Name: a proximal culture method to study paracrine signaling between cells
Uploaded: 2018-08-28T14:30:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

これらの実験の組織コレクションに参加するため患者さんにお世話になっております。国防総省 OCRP 卵巣癌アカデミー賞 (W81XWH-15-0253) および Anirban k. ミトラにコリーンの夢財団からパイロット賞は、この研究をサポートしました。

プロトコルは、インディアナ大学の制度的規制委員会のガイドラインに従います。
1. セル準備
<ol><li>
ひと中皮細胞の分離と培養
	<ol>
<li>分離ひと中皮細胞 (HPMCs)17,18を前述とのそれらを育てる人間の大網から完了成長培地 [ダルベッコ変更されたワシの媒体 (DMEM) 10% 牛胎児血清 1% を含むペニシリン-ストレプトマイシン, 1% 非本質的なアミノ酸、および 1% ビタミン] 37 ° C および 5% の CO2。 注: HPMCs は、通常 100% 合流点 (図 1 a) を栽培しています。</li>
	</ol>
</li>
	<li>
卵巣癌の細胞の成長条件
	<ol>
<li>37 ° C、5% CO2で完全培地に HeyA8 卵巣癌細胞を成長します。 注: 使用 HeyA8 セル成長する約 80% 合流 (図 1 b)。</li>
	</ol>
</li>
</ol>2. 細胞培養
<ol><li>
近位文化セットアップ
	<ol>
<li>使用 Transwell 0.4 μ m 孔 (108毛穴/cm2) と 4.67 cm2成長分野 10 μ m 厚い組織文化扱われるポリカーボネート膜 6 ウェル プレートを挿入します。必要な場合は、成長の表面積によると播種された細胞の数を拡大することで異なる挿入サイズの最適化します。温かみのある完全な成長媒体、トリプシン、およびリン酸緩衝生理食塩水 (PBS) 使用前に 37 ° c。</li>
	</ol>
</li>
	<li>
挿入の準備
	<ol>
<li>挿入の膜の下の表面の HeyA8 細胞をシードするには、滅菌ピンセットを使用してパッケージからドライブベイ カバーを取り外し、滅菌 15 cm 培養皿で反転配置。</li>
		<li>反転挿入に対応する適切な滅菌コンテナーとそれの代わりに深さがあり、15 cm 皿を使用します。実験によって 15 cm 皿に挿入の必要な数を配置します。</li>
		<li>3 つのコントロールと 3 つの実験条件をそれに応じてラベル挿入の端にマーカーで。</li>
	</ol>
</li>
	<li>
がん細胞を準備
	<ol>
<li>25 cm2フラスコ (10 の6セル × 2 ~) 80% 合流で育った HeyA8 細胞を trypsinize 40-60 s のトリプシン (0.25%) の 1 つの mL を追加し、6 ml の完全培のトリプシンを中和します。</li>
		<li>室温 (RT) 3 分間で 500 x gで細胞を遠心分離機、上澄みを廃棄し、5 ml の完全培細胞ペレットを再懸濁します。トリパン ブルー色素排除法を使用して生きているセルをカウントします。</li>
	</ol>
</li>
	<li>
癌細胞の播種
	<ol>
<li>100,000 セル/mL の完全培地に生きている HeyA8 のセルを中断します。慎重に種 80,000 HeyA8 細胞懸 (これは今直面している、それが反転するので) 挿入の下に完全な成長媒体の 800 μ L。</li>
		<li>ドームのような形を形成するので、セルの挿入 (図 1) のままに細胞を播きます。膜の中心から開始し、ゆっくりとピペッティングしながら同心円の外側に移動します。あらゆる媒体の流出を防ぐためにエッジから 3 mm 以上を行くことを避けてください。 注: セルの成長率と実験の期間に播種された細胞の数によって異なります。この近位文化のシード HeyA8 細胞数を最適化した、セルは、3 日目の終わりに 80-90% の合流になります。</li>
	</ol>
</li>
	<li>
癌細胞の接着
	<ol>
<li>カバー 15 cm 皿、CO2インキュベーターへの挿入を含む料理を慎重に移動します。培地およびこの手順で挿入のセルのドロップを破壊することが重要です。挿入にアタッチする癌細胞を許可する 4 h の 37 ° C で細胞を残します。</li>
	</ol>
</li>
	<li>
中皮細胞の準備
	<ol>
<li>約 4 時間後 100 %2 ml のトリプシン (0.25%) に 1-2 分の 75 cm2フラスコ (10 の6セル × 4 〜) で合流で成長して HPMCs を trypsinize、12 ml の完全培のトリプシンを中和します。</li>
