We thank Mr. O. Rios for his technical assistance.

この研究は、医療機関と国立工科大学の研究アドバンス研究センターの地域の倫理委員会によるヒト材料の使用が承認されました。 1.腫瘍サンプル取得<ol><li>トランス蝶形骨の外科処置14を介して患者から下垂体腺腫の組織生検を取得します。 </li><li>下垂体腺腫サンプルを採取し、新鮮な10％​​ウシ胎児血清（FBS）で富化199培地（M199）、0.6 mg / mlで、炭酸水素ナトリウム、2.4 mg / mlでHEPESおよび1％の50 mlを含む滅菌ガラス瓶に置きpH7.4での抗生物質（ペニシリン/ストレプトマイシン）。氷の上のガラス瓶にサンプルを維持します。 注：腫瘍組織は、手術後数時間を処理することができます。 </li><li>氷の上のガラス瓶で研究室に腫瘍サンプルを輸送します。 </li></ol>酵素組織解離ソリューションの調製、トリプシン阻害剤溶液とアルギン酸ソリューション注：前の細胞培養プロシージャに次の溶液を調製します。使用前に、0.22μmのメンブレンフィルターを使用して、すべてのソリューションをフィルタリングします。 <ol><li> M199-無血清5mlにコラゲナーゼ（タイプI）5mgを溶解させることによりコラゲナーゼ1％溶液を準備します。 </li><li> PBS-EDTA、0.3％の10mlのトリプシン0.01グラムに溶解することによりトリプシン0.01％の溶液を調製します。 </li><li> M199-無血清20mlに大豆トリプシンインヒビターの2ミリグラムを溶解し、0.1％トリプシン阻害剤溶液を準備します。 </li><li>トリプシン阻害剤溶液の4ミリリットルでのDNAse I 134 U / mlの20μlのを溶解することによりDNase溶液を準備します。 </li><li> 155 mMのNaClを40ミリリットルにEGTAの22.8ミリグラムを溶解することによりEGTA溶液（1.2 M）を準備します。 pHを7.4に調整します。 50ミリリットルの最終容量に進みます。 </li><li> 155 mMのNaClを50ミリリットルで238ミリグラムのHEPESを溶解させることによってHEPES溶液（20 mm）を準備します。 </li><li> 1.2を溶解して1.2％のアルギン酸塩溶液を調製しますHEPES溶液100mlのGアルギン酸ナトリウム。アルギン酸塩溶液をオートクレーブ。 </li><li> pHを7.4に調整する50mlのHEPES溶液中のCaCl 2の750ミリグラムを溶解させることによってカルシウム溶液（102ミリモル）を準備</li></ol> 3.初代細胞培養およびアルギン酸塩カプセル化<ol><li>クリーンベンチ内部には、約20ミリリットルを含まないPBSのCa 2+およびMg 2+、pH7.4の（PBS *）のを含む35ミリメートルペトリ皿に腫瘍組織を置きます。 </li><li>手術用ピンセットで組織を取り、新鮮なPBS *を含む別のペトリ皿に置き、静かに組織を洗います。赤血球および破片が削除されるまで、この手順を繰り返します。 </li><li>手術用ピンセットを使用して、下垂体腺腫の組織を保持し、小さな手術用ハサミで小片にそれをカット。丸みを帯びたエッジをパスツールピペットを用いて、RTで10分間68×gで、15mlチューブに遠心分離した組織フラグメントおよびPBS *を転送します。 注：パスツールのエッジを丸くするにはピペットは、わずかにブンゼンバーナーでピペットの先端を火炎。 </li><li>パスツールピペットを使用して、* PBSを削除し、コラゲナーゼ溶液（組織の65ミリメートル3に対して約5ミリリットル）3〜5mlのを追加し、反転させて2〜3回混ぜます。一定の回転で37℃で30分間コラゲナーゼで組織をインキュベートします。 </li><li> RTで10分間、68×gでチューブを遠心し、パスツールピペットでコラゲナーゼを削除します。 </li><li> DNアーゼの5ミリリットルを追加し、反転させることにより2〜3回混ぜ、次いで、室温で10分間68×gでチューブを遠心します。 注：組織が完全に解離していない場合は、37℃で3分間トリプシンで第二の酵素消化を行います。トリプシン阻害剤溶液の2倍量の添加によって消化を停止します。 </li><li>パスツールピペットで上清を吸引し、廃棄します。 EGTA溶液の1体積でペレットを再懸濁し、アルギン酸塩溶液の2倍量と混合。 </li><li>静かに細胞懸濁液とアルギン酸塩を混ぜますパスツールピペットでピペッティングすることもできます。 21 G針で無菌注射器を取り、プランジャーを削除します。アルギン酸塩および細胞溶液で注射器をロードするためにピペットを使用しています。 </li><li>ゆっくりシリンジにプランジャーを挿入し、慎重に約30ミリリットルのカルシウムが豊富なソリューションのを含有するガラスビーカーにアルギン酸細胞液滴ずつを分注します。 </li><li>約5cmまでのCaCl 2溶液に注射器の針を置きます。球状のビーズを形成するためのCaCl 2と接触するアルギネート細胞懸濁液ゲル。 5分間カルシウム溶液中にアルギン酸ビーズを保管してください。 注：ビーズがドロップされたガラスビーカーをかき混ぜます。互いに付着するのを防ぐために、円を描くように手でゆっくりと移動します。 </li><li>慎重にパスツールピペットでカルシウム溶液を除去し、FBSの20％で強化されたM199 3〜5ミリリットルで二回ビーズを洗浄します。 </li><li>定期的なT25組織培養にアルギン酸ビーズを転送無菌スパチュラを用いてフラスコを5％CO 2の加湿インキュベーター中、37℃でM199 4-5ミリリットル、20％FBSで富化インキュベート加えます。二日おきに培地を変更します。 </li></ol> 4.アルギン酸からの細胞を解放します注：前のアルギン酸ビーズから細胞を遊離するために、ポリ-D-リジンでコート直径13mmのカバースリップを。 注意：SULPHURIC ACIDは、腐食性の高い酸であり、APTSは目と皮膚への炎症を引き起こす可能性があります。これらの試薬を処理するためにゴム手袋を使用しています。 <ol><li> 、ドラフト内でポイントピンセットを用いて室温で1時間、20％の硫酸水溶液を含むガラスペトリ皿にカバースリップを浸します。 </li><li>硫酸をデカントし、5分毎に、蒸留水でカバーガラスを5回洗浄します。ポイントピンセット場所RT 15で5分間、0.1MのNaOHを含むペトリ皿の中のきれいなカバーグラスを使用。 </li><li> NaOHをデカントし、蒸留で1回洗います水を吸引し、カバースリップを乾燥、水を導きました。 </li><li> 4分間のAPTS（アミノプロピルトリエトキシシラン）で被覆するためにカバーグラスをマイクロピペットを使用して蒸留水でペトリ皿をフラッディング（6カバースリップのための500μLを使用します）。すばやく水を吸引し、水が下に浸透することを可能にする点ピンセットでカバースリップを移動し、蒸留水で洗浄し続けています。 </li><li>水を吸引し、カバースリップを乾燥、清潔なペトリ皿にカバースリップをポイントピンセットの場所を使用し、3回、45秒のそれぞれの間にマイクロ波の内側にそれらを滅菌します。ポリ-D-リジン（0.1 mg / mlで）を100μgに滅菌水1000μLを加えます。 </li><li>室温で5分間、ポリ-D-リジンの約50μlのカバースリップをインキュベートします。完全にポリ-D-リジンを吸引します。 注：培地中の可溶性ポリ-D-リシンが細胞増殖を阻害することができるので、これは重要です。 </li><li> 5分ごとにカバースリップを滅菌水で5回洗浄します。