This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

以下のプロトコルは、新生児仔ラット（10〜14匹）の1リットルから心筋細胞を分離するために設計されています。産子数が大幅に異なる場合は、手順は、補償するためにスケーリングされなければならないことがある。子犬は約48±6時間古いすべきである。すべての手順は、ここにノースカロライナ州立大学施設内動物管理および使用委員会（IACUC）によって承認されている。 1.細胞の単離の準備注：分離前の日には、次の手順を実行。 <ol><li>大はさみ、小さなはさみ、小さな鋭い鉗子、大鉗子：以下のオートクレーブ。 121℃、30分の乾燥時間を最小で60分間重力サイクルを使用する。 </li><li> O / Nを解凍する冷蔵庫で0.1％トリプシン溶液の40ミリリットルを置きます。冷蔵ハンクス平衡塩類溶液（HBSS）すでに寒さではない場合。 20％ウシ胎児血清（FBS）のストックメディア溶液を調製する。 <ol><li> Iscは400mlにオベ改変ダルベッコ培地（IMDM）は100ミリリットルFBS 5mlのL-グルタミン（200 mM）を2.5 mlのゲンタマイシン（10mgの溶液-1）、及び0.9ミリリットルの2-メルカプトエタノールを加える。真空フィルターを用いて滅菌する。最大3週間、4℃で保存メディア、これは小さくても大きくボリューム用のスケーラブルです。 10％のFBS NRCMメディアを生成するためにIMDMで1：20％のFBSの株式メディア1を希釈する。メディアだけの繰り返し加熱と冷却を防止するために、小さなバッチで作る。 </li></ol></li></ol> 2.細胞の単離1日目<ol><li>試料の汚染を防止するために、培養フード内で次の作業を行う。 O / Nインキュベーションがあるので、最高のタイミングについては、午後にこの作業を開始。 </li><li>無菌培養フードの内側にワークスペースを準備します。培養フードでベンチパッドを配置します。 70％エタノールで2 250ミリリットルのビーカーを記入し、その中に手術器具を置く。フード内氷の小さなバケツを置きます。それはいくつかのチューブと150ミリメートルディッシュを保持するのに十分な大きさであることを確認してください。 FOフードを殺菌するUVrは10分。氷上で40ミリリットルの0.1％トリプシンを置きます。 </li><li>母親から新生児ラットの子を取得します。使用するまで暖かい動物を維持するには、そのような暖かいそれらを保つためにベンチパッドに包まれたアイスバケットなどの断熱容器に配置します。 </li><li> 25ミリリットルHBSSで150ミリメートル培養皿を記入し、氷上に置きます。ワークエリア2-3ガーゼパッドと場所をアンラップ。首の後ろの皮膚によって子犬を持ち、フード内の70％エタノールと場所でそれをスプレー。注：最高の無菌性を維持するために、これはその後フードで洗浄子犬を置く別の人によって行うことができている。 </li><li>首の後ろの皮膚によって子犬を持ってください。その後、断頭により安楽死させる1迅速、強力なカットで、大規模なハサミを使用して。ガーゼで血を吸い取る。注：によりIACUC要件の差異に、むしろそれを噴霧よりワイプエタノールで子犬をきれいにするより適切である。このステップの彼らのガイドラインとして、大学動物実験委員会に確認してください。 </li><li> CHの前方側を伐採続行胸郭を通じてEST。心が表示されるまで胸郭を開く助けるために首に皮膚をつまんで。小さなハサミを使用して、氷上でHBSS培養皿に心と場所を削除します。他の子犬のために繰り返します。 </li><li>サンプルから任意の非心臓組織を削除し、心を洗って優しくHBSSを旋回。