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>

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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。

<table><tbody><tr><td>Growth Factor Reduced Matrigel</td><td>BD Biosciences</td><td>354230</td><td>Avoid repeat freeze thaw&amp;nbsp;</td></tr><tr><td>Lab-Tek glass hamber slides 8-well</td><td>Thermo Fisher Scientific</td><td>177402</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Lab-Tek glass chamber slides 2-well&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>177380</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Goat anti mouse F(ab&amp;acute;)&lt;sub&gt;2 &lt;/sub&gt;fragment</td><td>Jackson Immunoresearch</td><td>115-006-006</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Fluorescent conjugated Alexa antibodies</td><td>Molecular Probes</td><td></td><td>Please look at molecular probes website</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Molecular Probes</td><td>P36935</td><td></td></tr><tr><td>3D Culture Cell Harvesting kit</td><td>Trevigen</td><td>3448-020-k</td><td></td></tr><tr><td>Anti-Apha-6-integrin</td><td>Fisher Scientific</td><td>MAB1378</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-GM130</td><td>BD Biosciences</td><td>610822</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-phospho-histone 3 (Ser10)FITC Conjugated</td><td>Fisher Scientific</td><td>16-222</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-Cleaved caspase 3 (Asp 175)</td><td>Cell Signaling</td><td>9661s</td><td>1:50 dilution for IF</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>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

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

単球と細胞外マトリックスの主要な乳癌細胞間コミュニケーションを分析抽出 (ECME)-ベースの三次元システム

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

がん研究

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF -β誘発性浸潤を測定する回転楕円体アッセイ

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

医学

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

真似をして 3次元細胞培養の膵腺房の上皮化生を操作します。

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

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 ...

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 ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

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

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 ...