		<li>500 x gで RT 3 分で細胞を遠心分離機、10 mL の完全培に細胞ペレットを再懸濁します、トリパン ブルー色素排除法を使用して生きているセルをカウントします。</li>
	</ol>
</li>
	<li>
中皮細胞を播種
	<ol>
<li>完全培地にライブ HPMCs の 300,000 細胞/ml を中断します。6 ウェル プレートの各ウェルに新鮮な完全な成長媒体の 2.5 mL を追加します。うちバイオ セーフティ フードへの挿入を含む 15 cm の料理をもたらす慎重に。滅菌鉗子を使用して、挿入を反転し、6 ウェル プレートのウェルに配置、完全な成長媒体 (図 1) に浸漬下面に接続されている HeyA8 細胞直立。</li>
		<li>シード 450,000 HPMCs または HPMC コントロールの HeyA8 セルなし挿入 (図 1) 井戸の底の面に接続 HeyA8 細胞とインサートの内側の成長培地の 1.5 mL で中断します。細胞成長培地の 1.5 mL を HeyA8 コントロールに追加します。</li>
	</ol>
</li>
	<li>
近位の文化を育てる
	<ol>
<li>72 h の 37 ° C で 5% CO2 CO2インキュベーターで育つ近位文化をしましょう。必要な場合、媒体をとおり補充することができます。<ol>
<li>カバーの内側、750 μ L を削除し、完全な新鮮な成長媒体の 750 μ L を追加します。</li>
			<li>挿入 (6 ウェル プレート) の外、1 mL を削除し、新鮮な完全な成長培地 1 mL を追加します。1 mL は、1 つのステップの中での利便性のために選ばれました。に応じて、1.25 mL を削除、代わりに交換することができます。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
セルの収集
	<ol>
<li>近位文化の 72 時間後セルを収集します。セル HPMCs。 Trypsinize と HeyA8 細胞のクロスコンタミネーションを防ぐためにこの段階で特別な注意を取る、それらを遠心し、交差汚染を防ぐために膜に細胞を溶解ではなくペレットを溶解します。</li>
	</ol>
</li>
	<li>
細胞をトリプシン
	<ol>
<li>1x PBS で挿入の両側をすすいでください。</li>
		<li>新しい井戸に挿入を転送し、トリプシンを追加します。カバーの内側に 0.5 mL のトリプシンと挿入の下面に接続されているがん細胞を trypsinize に 6 ウェル プレートに 2 mL のトリプシンを追加します。37 ° C で 2 分間プレートを孵化させなさい</li>
	</ol>
</li>
	<li>
トリプシンを中和
	<ol>
<li>トリプシンを中和、ピペットの上下、および、細胞を取るし、ラベル コレクションの管にそれらを転送にカバーの内側に完全な成長培地 1 mL を追加します。中和を完全に完全な成長媒体の追加 2 mL を追加します。細胞のクロスコンタミネーションを防ぐためにピペッティングしながら細胞膜に穴を開けるように注意が必要があります。</li>
		<li>セルを挿入の下に収集、底面に 12.2 手順で追加した 2-3 x セル 6 ウェル プレートの底に落ちるように 2 mL のトリプシンをピペットします。6 ml の成長媒体のプレートの底にトリプシンを中和し、プレートの内容をラベル コレクション チューブに転送します。</li>
	</ol>
</li>
	<li>
収集し、細胞を溶解
	<ol>
<li>500 x g 3 分で細胞を遠心し、メディアを吸引します。再懸濁します、RNA 抽出用の溶解試薬のフェノールとグアニジン-チオシアン酸塩-ベースの 0.7 ml 細胞ペレットを溶解させます。任意の適切なキットを使用して RNA を分離します。</li>
	</ol>
</li>
</ol>3 量的なリアルタイムのポリメラーゼの連鎖反応
注: 次の手順は、近位の文化の結果として遺伝子の表現の変更を検討する、リアルタイムの量的なポリメラーゼの連鎖反応 (qPCR) をについて説明します。
<ol><li>QPCR の RNA のサンプルからの cDNA を準備します。対応するバッファーとランダム プライマーすべて mRNA の逆のトランスクリプションを確保するため任意の適切な逆転写酵素を使用 (材料の表を参照してください)。</li>
	<li>フィブロネクチン (FN1) と E-カドヘリン (CDH1) の mRNA 発現量の変化を評価し、内部統制としてグリセルアルデヒド-3-リン酸デヒドロゲナーゼ (GAPDH) と qPCR による成長因子 β 1 (TGFβ1) を変換します。標準化された市販遺伝子式の試金 (材料の表を参照) を使用するかプライマーを設計することが可能です。</li>
	<li>HeyA8 コントロールと HPMCs と HPMC コントロールと近位文化で育つ HPMCs 近位の文化で育った HeyA8 細胞を比較します。</li>
</ol>