新鮮なHBSSを含む別の培養皿に心臓組織を移し、さらなる組織を洗う。ピース1-3 mm 3になるまで、さらに組織を切断した後、トリプシン溶液に移す。 4℃の冷蔵庫のO / Nでロッカー上のトリプシン溶液を置きます。 </li></ol>細胞分離2日目注：以下のステップで吸引しながら、真空ラインではなく、ピペットを使用しています。組織は簡単に移動し、真空ラインは、依然として細胞を含む組織を詰まらせたり削除します。それは、代わりに吸引用のピペットを使用するのが最適です。組織は、ピペットを入力すると、ピペットはそれを削除するにはバックアウト。 <ol><li>トンを作る10％のNRCMメディアのHREE分量：15ミリリットルと25ミリリットルと1と2。 37℃の水浴中で媒体の一つの25mlチューブを置き。 5別の15ミリリットルコニカルチューブに4ミリリットルHBSSを追加し、氷上にすべてを置く。各15ミリリットルチューブ＃1-5にラベルを付けます。 </li><li> 50ミリリットルコニカルチューブに40ミリリットルHBSSを追加します。 、コラゲナーゼにHBSSの10ミリリットルを追加します。中断するピペット、および1mg mlのコラゲナーゼ-1溶液50mlを作成するためのソリューションを組み合わせる。 0.22μmの真空フィルターを用いて滅菌し、室温で保存。 </li><li>心臓組織/トリプシンチューブを取得します。トリプシンがクリアされている、と組織がふわふわ表示されていることを確認してください。組織がチューブと吸引の隅に限り液体できるだけを決済できるようにする。 </li><li>手でチューブと渦に25ミリリットル暖かい、10％FBS NRCMメディアを追加します。キャップおよび2分間37℃の水浴中で回転させる。再び吸引する液体。組織は、この段階でフロートべきである。もしそうであれば、管の底から吸引する。 </li><li> 10ミリリットルのコラゲナーゼを追加します。 2分間または溶液が濁るまで、水浴中で回転させる。屈原ickly液体を吸引。組織を含むチューブに10ミリリットル新鮮なコラゲナーゼを追加し、2分間水浴中で回転させる。ピペット、チューブ＃1に転送ソリューションを混合し、氷上に配置する。これは、全ての液体を除去することができない場合があります。繰り返して、他の3 15ミリリットルコニカルチューブ用3.2から3.6を繰り返します。チューブ＃5に残りの液体を転送します。 </li><li>遠心機8分間410 RCFで15 mlチューブ。遠心分離しながら、水浴中で15ミリリットルの10％FBS NRCMメディア管を配置。 50ミリリットルコニカルチューブの上に40μmのセルストレーナーを配置し、2ミリリットル冷HBSSで濡れた。 </li><li>スピンダウンした細胞からの上清を吸引。各チューブを再懸濁に6ミリリットル冷HBSSを追加します。を50mlコニカルにストレーナを介してピペット細胞懸濁液は、任意の組織を除去した。 3ミリリットル冷たいHBSSで各15ミリリットルチューブの側面をすすぎ、ストレーナに追加する。 10分間410は、rcfで遠心細胞。上清を吸引。 T-75フラスコに10ミリリットルの10％FBS NRCMメディア転送で再懸濁細胞ペレット。 5ミリリットルメディアと円錐をすすぎ、FLに追加お願いします。 </li><li> 37℃で1時間および5％CO 2でインキュベート。水浴中に置き、最後の15ミリリットルの10％FBS NRCMメディア管。 T-75の新しいT-175フラスコの内容を転送、15ミリリットルのメディアでT-75をすすぎ、T-175に追加する。 1時間インキュベートする。 </li></ol> 4.凍結保存<ol><li>氷上で凍結メディアを配置します。 