Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

マウスを安楽死（シンガポール国立大学の施設内動物管理使用委員会（IACUC）のガイドラインおよび実験動物研究のための国家諮問委員会（NACLAR）ガイドライン）まで機関のガイドラインに準拠して無菌状態で飼育しました。 1.マウス<ol><li>適切な遺伝的背景の一つにマウスを注文。このプロトコルは、1つのC57BL / 6マウス由来の組織に基づいています。 </li></ol>完全培地の調製<ol><li> 10％ウシ胎児血清（FCS）、50μM2-メルカプトエタノール、100μMのアスパラギン、2mMグルタミン、1％ペニシリン - ストレプトマイシン溶液：ロズウェルパーク記念研究所（RPMI）1640培地に、以下の成分を添加することによって、完全培地を調製します。 </li></ol>酵素液の調製<ol><li> 15ミリリットルコニカルチューブ中のコラゲナーゼD溶液を調製します。 <ol><li>コラゲナーゼD・ディ10mgを秤量4ミリリットルでssolveコラゲナーゼD完全培地。 </li></ol></li><li> 1.7ミリリットルのマイクロチューブにプロナーゼ溶液を調製します。 <ol><li>プロナーゼ10mgを秤量します。 </li><li> 1Mトリス緩衝液（pH8.0）の5μLを追加します。 0.5 M EDTA（pH8.0）を1μLを加えます。 </li><li>滅菌水494μlのアップトップ。 </li><li> 30分間、水浴中で37℃でのプロナーゼ溶液をインキュベートします。 </li></ol></li></ol>コラゲナーゼD-プロナーゼミックスの4準備（≤2尾） 注：無菌細胞培養フード内の後続の手順を実行します。 <ol><li>コラゲナーゼDの溶液4mlにプロナーゼ溶液の250μlのを追加します。 </li><li>滅菌15ミリリットルコニカルチューブに0.2μmのシリンジフィルターを通してコラゲナーゼD-プロナーゼ混合物を渡します。 </li></ol>耳としっぽ組織からの線維芽細胞の5抽出<ol><li>適切な機関のガイドラインに従ってマウスを安楽死させます。</li><li>細胞培養フード内でオートクレーブ処理手術器具（ハサミやピンセット）を配置します。 </li><li> 2つの10センチ細胞培養皿それぞれに10ミリリットルを完全培地を追加します。 </li><li>耳（〜1cmの半径）とはさみでマウスの（尾の先端から）尾の5センチカットし、滅菌50mlコニカル底チューブに40ミリリットルの70％エタノールで5分間インキュベートします。 </li><li>空気乾燥耳と尻尾5分間フード内で開いた10cmの細胞培養皿に置くこともできます。ステップ5.3で説明したように10ミリリットルを完全培地を含む「耳」と「テール」と表示された2つの培養皿に乾燥後、転送耳と尾の部分。 </li><li>はさみを使用して、耳と尻尾から髪を削除します。 </li><li>はさみを使用して、サイ​​ズが3ミリメートル未満の片に耳と尻尾を切ります。 </li><li> 「耳」と「テール」と表示された1.8ミリリットルクライオチューブバイアルにカット組織を移し、バイアルに1.8ミリリットルのマークに到達するために、ボリュームのために十分なコラゲナーゼD-プロナーゼ溶液を添加。 </li><li> Pシェーカー上で水平にクライオチューブバイアルレース、37℃で90分間、200rpmでサンプルを振ります。 </li><li> 90分間のインキュベーション後、シェーカーからクライオチューブバイアルを削除し、フード内でバイアルを配置します。 </li><li> 2つの10センチ細胞培養皿、標識された「耳」と「テール」それぞれに10ミリリットルを完全培地を追加し、各皿に70μmのセルストレーナーを置きます。 </li><li>ステップ5.11で製造さに応じてラベル皿に70μmのセルストレーナーで消化耳と尾の組織を置き、強制的に&gt; 5分間10mLのシリンジプランジャーを使用して組織を挽きます。セルストレーナーの外に細胞を洗浄するために培地中で時折セルストレーナーを振ります。 </li><li> 「耳」と「テール」というラベルの付いた2つの15ミリリットルコニカルチューブに各皿から細胞懸濁液をピペット。さらに10 mlの一品とストレーナーを洗浄し、完全培地および適切な15ミリリットルコニカルチューブに培地を追加します。 </li><li>細胞をスピンダウン〜580 XGと冷蔵細胞遠心機を用いて4℃で7分間のサスペンション。 </li><li>上清を除去し、15ミリリットルコニカルチューブ内の細胞ペレットに完全培地10ミリリットルを加えて細胞を懸濁します。 </li><li>繰り返しステップ5.14と5.15。 </li></ol>細胞混合物の6培養<ol><li>上清を取り除きます。細胞ペレットを邪魔されずに残っていることを確認してください。 </li><li> 10ml中の細胞完全培地を再懸濁し、「耳」と「テール」というラベルの付いた2つの10センチメートル細胞培養皿に、それぞれの混合物を追加します。 </li><li>培養液に10μlのアムホテリシンB溶液（250 / mlのストック溶液）を追加します。 </li><li>加湿した5％CO 2インキュベーター中37℃で細胞をインキュベートします。 </li><li>三日目に、10 mlの破片（ 図1および2）を除去するために、アムホテリシンBの10μLを含む新鮮な完全培地を培地を交換してください。 </li></ol>線維芽細胞の7サブカルチャー<ol><li> WHEn個の培養物は、約70％コンフルエンシー（日培養の3-4）培地を除去し、5 mlの滅菌1×リン酸緩衝生理食塩水（PBS）で細胞を洗浄するに至ります。 </li><li> PBSを削除し、セルに2ミリリットルの滅菌1×トリプシン-EDTA溶液を加えます。 </li><li>加湿した5％CO 2インキュベーター中、37℃で5分間、細胞をインキュベートします。 </li><li> 5分後、静かに培養皿をタップし、細胞に6ミリリットルを完全培地を追加します。 </li><li> 15 mlのコニカルチューブに細胞懸濁液を移し、冷蔵細胞遠心機を使用して〜450×gで、4℃で5分間チューブをスピン。 </li><li>上清を除去し、静かに5ミリリットルの完全培地中で細胞ペレットを再懸濁。 </li><li>シード2×10 10cmの細胞培養皿で5個の細胞と、手順7.6から7.1を繰り返す前に3~4日、37℃、加湿5％CO 2インキュベーター中で細胞を培養します。 </li></ol>出荷のための耳の8準備注意：Tを実行彼無菌細胞培養フード中でその後の工程。 <ol><li>適切な機関のガイドラインに従ってマウスを安楽死させます。 </li><li>細胞培養フードにオートクレーブ処理ハサミやピンセットを配置します。 </li><li> 50ミリリットルコニカルチューブに50ミリリットルを完全培地を追加します。 </li><li>はさみでマウスの耳（〜1cmの半径）をカットします。 </li><li>ピンセットを用いて（ステップ3で調製した）を50mlのコニカルチューブに耳を転送します。 </li><li>適切なボックスにRTで出荷する前にパラフィルムで50mlコニカルチューブを密封。 </li></ol>

