著者は、このwounderのプロトタイプを製造する際に氏タムポー・レオン氏ウォンチー金武とその技術的なスキルのための科学学部ワークショップの技術スタッフ、香港バプティスト大学、アドバイスに感謝したいと思います。

1. Wounderのパーツの概要（図1） <ol><li>等間隔に創傷のピンを固定することにより、ピンホルダーを準備します。ピペットチップを持ち、上下に調節可能な創傷ピンを移動させることにより、先端の高さを調整します。 </li><li> 96ウェル培養プレートの異なるブランドに調節可能なガイドバーを取り付け、創傷領域が固定位置にあることを確認してください。 </li><li>六角ネジを締めて、調整可能な創傷固定してください。両端の六角キャップを締めて位置の案内棒を固定します。 </li></ol> 2.ピンホルダーの幅を設定する（図2） <ol><li> M5六角レンチで六角キャップを緩めます。 </li><li>ガイドバーは、96ウェル培養プレートの幅に完全に収まるように、ピンホルダーの両側に調節リングの適切な数を挿入します。 注：コーニングといわきプレート大リングの1対の小さなリングの1組を必要とします。ファルコンとヌンクPLATEは、大きなリングの1ペアが必要。 </li><li>六角を締め案内バーを固定するキャップ。 </li></ol> 3.ガイディングバーを調整する（図3） <ol><li>滅菌10-μLプラスチックピペットチップで負傷ピンを取り付けます。 </li><li> M5六角レンチで六角キャップを緩めます。 </li><li> wounderを保持し、96ウェル培養プレートの1列に創傷ピンを浸します。 </li><li>ヒントのすべてがかろうじてウェルの底に触れるまで案内するバーの高さを調整します。 </li><li>六角キャップを締めます。 wounderは現在、完全に培養プレートに適合し、ピンをウェルの中央によく配置されていることをことを確認してください。 </li></ol>ピンの4キャリブレーション<ol><li>平らな無菌面に垂直なwounder（ すなわちシャーレ）を持ち、M3六角レンチですべての六角ネジを緩めます。 </li><li>ヒントのすべてが均等に平らな滅菌表面に接触するまでwounderをタップします。&lt;/李&gt; <li>位置にピンをロックするために、再び六角ネジを締めます。 </li><li>平らな無菌の表面を軽くwounderをタップすることで、各チップの均一性を確認してください。 </li></ol> 5.スクラッチ細胞単層（図4） <ol><li> 0.1％ゼラチンでコーティングした96ウェル細胞培養プレートにヒト臍帯血管内皮細胞を播種します。 20％熱不活化ウシ胎児血清、1％ペニシリン/ストレプトマイシン0.09グラム/ Lのヘパリンを添加した培地199中で培養細胞。一晩使用される前に、37ºCで5％二酸化炭素を含む加湿インキュベーター内で細胞を維持します。細胞が負傷する前に、100％の密集度に到達する必要があります。 </li><li>一番左（または右端）ウェル培養プレートの同じ列内の各側の創傷のヒントを配置します。ウェルの反対側に渡ってwounderをスライドさせます。創傷先端がウェルの底に触れていることを確認してください。 </li><li>すべての列のスクラッチ（ステップ5.2）を繰り返します。 </li><li>負傷後、トンを廃棄各ウェル中の培地彼と試験化合物を含む新鮮な培地と交換してください。 </li></ol> 6.データ収集と画像解析<ol><li>画像デジタルカメラ（ 例えば 10倍の対物レンズ）を備えた低倍率の顕微鏡を用いて細胞単層上の機械的創傷と全体だけでなく、すぐに（T = 0時間）創傷後。 </li><li>必要であれば、試験化合物を追加して、所望の時間インキュベートします。創傷領域と画像全体をよく再び希望のインキュベーション時間後（T =Δhを）。 </li><li>そのようなImageJのような画像解析ソフトウェアを（使用して<a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ）、フリーハンドツールを使用して、画像上の創傷領域を測定します。 </li><li>創傷閉鎖の割合を計算します。 <img alt="方程式" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., 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=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., 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=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