T-175〜50ミリリットルの円錐管から細胞培地を移し、そしてトリパンブルーを用いて細胞計数のために、細胞懸濁液10μlを集める。 5分間410 RCFで遠心分離する。遠心分離しながら、細胞カウンターを用いて細胞数を計測する。 </li><li> 1mlあたり2から4000000細胞の濃度で凍結培地中で吸引し、培養培地、および細胞を再懸濁する。極低温バイアルに1mlの細胞懸濁液を置き、次に場所は制御された冷却容器を凍結速度、及びCの冷凍庫O / N°-80の代わりバイアル。 1~2日以内に、液体窒素中にバイアルを転送する。 </li></ol> 5.解凍細胞<ol><li> 250μlの溶解することによりフィブロネクチンでコートディッシュ60; 9.75ミリリットルの水（最終濃度0.025 mgのミリリットル-1）へのフィブロネクチン（ストック濃度1mgのミリリットル-1）の。培養プレートをカバーし、少なくとも30分間、37°C ​​のでインキュベートするのに十分な溶液を加える。 <ol><li> 20％のメディアにDMSOを追加することによって、次のように4 15ミリリットルコニカルチューブでは、DMSO /メディアの混合物を作る。 500μlのDMSOで9.5ミリリットルメディア（5％）を追加します。 250μlのDMSOで（2.5％）9.75ミリリットルのメディアを追加します。 10ミリリットルメディア（これらのうちの2つを作る）を追加します。 </li></ol></li><li> 37℃の水浴中で、前述のメディア混合物を加熱する。液体窒素から細胞を取り出し、ドライアイス上に置きます。メディア混合物が温かいときにだけ解凍するまで、37℃の水浴中で細胞を解凍する。これは、細胞に損傷を与える可能性があるが長すぎるために水浴中で細胞を放置しないでください。 </li><li> 5％DMSO /メディア管に極低温バイアルの内容を転送します。 8分間410 RCFで遠心分離する。上清を吸引し、細胞ペレットを残す。細胞ペレットとrに2.5％DMSO /メディア管の内容を追加します。esuspend。 8分間410 RCFで遠心分離する。上清を吸引し、細胞ペレットを残す。 </li><li>細胞ペレットを再懸濁に1 0％DMSO /メディア管の内容を追加します。 8分間410 RCFで遠心分離する。上清を吸引し、細胞ペレットを残す。 </li><li>細胞ペレットを再懸濁する第0％DMSO /メディアの内容を追加します。細胞計数のために少量のサンプルを取る。 8分間410 RCFで遠心分離する。血球計数器で遠心分離カウント細胞がトリパンブルーを使用している間死んだ細胞を標識する。レコード細胞量、ならびに生存率。 </li><li>上清を吸引し、細胞ペレットを残す。 10％のNRCMのメディアに再懸濁（オプション：100μMのブロモデオキシウリジン（BrdU）の追加）フィブロネクチンコートディッシュ上とプレートの細胞を。 </li></ol> 6.細胞培養<ol><li> 1cm 2当たり約100Kの細胞でプレート細胞。文化の使用目的に基づいて、プレーティング密度を調整します。 1：10の割合でIMDM中の20％メディアを希釈することによって2％NRCMメディアを準備します。以下は、一般的な文化の概要である。&lt;/ LI&gt; <li> 1日目に、10％のNRCM（オプション100μMのBrdU）を追加し、暖かいIMDMで離れて死細胞をすすいでください。 2日目に、10％のNRCMを追加、暖かいIMDMで死細胞を洗い流す。 3日目 - 追加2％NRCMを。 4日目に2％NRCMを追加。 5日目に細胞がコンフルエントと鼓動であることを確認してください。約1週間、培養中の細胞を維持する。その時点で、線維芽細胞はかなりNRCMsを脱却し始める可能性があります。 </li></ol>