<ol>
	<li>Fitzpatrick, L. E., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+matrices+for+tissue+engineering+and+regenerative+medicine+applications.">Cell-derived matrices for tissue engineering and regenerative medicine applications.</a> Biomater Sci. 3 (1), 12-24 (2015).</li><li>Elenbaas, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+breast+cancer+cells+generated+by+oncogenic+transformation+of+primary+epithelial+cells.">Human breast cancer cells generated by oncogenic transformation of primary epithelial cells.</a> Genes Dev. 15 (1), 50-65 (2001).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> J Neuroinflammation. 9 (1), 115 (2012).</li><li>Lim, J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+transfection+of+HUVEC+and+MEF+cells+using+DNA+complexes+with+magnetic+nanoparticles+in+an+oscillating+field.">Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field.</a> J Genet. 91 (2), 223-227 (2012).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21 (11), 1527-1543 (2010).</li><li>Patel, M., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+reprogramming+somatic+cells+to+induced+pluripotent+stem+cells.">Advances in reprogramming somatic cells to induced pluripotent stem cells.</a> Stem Cell Rev. 6 (3), 367-380 (2010).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> J Invest Dermatol. 127 (5), 998-1008 (2007).</li><li>Shen, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-derived+cytosolic+DNA+mediates+type+I+interferon-dependent+rejection+of+B+cell+lymphoma+cells.">Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.</a> Cell Rep. 11 (3), 460-473 (2015).</li><li>Seluanov, A., Vaidya, A., Gorbunova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> J Vis Exp. (44), (2010).</li><li>Baglole, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+phenotypic+characterization+of+lung+fibroblasts.">Isolation and phenotypic characterization of lung fibroblasts.</a> Methods Mol Med. 117, 115-127 (2005).</li><li>Alt, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+share+mesenchymal+phenotypes+with+stem+cells,+but+lack+their+differentiation+and+colony-forming+potential.">Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential.</a> Biol Cell. 103 (4), 197-208 (2011).</li><li>Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essence+of+senescence.">The essence of senescence.</a> Genes Dev. 24 (22), 2463-2479 (2010).</li><li>Lander, M. R., Moll, B., Rowe, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+culture+of+cells+from+mouse+tail+biopsies:+brief+communication.">A procedure for culture of cells from mouse tail biopsies: brief communication.</a> J Natl Cancer Inst. 60 (2), 477-478 (1978).</li><li>Moore, C. B., Allen, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ear+fibroblast+derivation+from+mice.">Primary ear fibroblast derivation from mice.</a> Methods Mol Biol. (1031), 65-70 (2013).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+pluripotent+stem+cells+from+NOD+mouse+tail-tip+fibroblasts.">Generation of stable pluripotent stem cells from NOD mouse tail-tip fibroblasts.</a> Diabetes. 60 (5), 1393-1398 (2011).</li></ol>