私たちの細胞wounderは、従来の細胞遊走アッセイの問題を解決するにはいくつかのユニークな特徴を持っています。 8チャンネル機械細胞wounderは、オートクレーブにより滅菌することができる長寿命で高品位のステンレス鋼（スチール304）で構成されています。市販の96ウェル培養プレートのほとんど全ての商業ブランドがあるため、調整可能なガイドバーのこの機械的な細胞wounderと共に使用することができます。調整可能なガイドバーの設計はまた、スクラッチ領域は、各ウェルの中心にあることを保証します。ウェルの中心位置における創傷は、顕微鏡を用いて撮影を容易にすることができます。異なる時点で採取しながら、画像はいくつかのバリエーションを持っているが、ウェル領域のほぼ90％は低倍率の対物レンズ（ すなわち 10倍）で顕微鏡で見ることができるので、彼らは、データ解析には影響を与えません。したがって、準ハイスループット分析は、&lt;、96ウェル培養プレート7上で実施することができますSUPクラス= &quot;外部参照&quot;&gt; 8。しかし、この方法は、依然として長いデータ分析を必要とする画像を多数必要とします。自動画像取得システムが利用可能である場合、これは解決することができます。 wounderの個々の調整可能なピンの設計は、さらに、細胞単層に、同じ滑らかで鋭い創傷を達成するために、96ウェルプレートの表面に接触可能となります。引っかき傷のピンはすべての単一のスクラッチ後に滅菌して配置することができる商業プラスチック製ピペットチップ、であり、機械的な損傷を与えることなく、ウェル表面に接触するように偉大な弾力性を提供することができます。これらの利点は、さらに実験と剥皮領域にわたって細胞運動への影響の間にサンプルのクロスコンタミネーションを最小限に抑えることができます。今後は、ピンの高さ調整工程を省略することができ、スプリング制御ピンを開発します。このデバイスを使用するための最も重要なステップは、スクラッチ時の細胞単層に加えられる力です。 wounder必要があります8ピペットチップは、ウェルの底に触れているように、培養プレートに対して垂直に保持されます。実際、wounderは、高価で複雑な装置を使用せずに細胞移動アッセイを実行するための方法を提供します。このwounderためのプロトコルは単純明快です。 結論として、私たちの8チャンネル機械wounderは、特別なスキルを必要としない細胞遊走アッセイのためのユーザーフレンドリーなデバイスです。ユーザは、迅速かつ簡単な方法9、10、11サンプルの比較的大きな数の96ウェルプレート中の細胞移動アッセイを実行することができます。

細胞遊走は、胚発生、神経発生、血管新生、創傷治癒、粘膜修復、正常な発達と病気の状況1下上皮-間葉遷移などの細胞プロセスにおいて重要な役割を果たしています。これは、シグナル伝達分子間相互作用、細胞極性、細胞骨格の再編成、マトリックスリモデリング、膜の突起と動的細胞間接着の変調2を含む多数の間および細胞内のイベントの調整を必要とする複雑なプロセスです。細胞遊走を研究することによって、細胞運動およびその関連する生化学的経路をもたらす化学物質または生体分子の発見および検証を決定することができます。この基本的なプロセスは、このような疾患の治療および薬物標的化のような種々の用途のためのプロキシとして有利に使用することができます。 創傷アッセイは、細胞遊走アッセイ3の一つです。ここでは、細胞の個々のサンプルは、部分的に、細胞が領域をカバーするように移動する露出領域を生成するために機械的手段によって除去されます。特定の時点で回収率が監視されます。多数のサンプルを取り扱う場合、そのようなサンプル内の実験的なコストと交差汚染などの問題は、問題となる可能性があります。多くの商用ツールおよびアッセイは、細胞遊走4、5、6を研究するために利用可能であるが、それらの多くは、高価で精巧な装置を必要とし、改善が依然として必要とされています。これらの理由のために、8チャンネル機械細胞wounder（ 図1）が開発されています。 我々の8チャンネル機械細胞wounderは、上記課題を解決するにいくつかのユニークな特徴を組み込んでいます。これは、96ウェル培養プレートフォーマットでの細胞遊走/創傷アッセイを実行するための柔軟性を提供します。調整可能な区idingバーのデザインは、スクラッチ領域は、各ウェルの中央の位置にあることを保証します。 wounder培養プレートの様々なブランドのために適用可能であるように、また、ガイドバーの高さを調整することができます。最後に、調整可能な創傷ピンの設計は、同時かつ再現性の創傷7、8を達成するために、培養皿の表面にピペットチップであっても接触を可能にします。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td>Other brands of 96-well cell culture plate can also be used</td>
		</tr>
		<tr>
			<td>P10 pipette tips</td>
			<td>Axygen</td>
			<td>301-03-051</td>
			<td>Short P10 pipette tip is more easy to create a clear wound</td>
		</tr>
		<tr>
			<td>Wounder</td>
			<td>R&#38;P Technology Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M2520-1L</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
	</tbody>
</table>

a device for performing cell migration/wound healing in a 96-well plate

細胞遊走は/アッセイを負傷して細胞遊走および血管新生および腫瘍転移などの他の生物学的プロセスを研究するために一般的に用いられる方法です。このアッセイでは、細胞は、コンフルエントな単層を形成するために増殖させ、機械的な傷は、デバイスとのスクラッチによって作成されます。次いで、露出領域に向かって細胞の移動速度は、イメージングによってモニターすることができます。当社の8チャンネル機械wounderは、細胞遊走アッセイに関連する問題の多くに対処するように設計されています。まず、私たちのwounderは簡単にオートクレーブ処理によって、あるいは共通の消毒剤で滅菌することができます。第二に、個別の調整可能なピンでもシャープで再現性の創傷を作成することができるように、細胞培養プレートとの接触を可能にします。第三に、wounderの両側の案内バーは、各ウェル中に一貫性のある創傷の位置を確認してください。さらにwounderのより良いハンドリングを提供できるだけでなく、クロスcontaminatを最小限に抑えるために負傷のための使い捨てのプラスチック製ピペットチップの使用イオン。結論として、我々の細胞wounderは、標準的な96ウェル培養プレートを用いて細胞遊走アッセイを行うためのユーザーフレンドリーで再現可能なデバイスと研究者に提供することができます。