<ol>
	<li>Hanahan, D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21 (3), 309-322 (2012).</li><li>Cupedo, T., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+interactions+in+lymph+node+development.">Cellular interactions in lymph node development.</a> The Journal of Immunology. 174 (1), 21-25 (2005).</li><li>Suvas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Substance+P+Neuropeptide+in+Inflammation,+Wound+Healing,+and+Tissue+Homeostasis.">Role of Substance P Neuropeptide in Inflammation, Wound Healing, and Tissue Homeostasis.</a> The Journal of Immunology. 199 (5), 1543-1552 (2017).</li><li>Gnecchi, M., Danieli, P., Malpasso, G., Ciuffreda, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+Mechanisms+of+Mesenchymal+Stem+Cells+in+Tissue+Repair.">Paracrine Mechanisms of Mesenchymal Stem Cells in Tissue Repair.</a> Methods in Molecular Biology. , 123-146 (2016).</li><li>Lionetti, V., Bianchi, G., Recchia, F. A., Ventura, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+autocrine+and+paracrine+myocardial+signals:+an+emerging+therapeutic+strategy+in+heart+failure.">Control of autocrine and paracrine myocardial signals: an emerging therapeutic strategy in heart failure.</a> Heart Failure Reviews. 15 (6), 531-542 (2010).</li><li>Nicosia, R. F., Zorzi, P., Ligresti, G., Morishita, A., Aplin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+angiogenesis+by+different+cell+types+in+the+aorta+ring+model.">Paracrine regulation of angiogenesis by different cell types in the aorta ring model.</a> International Journal of Developmental Biology. 55 (4-5), 447-453 (2011).</li><li>Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+cancer+stem+cells+-+what+challenges+do+they+pose.">Tackling the cancer stem cells - what challenges do they pose.</a> Nature Reviews Drug Discovery. 13 (7), 497-512 (2014).</li><li>Plaks, V., Kong, N., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+niche:+how+essential+is+the+niche+in+regulating+stemness+of+tumor+cells.">The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells.</a> Cell Stem Cell. 16 (3), 225-238 (2015).</li><li>Elenbaas, B., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotypic+signaling+between+epithelial+tumor+cells+and+fibroblasts+in+carcinoma+formation.">Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.</a> Experimental Cell Research. 264 (1), 169-184 (2001).</li><li>Wilson, K. 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=EGFR+ligands+exhibit+functional+differences+in+models+of+paracrine+and+autocrine+signaling.">EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.</a> Growth Factors. 30 (2), 107-116 (2012).</li><li>Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling.">Notch signaling.</a> Cold Spring Harbor Perspectives in Biology. 4 (10), (2012).</li><li>Singh, A. B., Sugimoto, K., Harris, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacrine+activation+of+epidermal+growth+factor+(EGF)+receptor+by+membrane-anchored+heparin-binding+EGF-like+growth+factor+protects+epithelial+cells+from+anoikis+while+maintaining+an+epithelial+phenotype.">Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype.</a> Journal of Biological Chemistry. 282 (45), 32890-32901 (2007).</li><li>Swartz, M. A., 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=Tumor+microenvironment+complexity:+emerging+roles+in+cancer+therapy.">Tumor microenvironment complexity: emerging roles in cancer therapy.</a> Cancer Research. 72 (10), 2473-2480 (2012).</li><li>Mitra, A. K., 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=MicroRNAs+reprogram+normal+fibroblasts+into+cancer-associated+fibroblasts+in+ovarian+cancer.">MicroRNAs reprogram normal fibroblasts into cancer-associated fibroblasts in ovarian cancer.</a> Cancer Discovery. 2 (12), 1100-1108 (2012).</li><li>Nieman, K. 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nature Medicine. 17 (11), 1498-1503 (2011).</li><li>Salimian Rizi, 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=Nitric+oxide+mediates+metabolic+coupling+of+omentum-derived+adipose+stroma+to+ovarian+and+endometrial+cancer+cells.">Nitric oxide mediates metabolic coupling of omentum-derived adipose stroma to ovarian and endometrial cancer cells.</a> Cancer Research. 75 (2), 456-471 (2015).</li><li>Mitra, A. K., 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=Microenvironment-induced+downregulation+of+miR-193b+drives+ovarian+cancer+metastasis.">Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis.</a> Oncogene. 34 (48), 5923-5932 (2015).</li><li>Tomar, S., 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=ETS1+induction+by+the+microenvironment+promotes+ovarian+cancer+metastasis+through+focal+adhesion+kinase.">ETS1 induction by the microenvironment promotes ovarian cancer metastasis through focal adhesion kinase.</a> Cancer Letters. 414, 190-204 (2018).</li><li>Ovarian Cancer Metastasis: A Unique Mechanism of Dissemination. Tumor Metastasis Available from: <a target="_blank" href="https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination">https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination</a> (2016)</li><li>Iwanicki, M. P., 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=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discovery. 1 (2), 144-157 (2011).</li><li>Kenny, H. A., 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=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> Journal of Clinical Investigation. 124 (10), 4614-4628 (2014).</li><li>Boelens, M. 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=Exosome+transfer+from+stromal+to+breast+cancer+cells+regulates+therapy+resistance+pathways.">Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways.</a> Cell. 159 (3), 499-513 (2014).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+function+of+exosomes+in+cancer.">The biology and function of exosomes in cancer.</a> Journal of Clinical Investigation. 126 (4), 1208-1215 (2016).</li><li>Kohlhapp, F. J., Mitra, A. K., Lengyel, E., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+mediators+and+communicators+between+cancer+cells+and+the+tumor+microenvironment.">MicroRNAs as mediators and communicators between cancer cells and the tumor microenvironment.</a> Oncogene. 34 (48), 5857-5868 (2015).</li></ol>