目を吸引erile水とカバースリップは、クリーンベンチ内部に乾燥させます。 </li><li>アクチン細胞骨格を分析するために、アルギン酸塩から下垂体細胞を遊離させます。 1ミリリットルピペットチップを用いて培養フラスコからアルギン酸ビーズの一部を取り、15mlチューブに入れます。 </li><li> 55 mMクエン酸ナトリウムの5 mlを加え、室温で10分間、68×gで遠心分離（323.6のNa 3 C 6 H 5 O 7 mgの、2 H 2 O 20 mLのHEPES溶液を溶解します）。上清を吸引し、廃棄します。 </li><li> 20％FSBで富化暖かいM199の1ミリリットル中にペレットを再懸濁;静かにパスツールピペットで細胞懸濁液を混ぜます。 注意：ファロイジンおよびDAPIは有毒です。これらの試薬を扱うために手袋を使用します。 </li><li>細胞をカウントし、1.5ミリリットルチューブに0.4％トリパンブルー溶液の125μlのを転送します。 M19975μlのと最後のステップ（希釈係数= 5）で得られた50μlの細胞懸濁液を追加します。完全に混合。 </li><li> PLACするためにマイクロピペットチップを使用します血球計数器チャンバー内のトリパンブルー細胞懸濁液の電子約10μlの。 </li><li>位相コントラスト照明付き倒立顕微鏡を用いて、血球計数器カウンターで細胞をカウントします。 1ミリメートルの中央広場、4 1ミリメートル角の正方形のすべてのセルを数えます。希釈係数により平方あたりの平均数を乗算してミリリットル当たりの細胞密度を得る- 。10 4、例えば平方あたりの平均数は45セル×5×104 = 2.25×10 6細胞/ mlである場合。 注：血球計数器室の10乗で細胞をカウントします。下と右の両側に中央の線に触れて細胞をカウントしないでください。 </li><li>ポリ-D-リジンで被覆された直径13mmのカバーガラス上で35,000細胞をシード。 </li></ol> 5.アクチン細胞骨格のアレンジメント<ol><li>培養48時間後、パスツールピペットで培地を除去し、素早くPBSのカバースリップあたり500μlを添加、直ちにPBSを削除します。 </li><li>トンを修正し、透過処理37℃で10分間固定緩衝液（0.1M PIPES、pHが6.75、4％PEG-6000、1mMのEGTA、1mMの硫酸マグネシウム、0.5％トリトンX-100および2％ホルムアルデヒド）のカバースリップあたり500μlの彼の細胞C言語</li><li>固定バッファを削除し、RTで30分間、PBS中の3.5％パラホルムアルデヒドのカバースリップあたり500μlを添加します。パラホルムアルデヒド溶液を除去します。 </li><li> 5分ごとに、PBSのカバースリップあたり500μLを添加することにより、カバーグラスを3回洗浄します。一定の撹拌で、室温でカバースリップを洗い流します。 </li><li> 、10分間、50mMの塩化アンモニウムで細胞をインキュベート塩化アンモニウム溶液を吸引し、ステップ5.4を繰り返します。 </li><li> 、30分間、PBS中の1％BSA（IgGのフリー、プロテアーゼフリー）で細胞をインキュベートし、BSA溶液を除去し、ステップ5.4を繰り返します。 </li><li> RT、リピートステップ5.4で7分間、ローダミン結合ファロイジンの50μMで細胞をインキュベートします。 </li><li> RT、リピートステップ5.4で5分間、PBSで希釈したDAPIの255μMで細胞をインキュベートします。最後にトンをマウント彼は、封入剤でカバースリップし、マニキュアでそれらを封印。 </li><li> 541 nmの励起波長で63X対物レンズを用いて共焦点レーザー走査顕微鏡を用いて、アクチン細胞骨格の構成の画像を取得します。 </li></ol>

