この作品は、NIHの助成金R01 CA107469、R01 CA125577、およびCGのKleer、ミシガン州のがんセンターのサポート大学がU01CA154224によってサポートされていました。

1。材料の作製と培養培地<ol><li> 4℃のCO / Nの減少融解成長因子（GFR）マトリゲル</li><li> 37℃での必要な細胞株について完全なメディアを温めるMCF10A-ATCCは、メディアを指定していました。 5％ウシ胎児血清を補充したHME-HamのF-12培地、1μg/ mlのヒドロコルチゾン、5μg/ mlのインスリン、10 ng / mlの上皮増殖因子、および100 ng / mlのコレラ毒素; 5％FBS、2μg/ mlのヒドロコルチゾン及び5μg/ mlのインスリンを補充したSUM149-ハムF-12培地;およびMDA-MB-231から10パーセントFBS-DMEM </li><li>氷上で8ウェルチャンバースライドを予冷。 </li><li>組織培養フード、用品を準備します。 </li></ol> 8ウェルチャンバースライド中の2。三次元培養<ol><li>コー​​トGFRマトリゲル40μlの8ウェルスライドの各ウェル。スライドの中央にマトリゲルを追加してから、P200のピペットチップを使用して拡散。につながる可能性気泡やoverspreadingを避け、できるだけ均等に広がるようにしてくださいメニスカス形成。 </li><li> 5％CO 2下、37℃でスライドをインキュベートする。マトリゲルが固化されているときに、細胞をトリプシン処理し、収集し4分間組織培養遠心分離機で150×gでスピン。 </li><li>細胞を計数し、各細胞株の完全培地中12,500細胞/ mlに希釈する。 2％GFRマトリゲルを追加し、マトリゲル予めコーティングチャンバースライドの各ウェルに400μLを加える。 </li><li> CO 2インキュベータ内でスライドをインキュベートします。 15日まで、すべての4 日目の新鮮な完全なメディアの変化を与える。 </li><li> 薬理学的阻害剤を用いた治療：研究の阻害剤は、GFRマトリゲル上で細胞を成長させながら、3日ごと阻害剤を補充した新鮮な培地の変化に続いて、メッキの際に完全なメディアへの小分子阻害剤を追加します。 </li><li> 精製タンパク質を用いた治療： <ol><li>細胞プレート次は2.1から2.3を繰り返します。培養物は、血清starvati続く4日間増殖することができます16時間に。 </li><li> 5日目に、FBSは、血清飢餓細胞へのメディアの補足0.1％で精製されたタンパク質の適切な濃度で細胞を処理する。 2日後に精製したタンパク質を含む新鮮な培地交換を与えます。 </li><li> 10日目での完全なHMEメディアに馴化培地を交換してください。培養物は、別の5日間増殖することができます。 </li></ol></li><li> マトリゲル上で増殖させた腺房構造の免疫蛍光染色。 <ol><li> 2％パラホルムアルデヒド、0.5％トリトンX-100および1×PBS、pH7.4中の100mMグリシンを調製する。 0.1％ウシ血清アルブミン、0.2％トリトンX-100、0.05％のTween-20を含む1×PBSを含む、免疫蛍光緩衝液（緩衝液IF）を調製する。 </li><li>慎重に条件培地を吸引するP200のピペットチップを使用してください。 </li><li> 20分間室温（RT）で2％、新たに調製したホルムアルデヒドで腺房の構造を修正します。 </li><li> 4℃で10分間、0.5％トリトンX-100で細胞を透過</li><li>文化を洗う100mMグリシンエチルで3回、各洗浄のために10〜15分だ。 </li><li> RTで1時間、一次ブロックと呼ば​​IF緩衝液中の10％ヤギ血清で細胞をブロックする。 </li><li> 20μg/ mlのヤギ抗マウスのF（ab &#39;）30分間の一次ブロックバッファ内の2フラグメントと文化をインキュベートする。このステップは、マトリゲルで免疫反応性マウスIgG種をブロックすることが重要です。 </li><li>第二のブロッキング溶液中で一次抗体と共にインキュベートし、4℃で（一次ブロック+20μg/ mlのヤギ抗マウスF（ab &#39;）2フラグメント）O / N </li><li> 10〜15分緩やかに揺り動かしながらそれぞれのIF緩衝液で3回洗浄する。 </li><li> 45分間のIFバッファ内の蛍光結合二次抗体でインキュベートする。 </li><li> 10分間の洗浄を3回穏やかに揺らしながらIF緩衝液を用いて、それぞれ。 </li><li>核を可視化するために、DAPIを含有する抗退色試薬でスライドをマウントします。 </li><li>スライドを室温でO / Nを乾燥することができます。 </li><li>画像収集：コロニー周辺で共焦点画像を取得マトリゲルの上に増殖した細胞のdsection。詳細については、Zスタッキングにより腺房構造の全長にわたって光学一連のセクションを取得する。 </li></ol></li></ol> 3。イムノ用2ウェルチャンバースライドで三次元培養<ol><li>氷上で2ウェルチャンバースライドを予冷。 2.1〜2.4がマトリゲル160μlの2ウェルチャンバースライドなどのコートのための試薬 ​​をスケールアップし、2ウェルスライドの各ウェルに混ぜ、細胞/マトリゲルの1.6ミリリットルを追加する手順に従います。 </li><li>製造業者の説明書に従って市販の3D培養の細胞の回収キットを用いてマトリゲルから腺房構造を収穫。 </li><li> 1Xと4℃に予冷する細胞洗浄および細胞の回収バッファーを希釈マトリゲルの洗浄および脱落を氷上で行うべきである。 </li><li>細胞洗浄用緩衝液を用いて3倍腺房構造を洗ってください。 </li><li>細胞収穫BUを使用して15ミリリットル遠心チューブに腺房を収集ffer 4℃でロッカーで30分間インキュベートした1ミリリットルピペットでよく懸濁物を混ぜる。これを小片にマトリゲルを破壊し、細胞収穫バッファに細胞の大部分を解放します。 </li><li>培養遠心機で200×gで細胞をペレット化。 P200のピペットチップでペレット化した細胞からのヒドロゲルの浮遊層を除去する。 </li><li>キットで提供SDSサンプル緩衝液中で溶解物を再懸濁する。 RIPA溶解緩衝液を使用することもできる。 </li></ol>悪性対良性乳房表現型の4。定量<ol><li>ステップ2.1から2.4以下の各治療セットに対して三連で細胞を成長させる。 </li><li>スライドシフタと、コンピュータに取り付けられた位相差顕微鏡を用いて5倍または10倍の解像度で各ウェルから腺房構造の位相コントラスト画像を撮影する。別のフレームからスライドを移動するため、チャンバースライドのウェル全体を覆うことにより、画像を取る。 </li><li>腺房構造の合計数をカウントし、シングルラウンドフラット腺房およびmultiacinar構造や組織を欠いて無計画に成長している構造の数の、それから発せられる分岐プロセスと数。 </li><li>棒グラフのような単一のラウンドフラット腺房構造の割合対悪性の構造とプロットを計算します。スチューデントのt検定で有意差を計算します。 </li></ol>