著者が明らかに何もありません。

パラクリンの juxtacrine 細胞間シグナル伝達機構を理解することは、正常組織の恒常性と病気の条件7,8のよりよい知識を開発するため不可欠です。ほとんどパラクリン シグナルにより収集実施エアコンから 1 つのセルの種類とその他の細胞のタイプの治療に使用中。このメソッドでは、その本来のシンプルさの利点があります。しかし、それはない?...

がん細胞と腫瘍の進行がん微小環境との間の生産的な相互作用の役割は、定着させてきた、がん生物学1の研究の主要な焦点となっています。双方向信号のような場合、重要な創傷治癒、免疫反応、血管新生、幹細胞ニッチ中および開発2,3,4,5,6,7,8. これらのすべての生物学的プロセスに共通するテーマは、細胞が様々 な細胞の運命、組織生理学、病気の進行を決定するの細胞外のキューにその微小環境からの方法で応答します。したがって、このような細胞間コミュニケーションに関与するメカニズムの理解に向けてますますなっています。このような相互作用の大部分は、パラクリンや juxtacrine 細胞間シグナル伝達を伴います。Juxtacrine に対し9,10、それの応答をトリガー、近くの別のセルの対応する受容体によって感知される 1 つのセルによって特定のシグナル伝達因子の分泌を含むパラ分泌シグナル信号 2 つのセル関係11,12細胞成分の直接の接触が必要です。
このような信号だけでなく、腫瘍微小環境における組織の恒常性の重要なコンポーネントです。がん細胞の恩恵癌関連付けられている線維芽細胞 (CAFs)、免疫細胞、脂肪細胞13,14,,1516など、腫瘍・間質の細胞からパラクリン juxtacrine 要因。パラクリン シグナル並置配位子と Notch シグナル、またはインテグリンと、それぞれの相互作用と同様に受容体を含む juxtacrine シグナリング、成長因子、サイトカイン、ケモカインなどによって媒介されることができます。細胞外マトリックス蛋白質。我々 は、腫瘍の進展と転移14で卵巣癌のセルと CAFs との相互作用の重要性を実証しました。キーのマイクロ Rna と癌細胞転移増殖17,を促進する転写因子同様に、転移性卵巣癌細胞転移を覆う中皮細胞の相互作用を調節します。18。
パラクリン シグナル伝達に関するほとんどの研究と 2 番目のセルのタイプの治療に 1 つのセル型から収集した馴化培地の使用を含みます。このアプローチは広く使用されて、それは効果的に受容細胞の微小環境における分泌因子のローカライズされた高濃度を複製してないです。また、1 つのセルによって生成されて分泌因子の連続的な流れの動態を再現するは失敗し、隣接セルによって受信されました。パラクリン シグナル分泌因子、ソース セルの近くでのみ必要な濃度で拡散し、を希釈する距離が長くなる傾向があると短い距離で効果的です。このローカライズされた高濃度分泌因子の受容細胞の応答をトリガーに不可欠です。また、受信者の細胞応答も新しく分泌因子と劣化、バインド、および受信者のセル、ソース セルから拡散の国際化を通じて継続的な枯渇のバランスに依存しているです。馴化培地は、微小環境に存在高いローカライズされた濃度のアカウントに集中することができますが、ことは、正確な濃度を正確に複製することはできません。さらに、生産の自然動態と関与する因子の枯渇を模倣することはできません。正確にパラ分泌シグナルをレプリケートし、juxtacrine シグナリング メカニズムから分離、我々 は多孔質膜のいずれかのサーフェスの 2 つのセルのタイプを育てることを含む近位培養法を考案しました。毛穴が小さく、juxtacrine やり取りを防ぐためし、まだローカライズされた高濃度に分泌因子の交換を許可します。その方法では、このシステムは、生産の動力学とパラクライン因子の枯渇を保持します。