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The authors have nothing to disclose.

このプロトコルは、いくつかの重要なステップがあります。最初は、細胞培養のすべての中の栄養素とガスの拡散の同じ条件を維持するために必要とされるビーズサイズの均一性です。我々の経験では、アルギン酸ビーズを作るために21 G針の使用は、均一なビーズサイズの効率的な文化の取得を可能にします。第二の重要な要素は、針からの距離です。この距離は、カルシウム溶液の表面に...

それらは、培養細胞および細胞-細胞コミュニケーション1の維持のための三次元構造的支持を提供するための三次元足場は、広範囲の細胞培養に使用されてきました。天然由来のポリマー材料は​​、I型コラーゲン、キトサンおよびアルギン酸塩を含む三次元の足場を作るために使用されています。アルギン酸塩は、褐色海藻Macrocystispyrifera（昆布）に由来する天然高分子です。このポリマーは、β-D-マンヌロン酸（M）及びα-L-グルロン酸（G）は、カルシウム及びバリウムのような特定の二価カチオンの存在下で、2及びフォーム安定なゲルの反復単位で構成されています。 塩化カルシウム（CaCl 2）溶液は、一般的にアルギン酸ビーズを形成するために、架橋試薬として使用されます。アルギン酸ソリューションは、すぐに3次元球状ゲルを形成するのCaCl 2に低下しました。アルギン酸塩溶液を細胞と混合されると、細胞は、アルギネートBに封入されていますEADS 3。 アルギン酸塩は、軟骨4を含む細胞、骨格筋芽細胞5及び神経幹細胞6の種々のカプセル化のためのマトリックスとして使用することを可能にしている特性を有します。これは、不活性材料であり、栄養素、酸素及び細胞の生存および機能を維持する代謝産物の拡散を可能にします。また、他のゲルベースの培養系とは異なり、アルギン酸ビーズ内の培養細胞は、例えば、クエン酸ナトリウムなどのカルシウムキレート化剤を使用して、骨格から遊離することができ、次いで細胞をさらに調査7のために回収することができます。 下垂体腺腫は、典型的には、低増殖速度と良性腫瘍です。ラット下垂体腺腫細胞系は、二次元系8で正常培養しました。しかし、分泌ヒト下垂体細胞腫瘍のこの文化は、効率的な開発ではありません。分散した腫瘍下垂体細胞は成長が悪いです培養において、細胞は、制限された付着および拡散を示し、細胞は、典型的には、培養皿9,10浮遊凝集体を形成します。 いくつかの試みは、酵素的、機械的分散手法11、単に機械的分散法12と腫瘍外植片10の培養を含む、腫瘍、下垂体細胞懸濁液を得るために行われています。これらのアプローチでは、別の著者は、異なる期間のために実行可能な接着培養を得ました。これらの2次元系で生き残るために腫瘍分泌細胞の能力は、腫瘍の種類とそれらの増殖率9に依存します。しかし、長期培養において、線維芽細胞の表現型を有する細胞が11,13が優勢 。本論文では、さらに以下の詳細な分析を可能にアルギン酸足場からそれらを遊離アルギン酸ビーズ中にカプセル化された下垂体腺腫細胞からの初代培養を得るための方法を説明しその細胞生物学、 例えば 、それらの細胞骨格の配置の側面。

