Name: a rapid filter insert-based 3d culture system for primary prostate cell differentiation
Uploaded: 2017-02-13T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

本研究では、T32（CA 9686から18）と、TL1（TL1TR001431）ポスドクの訓練助成賞（LT）、DOD PC140268（CA）、W81XWH-13-1-0327（CA）TR000102-04（CA）と同様でサポートされていましたU01 PAR-12から095（クマー）とP30 CA051008-21（ウェイナー）。サンプルの固定、切片と染色はロンバルディ総合がんセンター組織学および組織共有リソースで行いました。私たちは便利な議論のためのリチャード・シュレーゲルに感謝します。内容はもっぱら著者の責任であり、必ずしも国立がん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。

3D細胞培養インサートシステムの1.設立<ol><li> 6ウェルプレート（ 図1A）にダウン逆方向（フィルタ側まで）で2ポリカーボネート細胞培養インサートを配置します。 </li><li>インサートの底面側に水中0.1％ゼラチンの薄い層を適用し、無菌性を維持するために、生物学的安全キャビネットの中で（10〜20分間）乾燥させます。 </li><li>手順を繰り返し1.2インサートに0.1％ゼラチンの3アプリケーションの合計2回以上。 注：ゼラチンコーティングは、チャンバ間の拡散を制限するのに役立ちます。 </li><li>培養フラスコを洗浄するためのPBS 5 mLを加え、標準的な培養条件から主CRCを収集します。 <ol><li> 37℃で5分間、0.25％トリプシン-EDTA（T-75のためのT-25及び2mLのために1 ml）を用いて細胞をトリプシン処理。細胞を穏やかに取り除くのを助けるために、フラスコの側面をタップします。その後トリプシン反応を停止し、完全DMEMを5ml加えることにより細胞を収集するために進みます。 </li><li>集めます<html> 15 mLチューブ内の細胞。 4℃、188×gでチューブを遠心し、自動細胞カウンターまたは血球計数器を使用してカウントされます。 </li><li>メディア（CM）を条件付け250μL60万CRCを再懸濁し、適切に配向（フィルタ側を下に）カルチャーインサート（ 図1B）に追加します。これは、内部チャンバと呼ばれています。 注：CMは、照射J2細胞によって条件付けF-メディアであると以前に7、8、9、11を説明したように10μmのY-27632が含まれています。 </li></ol></li><li> 6ウェルプレートの外側室にCMの2ミリリットルを適用し、（少なくとも18時間）、37℃で一晩インキュベートします。 </li><li>慎重に1ミリリットルまたは200μLピペットを用いて内部チャンバにCMを削除して、ゆっくりと完全になるまで1 mLのピペットで内室に分化培地（DM）を追加します。細胞層を乱さないように注意してください。 </li></ol>3Dの文化のル &quot;&gt; 2。メンテナンス<ol><li>毎日文化を確認してください。 図2に示すように、健康な細胞が出現します。 </li><li>外側チャンバ内のCM毎日または細胞の代謝に応じて一日おきに変更します。 </li><li>内部チャンバのメディアは、色（黄色）を変更する場合は、慎重に1ミリリットルまたは200μLピペットで内部フィルター室にメディアを削除します。 </li><li>吸引先端を有する外側6ウェルプレート室でメディアを吸引除去します。 </li><li> 1 mLのピペットを使用して、ゆっくりと完全になるまで、内側室に新鮮なDMを適用します。こすりまたはその他の細胞層を落とさないように注意してください。 </li><li>新鮮なCM 2mLで外側室でメディアを交換してください。 </li><li> 2週間の文化を維持し、目的の生体分子の分離のための処理に進みます。 注：6ウェルプレートの各行は、6インサート（ 図1B）の合計は、DNのための十分な細胞を取得するために、実験ごとに処理する必要があります分析のために、RNAまたはタンパク質。 </li><li>外側チャンバ内を吸引メディアとは、2 mLのリン酸緩衝生理食塩水（PBS）ですすいでください。 </li><li>慎重に1ミリリットルまたは200μLピペットで内側チャンバからメディアを取り出し、500μLのPBSを追加します。スクレイピングまたはそうでなければ、膜上の細胞を乱すことは避けてください。 </li><li>吸引除去し、PBS外側チャンバから、慎重に1ミリリットルまたは200μLピペットで内部チャンバーからPBSを削除します。 PBSを除去した後、以下に詳述する適切なアプリケーションに直接進みます。膜を乾燥させないでください。アプリケーションを開始する準備ができるまで、文化はPBSで簡単に維持することができます。 </li></ol> 3.ペレットバンキング用フィルターの処理<ol><li>各内部チャンバ100μL、0.25％トリプシン-EDTAを加え、37℃で5分間インキュベートします。 </li><li> （puncを避ける上下にピペッティングとしながら、200μLピペットで膜表面上の細胞をこすり/簡単に言えば、慎重に攪拌）膜をチューリング。 37℃で1分間インキュベートします。 </li><li>フィルターを洗浄するために静かに上下最初の内室とピペットにCMの250μLを追加します。 </li><li>転送は、同じメディア条件が含まれており、上下にピペッティングを繰り返し、隣接するフィルタ室に細胞を再懸濁しました。 </li><li>単一条件の挿入ごとに、この手順を繰り返します。収集し、1.5 mL遠心チューブに細胞を移します。 </li><li>残りの細胞を収集するために、CMの追加の100-200μLで各内側チャンバを洗い流します。ステップ3.5で1.5 mLの遠心分離管に追加します。 </li><li> 5分間、4℃で微量で423×gで細胞をスピン。 </li><li>上清を吸引除去し、1000年μLPBSで一度細胞ペレットを洗浄します。 </li><li> 5分間、4℃で微量で423×gで再びスピン。 </li><li>吸引し、PBS、後で使用するために-80℃で凍結。 </li></ol> 4. RNAまたはDNAの単離のためのフィルタの処理<ol&gt; <li>内室にグアニジンチオシアネート - フェノール - クロロホルムまたは類似の抽出試薬の200μLを加え、簡単に上下にピペッティングすることにより、膜表面上の細胞を攪拌。 </li><li> 外室の乾燥を用いて室温で5分間抽出試薬でインキュベートします。 <ol><li>フィルタは、挿入から解離することができるように、上下に抽出溶液をピペットで解離し、フィルター膜を洗浄してください。 6ウェルプレートを傾けると、残留液体を吸引することにより、可能な限り抽出試薬をできるだけ多く収集します。 </li></ol></li><li>精製およびDNA、RNAまたはタンパク質の回収のための製造業者のプロトコルに従ってください。 </li></ol> 5.タンパク質単離のためのフィルタの処理<ol><li>氷上で6ウェルプレートにフィルタインサートを置きます。内側チャンバーに、10μLの1mMフッ化ナトリウム（NaF）、1mMのバナジン酸ナトリウム（NAV）、100mMのdithiothreを補充したタンパク質の溶解緩衝液を追加ITOL（DTT）および抗プロテアーゼカクテルの100μL。 </li><li> /攪拌上下にピペッティングしながら20μLピペットで膜表面上の細胞をこすり取ります。溶解緩衝液中に気泡を発生させることは避けてください。 </li><li> 10分間氷上でフィルターインサートで6ウェルプレートをインキュベートします。 </li><li>掻き繰り返し、次いで溶解緩衝液で膜表面をすすぎます。 </li><li> 6インサートからの溶解物を収集し、氷上で1.5 mL遠心管に移します。 </li><li>溶解緩衝液の追加の10μLと第一の膜をすすぎ、次のフィルタに転送します。 </li><li>すべての挿入を繰り返しステップ5.6、任意の残留溶解緩衝液を収集し、1.5 mLのチューブ（ステップ5.5）に追加します。 </li><li>さらに5分間氷上でサンプルをインキュベートします。 </li><li> 4℃で15分間、最高速度（テーブルトップ遠心分離機で16873×gで）でスピン。 </li><li>新鮮な1.5 mLのチューブにピペット溶解液上清。 </li><li>タンパク質濃度やSTOの分析に進みます-80℃での溶解物の再。 </li></ol> 6. H＆Eの処理及び免疫染色<ol><li>内側と外側のチャンバーに500μL、10％NBF（中性緩衝ホルマリン）の1 mLを加え、4℃で一晩インキュベートしましょう。 </li><li>外側室からNBFを吸引し、1 mLのヒドロキシエチルアガロース（HEA）1.5 mL遠心に処理ゲルを追加します。ゆっくりと低消費電力を使用して電子レンジでアガロースを融解し、アガロースが溶けるまで、10から20秒のパルスを繰り返しました。 </li><li>使用する準備ができるまでの凝固を防ぐために、37℃の温浴中でHEAを保管してください。 </li><li>内側チャンバからNBFを外し、内部チャンバに溶融HEAの25μLを適用し、アガロースは、2-5分間固化することができます。 </li><li>ウェット2発泡組織学パッドを10％NBF中と包埋カセットに一つのパッドを配置する（ 図3Dを参照）。 </li><li>インサートの底面側からフィルタを獲得するために（非常に鋭い、微尖った刃を持っている）＃11ブレードメスを使用しますチャンバーは、部分的にプラスチック製のインサート室からそれを解放します。 </li><li>ペトリ皿にNBFの少量（100〜200μL）を配置し、NBF（ 図3A）で部分的に外れフィルタを浸します。 </li><li> ＃10ブレードメスで、静かに完全に挿入バレル（ 図3B）からフィルタを取り除くために、インサートチャンバーの内側から、フィルタの中央押し付けます。まだバレルに取り付けられているフィルタの任意の部分がある場合には、メスを用いてそれを切断します。 </li><li> ＃10ブレードメス（ 図3C）で半分にHEAコーティングされたフィルタをカットします。 </li><li>ステップ6.4（ 図3D）で作製した組織学カセットにフォームパッドの上にフィルタの各半分を置きます。 </li><li>フィルタを挟んで、カセットに2つ目の10％NBF浸したスポンジを追加します。 </li><li>スナップNBFカセット、場所を密封し、一晩インキュベートします。 </li><li>切片や染色などのためにパラフィンブロックへのプロセスフィルター以前12記載。 </li></ol>