<ol>
	<li>Arendt, L. M., Rudnick, J. A., Keller, P. J., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma+in+breast+development+and+disease.">Stroma in breast development and disease.</a> Semin. Cell Dev. Biol. 21, 11-18 (2009).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30, 256-268 (2003).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-365 (2007).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-828 (2010).</li><li>Rosen, P. P., Rosen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rosen's breast pathology. , (2008).</li><li>Weaver, V. M., Fischer, A. H., Peterson, O. W., Bissell, M. 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+importance+of+the+microenvironment+in+breast+cancer+progression:+recapitulation+of+mammary+tumorigenesis+using+a+unique+human+mammary+epithelial+cell+model+and+a+three-dimensional+culture+assay.">The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay.</a> Biochem. Cell Biol. 74, 833-851 (1996).</li><li>Kenny, P. 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=The+morphologies+of+breast+cancer+cell+lines+in+three-dimensional+assays+correlate+with+their+profiles+of+gene+expression.">The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression.</a> Mol. Oncol. 1, 84-96 (2007).</li><li>Pal, A., Huang, W., Li, X., Toy, K. A., Nikolovska-Coleska, Z., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+modulates+BMP+signaling+via+the+Smad-independent+TAK1/p38+pathway,+acting+to+suppress+metastasis+of+breast+cancer.">CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer.</a> Cancer Res. 15, 4818-4828 (2012).</li><li>Pal, A., Huang, W., Toy, K. A., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+knockdown+disrupts+acinar+organization+of+breast+cells+in+three-dimensional+cultures+through+up-regulation+of+type+III+TGF-&#946;+receptor.">CCN6 knockdown disrupts acinar organization of breast cells in three-dimensional cultures through up-regulation of type III TGF-&#946; receptor.</a> Neoplasia. 14, 1067-1074 (2012).</li><li>Wang, F., 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=Phenotypic+reversion+or+death+of+cancer+cells+by+altering+signaling+pathways+in+three-dimensional+contexts.">Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.</a> J. Natl. Cancer Inst. 94, 1494-1503 (2002).</li><li>Weaver, V. 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=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J. Cell Biol. 137, 231-245 (1997).</li><li>Wang, R., 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=Three-dimensional+co-culture+models+to+study+prostate+cancer+growth,+progression,+and+metastasis+to+bone.">Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone.</a> Semin. Cancer Biol. 15, 353-364 (2005).</li><li>Vaughan, M. B., Ramirez, R. D., Wright, W. E., Minna, J. D., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+model+of+differentiation+of+immortalized+human+bronchial+epithelial+cells.">A three-dimensional model of differentiation of immortalized human bronchial epithelial cells.</a> Differentiation. 74, 141-148 (2006).</li><li>Fata, J. E., Werb, Z., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mammary+gland+branching+morphogenesis+by+the+extracellular+matrix+and+its+remodeling+enzymes.">Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.</a> Breast Cancer Res. 6, 1-11 (2004).</li><li>Luca, A. 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=Impact+of+the+3D+microenvironment+on+phenotype,+gene+expression,+and+EGFR+inhibition+of+colorectal+cancer+cell+lines.">Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.</a> PLoS One. 8, 1-11 (2013).</li></ol>

