僧帽弁07CVD04：本研究では、ハートウェル財団、米国心臓協会（サイエンティスト開発グラント＃0830384N）と財団Leducqによってサポートされています。

1。準備<ol><li>湿度60％で（約2日間）または25（約4.5日）と37℃ -肥沃なウズラやニワトリの卵ステージ14までをインキュベート</li><li>無菌アールの平衡塩溶液を準備します（EBSS） <ol><li> 600mLの18MΩ水を100 mLの10X EBSSを追加</li><li> 2.2グラムの重炭酸ナトリウムを追加</li><li> 7.2溶液のpHを調整する</li><li> 1000mLとするソリューションを育てる</li><li> 0.2μmのフィルターを通過してソリューションを滅菌</li></ol></li><li>無菌M199培地を準備します。 <ol><li> 700mlの18MΩ水にM199粉の1パッケージを追加します。 </li><li> 2.2グラムの重炭酸ナトリウムを追加</li><li> 7.2〜溶液のpHを調整します。 </li><li> 10mLの（1％）ペニシリン - ストレプトマイシンを追加</li><li> 1000mLとするソリューションを育てる</li><li> 0.2μmのフィルターを通過してソリューションを滅菌</li><li> 1 mLの滅菌100Xインスリン - トランスフェリン - セレン- Gのサプリメントを追加</li><li> 10mLの（1％）無菌ニワトリ血清を追加</li></ol></li><li>は1.5 mg / mLのコラーゲン濃度での三次元コラーゲンゲルを準備します。私たちの研究室の前の仕事は、以上1.5 mg / mLのコラーゲン濃度は、細胞が内を移動するためにはあまりにも生体力学的に硬いですマトリックスを提供することが示されている。コラーゲンゲルの濃度が低いよりも1.5 mg / mLのは、細胞集塊が細胞の島を生産する、局所的に繊維を細断処理できるようにいくつかのコラーゲン繊維を持っている。 <ol><li>順番に滅菌15 ml遠心チューブに以下の氷冷試薬を追加します。 <ul><li> 3X M199：=合計容積必要/ 3巻</li><li>滅菌18MΩ水：ボリュームに必要=総容積（M199音量+ニワトリ血清ボリューム+ 0.1MのNaOHの体積+コラーゲンの量） </li><li>無菌ニワトリ血清：* 0.01必要な量=総体積</li><li>ラット尾コラーゲンI：ボリューム=（コラーゲンゲルの濃度*合計容積必要）/コラーゲン原液濃度</li><li>滅菌0.1MのNaOH：ボリューム=コラーゲンの量* 0.2 注：水の量は、0.1 M NaOHおよびコラーゲンは、コラーゲン原液の濃度によって異なりますが追加。 </li></ul></li><li>滅菌ピペットでよくコラーゲン溶液を混ぜる。 </li><li>すぐに1.9センチメートル2成長領域のウェルにコラーゲン溶液0.3 mLを転送する。これは完全に井戸をカバーし、約3mmの厚さのゲルを提供するために十分なコラーゲン溶液です。少なくとも250μmのゲルの厚さは細胞浸潤の研究に必要です。 </li><li> 37℃で少なくとも1時間ゲルのコラーゲン溶液を許可° C、5％CO 2、および100％の湿度。 </li></ol></li></ol> 2。プレEMT心内膜の細胞分離と文化<ol><li>胚の分離<ol><li>卵の大きい方の端を上の実験室でのテープの1インチ長いピースを置きます。空気のセルが配置されているところです。テープは壊れやすいウズラの卵の殻を安定させます。 </li><li>ゆっくりと穿刺滅菌湾曲したハサミの先端で空気のセル内の卵のテープ面積。 </li><li>卵黄の通過を許可するのに十分な大きさの卵のテープ部分に穴を開けます。パンクの穴を起動し、らせん状に外側にカット。外側の膜をカットしますが、卵黄にピアスを避けるようにしてください。 </li><li>シェルの穴が完了すると、ウズラ胚を可視化する。胚は、通常、卵黄の上面に位置しています。 </li><li>片手で卵の穴の傷口から卵黄を注ぎ始める。一方で第55ピンセットを保持する。卵黄の上面上に胚が出ると卵のように、そっと胚で第55ピンセットをスライドさせて卵黄から取り外します。注：鶏の卵は、胚の分離は、シャープ、きれいな表面に鶏の卵をクラッキングおよび100mmペトリ皿に卵を壊す伴います。胚は卵黄の上面側に配置する必要があります。ゆっくりと胚の下第55ピンセットをスライドさせて卵黄から取り外します。 </li><li>氷冷、滅菌EBSSを含む100mMシャーレに胚を置きます。 </li></ol></li><li>ハンバーガーとハミルトン1の基準に従って胚をステージングします。ステージ14での胚-過去のステージ13ですが、まだステージ15で、20から21体節と尾芽を持つ。ステージ14の主要な特徴-胚は、体に90 °よりも若干大きいヘッドの角度、です。 </li><li>ステージ14でのウズラ胚から心を取り除く-第55ピンセットでAV運河とOFTが胸壁に接触する場所横方向にカットして。ペトリ皿の片側に一緒に心を置きます。 </li><li>無菌トランスファーピペットで分離された心臓を収集し、滅菌、氷冷EBSSで満たされた新しい100ミリメートルペトリ皿に入れます。 </li><html><li> ＃55ピンセットで心室から横方向にOFTsとAV運河をカット。 </li><li>セクションでは、残りのAV運河及び/または縦方向に流出地帯（OFTs）。 </li><li> 20μlの体積にピペットを設定します。 AV運河とOFTsを縦方向にカット6つの吸引には、このピペットを使用してください。 </li><li>コラーゲンゲル上にAV運河とOFTsを追放する。ゲルの表面から余分なEBSSを吸引除去する。 </li><li>彼らはフラットになるように向きを外植片に第55ピンセットを使用し、その内腔の側には、コラーゲンゲルに直面している。