本研究では、国立衛生研究所（CA148828）、ミシガン胃腸ペプチドセンターの大学、ミシガン大学総合がんセンターのジェフリーA.コルビーコロン癌研究とトム劉メモリアル基金からYMSへの補助金によってサポートされていました。

1。大腸腫瘍の単離と細胞解離<ol><li>腸の腫瘍は、任意の散発または治療によって誘発される大腸癌モデルから単離することができる。マウスはCO 2で安楽死させなければならない。コロン次いで、冷リン酸緩衝食塩水（PBS）でフラッシュし、収集され、長手方向に開口している。実体顕微鏡を使用して腫瘍を含む領域を特定し、ハサミで出て解剖し、冷PBSで洗浄する。 </li><li> EDTAキレート化緩衝液（2 mMのEDTA、5.6ミリモル/ LのNa 2 HPO 4、8.0ミリモル/ L KH 2 PO 4、96.2ミリモル/ LのNaCl、1.6ミリモル/ LのKCl、43.4ミリモル/ Lスクロース、内腫瘍を含む腸断片をインキュベート氷上で60分間、D-ソルビトール54.9ミリモル/ L、蒸留水で0.5ミリモル/ L DL-ジチオスレイトール）。 </li><li>腫瘍細胞が間葉に取り付けたままになりつつキレート化した後、正常な腸上皮細胞のほとんどは、切り離される。正常上皮細胞を含むキレート化バッファをオフに吸引5mlの冷たいキレート化バッファーで残留腫瘍断片をもう一度洗う。 </li><li>キレート化バッファをオフに吸引、5ミリリットル冷たい1×PBSで腫瘍フラグメントを洗う。 </li><li> 1×PBS、消化バッファー（2.5％ウシ胎児血清、ペニシリンの1単位/ ml、ストレプトマイシンを1μg/ mlであり、2.5 ngのアムホテリシンB、200 U / mlのIV型コラゲナーゼの/ mlでインキュベート腫瘍フラグメントをオフ吸引37℃で2時間125ダルベッコ改変イーグル培地でμgの/ mlのII型ディスパーゼ）℃、 </li><li>腫瘍フラグメントは1分間通常の重力下で落ち着く、と15ミリリットルファルコンチューブに上清を回収することができます。ペレット3分間200×gで遠心分離し、3分間200×gで遠心分離することによって5ミリリットルPBSで1回洗浄することにより、単一細胞腫瘍サスペンション上澄み。 </li></ol> 2。腸腫瘍の文化<ol><li> 500μlのPBSで腫瘍細胞ペレットを再懸濁し、血球計を用いて単離し、単一腫瘍細胞を数える。 </li><li>セントによってペレット腫瘍細胞3分間200 xgでrifuging、およびウェルあたりマトリゲル50μlの当たり15,000細胞で24ウェルプレートに氷と皿の上に5 mg / mlのマトリゲルでそれらを再懸濁します。 </li><li>マトリゲルは、37℃で15分間重合し、500よく基底/μlの培地（ペニシリン1単位/ ml、ストレプトマイシンを1μg/ mlの、および2.5 ngの/アムホテリシンB mlの、10ミリモル/ L HEPES追加してみましょう、2mMのグルタマックス、1X N2サプリメント、1X B27サプリメント、アドバンストダルベッコ改変イーグルMedium/F12 1ミリモル/ L N-アセチルシステイン）は50 ngの/ mlのマウスEGFを含む。 </li></ol> 3。設立オルガノイドのメンテナンス<ol><li>週に一度、EGF 2日おきと通路オルガノイド1:5に含む基礎培地を変更します。 </li><li>継代のために、新鮮な基礎培地に培地を交換してください。機械的に遮断するヒントP1000ピペットを用いオルガノイドとマトリゲルを混乱させると15ミリリットルファルコンチューブに移す。さらに、機械的な解離は、火災磨かパスツールパイプを用いて達成されるって。 </li><li> 2分間200 xgで基礎培地と遠心分離機5mlの解離オルガノイドを洗ってください。 </li><li>上清を捨て、マトリゲルでペレットを再懸濁し、上記のように培地を追加します。 </li></ol> 4。ストレージと設立オルガノイドの回収<ol><li>長期保存の場合は、少なくとも2年間安定である液体N 2中オルガノイドを凍結。凍結オルガノイドの場合、ヒントはカットオフと15ミリリットルファルコンチューブに移すとP1000ピペットを使用して混乱させる。 </li><li> 2分間200 xgで基礎培地と遠心分離機5mlの解離オルガノイドを洗ってください。 </li><li> 20％ウシ胎児血清（FBS）および10％ジメチルスルホキシド（DMSO）を含むダ​​ルベッコ改変イーグル培地（DMEM）でペレットを再懸濁し、上清を捨てる。 </li><li> 1.5 mlのクライオチューブに転送細胞は、その後℃の冷凍庫で冷却速度を達成するために-80でナルゲンミスターフロスティ冷凍コンテナとストアにチューブを入れて-1の°C /分。一晩インキュベートした後、液体N 2にチューブを移す。 </li><li>回復のために、冷凍オルガノイドは急速に、上記の条件をマトリゲルと文化の中で再懸濁し、スピンダウン、基礎培地で洗浄し、37℃の水浴中で解凍されています。 </li></ol> 5。 RNA抽出、タンパク質抽出と免疫組織化学<ol><li> RNA抽出：通路について上述したように細胞を収集する。 RNAは、製造業者の指示に従ってPicoPure TM RNAアイソレーションキットで分離されています。 </li><li>タンパク質抽出：細胞ペレットを上記のように、100μlの放射線免疫アッセイ緩​​衝液（RIPAで溶解集められ、50ミリモル/ LのTris-HCl pH7.5で、150ミリモル/ LのNaCl、2ミリモル/ L EDTA、1％NP-40、0.1 1xのプロテアーゼ阻害剤％SDS）。 </li><li>免疫組織化学：細胞培養​​培地をオフに吸引し、クライオ金型にカットオフヒントをP1000ピペットで腫瘍オルガノイドを転送し、TISですぐに凍結ドライアイスで凍結培地（OCT）を訴える。凍結切片は7μmで ​​切断し、以前26説明したように染色した。 </li></ol>