著者らは、競合する経済的利益を宣言していません。

ここでは、再構成基底膜上の乳房上皮細胞の三次元培養および悪性対良性乳房表現型を区別するために、このシステムの有用性を記載している。このシステムはまた、他の腺組織由来の培養物の異なる細胞型に使用することができ、いくつかの正常12-14に名前を付けるために、培養前立腺上皮、気管支上皮、および甲状腺上皮細胞に使用されてきたことが報告されている。 3D?...

再構成基底膜上の乳房上皮細胞の三次元培養（3D）は、複雑な表現型および正常乳房および乳癌2,3の関連するシグナリングを研究するための重要なモデル系である。乳房の機能ユニットは、腺房へ分岐する小さなダクトで構成され、端末のダクト小葉単位（TDLU）です。腺房は、基底膜によって囲まれている5つの細胞層、上皮細胞および筋上皮細胞からなる高度に組織化された構造である。正常な腺房の最も顕著な特徴は、細胞の分極、基盤となる基底膜への付着や、特殊な細胞間接触である。この複雑な組織は浸潤癌で破壊されている。細胞が単層として成長させた広く使用されている2次元培養は、腺房の形成を可能にしないでください。このように、単層培養上皮細胞との間の複雑な関係を捕捉するシステムを提供することに欠けている正常な乳房と乳がんでの規制緩和にある。乳房細胞の機能的単型上皮培養物をいくつかの研究室2,3に開発されている。三次元培養は、Engelbreth-Holm-Swarmマウス肉腫細胞由来の可溶化抽出物であり、市販されてマトリゲル上の乳房細胞の増殖を伴う。コラーゲンのような他の3D基層はIもまた使用される。乳癌細胞は、細胞がマトリゲル2で培養埋め込 ​​まれている3D埋め込 ​​みアッセイにより3次元で培養することができる。あるいは、細胞はまた、マトリゲルより少ない量を必要とし、位相コントラスト撮影によってコロニー形成の時間経過のモニタリングを容易にし、 その場イメージング 2 のために理想的であるように費用対効果のある、3Dオーバーレイアッセイ、いわゆる、トップアッセイに3Dで培養することができる-3。上部アッセイに3Dは腺房表現型および遺伝子発現プロファイル7との間の相関を定義するために使用されている。 3D文化がeffectivすることができますエリー浸潤癌から正常および良性の細胞を区別するために使用される。通常の良性乳腺細胞が浸潤癌細胞が内腔の無いクリアで多産し、無計画に成長する一方で成長がマトリゲル上の中心部にある、明確に定義されたルーメンと偏光構造を逮捕形成している。 E6/E7不死化ヒト乳腺上皮細胞および線維嚢胞性変化を有する36歳の患者から単離された自然発生的に不死化細胞株であるMCF10A細胞株は、正常な3D 3,8における良性乳房表現型を再捕捉するために使用されているとして役立ったいくつかの分子8,9の発癌と腫瘍抑制機能を研究するためのモデルシステム。これらの良性の細胞は、レンチウイルス/レトロウイルスを導入することによって、目的の遺伝子（単数または複数）を操作するために操作することができる。目的の遺伝子（群）は、腫瘍抑制因子である場合は、目的の遺伝子が良性細胞では癌遺伝子の過剰発現であるかのに対し、shRNAを媒介ノックダウンは、悪性形質転換につながる悪性の表現型を示すべきである。どちらの場合も、3Dで形成された腺房構造は、悪性形質転換をキャプチャすることができます。 3Dでメッキ良性単一細胞は増殖し、球状構造ラウンドフォルムを開始します。一日5-8による細胞のこの球状凝集体は、マトリゲルと接触している細胞の周囲の偏光層と、マトリゲルとの接触を欠く少ない極性細胞の内塊へと分化する。次の10〜15日に、内側の細胞はアポトーシスが明らかに管腔を形成することにより、死に始めます。悪性細胞は、それらの極性を失って無計画に成長します。悪性度の高い細胞は、通常、分岐構造を形成する。表現型におけるこれらの差異は、タイムラプス位相コントラスト撮影によりその場で関連する分子マーカーを用いた免疫染色によって捕捉することができる。最も一般的に使用されるマーカーは、増殖マーカーホスホ - ヒストン3およびapoptotと組み合わせて、α-6インテグリン、基底極性マーカーであるでICマーカーはカスパーゼ-3を切断した。後者は、腺房の中心、正常および良性の乳房細胞の機能における内腔の形成があるかどうかを決定することが重要である。他方の極性と細胞間接触マーカーはGM130、ラミニン、ERM、E-カドヘリンが含まれる。交互に乳癌細胞株は、目的の遺伝子を操作するために操作することができる。この場合、それ以上の成長への復帰は、良性の表現型は癌表現型と区別するために読むようにして使用することができ逮捕した。 三次元培養はまた、悪性形質転換注意深く10-11に関与するシグナル伝達経路を描写するために使用することができる。使用して、上記リードアウト、薬理学的阻害剤、遮断抗体又は組換えタンパク質を挙げるが悪性形質転換をもたらすシグナル伝達分子で突き止めることに使用することができる。種々の研究室からの継続的な努力とそれがRNAを単離し、また、免疫ブロット法および免疫のための溶解物を調製するために3Dから細胞を抽出することが可能であるnoprecipitation。これらの戦略は、プロトコルの段階的かつ詳細に説明されています。