The authors declare no conflict of financial interests.

ここでは、マウスの耳と尾からの一次線維芽細胞培養物を確立するために、単純な、安価で高速な実験手順を提供します。抽出は、組織の3日後に分離以内に付着し、急速に分裂する線維芽細胞を生じるはずです。初代細胞の重要な制限は、老化、永久的な増殖停止15です。プロトコルを使用して、線維芽細胞培養物は、サイズの増加（2〜3倍の増加）と拡大に失敗、線維芽細胞が老化...

初代細胞は 、in vitro条件下で組織培養を生きから誘導されます。それは一般的に一次細胞は、より密接に生理学的状態、それらが不死化より由来する組織または腫瘍細胞株1の遺伝的背景に似ていることが想定されます。そのため、一次細胞は、生物学的な質問の2,3を研究するための有用なモデルを表します。しかし、無制限に成長する樹立細胞株とは異なり、一次細胞培養物は、最終的に老化を受け、頻繁に再確立される必要があります。 一般的に使用される一次細胞は、線維芽細胞、上皮細胞、内皮細胞、T細胞、B細胞、骨髄由来マクロファージ（BMDM）及び骨髄由来樹状細胞（BMDC）を含みます。線維芽細胞は、多くの場合、一次細胞培養モデルとして利用されます。彼らは、他の一次細胞を超える重要な利点を提供します。細胞培養物は、簡単に容易に維持し、設立され、何のpurificaを必要とされていません培養前の細胞の化。彼らは、迅速な初期の増殖および特殊な媒体や活性化のプロトコルのための要件が​​あります。線維芽細胞は、効率的に4,5、生物学的、化学的、および物理的なプロトコルを使用してトランスフェクトすることができます。細胞培養を確立する前に、室温で最大10日間の耳を格納する可能性があります。線維芽細胞培養物は、細胞質プロセスの可視化に資すると人工多能性幹（iPS）細胞6に再プログラミングするのに適しています。 線維芽細胞は、コラーゲンタンパク質及び細胞外マトリックス7を分泌する結合組織の重要な細胞です。彼らは多くの組織8の構造フレームワークを提供し、創傷治癒および組織修復9,10において重要な役割を果たしています。 ここでは、マウス11の耳と尾から線維芽細胞培養物を確立するための簡単かつ迅速（&lt;4時間）プロトコルを記述します。プロトコルは、最小限のマウスが必要です経験は、（他のプロトコル12,13とは対照的に）組織を採取し、最大10日間室温で媒体に記憶された耳からの培養物を確立するために使用することができます。