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	<li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141, 1762-1772 (2011).</li><li>Creamer, B., Shorter, R. G., Bamforth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+turnover+and+shedding+of+epithelial+cells.+I.+The+turnover+in+the+gastro-intestinal+tract.">The turnover and shedding of epithelial cells. I. The turnover in the gastro-intestinal tract.</a> Gut. 2, 110-118 (1961).</li><li>Crosnier, C., Stamataki, D., Lewis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizing+cell+renewal+in+the+intestine:+stem+cells,+signals+and+combinatorial+control.">Organizing cell renewal in the intestine: stem cells, signals and combinatorial control.</a> Nat. Rev. Genet. 7, 349-359 (2006).</li><li>Medema, J. P., Vermeulen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+stem+cells+in+intestinal+homeostasis+and+cancer.">Microenvironmental regulation of stem cells in intestinal homeostasis and cancer.</a> Nature. 474, 318-326 (2011).</li><li>Korinek, V., 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=Depletion+of+epithelial+stem-cell+compartments+in+the+small+intestine+of+mice+lacking+Tcf-4.">Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.</a> Nat. Genet. 19, 379-383 (1998).</li><li>Pinto, D., Gregorieff, A., Begthel, H., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canonical+Wnt+signals+are+essential+for+homeostasis+of+the+intestinal+epithelium.">Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.</a> Genes Dev. 17, 1709-1713 (2003).</li><li>Kim, K. 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=Mitogenic+influence+of+human+R-spondin1+on+the+intestinal+epithelium.">Mitogenic influence of human R-spondin1 on the intestinal epithelium.</a> Science. 309, 1256-1259 (2005).</li><li>Reya, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+stem+cells+and+cancer.">Wnt signalling in stem cells and cancer.</a> Nature. 434, 843-850 (2005).</li><li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2012.">Cancer statistics, 2012.</a> CA Cancer J. Clin. 62, 10-29 (2012).</li><li>Sesink, A. L., Termont, D. S., Kleibeuker, J. H., Vander Meer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+meat+and+colon+cancer:+the+cytotoxic+and+hyperproliferative+effects+of+dietary+heme.">Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme.</a> Cancer Res. 59, 5704-5709 (1999).</li><li>Fearon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+colorectal+cancer.">Molecular genetics of colorectal cancer.</a> Annu. Rev. Pathol. 6, 479-507 (2011).</li><li>Nishisho, I., 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=Mutations+of+chromosome+5q21+genes+in+FAP+and+colorectal+cancer+patients.">Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.</a> Science. 253, 665-669 (1991).</li><li>Xue, X., 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=Hypoxia-inducible+factor-2alpha+activation+promotes+colorectal+cancer+progression+by+dysregulating+iron+homeostasis.">Hypoxia-inducible factor-2alpha activation promotes colorectal cancer progression by dysregulating iron homeostasis.</a> Cancer Res. 72, 2285-2293 (2012).</li><li>Liu, W., 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=Mutations+in+AXIN2+cause+colorectal+cancer+with+defective+mismatch+repair+by+activating+beta-catenin/TCF+signalling.">Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling.</a> Nat. Genet. 26, 146-147 (2000).</li><li>Morin, P. 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=Activation+of+beta-catenin-Tcf+signaling+in+colon+cancer+by+mutations+in+beta-catenin+or+APC.">Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.