、この時間を小さなアリコートを取り、使用してください。良い準備が鮮明な縞を持つ細胞のような棒の割合が高い（&gt; 80％）になります。 </li><li>細胞が定着した後、洗浄バッファー＃2で上清と穏やかに懸濁し、細胞を捨てる。細胞は室温で沈殿させ、これは再び10〜20分かかりますし、上清を破棄すべきである。 </li><li>第3洗浄バッファーで一回以上の洗浄工程を繰り返し、細胞は培養のための準備が整います。 </li></ol>パートIV。筋細胞の細胞培養<ol><li>穏やかに5％血清培地で細胞を懸濁します。組織培養プレートに細胞を転送する前に、1 × SFMでプレートを洗浄。 3-5時間のためにCO 2組織培養インキュベーター内で所望の濃度とインキュベーションで細胞を移す。 </li><li> 1X SFMにメディアを切り替える。 </li><li>細胞は4日間まで培養し、などの実験に必要な使用することができます。 </li></ol>パートV.代表的な結果/成果プロトコルが正しく行われている倒立顕微鏡下で70％以上のライブ心筋細胞があるはずです。 <img src="/files/ftp_upload/1308/fig1.jpg" alt="図1" /> 表1：1 L 10倍のCa 2 + -フリーKHバッファのためのソリューションレシピ： <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>質量</td><td>最終濃度</td></tr><tr><td> NaClの</td><td> 68.96グラム</td><td> 1180 mMの</td></tr><tr><td>塩化カリウム</td><td> 3.58グラム</td><td> 48 mmの</td></tr><tr><td> HEPES </td><td> 59.58グラム</td><td> 250mMの</td></tr><tr><td> K 2 HPO 4 </td><td> 2.85グラム</td><td> 12.5 mmを</td></tr><tr><td> MgSO 4を </td><td> 3.08グラム</td><td> 12.5 mmを</td></tr></tbody></table> 表2：1 Lのバッファーのためのソリューションレシピ： 100 mLの10X KHのバッファーに1.98グラムのブドウ糖を追加し、1リットルの最終容量は、グルコースと近い生理学の条件（〜300）への浸透圧を調整するためにもたらす。 NaOHでpH7.4に持参。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td>質量</td><td>最終濃度</td></tr><tr><td> 10X KHバッファー</td><td> 100mLの</td><td> 100ミリリットル</td></tr><tr><td>グルコース</td><td> 1.98グラム</td><td> 11 mmの</td></tr><tr><td> 1 Lへ移動</td><td></td><td></td></tr><tr><td> NaOHでpH7.4に持参</td><td></td><td></td></tr><tr><td colspan="3">グルコースと近い生理的条件（〜300）への浸透圧を調整します。 </td></tr></tbody></table> 表3：酵素のバッファのためのソリューションのレシピ<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>質量</td><td>最終濃度</td></tr><tr><td>コラゲナーゼタイプII </td><td> 25mgの</td><td> 0.05％（w / v）の</td></tr><tr><td>プロテアーゼXIV </td><td> 10 mgの</td><td> 0.02％（w / v）の</td></tr><tr><td> BDM </td><td> 0.025グラム</td><td> 5mMの</td></tr><tr><td>カルニチン</td><td> 0.020グラム</td><td> 2mMの</td></tr><tr><td>タウリン</td><td> 0.031グラム</td><td> 5mMの</td></tr><tr><td>グルタミン酸</td><td> 0.020グラム</td><td> 2mMの</td></tr><tr><td> 0.1 M 塩化カルシウム</td><td> 12.5 uLを</td><td> 25μMの</td></tr><tr><td>バッファ</td><td></td><td> 50mlに埋める</td></tr></tbody></table> 表4：25X BDM /タウリン/ BSA（B / T / B）のためのソリューションレシピ<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>質量</td><td>最終濃度</td></tr><tr><td> BDM </td><td> 0.076グラム</td><td> 125ミリメートル</td></tr><tr><td>タウリン</td><td> 0.094グラム</td><td> 125ミリメートル</td></tr><tr><td> BSA </td><td></td><td> 0.1％（w / v）の</td></tr><tr><td>バッファ</td><td></td><td> 6 mLのに埋める</td></tr></tbody></table> 表5：バッファBとウォッシュのバッファのためのソリューションのレシピ<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> バッファ： </td><td> B </td><td> 第1位 </td><td> ＃2 </td><td> 第3位 </td></tr><tr><td> 追加： </td><td></td><td></td><td></td><td></td></tr><tr><td> 0.5 M 塩化カルシウム</td><td> 0.4 mLの</td><td> 0.1 mLの</td><td> 0.125 mLの</td><td> 0.15 mLの</td></tr><tr><td> CaCl 2の終濃度 </td><td> 1mMの</td><td> 1mMの</td><td> 1.25 mMの</td><td> 1.5mMの</td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 mLの</td><td> 2 mLの</td><td> 2 mLの</td></tr><tr><td>最終容量にバッファ</td><td> 200 mLの</td><td> 50 mLの</td><td> 50 mLの</td><td> 50 mLの</td></tr></tbody></table> 表6：培養のメディアのためのソリューションのレシピ<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X無血清培地（SFM） </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> 最終濃度 </td></tr><tr><td> （希釈後） </td></tr><tr><td>カルニチン</td><td> 0.099グラム</td><td> 10mMの（5mM）の</td></tr><tr><td>タウリン</td><td> 0.063グラム</td><td> 10mMの（5mM）の</td></tr><tr><td>クレアチン</td><td> 0.075グラム</td><td> 10mMの（5mM）の</td></tr><tr><td>抗生物質/抗真菌剤</td><td> 0.5ミリリットル</td><td> 1％（0.5％）（V / V） </td></tr><tr><td>メディア199 </td><td> 45mlの</td><td> 50 mLに埋める</td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> 最終濃度 </td></tr><tr><td> 2X SFM </td><td> 25 mLの</td><td> 1X </td></tr><tr><td>メディア199 </td><td></td><td> 50 mLに埋める</td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X 5パーセント血清メディア </td><td></td><td></td></tr><tr><td></td><td></td><td> 最終濃度 </td></tr><tr><td> 2X SFM </td><td> 25 mLの</td><td> 1X </td></tr><tr><td> FBS </td><td> 2.5mLの</td><td> 5％（v / v）の</td></tr><tr><td>メディア199 </td><td></td><td> 50 mLに埋める</td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