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初代細胞株は癌研究のための重要かつ急速に発展プラットフォームです。カルチャーインサートベースの3D培養系は、2週間の期間内に原発性前立腺のCRCの分化をサポートしています。 CRCフィルタ法は、前立腺の研究のための新しい媒体スループット法を表します。既存のマウスPDXモデルは時間がかかり、非常に高価であり、PDXサンプルの多くは、研究者によって開始実験とその有用性を制限...

個人にパーソナライズされた癌治療の同定および使用は、癌研究における第一の目標です。最近、新しいアプローチが開発されており、潜在的に識別し、パーソナライズされた治療法をテストし、両方の方法を提供し、初代細胞培養の確立でより容易にするために可能にします。例えば、Rスポンジンベース前立腺オルガノイドアプローチ1は、セルの条件付き再プログラミング（CRC）法が、商業外マトリクス（ 例えば 、マトリゲル）中の正常および転移性前立腺癌細胞の三次元（3D）培養を可能にしますジョージタウン2、3で開発は、より標準的な2D培養条件を利用します。具体的には、Rhoキナーゼ阻害剤（Y-27632）、照射J2マウス線維芽細胞フィーダー細胞の組み合わせは、ケラチノサイトのCRC 2の不定培養をもたらします。 CRC方法であります極めて堅牢、初代細胞株が正常に確立し、前立腺癌および他の多くの正常および悪性上皮組織3から無期限に維持したまま。重要なことは、私たちのCRC技術は、以前の薬物治療の数を失敗していた患者における再発性呼吸器乳頭腫症のための病因の基礎の迅速な同定を可能にしました。また、正常および腫瘍由来のCRCを使用して、FDAの成功同定は、薬物、ボリノスタットは、初期の組織生検の2週間以内に行われた承認されました。患者は、その病気4の成功した治療の結果、ボリノスタット上に置きました。 正常な前立腺は管腔、基底と希少な神経内分泌細胞5から構成されています。管腔細胞が腺の円柱上皮層を形成し、アンドロゲン受容体（AR）、ならびにサイトケラチン8と18とプロのような他の管腔マーカーを発現します状態特異的抗原（PSA）6。逆に、基底細胞が管腔層の下に局在し、サイトケラチン5およびP63が、AR 5を低レベルで発現しています。私たちは、7、8、9、その他10が正常に前臨床メカニズムの薬剤感受性調査で前立腺CRCを使用しています。標準2D組織培養条件下で増殖させた場合しかし、これらの細胞は、完全に10シグナリング ARに係合することができません。免疫不全マウスの腎被膜下に置かれたとき重要なのは、CRCは許容する環境に置かれたとき、前立腺CRCがその系譜コミットメントを維持していることを示す正常前立腺腺のアーキテクチャと機能を取り戻しました。ここで説明したフィルタインサート基づく細胞培養系の開発が急速に（2週間）のために証明さbと前立腺のCRCのインビトロ分化を可能にします増加したARとAR標的遺伝子の発現と同様には、P63のレベルを減少させたY。 使用されるフィルタインサートは、哺乳動物細胞の培養を支持することができるポリカーボネート膜（孔径0.4μm）を含みます。システムは、開発され、正常および悪性前立腺のCRC、6ウェル培養皿とフィルタインサートを利用します。 J2セル11によって調整細胞培養培地を上部チャンバーに下部チャンバと前立腺分化培地中に置かれます。本明細書に記載の技術は、個別化医療の目標と一致して2週間の時間枠内に前立腺管腔細胞分化をサポートしています。文化の総合的、分子遺伝学および細胞プロファイリングを可能にする方法論を開発することも不可欠でした。フィルター表面から放出された細胞からのDNA、RNAおよびタンパク質を単離するための手法が開発され、正確な反復サンプル処理のための合理化されています。フィナLLY、埋め込みを有効にするフィルタを削除切片とH＆E、免疫組織化学および免疫蛍光染色のために必要な方法論は、完全に記載されています。