</a> Science. 275, 1787-1790 (1997).</li><li>Brattain, M. G., Fine, W. D., Khaled, F. M., Thompson, J., Brattain, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+malignant+cells+from+a+human+colonic+carcinoma.">Heterogeneity of malignant cells from a human colonic carcinoma.</a> Cancer Res. 41, 1751-1756 (1981).</li><li>Leibovitz, 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=Classification+of+human+colorectal+adenocarcinoma+cell+lines.">Classification of human colorectal adenocarcinoma cell lines.</a> Cancer Res. 36, 4562-4569 (1976).</li><li>Willson, J. K., Bittner, G. N., Oberley, T. D., Meisner, L. F., Weese, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+human+colon+adenomas+and+carcinomas.">Cell culture of human colon adenomas and carcinomas.</a> Cancer Res. 47, 2704-2713 (1987).</li><li>Brattain, M. G., Strobel-Stevens, J., Fine, D., Webb, M., Sarrif, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+mouse+colonic+carcinoma+cell+lines+with+different+metastatic+properties.">Establishment of mouse colonic carcinoma cell lines with different metastatic properties.</a> Cancer Res. 40, 2142-2146 (1980).</li><li>Ikubo, A., Aoki, Y., Nagai, E., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+metastatic+variant+of+a+mouse+colon+carcinoma+cell+line,+LM17+and+its+response+to+GM-CSF+gene+therapy.">Highly metastatic variant of a mouse colon carcinoma cell line, LM17 and its response to GM-CSF gene therapy.</a> Clin. Exp. Metastasis. 17, 849-855 (1999).</li><li>Basant, S. K., Rinesh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+Of+Animal+Cell+Culture.">Principles Of Animal Cell Culture.</a> Student Compendium. Textbook Student Edition. 6, 61-96 (2008).</li><li>Vachon, P. H., 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=Differentiation+state-selective+roles+of+p38+isoforms+in+human+intestinal+epithelial+cell+anoikis.">Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis.</a> Gastroenterology. 123, 1980-1991 (2002).</li><li>Darido, 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=Defective+claudin-7+regulation+by+Tcf-4+and+Sox-9+disrupts+the+polarity+and+increases+the+tumorigenicity+of+colorectal+cancer+cells.">Defective claudin-7 regulation by Tcf-4 and Sox-9 disrupts the polarity and increases the tumorigenicity of colorectal cancer cells.</a> Cancer Res. 68, 4258-4268 (2008).</li><li>Sato, T., 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+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Yui, 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=Functional+engraftment+of+colon+epithelium+expanded+in+vitro+from+a+single+adult+Lgr5(+)+stem+cell.">Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell.</a> Nat. Med. 18, 618-623 (2012).</li><li>Anderson, E. R., Xue, X., Shah, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+hypoxia-inducible+factor-2alpha+(HIF-2alpha)+is+critical+for+efficient+erythropoiesis.">Intestinal hypoxia-inducible factor-2alpha (HIF-2alpha) is critical for efficient erythropoiesis.</a> J. Biol. Chem. 286, 19533-19540 (2011).</li><li>Sato, T., 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=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469, 415-418 (2011).</li><li>Kondo, 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=Retaining+cell-cell+contact+enables+preparation+and+culture+of+spheroids+composed+of+pure+primary+cancer+cells+from+colorectal+cancer.">Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 6235-6240 (2011).</li><li>Clevers, H., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+and+disease.">Wnt/beta-catenin signaling and disease.</a> Cell. 149, 1192-1205 (2012).</li></ol>