<table><tbody><tr><td>24 mm Transwell permeable support with 0.4 &amp;micro;m Pore Polycarbonate Membrane Insert</td><td>Corning (Costar)</td><td>3412</td><td>&amp;bull; 10 &amp;micro;m thick translucent polycarbonate membrane&lt;br/&gt; &amp;bull; Treated for optimal cell attachment&lt;br/&gt; &amp;bull; Packaged 6 inserts in a 6 well plate, 4 plates per case&lt;br/&gt; &amp;bull; Membrane must be stained for cell visibility&lt;br/&gt; &amp;bull; Sterilized by gamma radiation</td></tr><tr><td>6 well plate</td><td>Corning (Falcon)</td><td>353046</td><td>Flat Bottom, TC-treated, sterile, with Lid</td></tr><tr><td>15 cm culture dish</td><td>Corning (Falcon)</td><td>353025</td><td>Sterile, TC-treated Cell Culture Dish</td></tr><tr><td>DMEM</td><td>Corning (Cellgro)</td><td>10-013-CV</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning (Cellgro)</td><td>25-025-CI</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning (Cellgro)</td><td>25-020-CI</td><td></td></tr><tr><td>0.25% Trypsin, 2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Pipets</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>CO2 Incubator</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>Biosafety level II cabinet</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>FN1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01549976_m1</td><td></td></tr><tr><td>TGFB1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs00998133_m1</td><td></td></tr><tr><td>CDH1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01023895_m1</td><td></td></tr><tr><td>GAPDH TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs99999905_m1</td><td></td></tr><tr><td>miRNeasy mini RNA isolation Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher Scientific</td><td>43-688-13</td><td></td></tr><tr><td>HeyA8 ovarian cancer cells</td><td>Obtained from Ernst Lengyel Lab, University of Chicago</td><td></td><td></td></tr><tr><td>TGF&amp;beta; Neutralizing Antibody</td><td>R&amp;amp;D Systems</td><td>MAB1835-100</td><td></td></tr></tbody></table>

a proximal culture method to study paracrine signaling between cells

細胞間相互作用は、腫瘍の進行、免疫反応、血管新生、開発など多くの生物学的プロセスに重要な役割を果たします。パラクリンや juxtacrine シグナリングは、このような相互作用を仲介します。馴化培地と培養研究の使用は相互作用のこれらの 2 つのタイプを区別する最も一般的な方法です。ただし、パラクリン作用中に、微小環境における分泌因子のローカライズされた高濃度の効果は馴化培地によって正確にそうはいないと、したがって、不正確な結論につながる可能性があります。この問題を克服するために我々 は、パラクリン シグナルを研究する近位部の培養法を考案しました。2 つのセル型は、0.4 μ m 孔を持つ 10 μ m 厚ポリカーボネート膜のいずれかのサーフェスに栽培しています。毛穴は分泌因子の交換を許可して、同時に juxtacrine シグナリングを禁じる。細胞を収集、パラクリン シグナル伝達の効果を決定するエンドポイントで分離できます。分泌因子の局在の濃度勾配をできるだけでなく、この方法は長時間文化だけでなく、阻害剤の使用を含む実験を受けやすい。このメソッドを使用して、卵巣癌細胞と中皮細胞の転移のサイトで出会う彼ら間の相互作用を研究する、パラクリン シグナル、様々 な分野の研究に研究の 2 つ付着性のセル型に合わせることができます。開発、免疫学、腫瘍微小環境を含みます。

転移性卵巣癌細胞19腹腔内転移の部位に中皮細胞が発生します。成功した転移17,18,20,21 を有効にすると卵巣癌のセルの適応応答の誘導に中皮細胞の助けを借りて生産パラクリンと juxtacrine 相互作用.近位培養法の有効性をテストするため HeyA8 卵巣癌細胞と...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

傍分泌と juxtacrine 細胞間相互作用は、腫瘍の進行、免疫反応、血管新生、開発など多くの生物学的プロセスに重要な役割を果たします。ここでは、近位文化メソッドを細胞の直接接触を防止しながら分泌因子のローカライズされた濃度を維持する、パラクリン シグナル伝達研究に使用します。