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			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
</table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

浸潤性乳癌の転移に隣接する組織や性向の侵入を特徴とする悪性上皮性腫瘍の一群である。癌細胞とそれらの微小環境との間の信号の相互作用は、乳癌の成長および生物学的挙動1に強力な影響を与える。しかし、細胞外マトリックスからのこれらの信号の大部分が失われたり、細胞を単層として2次元培養（2D）で栽培されている場合、その関連性が代役されています。近年では、再構成基底膜上の3次元（3D）培養物は、良性および悪性の乳房細胞の組織構造を再現するための選択の方法として浮上している。 3Dで増殖した細胞は、細胞外マトリックスからの重要な手がかりを保持し、生理学的に関連するex vivoでのシステム2,3を提供する。注目すべきは、2次元4と比較して、3Dで増殖させたときの細胞は異なる挙動を示すことを示唆している証拠が増えている。三次元培養効果的に3関与良性乳房表現型から悪性表現型を区別するための手段として、および細胞および分子のシグナル伝達を支えるために使用することができる。良性の上皮細胞の顕著な特徴の1つは、頂端細胞質内腔に向かってであり、基底細胞質は基底膜上に載るように、それらが偏光されることである。このapico - 基底極性がでたらめに周囲の間質に浸透細管を吻合及び分岐の細胞崩壊と形成を特徴としている浸潤性乳癌で失われます。良性腺と浸潤癌との間に、これらの病理組織学的な違いは、3D 6,7で再生することができる。単一ラウンド腺房構造の定量のような適切なリードアウトを用いて、又は他の分子および細胞生物学技術と組み合わせて、細胞増殖、およびアポトーシスの極性が検証分子マーカーの発現を差動に、3D culturEは、より良い悪性形質転換の際に、責任あるシグナリングの輪郭を描くための細胞の変化を理解するための重要なツールを提供することができます。

3D培養物を効果的に良性の乳房からのヒト乳房上皮細胞の悪性の表現型を区別するために使用することができる。マトリゲル上にプレートし、単一の良性細胞が簡単に位相コントラストイメージングを用いて1つの平面内でラウンドフラット腺房として画像化することができる組織化球状構造として成長。マトリゲル上に播種単一の悪性細胞が、偶然に成長する非常に高い屈折率を示し、一平...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

再構成基底膜上の乳房上皮細胞の三次元培養は、良性乳房のインビボアーキテクチャを再現するために、良性乳房表現型から悪性の表現型を区別するために有用な方法である。重要なことに、このシステムは、他の組織に浸潤癌を研究するために適用することができる。

三次元文化：乳細胞の悪性形質転換正常対腺房アーキテクチャを研究するためのツール

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

three-dimensional-cultures-tool-to-study-normal-acinar-architecture

Research

JoVE Journal

Medicine

19.8K ビュー.  University of Michigan Comprehensive Cancer Center. 再構成基底膜上の乳房上皮細胞の三次元培養は、良性乳房のインビボアーキテクチャを再現するために、良性乳房表現型から悪性の表現型を区別するために有用な方法である。重要なことに、このシステムは、他の組織に浸潤癌を研究するために適用することができる。 

ビデオ: Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

医学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

正常および病的脳機能を研究するために脳深部刺激でSaccadometryを使用します

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.