我々は、開示することは何もありません。

このプロトコルで説明する実験手順は、プライマリマウス大腸腫瘍の単離および培養のためにできるようになります。プロトコルは博士Cleversグループ1,24,27によって行わ精液の仕事から構成されている。我々は、腫瘍オルガノイドの良い収量を得るために消化時間とコラゲナーゼ濃度を最適化しました。重要なステップは、単一のセル、マトリゲル再懸濁、および選択培養への腫瘍細...

腸管上皮細胞が増殖し、脊椎動物の体2,3の他のすべての組織を上回る、臨時の速度で裏返します。腸管幹細胞（ISC）と通過増幅セルを含む分裂細胞、分泌（ゴブレット、パネートおよび腸内分泌細胞）または腸3のいずれかに分化する。 ISCは、陰窩の底に位置しています。パネート細胞は陰窩の一番下まで移動し、他の系統は絨毛3,4に上向きに移行するのに対し、長寿命です。ここでは細胞が微生物を含む腸の内容にさらされているとアノイキス誘発性アポトーシスのメカニズムを介して絨毛先端から流している。コロンは絨毛とパネート細胞を欠いていますが、恒常性を維持するためのメカニズムは4と似ています。 Wntシグナル伝達経路は、腸の増殖およびISCメンテナンス4において重要な役割を果たしているに関与している。番目の削除電子転写因子TCF4、Wntシグナルの下流エフェクターは、腸管幹細胞および組織破壊5の後続の損失につながる。同様に、Wntシグナル阻害剤DKK1のトランスジェニック発現は、上皮増殖を減少させ、分泌細胞系統6を枯渇させる。逆に、WntシグナルアゴニストR-スポンジン-1の過剰発現は、腸陰窩細胞7の強力かつ迅速な増殖を誘導する。 腸のホメオスタシスのためのWntシグナル伝達の重要性が与えられ、Wnt経路の変異は、しばしば結腸癌8において観察されている。大腸癌は、米国9のがんによる死亡の3番目の主要な原因である。赤身の肉やアルコールを過剰摂取は、身体活動の減少、そして継承と体細胞突然変異は結腸癌10,11のリスク要因であると考えられている。大腸腺腫性ポリポーシス（APC）遺伝子、キーWntシグナル伝達因子は、PAの大部分で変異されている12,13、家族散発、および大腸炎関連大腸癌とtients。 Axin2およびβ-カテニンを含むWntシグナル伝達経路に関与する他の因子の変異はまた、大腸癌14,15が観察される。しかしながら、結腸癌のための正確なメカニズムで効果的な治療法はまだ欠けている。結腸癌の分子機構の調査を容易にするために、積極的な細胞型に良性の癌の進行の異なる段階を示すヒト結腸癌細胞株は、16〜18が確立されている。別の転移特性を有するマウスの大腸癌細胞株もまた19,20入手可能である。彼らは密接に生体状態の模倣や、その他の生理学的に関連データ21を生成するので、それにもかかわらず、一次電池またはオルガノイド培養は、形質転換細胞株よりも好ましい。ほとんどの大腸癌由来の細胞ラインはlackin、板に取り付け単層としてまたは細胞懸濁液のような成長頂端 - 基底外側向きと細胞間のタイトジャンクショ​​ンのgの。分化した細胞は、絨毛の先端に到達し、22を流しているとしても、 生体内での正常および腫瘍腸上皮細胞はアポトーシスと呼ばれるアノイキスの自発的な形を受ける。これらの機能は、細胞株で要約することが困難であるが、大腸癌23の発達過程において重要である。これらの機能は、プライマリオルガノイドに保持されます。また、腫瘍オルガノイド培養はin vivo試験に比べて遺伝子の腫瘍の自律機能を評価するための効率的なシステムを提供する。腸のin vivoでの遺伝子操作は、腸固有のドライバを使用したトランスジェニックおよび/ ​​またはノックアウトマウスの作成 ​​を通じ、主に時間のかかるプロセスです。しかし、腫瘍オルガノイドは容易にウイルス媒介遺伝子操作の影響を受けやすいので、正確な分子メカニズムを評価するための素晴らしいツールです。プライマリ腸腫瘍オルガノイド培養HAVeが可能と強力な手法であることが実証されて。プライマリ腸細胞培養は、単一の成体LGR5 +幹細胞24からin vitroでの陰窩·絨毛構造を持つ機能的な腸オルガノイドを確立することができます。これらオルガノイドは、再生25損傷結腸組織に移植し、移植することができます。培養条件のさらなる適応は、マウスとヒトの大腸小腸と1実現可能な大腸から類似の上皮オルガノイドをしていた。基礎培地とEGFがプライマリマウス結腸腫瘍オルガノイド1を成長させるために十分であるのに対し、一次正常な結腸上皮培養、基礎培地と同様にEGF、ノギン、R-スポンジンとWnt3aは含めて成長因子のために、必要不可欠である。ここでは、単離するための詳細なプロトコルを記述し、文化、および結腸腫瘍オルガノイドを生成します。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

