Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
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Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

私たちはシェリルSchaeberleによって社説貢献を大幅に感謝しています。この研究は、エイボン補助金＃02-2009-093と02-2011-095、およびAMSへNIEHS / NIH ES 08314によってサポートされていました。内容はもっぱら著者の責任であり、必ずしも環境健康科学研究所や国立衛生研究所の公式見解を示すものではありません。

試薬の調製<ol><li> 10 -3 Mストック溶液を作るために、エタノール中で合成黄体ホルモンのプロメゲストン（R5020）および17-βエストラジオール（E2）を溶解します。 1mg / mlのストック溶液を作製するために蒸留脱イオン水にプロラクチンを溶解します。 10 -2 Mの原液を作るためにDMSOに抗エストロゲンICI 182,780を溶解します。最大6ヶ月間-20℃でこれらのソリューションを保管してください。 </li><li>チャコールデキストラン（CD）は、血清およびCDFBS媒体を剥奪しました： <ol><li> 30分間57℃の水浴中でウシ胎児血清（FBS）を熱不活性化します。 注：このプロトコルの残りの部分のみを使用して酸洗浄ガラス製品を。 </li><li>必要な炭の量を計算します。粉末活性炭は、炭による変位を補償するために、血清の量よりも10％以上のボリュームを5％重量/体積を計量使用します。 </li><li>血清の量と同じ量を使用して、冷脱イオン蒸留水で2回木炭を洗ってください。各洗浄cの木炭をペレット化するために2分間50×gでentrifuge。洗浄の間に水を除去します。最後の炭ペレットに0.5％重量/体積デキストランT-70を追加します。 5分間100×gで蒸留、脱イオン水および遠心分離機で再懸濁します。水を吸引。 </li><li>ペレットに血清の適切な量を追加し、血清中に木炭デキストランペレットを再懸濁。 60分間37ºCで、（6 RPM）転動しながら、インキュベートします。 20分間、2000×gで遠心分離します。上清が濁って表示された場合は、遠心分離を繰り返します。しかし、血清は、現時点では完全には明らかではないかもしれないが、濾過中にクリアされます。 </li><li>より大きな粒子状物質を除去するために0.45ミクロンフィルターを通して再び0.80ミクロンフィルターを通して濾過し、。最後に、0.20ミクロンフィルターで濾過滅菌します。ストアは、-20ºCでガラス管またはT-25培養フラスコ内CDFBSを濾過しました。 </li><li>チャコールデキストランウシ胎児含有培地（CDFBSメディア）：フェノールレッドを含まないDMEMの375ミリリットルとDMEMの125ミリリットルを混ぜます/ F-12。 37.5ミリリットルを取り外し、チャコールデキストランウシ胎児血清の同じボリュームに置き換えます。 2 mMの最終濃度に10 6 U / mlのペニシリンおよびL-グルタミンを追加します。得られた培地は、75％のDMEM、25％DMEM / F-12、2mMのL-グルタミン、及び10 6 U / mlのペニシリン、FBS剥離7.5％チャコールデキストランです。 </li></ol></li><li> 10X PBS、1N NaOH及び蒸留脱イオン水を準備します。必要とされるコラーゲン溶液の量に応じて各の十分なボリュームを準備します。フィルタリングによって、すべてのソリューションを滅菌します。 1ヶ月まで4℃で10×PBSと滅菌蒸留脱イオン水を保管してください。 1N NaOH溶液を保管しないでください。 </li><li>製造元によって提供される「コラーゲンI、ラットの尾の代替ゲル化手順」のプロトコルに従って、1mg / mlの最終濃度でコラーゲン溶液を準備します。各ゲルは、コラーゲン溶液1.5mlで作られています。例えば、準備するコラーゲン溶液18.5ミリリットル（ピペット操作の間の損失を考慮して0.5ml）で調製12ゲル。すぐにコラーゲン溶液を使用するか、最大2-3時間、氷上で保持します。 </li><li>カーマインミョウバン染料： <ol><li>蒸留水500ミリリットルで1グラムカーマインと2.5グラム硫酸アルミニウムカリウムを兼ね備​​えています。 20分間撹拌しながら沸騰。吹きこぼれを避けるために注意して観察してください。バック追加の蒸留水で500ミリリットルへの最終的な音量を調整します。 </li><li>抗菌活性のためのチモールの結晶を追加し、光から保護するためにアルミホイルで瓶をカバーしています。各使用前に実験室グレードの濾紙を通して濾過します。までの3ヶ月間のカルミンソリューションを再利用します。光から保護し、室温で保存し、。 </li></ol></li><li>コラゲナーゼ溶液：w / vの0.125％の濃度でDMEM / F12培地中でコラゲナーゼを溶解します。このソリューションを保管しないでください。 </li></ol>ラットテールコラーゲンタイプでT47D細胞の2次元文化1ゲルおよびホルモン治療注：4℃で滅菌した血清学的ピペットとピペットチップを保持します。 <ol><li> Prepa単一細胞懸濁液を再度、細胞カウントを行います。分割T47D細胞培養物は、それらが70％コンフルエンスに到達する前に。