ピンセットで穿刺コラーゲンゲルをしないようにしてください。 </li><li> ℃、5％CO 2 37 2時間後、第55ピンセットで植から心筋を削除します。心内膜細胞は、コラーゲンゲルの表面に置き去りにされるべきである。また、心筋は、外植片にしておくことができます。心筋が削除されたため、と心内膜細胞が介入しなくてもEMTを受けない。心筋が存在すると心内膜細胞のサブセットは、間充織に変換しますが、変形の度合いは、外部要因で変調することができます。注：培地が追加される前に、心筋の上に残される場合でも、外植片は、少なくとも2時間は、ゲルへの接続を許可する必要があります。 </li><li>各ウェルを含む植片を0.4 mLのM199培地を追加。 </li><li>文化37細胞° C、5％CO 2、および100％の湿度。 </li></ol> 3。ポストEMT AVクッション間葉細胞の単離と文化<ol><li>シャープ、清浄表面上に鶏の卵を割るおよび100mmペトリ皿に卵を破る。胚は卵黄の上面側に配置する必要があります。胚を包む膜をつかんで＃5ピンセットで胚をピックアップし、氷冷、滅菌EBSSで満たされた100ミリメートルシャーレに胚を置く。 </li><li>ハンバーガーとハミルトン1の基準に従って胚をステージングします。ステージ25胚の特徴は、別個の肘と膝関節とわずかな眼の色素が含まれています。 </li><li>すべてのHH25胚から心臓を隔離し、ペトリ皿の片側に一緒に心を置く。 </li><li>無菌トランスファーピペットで分離された心臓を収集し、滅菌、氷冷EBSSで満たされた新しい100ミリメートルペトリ皿に入れます。 </li><li>一度にすべての心からAVの領域を分離する。ペトリ皿の片側に一緒にAVを置きます。ゆっくりと細胞の生存に影響を与えることができるすべての血液を、削除するためにAVの領域に撹拌。 </li><li>無菌トランスファーピペットで分離された視聴覚資料を収集し、滅菌、氷冷EBSSで満たされた新しい100ミリメートルペトリ皿に入れます。 </li><li>順次AVからクッションを分離する。 AVクッションには心筋の存在がないことを確認してください。 </li><li>無菌トランスファーピペットで分離されたクッションを収集し、滅菌15 ml遠心チューブに入れます。 </li><li> 3分15 × gで遠心分離管の底にクッションを回転させる。 </li><li>クッションを乱すことなくできるだけEBSS上清をできるだけ多く除去する滅菌1000mLのチップを使用してください。 </li><li> 37事前に暖められている0.25％トリプシン- EDTAを1 mL℃を追加それらが完全に溶解するまでクッションがトリプシンで培養することを許可する、これは3-5分程度、通常です。 </li><li>トリプシンをクエンチするために遠心分離管に鶏の血清100μLを加える。 </li><li> 5分間で160 × gでペレットは細胞。滅菌、1000μLチップで上清を取り除きます。 </li><li> M199培地とピペッティングにより混和する2mLに細胞を再懸濁します。血球計でカウントし、単離されているセルの合計数を決定するために細胞懸濁液の10μLを取る</li><li>コラーゲンゲルを単離した細胞の数に基づいて行われる数を決定します。細胞は250μLゲル体積の2 × 10 5細胞/ mLの密度で播種する必要があります。 </li><li> 5分間で160 × gでペレットは細胞。滅菌、1000μLチップで上清を取り除きます。 </li><li>上記のプロトコルを使用して、細胞ペレットに、そのためには3X M199、水、鶏血清、コラーゲン、および0.1MのNaOHを、追加してください。穏やかなピペッティングにより混和する。 </li><li>よく各1.9センチメートル2成長領域に250μLを追加。 </li><li> 37℃で少なくとも1時間ゲルのコラーゲン溶液を許可° C、5％CO 2、および100％の湿度。 </li><li>培地0.4 mLを加え、一晩インキュベートする。 </li><li>ストレスのない条件下で培養ゲルを続行するには、よく滅菌、200μLピペットチップを使用して文化の側面からゲルを解放する。機械的に制約の文化はよく文化の側面に取り付けられたままにゲルである。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/2159/2159fig1.jpg" alt="図1" /> 図1：胚の例 （A）HH14 -ウズラの胚、及び（B）HH25ニワトリ胚がここに示されている。 fig2.jpg&quot;ALT =&quot;図2&quot;/&gt; 図2：HH14 -弁心内膜心筋の存在と文化の2時間後に植 （）、または（B）心筋では削除されます。 <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="図3" /> 図3：HH14 -弁心内膜片 （）現在の心筋では、細胞の多くは、間葉転換に心内膜受けると文化の48時間後の紡錘状の、間葉系の表現型を持っている。 （B）心筋細胞のすべてを削除すると文化の中で48時間後の石畳の形をした、心内膜表現型を維持する。 <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="図4"/> 図4：。HH25バルブ間葉系細胞をコラーゲンゲル内に細胞を播種直後に丸められます。 <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="図5"/> 図5：。文化のHH25間葉系細胞は （A）制約コラーゲンゲル内培養の48時間後HH25間葉系細胞が普及し始めている。 （B）7日の解放後に播種し、6日後のストレスのないコラーゲンゲルでのHH25細胞は、元の面積の約50％にゲルを圧縮している。