この8チャンネル機械wounderは、細胞遊走アッセイを実行するために、細胞単層を傷つけるように構成されています。それは、特別な訓練なし分未満で96ウェルプレート中の細胞引っ掻きアッセイを行うことができるユーザーフレンドリーなデバイスです。このwounderは600μm程度の均一な幅とし、シャープなエッジで細胞単層上の創傷領域を導入することができる（ 図5および6）。傷の幅は、ピペットチップのブランドとユーザーのパフォーマンスに依存します。創傷幅のこの範囲は、一般的な画像の低電力倍率で捕捉する（ 図7）に適しています。画像を捕捉した後、創傷閉鎖の割合を計算することができます。この細胞wounderは、細胞遊走アッセイで直面する問題を解決するために様々な戦略が組み込まれました。また、様々な96ウェル培養PLにおける細胞遊走アッセイを実行する際に柔軟性を提供しますフォーマットを食べました。 <img alt="図1" src="/files/ftp_upload/55411/55411fig1.jpg" /> 図1：8チャンネルの機械的細胞wounder。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/55411/55411fig2.jpg" /> 図2：ピンホルダーの幅を調整します。 96ウェルプレートに適合するために、調整リングを挿入します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/55411/55411fig3.jpg" /> 図3.異なるプレートフォーマットに適合するように案内するバーを調整します。 </stronグラム&gt; 96ウェルプレートに合わせて上下のガイドバーをスライドさせます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/55411/55411fig4.jpg" /> 図4：先端の方向を掻きます。井戸底の反対側に井戸底の一方の側からwounderをスライドさせることによって細胞を傷つけます。 <img alt="図5" src="/files/ftp_upload/55411/55411fig5.jpg" /> 図5：創傷後、96ウェルプレートの概要。細胞単層は、約600ミクロンの均一な幅とシャープなエッジを生成wounderにより負傷されます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> &lt;<html> / P&gt; <img alt="図6" src="/files/ftp_upload/55411/55411fig6.jpg" /> 図6：行の代表的な画像とすぐに引っ掻き後のウェルの列。各創傷の幅の定量分析。画像は、10倍の対物レンズ下で撮影されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig6large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図7" src="/files/ftp_upload/55411/55411fig7.jpg" /> 図7：トン= 0時間およびt =Δhのでキャプチャした創傷画像（10倍対物レンズ）。点線は、創傷領域を示し、実線は、細胞遊走の領域を示しています。 A）ヒト臍帯静脈内皮細胞。 B）ヒト皮膚線維芽細胞。= &quot;_空白」を取得&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

ここでは、96ウェル細胞培養プレートに半ハイスループット細胞遊走アッセイを実行するためのプロトコルを提示します。このプロトコルは、細胞単層上で一貫性のあるスクラッチ傷を作成するには、高速、シンプルかつ経済的な方法です。

96ウェルプレートに細胞遊走/創傷治癒を行う装置

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.4K ビュー.  Hong Kong Baptist University. ここでは、96ウェル細胞培養プレートに半ハイスループット細胞遊走アッセイを実行するためのプロトコルを提示します。このプロトコルは、細胞単層上で一貫性のあるスクラッチ傷を作成するには、高速、シンプルかつ経済的な方法です。 

ビデオ: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

簡体字を使用した初期の器官形成の理解<em&gt;その場で</em&gt;ハイブリダイゼーションプロトコルで<em&gt;アフリカツメガエル</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

発生生物学

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

ロボット液体ハンドリングシステムを用いたiPS細胞培養および生産ベースのスケーラブルな96ウェルプレート

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

臨床的に関連の確立<em&gt;エクスビボ</emネイティブ微小環境における上皮創傷修復を調査するための&gt;モック白内障手術モデル

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

糖尿病性角膜症のためのアデノウイルス遺伝子治療：ヒト臓器培養角膜と​​輪部上皮細胞における創傷治癒に及ぼす影響と細胞マーカーの発現を幹

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

頭蓋冠欠損治癒モデルで使用するためのBMPR-IB +脂肪由来間質細胞の迅速な単離

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

マウス膵臓前駆細胞の in Vitroからスキャフォールド フリー、三次元のインスリンを表現する Pancreatoids の生成

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...