いくつかのヒトおよびマウスの大腸癌細胞株が確立された、そのような複数の細胞層、基底頂端極性、分化する能力、およびアノイキスなどの結腸腫瘍の生理学的完全性は大腸癌由来の細胞株で維持されていない。本研究では、佐藤T ら 1、大腸腫瘍の重要な生理機能を保持してから適応培養マウスの主結腸腫瘍オルガノイドするための方法を示しています。この方法は、腫瘍細胞が正常に比べて栄養素の条件を制限する上での成長を維持するという原理に基づいて、マウスの結腸腫瘍組織収集、隣接する正常結腸上皮解離、単一細胞に結腸腫瘍細胞を消化し、マトリゲルに結腸腫瘍細胞を包埋し、選択培養から成り上皮細胞。 原発腫瘍オルガノイド遺伝子組み換えマウスから単離した場合腫瘍自律FUNCTを評価するために非常に有用なシステムを提供特定の遺伝子のイオンである。また、腫瘍オルガノイドは遺伝子送達を瞑想ウイルスによる遺伝子操作に適している、大腸の腫瘍形成に関与し、したがってシグナル伝達経路は、また広範囲に過剰発現やノックダウンして調べることができます。原発腫瘍オルガノイド文化は、結腸腫瘍形成のためのメカニズムと治療法を研究する生理学的関連性の実現可能な手段を提供します。

/ +マウスは、 図1に示されている3ヶ月のApc 分から結腸腫瘍オルガノイド形成の時間経過。 0日目に、単一細胞をプレーティング（ 図1A）に続く数時間を観察することができた。 1日目、で耐火核を有する上皮細胞を観察することができた結腸腫瘍を生き延びた。 3日目に、細胞の大きさは倍増した。 6日目、オルガノイドの大きさは10倍以上に?...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

一次マウス結腸腫瘍オルガノイドを確立するための簡単​​な方法が記載されている。通常のコロンは上皮ないのに対し、この方法では、結腸腫瘍細胞が生き残ると限られた成長因子を含む培地中でオルガノイドに成長するという特徴を利用しています。

プライマリマウス大腸腫瘍のin vitroオルガノイド培養で

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K ビュー.  University of Michigan. 一次マウス結腸腫瘍オルガノイドを確立するための簡単​​な方法が記載されている。通常のコロンは上皮ないのに対し、この方法では、結腸腫瘍細胞が生き残ると限られた成長因子を含む培地中でオルガノイドに成長するという特徴を利用しています。

ビデオ: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

医学

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人間の前庭神経鞘腫の初代培養

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

エキソビボ処理前臨床のための原発腫瘍および/ ​​または関連した転移の応答と治療薬の臨床開発

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

マウス大腸求心性終末の体外機能解析では、

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNAを用いた乳がん組織におけるmiRNA発現の臨床病理学的解析<em&gt;その場で</em&gt;ハイブリダイゼーション

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

筋肉の不均衡: テストと競技人口の機能的な偏心ハムスト リングの筋力トレーニング

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

メタボロミクス解析とリピドミクス解析のための脳腫瘍の現場でのサンプリングと抽出

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...