<table><tbody><tr><td>Corning Costar Snapwell Culture Inserts</td><td>Corning</td><td>3801</td><td></td></tr><tr><td>Millicell Cell Culture Inserts</td><td>Millipore</td><td>PIHP01250</td><td></td></tr><tr><td>Multiwell 6 Well</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Gelatin 0.1% in water</td><td>Stemcell Technologies</td><td>7903</td><td></td></tr><tr><td>Sterile Saftey Scapel 10 Blade</td><td>Integra Miltex</td><td>4-510</td><td></td></tr><tr><td>Sterile Standard Scalpel 11 Blade</td><td>Integra Miltex</td><td>4411</td><td></td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>Thermofisher Scientific</td><td>89900</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Fishcer Scientific</td><td>S299-100</td><td></td></tr><tr><td>Sodium Vanadate</td><td>Fishcer Scientific</td><td>13721-39-6</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3483123</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A)</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Thermofisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>Glutamine</td><td>Thermofisher Scientific</td><td>25030081&amp;nbsp;</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>F-12 Nutrient Mix Media</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Y-27632 dihydrochloride Rock inhibitor</td><td>Enzo</td><td>ALX-270-333-M025</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td></td></tr><tr><td>EGF</td><td>Thermofisher Scientific</td><td>PHG0315</td><td></td></tr><tr><td>Insulin</td><td>Thermofisher Scientific</td><td>12585-014</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C3012</td><td></td></tr><tr><td>Gentamicin</td><td>Thermofisher Scientific</td><td>15710-064</td><td></td></tr><tr><td>Fungizone</td><td>Fisher Scientific</td><td>BP264550</td><td></td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>Histogel Specimen Processing Gel (hydroxyethyl agarose (HEA))</td><td>Thermo Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Non-Adherent Dressing</td><td>Telfa</td><td>KDL2132Z</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Sigma</td><td>SIAL0167</td><td></td></tr><tr><td>HISTOSETTE II Tissue Processing / Embedding Cassettes</td><td>Crystalgen</td><td>CG-M492</td><td></td></tr></tbody></table>

a rapid filter insert-based 3d culture system for primary prostate cell differentiation

条件付きで再プログラムされた細胞（CRCが）初代培養細胞と患者由来細胞株の大規模な「生きたバイオバンク」を開発する能力のための持続可能な方法を提供します。上皮細胞の多くのタイプのために、様々な3次元（3D）培養アプローチは、改善された分化状態をサポートすることが記載されています。 CRCが、それらが分離されている、そこから組織への系統コミットメントを保持するが、それらは通常の2次元（2D）培養条件下で増殖させた場合起源の組織に関連分化マーカーの多くを発現しません。前立腺癌の研究のための患者由来のCRCのアプリケーションを強化するために、3D培養形式は、正常および腫瘍由来の前立腺上皮細胞の両方の急速な（2週間合計）管腔細胞分化を可能にするように定義されています。ここで、フィルタインサートベースのフォーマットは、正常および悪性の前立腺のCRCの両方の培養および分化のために記載されています。デ免疫組織化学および免疫蛍光染色のための細胞の収集および処理に必要な手順の尾説明が提供されます。総称して、プライマリCRCラインと組み合わせて説明した3D培養フォーマットは、生物試料ベースの前立腺研究のためのスループットモデルシステムを高するための重要な中長期を提供します。

細胞培養インサートに基づくシステムは、管腔細胞分化をサポートしている前立腺のCRCの3D培養物を作製するための比較的単純かつ迅速な方法です。システムの概略フィルタインサートの下面にゼラチンコーティングの適用を強調し（ 図1A）が示されています。インサートはゼラチンのみの適用のために覆されています。 図1Bにフィルタが培?...

Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

原発性前立腺細胞の分化のための迅速なフィルターを挿入ベースの3D培養システム

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

a-rapid-filter-insert-based-3d-culture-system-for-primary-prostate

Research

JoVE Journal

Cancer Research

8.5K ビュー.  Lombardi Comprehensive Cancer Center. Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

ビデオ: A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

前立腺癌および他の年齢に関連した病状または代謝疾患における生物物理トリガーとして基底膜剛性を研究する方法

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

がん研究

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

培養 3 D 回転楕円体モデルでのマウス前立腺の葉の解剖組織学的特性及び

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

前立腺癌の遺伝子組み換えマウスモデルからの腫瘍オルガノイドの生成

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遺伝学

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

オルガノイド培養を用いたマウス前立腺上皮細胞の分化能力の評価

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

遺伝子型と突然変異プロファイルを薬理学的応答に変換するツールとしての前立腺オルガノイド培養

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3次元スフェロイド培養からの幹様細胞の分離

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

処理と人間プライマリ前立腺培養の評価

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

生物工学

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

ヒト血液脳関門をモデル化した三重培養細胞系

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

生物学

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...