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The authors have nothing to disclose.

このプロトコルは、凍結保存され、単離されるべきNRCMsを可能にし、解凍した。細胞を解凍すると、手順の重要な部分である。 DMSOの希釈系列をゆっくりとセル14からDMSOを除去するために使用される。これは、細胞が、解凍後すぐに死ぬに特に敏感であるようにDMSOの抽出を迅速に行うことが重要である。多かれ少なかれ細胞は解凍のために必要とされる場合は、必要に応じて、DMSO溶?...

心筋細胞の細胞培養は、現代の心臓の研究における重要なツールである。単離および培養成体ラット心筋細胞1よりも容易であるので、新生児ラット心筋細胞（NRCMs）が一般的に使用される。 NRCM方法はまだ長い分離手順とディッシュ内の限られた細胞増殖を含む、いくつかの制限を有する。最も一般的な作業2-6の4-48時間を必要とNRCMsの単離のための多数のプロトコルがあります。さらに、細胞はしばしば2,4-7 1~2日齢のラットの仔から単離される。出産のタイミングは予測できないと研究室で他の作業と競合することができます。細胞のわずかな量は、実験に必要とされる場合に単離が非効率的で無駄にすることができる。分離時間を短縮することにワークフローのフォーカスを改善する上でほとんどの努力は、まだこれは子犬の出産のタイミングの問題を解決しない。 代替案として、多くのラボでは、利用胚性幹細胞（ESC）または人工多能性幹細胞（iPS細胞）に由来する心筋細胞。しかし、再プログラミングおよび/または分化のプロセスも非常に時間がかかり、コストがかかる可能性がある。 インビトロ筋細胞モデルとして、これらの細胞を使用した場合、他の問題が存在し得る。両方ESC-とiPSC-由来の心筋細胞を初代心筋8-10からの電気の違いを示すことが示されている。 解離NRCMs冷蔵11を用いて、数日間保存することが可能であり、まだこれは、長期保存を可能にしない。液体窒素は、典型的には、時間の大きな期間、細胞を保存するために使用されるが、ジメチルスルホキシド（DMSO）などの凍結防止剤を必要としている。これまでの研究では、凍結メディア5-10％DMSOの間の理想的な濃度はNRCMsの凍結保存を可能にすることが示されている、まだその時でさえ生存率は低い12のまま。 DMSOは、無料の間に細胞を保護するのに役立ちますが、熱意は、それは1.5％13以上の濃度で細胞に対して毒性であり得る。以前の研究は、ゆっくり細胞からDMSOを除去し、細胞生存率14を改善することができることを示している。 我々は、単離後に凍結保存細胞によるNRCM細胞ベースのアッセイの効率を改善しようとした。これは、細胞を解凍し、必要に応じて、単離および動物の消費の頻度を低減する際に使用することを可能にする。この方法を使用して、我々はそれがNRCMsを凍結保存し、後で使用するために解凍することが可能であることを示す。解凍後、細胞は、許容可能な生存能力を維持し、自然発生的にα-筋節アクチニン（α-SA）との契約について陽性であるNRCM文化を作り出す。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
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		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
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		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

新生児ラット心筋細胞の単離後、細胞を液体窒素中に凍結しており、少なくとも数ヶ月間保存することができる。典型的に解凍すると、トリパンブルー分析により決定生存率は40〜60％の間であろう。にもかかわらず、これは適切な播種し、培養して、細胞が（ 図1）を増殖する、他の細胞型より低い。収縮性を確認するために、細胞を、位相差顕微鏡を使用して画像化すること?...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Research

JoVE Journal

Developmental Biology

12.4K ビュー.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

ビデオ: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

成体ラットおよびヒト脊髄の脳室周囲領域から神経幹/前駆細胞の単離

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

発生生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

ラットヘッドBranchiomeric筋肉から衛星細胞の単離とキャラクタリゼーション

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

GATA4、MEF2C及びTBX5の最適な比率を表現ポリシストロン構築物を用いて誘導心筋細胞の改善された世代

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

新生児マウスの膵臓からの機能性膵島の効果的な分離

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

生後心筋細胞における細胞周期変化の可視化と核の決意

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

バンドウイルカ イルカ (バンドウイルカ) 精子: 異種には体外受精、凍結保存、コレクション

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

初代ラットの分離と性状バルブ間質細胞: 大動脈弁石灰化を研究するための新しいモデル

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

処理と治療の男性欧州ウナギ (アンギラ) ホルモンの成熟、精子凍結保存

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...