<table><tbody><tr><td>RPMI-1640</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Asparagine</td><td>Sigma-Aldrich</td><td>A4159</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Ethanol</td><td>Merck Millipore</td><td>107017</td><td>Absolute for analysis</td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics</td><td>11088866001</td><td>From &lt;em&gt;Clostridium histolyticum&lt;/em&gt;, lyophilized, non-sterile</td></tr><tr><td>Pronase protease</td><td>Merck Millipore</td><td>53702</td><td>From &lt;em&gt;Streptomyces griseus&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Tris buffer (pH 8)</td><td>1st BASE</td><td>1415</td><td>Ultra pure grade</td></tr><tr><td>0.5M EDTA (pH 8)</td><td>1st BASE</td><td>BUF-1053</td><td>Biotechnology grade</td></tr><tr><td>10X Phosphate Buffered Saline (PBS)</td><td>1st BASE</td><td>BUF-2040-10X4L</td><td>Ultra pure grade</td></tr><tr><td>Trypsin-EDTA solution 10X</td><td>Sigma-Aldrich</td><td>59418C-100ML</td><td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2492-20ml</td><td>250 &amp;mu;g/ml in deionized water, sterile-filtered</td></tr><tr><td>Scissors</td><td>Aesculap</td><td></td><td></td></tr><tr><td>Forcep</td><td>Aesculap</td><td>AE-BD312R</td><td></td></tr><tr><td>0.2 &amp;mu;M syringe filter</td><td>Sartorius Stedim</td><td>16534</td><td></td></tr><tr><td>70 &amp;mu;M cell strainer</td><td>SPL</td><td>93070</td><td></td></tr><tr><td>Syringe plunger</td><td>Terumo</td><td>SS+10L</td><td></td></tr><tr><td>Cryovial tube</td><td>NUNC</td><td>368362</td><td></td></tr><tr><td>1.7 ml microcentrifuge tube</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Greiner</td><td>664160</td><td>Cell culture treated dish&amp;nbsp;</td></tr><tr><td>15 ml conical bottom tube</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>50 ml conical bottom tube</td><td>Greiner</td><td>227261</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1002</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Incubation shaker</td><td>Sartorius Stedim</td><td></td><td>Certomat-BS1</td></tr><tr><td>Zeiss Axiovert 25 light microscope</td><td>Carl Zeiss AG</td><td></td><td></td></tr></tbody></table>

generating primary fibroblast cultures from mouse ear and tail tissues

初代細胞は、組織から直接誘導され、確立された細胞株よりも、インビボでの細胞の生理学的状態をより代表すると考えられています。しかし、初代細胞培養は通常、有限の寿命を有しており、頻繁に再確立される必要があります。線維芽細胞は、初代​​細胞の容易にアクセス可能な供給源です。ここでは、マウスの耳と尾からの一次線維芽細胞培養物を確立するための簡単​​かつ迅速な実験手順を説明します。プロトコルは、最大10日間室温で保存された耳からの一次線維芽細胞培養物を確立するために使用することができます。プロトコルを注意深く続く場合、汚染は、場合によっては長時間保存された非滅菌組織を使用しても発生しにくいです。線維芽細胞は、培養液中で急速に増殖し、複製老化を受ける前に、かなりの数に拡張することができます。

組織破片のかなりの量の組織の結果からの線維芽細胞の抽出（ 図1）。組織片とは対照的に、線維芽細胞を1日培養の3との間の組織培養プラスチック表面に付着します。線維芽細胞培養物の培地は、安全に大幅文化（ 図2）に存在するデブリのレベルを減少させるべきである文化、の3日目に変更することができます。線維芽細胞は、細長い形態とはっきりと見える細?...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

我々は、マウスの耳と尾から初代線維芽細胞を生成するための簡単​​かつ迅速に実験手順を説明します。手順は、特別の動物の訓練を必要とせず、最大10日間室温で保存された耳からの線維芽細胞培養物の生成のために使用することができます。

マウスの耳としっぽの組織から生成初代線維芽細胞培養

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Research

JoVE Journal

Developmental Biology

33.7K ビュー.  Yong Loo Lin School of Medicine, National University of Singapore. 我々は、マウスの耳と尾から初代線維芽細胞を生成するための簡単​​かつ迅速に実験手順を説明します。手順は、特別の動物の訓練を必要とせず、最大10日間室温で保存された耳からの線維芽細胞培養物の生成のために使用することができます。 

ビデオ: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

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 ...

発生生物学

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

原発性マウスメラノサイトと線維芽細胞培養の急速な生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

がん研究

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

生物工学

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

リネージュの赤芽球系前駆細胞に成体マウス線維芽細胞のリプログラミングを直接します。

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

脱細胞肺足場を使用してESC由来のマウス気道上皮細胞の生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions 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 ...

生物学

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超音波増強原発性成人線維芽細胞分離

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...