必要な細胞の量を含むボリュームを遠心。 1つのゲルは、約75,000 T47D細胞を含むべきであることを考えてみましょう。 </li><li>コー​​ルド・ピペットを使用して、コラーゲンの適切な量で細胞ペレットを再懸濁します。各ゲルはコラーゲン1.5mlので構成されていることを考えます。気泡を導入することを避けるために穏やかに細胞とコラーゲンを混ぜます。 </li><li>細胞分布上の静電気の影響を防止するために、プレートを開梱した後、静的低減ブラシで12ウェルプレートの境界、上部と底部を磨きます。また、静的低減brush.Gentlyでブラッシングされた別の12ウェルプレート（ブランクプレート）の上に、このプレートを置き、フードの作業面との接触時の静電荷の蓄積を回避するために、ミックスの1.5ミリリットルを注ぎます12ウェルプレートの各ウェルに。 </li><li> 30分間、37℃のインキュベーターでプレートを置きます。目を削除しますインキュベーターからの電子プレートとフードの中に戻って置きます。 </li><li> P200の滅菌チップを用いて、慎重に井戸の国境から緩やかな円を描くようにゲルを切り離します。ゲルはまた、井戸の底から切り離す必要があります。 </li><li> CDFBS培地で希釈したホルモンをウェルに添加します。 10 -7 Mの最終濃度で、10 -10 Mの最終濃度およびプロラクチンでE2とR5020を使用してください。対照として、追加されたホルモンなしCDFBSメディアを使用していますが、まだ使用ホルモン株式に等しいDMSOの最高のボリュームを含みます。各ウェルは、メディアの1.5ミリリットルが含まれていることを確認してください。 37℃、5％CO 2、100％の内部3D培養物を置き 加湿組織培養インキュベーター。 2日おきにメディアを変更し、1または2週間の培養を維持します。 </li></ol>全体のマウント3.ゲル法<ol><li>培養培地を除去し、各ウェルにPBSの1.5ミリリットルを追加します。 </li><li>スライドにゲルを転送します。曲線を用いて半分にゲルをカットD手術用ブレード。半分で処理を継続し、組織学的分析のために残りの半分を保存します。 </li><li>全体スライドにマウントするための半ゲルを転送します。 5分間ゲル乾かした後、10％ホルマリンを含むコプリンジャーにスライドを転送します。 注：すべてのインキュベーションは室温で行われるべきです。 </li><li>シェーカーO / N上のコプリンジャー中でゲルを修正しました。 </li><li>ウォッシュは、シェーカー上で10分間ずつ2回PBSでスライドします。転送が70％エタノールにスライドします。シェーカーで1時間インキュベートします。カーマインミョウバンO / Nへの転送スライド。 </li><li>次の日は、1時間ごとに70％エタノール、95％エタノールおよび100％EtOH中でシェーカー上でスライドを脱水。転送は1時間ごとに二回キシレン、シェーカー上で維持するためにスライドします。ゲルおよび厚さ1.5mmのカバースリップをマウントするために、トルエンベースの封入剤を使用してください。 </li><li>定期的な光（顕微鏡やステレオスコープ）の下で全体のマウントを調べます。形状および内腔のより詳細な分析のために、共焦点顕微鏡を使用し、ヘリウムネオン633nmのレーザーでカーマイン色素を可視化。 </li></ol>組織学分析のための処理4.ゲル法<ol><li> 10％ホルマリンを含む20ミリリットルのガラスバイアルにゲルの残りの半分を転送し、シェーカーO / Nで固定します。 </li><li>シェーカー上で10分間ずつ2回PBSでゲルを洗浄してください。パラフィン包埋のために処理カセットに置きゲル。 </li><li> 70％エタノールに移しゲルは、1時間インキュベートし、1時間95％EtOHで、次の1時間の新鮮な95％エタノールに変更し、100％エタノールを1時間、および1時間の新鮮な100％エタノールに変更します。 1時間の新鮮なキシレンで再びこの時点では、ゲルは、室温での100％EtOH O / Nに格納されてもよいし、1時間キシレンでインキュベートし。 </li><li>転写カセットをさらに2回、新鮮なパラフィンで繰り返し、真空インキュベーター中で60℃で1時間パラフィンします。 </li><li>きれいなパラフィンでプラスチック金型を埋めます。 、カセットを開き、ゲルを除去し、パラフィンを含むプラスチック金型に入れてください。パラフィンでいっぱいにし、上部にあるラベルを含むカセットの上部を追加し、ブロック冷まします。 </li><li>プラストを削除しますICカビやセクションミクロトームを使用してブロック。 HE染色および免疫細胞化学のために5μmの厚さの切片を取得します。 </li></ol>コラゲナーゼ処理を用いてゲルからの細胞からの細胞の5抽出<ol><li>井戸からゲルを外し、PBSを含むペトリ皿に移します。簡単にゲルをすすぎます。 </li><li> 0.125％コラゲナーゼの2ミリリットルを含む15ミリリットルチューブにゲルを転送します。小片にゲルを破壊するために、アップピペットダウン4-5回。 </li><li> 25分間37℃でインキュベートする（ゲルは溶解し、細胞が懸濁液中にあるまで） </li><li>血清添加した培養培地2mlを追加します。遠心分離（千×gで、5分）、上清を捨てて細胞を収集し、細胞を再懸濁し、カウントされます。 </li></ol>