重要なステップは、単離心臓を灌流システム上でハングアップされる速度です。酵素消化の期間の長さは、各ラットのために少し異なる場合があります。調整は、心臓、消化の定期的な期間の後になるとどのようにソフトに依存します。酵素消化後のCa 2徐々に回復+は、Ca 2 +トレラント健康な細胞を得るために不可欠です。 モルモットの場合、プロトコ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
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<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of adult rat heart myocytes

培養一次成人の齧歯類の心臓の細胞は、心臓血管研究のための重要なモデル系である。それにもかかわらず、堅牢な、実行可能な培養成人心筋細胞の確立は、多くの研究者のための技術的に困難な、律速段階であることができる。ここでは、数日間培養中で実行可能なまま成体ラットの心筋細胞の高い収量を得るためのプロトコルを説明した。心臓は、低Caで単離され、コラゲナーゼやプロテアーゼで灌流され<sup&gt; 2 +</sup&gt;条件は、単一の筋細胞を回復する。のCa<sup&gt; 2 +</sup&gt;耐性細胞は、細胞外Caで段階的に増加することによって得られる<sup&gt; 2 +</sup以降3回の洗浄ステップで&gt;集中。細胞を濾過し、培養培地中に再懸濁し、ラミニンコートしたガラス上でメッキされています。このプロトコルを使用して得られた培養心筋細胞では最長4日間生存可能と電気生理学、生化学、イメージングと分子生物学を含むほとんどの実験に適しています。

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

本稿では、成体ラットの心筋細胞を分離、培養に一般的な方法を説明した。コラゲナーゼとプロテアーゼは、単一の心筋細胞を消化して単離するために使用されています。心筋細胞は、ほとんどの実験の要件を満たすこのプロトコルに従って培養した。

成体ラットの心筋細胞の初代培養

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

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JoVE Journal

Biology

20.2K ビュー.  Columbia University. 本稿では、成体ラットの心筋細胞を分離、培養に一般的な方法を説明した。コラゲナーゼとプロテアーゼは、単一の心筋細胞を消化して単離するために使用されています。心筋細胞は、ほとんどの実験の要件を満たすこのプロトコルに従って培養した。

ビデオ: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

大人のウサギの網膜の器官培養

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

生物学

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

げっ歯類からプライマリ大人の線維芽細胞培養の確立

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

ミトコンドリアは密結合のラットの心臓の準備

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

成体マウスから洞房結節筋細胞の単離、培養、および機能解析のための方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

初代培養ラット副腎皮質細胞とステロイド ホルモン産生機能の試金

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...