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

利害の衝突は宣言されません。

ステージHH14から心内膜細胞を単離する方法-もともとラニアンとMarkwaldによって開発された心臓は、胚性EMT 2を開始し、変調の要因を研究するためのin vitro環境で 、制御を提供します。 HH14 -心筋なしで培養した心内膜片は、生体力学的または生化学的な介入なしにEMTを起こさない。心筋が植片上に残っている場合、それは間葉転換を受けるために心内膜細...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
<tr>
<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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isolation and culture of avian embryonic valvular progenitor cells

胚の心臓弁の適切な形成と機能は、発達の進行に重要です。初期胚の心臓は心筋に囲まれた心内膜のU字型チューブです。心筋分泌心臓ゼリー、ヒアルロン酸が豊富なゼラチン状のマトリックス、房室（AV）ジャンクショ​​ンと流出路（OFT）腔へ。段階でHH14 valvulogenesisは心内膜細胞のサブセットは、間葉転換（EMT）への心内膜心筋からの信号を受ける受信、および心臓のゼリーに侵入するところから始まります。段階HH25で弁膜クッションは完全にmesenchymalized、そしてそれは最終的に心臓の弁膜症と中隔装置を形成するこの間充織であるされています。 EMTと細胞分化のプロセスを開始し、調節するメカニズムを理解するために重篤な先天性心疾患への接続が重要です。本研究では、prevalvularクッションの2つの異なる細胞表現型であるプレEMT心内膜とポストEMT間葉系細胞を単離する方法を提示する。プレEMT心内膜細胞が心筋の有無に関係なく培養することができる。ポストEMT AVクッション間葉系細胞は内部に機械的制約やストレスのないコラーゲンゲルを培養することができる。 in vitroモデルこれらの3Dは、主要な弁の形態形成のイベントを模倣し、初期および後期のvalvulogenesisのメカニズムを解体するのに便利です。

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

この記事では、単離するための方法および培養ウズラやニワトリHH14を提供します<sup&gt; -</sup&gt;弁心内膜細胞とHH25バルブクッション間葉系細胞。

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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10.1K ビュー.  Cornell University. この記事では、単離するための方法および培養ウズラやニワトリHH14を提供します -弁心内膜細胞とHH25バルブクッション間葉系細胞。

ビデオ: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

ポストナタールマウス小脳顆粒ニューロン前駆細胞と神経細胞の単離と培養

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

生物学

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

弁内皮細胞の単離

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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 ...

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 ...