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 border="0" cellpadding="0" cellspacing="0" width="1344">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">12-well Tissue Culture Plates (Falcon)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">08-772-29</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">15 ml polystyrene conical Tubes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">14-959-49D</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Activated Charcoal</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C-5510</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Carmine Alum</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C1022-100G</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Collagenase, Type 3</td>
			<td style="width:249px;">Worthington</td>
			<td style="width:171px;">S0C11784</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Confocal Microscope</td>
			<td style="width:249px;">Zeiss</td>
			<td style="width:171px;">LSM510</td>
			<td style="width:488px;">Equiped with HeNe 633nm laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Dextran T-70</td>
			<td style="width:249px;">Abersham/Pharmacia</td>
			<td style="width:171px;">17-0280-01</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">DMEM/F-12, HEPES, no phenol red&#160;</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11339-021</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:436px;">DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11054-020</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">17-&#946;-Estradiol</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:171px;">3301</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Ethanol</td>
			<td style="width:249px;">Koptec</td>
			<td style="width:171px;">V1001</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Fetal Bovine Serum</td>
			<td style="width:249px;">Hyclone</td>
			<td style="width:171px;">SH30070.03</td>
			<td style="width:488px;">For use with hormones, must be Charcoal Dextran stripped&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:436px;">Filters (115ml)</td>
			<td style="width:249px;">Nalgene</td>
			<td style="width:171px;">380-0080, 245-0045, 120-0020</td>
			<td style="width:488px;">0.88, 0.45, 0.20 micron, respectively&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Formalin, 10%</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SF93-20</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">L-Glutamine (200 mM)</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">25030-081</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">ICI 182,780 (fulvestrant)</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">I4409-25MG</td>
			<td style="width:488px;">Dissolved in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Microtome&#160;</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">RM2155</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue embedding media</td>
			<td style="width:249px;">McCormick Scientific</td>
			<td style="width:171px;">39502004</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Penicillin</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">7794-10MU</td>
			<td style="width:488px;">Dissolved in 10 ml of distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Permount</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SP15-500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">P3813-10PAK</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Prolactin</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">L4021-50UG</td>
			<td style="width:488px;">Dissolved in distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Promegestone</td>
			<td style="width:249px;">Perkin Elmer</td>
			<td style="width:171px;">NLP004005MG</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Rat-Tail Collagen&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">354236</td>
			<td style="width:488px;">Lots may contain varying concentrations, note accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Scalpel</td>
			<td style="width:249px;">Miltex</td>
			<td style="width:171px;">4311</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Semi-enclosed Benchtop Tissue Processor</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">TP1020</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sodium Hydroxide</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">S5881</td>
			<td style="width:488px;">Prepare 1N NaOH stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">StaticMaster Anti-static brush&#160;</td>
			<td style="width:249px;">Amstat</td>
			<td style="width:171px;">C3500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Stripette Serological Pipettes&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">4101</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">T-25 flasks</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">430168</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue Cassettes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">15-200-403E</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Wheaton Vials, Glass, 20mL</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">03-341-25D&#160;</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Xylene&#160;</td>
			<td style="width:249px;">VWR</td>
			<td style="width:171px;">95057-822</td>
			<td style="width:488px;"></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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D楕円体の生成のための簡単​​なハンギングドロップ細胞培養プロトコル

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

生物工学

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

ウサギの乳腺への管内配達

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

ひと膵臓の神経島国的なネットワークの高解像度の 3 D イメージング

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 D ダイナミック文化システムの多層間葉系幹細胞シートの構築

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

乳腺からの照射オルガノイドの成長とキャラクタリゼーション

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

ヒト乳腺上皮のホルモン応答性の3D培養モデル

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この動画の章

3D楕円体の生成のための簡単なハンギングドロップ細胞培養プロトコル

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

人間の3次元の組織工学

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

ウサギの乳腺への管内配達

ひと膵臓の神経島国的なネットワークの高解像度の 3 D イメージング

3 D ダイナミック文化システムの多層間葉系幹細胞シートの構築

乳腺からの照射オルガノイドの成長とキャラクタリゼーション

ヒト乳腺上皮のホルモン応答性の3D培養モデル

要約

さらに動画を探す

この動画の章

3D楕円体の生成のための簡単​​なハンギングドロップ細胞培養プロトコル

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

人間の3次元の組織工学

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

ウサギの乳腺への管内配達

ひと膵臓の神経島国的なネットワークの高解像度の 3 D イメージング

3 D ダイナミック文化システムの多層間葉系幹細胞シートの構築

乳腺からの照射オルガノイドの成長とキャラクタリゼーション

3D楕円体の生成のための簡単なハンギングドロップ細胞培養プロトコル