パラ分泌細胞間シグナル研究に近位の培養法

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

a-proximal-culture-method-to-study-paracrine-signaling-between-cells

Research

JoVE Journal

Biology

9.4K ビュー.  Indiana University School of Medicine. 傍分泌と juxtacrine 細胞間相互作用は、腫瘍の進行、免疫反応、血管新生、開発など多くの生物学的プロセスに重要な役割を果たします。ここでは、近位文化メソッドを細胞の直接接触を防止しながら分泌因子のローカライズされた濃度を維持する、パラクリン シグナル伝達研究に使用します。

ビデオ: A Proximal Culture Method to Study Paracrine Signaling Between Cells

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

がん研究

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

透過性膜支持体との共培養を用いたマクロファージ活性化に及ぼす腫瘍分泌パラクリンリガンドの効果の研究

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

歯髄パラクリン信号に反応した神経突起の成長を研究する共培養法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

発生生物学

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

in vitroでのパラクリン非標準Wntシグナル伝達のモデリング

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

免疫・感染学

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

抵抗血管の細胞培養モデル

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

医学

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...

生物学

3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

Source: Espinoza-S&#225;nchez, et al. <a href="https://www.jove.com/v/56589/analyzing-communication-between-monocytes-primary-breast-cancer-cells">Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System.</a> J. Vis. Exp. (2018).
This video describes the 3D co-culture of breast cancer cells with monocytes to study their interaction in an extracellular matrix extract (ECME) based environment. This method helps in studying specific features such as collagen degradation, immune cell recruitment, and cell invasion.

直接相互作用による3D共培養:がん細胞と単球の共培養による相互作用の研究

Source: Espinoza-S&#225;nchez, et al. Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ...

JoVE Encyclopedia of Experiments

乳がん

試験管研究

Proximal Culture System: An In Vitro Culture Technique to  Study Paracrine Signaling Between Cells

Source: Dasari, S. et al. <a href="https://www.jove.com/t/58144">A Proximal Culture Method to Study Paracrine Signaling Between Cells.</a>&#160;J. Vis. Exp. (2018)
In this video, we develop a proximal co-culture model of ovarian cancer cells and primary human mesothelial cells. This physiologically relevant model allows the study of paracrine signaling, where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

近位培養システム:細胞間のパラクリンシグナル伝達を研究するためのin vitro培養技術

Source: Dasari, S. et al. A Proximal Culture Method to Study Paracrine Signaling Between Cells.&#160;J. Vis. Exp. (2018)
In this video, we develop a ...

パラ分泌細胞間シグナル研究に近位の培養法

要約

さらに動画を探す

この動画の章

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

透過性膜支持体との共培養を用いたマクロファージ活性化に及ぼす腫瘍分泌パラクリンリガンドの効果の研究

歯髄パラクリン信号に反応した神経突起の成長を研究する共培養法

in vitroでのパラクリン非標準Wntシグナル伝達のモデリング

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

抵抗血管の細胞培養モデル

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

直接相互作用による3D共培養:がん細胞と単球の共培養による相互作用の研究

近位培養システム:細胞間のパラクリンシグナル伝達を研究するためのin vitro培養技術

パラ分泌細胞間シグナル研究に近位の培養法

要約

さらに動画を探す

この動画の章

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

透過性膜支持体との共培養を用いたマクロファージ活性化に及ぼす腫瘍分泌パラクリンリガンドの効果の研究

歯髄パラクリン信号に反応した神経突起の成長を研究する共培養法

in vitroでのパラクリン非標準Wntシグナル伝達のモデリング

X 線間のクロストークを解析のための共培養法照射 caco-2 細胞と PBMC

抵抗血管の細胞培養モデル

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

直接相互作用による3D共培養:がん細胞と単球の共培養による相互作用の研究

近位培養システム:細胞間のパラクリンシグナル伝達を研究するためのin vitro培養技術

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単な方法：腫瘍微小環境のミミック