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The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.</...

標準的な2Dの文化とは異なり、3次元細胞培養サロゲートモデルは、生理学的に関連するコンテキスト、1組織に似における上皮細胞の挙動の研究を可能にします。乳腺の3D培養物は、乳腺の発達と腫瘍の多くの側面を解明に役立ちました。しかし、現在入手可能な三次元培養モデルのほとんどは、タスクに使用したヒト上皮細胞株は、ホルモン受容体の発現6,7,9を欠いているので、ホルモン作用を研究するためには不適当です。 ここで、我々はホルモン作用12を研究するのに適しているヒト乳房上皮の三次元培養モデルについて説明します。このモデルは、もともと乳房の浸潤性乳管癌で54歳の女性患者から得られた胸水から由来した市販のホルモン応答ヒト乳房上皮細胞株、T47D 3,11,13を 、使用しています。私たちは、マトリックスとしてラット尾コラーゲンタイプ1を使用します。この3DカルトUREモデルは、ヒト乳房上皮細胞上の3つの主要なmammotropicホルモン（エストラジオール、プロメゲストン（プロゲステロンの類似体）、およびプロラクチン）の作用の研究に適しています。ホルモン誘導性の上皮形態は、形態学的分析12時間にわたって定量的に評価することができます。 適切な播種密度は、これらの3D培養物は2週間保持することができます。この時点で、構造物の開発は、上皮形態に対するホルモン作用の堅牢な定量的評価のために十分です。ゲルは、細胞増殖のための以前の時点で回収してもよいし、遺伝子発現解析します。さらに、このモデルは、連続ホルモン療法の効果を試験するのに適しています。例えば、次の週の間のホルモンの他のホルモン/組み合わせで最初の週と交換時のエストラジオールで処理した後。このようなICI 182,780などのエストロゲン様化合物および抗エストロゲンの効果は、また、STUすることができますこの三次元培養モデル12を使用して死亡しました。

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

図1は、ホルモン感受性3D培養液を調製するための手順をまとめたものです。上皮構造は、単独E2の存在下および他のホルモンとの組み合わせで2週間培養ゲルの全マウントに観察されます。何のホルモンを培地（CDFBS培地）（ 図2）に追加されていない場合にのみ、単一の細胞または2-3細胞のグループが存在しています。この状態は、陰性対照としての役割を果たす。 ...

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

ヒト乳腺上皮のホルモン応答性の3D培養モデル

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

a-hormone-responsive-3d-culture-model-human-mammary-gland

Research

JoVE Journal

Bioengineering

8.7K ビュー.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

ビデオ: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

マウス乳房上皮HC11細胞とEpH4細胞の分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

がん研究

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 ...

医学

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

三次元培養におけるリン脂質メディエーター誘導形質転換

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

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

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

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 ...

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 ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

継代なしの正常および腫瘍乳腺組織からのマウスおよびヒト上皮オルガノイドの生成とイメージング

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

生物工学

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

微小トリプシン法によるモザイク乳腺オルガノイドの生成

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

発生生物学

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...