<table><tbody><tr><td>&lt;strong&gt;Reagent&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>199 culture medium&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>31100-027</td><td>For culture, warm in a 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA</td><td>A15-751</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>106329</td><td></td></tr><tr><td>N-(2-Hydroxyethyl) piperazine-N&amp;acute;-2ethanesulfonic acid (HEPES)</td><td>Sigma&amp;nbsp;</td><td>H-4034</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Invitrogen&amp;nbsp;</td><td>21600-044</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>27250-018</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Research Organics</td><td>3002E</td><td></td></tr><tr><td>Sorbean trypsin inhibitor&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>17075-029</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>2139</td><td></td></tr><tr><td>Alginic acid, From Macrocystis Pyrifera (Kelp)</td><td>Sigma&amp;nbsp;</td><td>A-2158</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>J.T. Baker</td><td>1332</td><td></td></tr><tr><td>Sodium citrate (Na3C6H5O7)</td><td>J.T. Baker</td><td>3646</td><td></td></tr><tr><td>Piperazine-N,N&amp;prime;-bis(2-ethanesulfonic acid) (PIPES)</td><td>Research Organics</td><td>9624P</td><td></td></tr><tr><td>Polyethylene Glycol 6000 (PEG 6000)</td><td>Calbiochem</td><td>528877</td><td></td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>Research Organics</td><td>9574E</td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>2500</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Formaldehyde</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>J.T. Baker</td><td>660</td><td></td></tr><tr><td>BSA Bovine Serum Albumin IgG-Free</td><td>Jackson Immuno Research</td><td>001-000-161</td><td></td></tr><tr><td>Phalloidin&amp;ndash;Tetramethylrhodamine B isothiocyanate</td><td>Sigma</td><td>P1951</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>Invitrogen&amp;nbsp;</td><td>D1306</td><td>Toxic. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>J.T. Baker</td><td>9681</td><td>Highly corrosive. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Sodium hydroxide (NAOH)</td><td>Merck</td><td>1.06498</td><td>Can cause eye and skin irritation.Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>(3-Aminopropyl)triethoxysilane (APTS)</td><td>Sigma</td><td>A-3648</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Toxic: May cause cancer. Use gloves to handle this reagent&amp;nbsp;</td></tr><tr><td>Rotator&amp;nbsp;</td><td>Boekel Scientific</td><td>Model 230300</td><td></td></tr><tr><td>Centrifuge</td><td>DuPont Corporation&amp;nbsp;</td><td>Model sorvall TC6</td><td></td></tr><tr><td>shaker&amp;nbsp;</td><td>Lab-line Instruments</td><td>Model 314-820&amp;nbsp;</td><td></td></tr></tbody></table>

three-dimensional alginate-bead culture of human pituitary adenoma cells

三次元培養法が記載されている主要な下垂体腺腫細胞をアルギネートビーズ中で増殖させます。アルギン酸塩は茶色の海藻由来ポリマーです。簡潔には、腫瘍組織を小片に切断し、コラゲナーゼ及びトリプシンで酵素消化に供します。次に、細胞懸濁液を得ました。腫瘍細胞懸濁液を1.2％アルギン酸ナトリウムと混合し、 塩化カルシウム溶液に滴下し、そしてアルギネート/細胞懸濁液を球状ビーズを形成するためのCaCl 2と接触してゲル化されます。アルギン酸ビーズに埋め込まれた細胞を、20％FBSを富化培地によって提供栄養分が供給されます。アルギン酸ビーズ内の三次元培養物は、4ヶ月まで、長期間腺腫細胞の生存率を維持します。また、細胞は、クエン酸ナトリウムでビーズを洗浄することにより、アルギン酸塩から遊離し、さらに免疫細胞化学的分析のためにカバーガラス上に播種することができます。 CELの使用リットル培養モデルは、最小限の解体とアクチン細胞骨格の固定と可視化を可能にします。要するに、アルギン酸ビーズは、下垂体腺腫細胞の維持のための信頼できる培養系を提供します。

このプロトコルは、異なる期間のためアルギン酸ビーズに腺腫下垂体細胞の培養および維持に正常に適用された3ヶ月後、アルギン酸ビーズ中の細胞を埋め込 ​​ま1に示す図 。これらの細胞は、倒立光学顕微鏡（ 図1）の下で、双屈折丸みを帯びた形状を示す。 図2は、アルギン酸塩に埋め込 ​​まれたラット下垂体腺腫細胞に?...

Watch this Scientific Journal Video about Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells at JoVE.com

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

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

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

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

three-dimensional-alginate-bead-culture-human-pituitary-adenoma

Research

JoVE Journal

Medicine

9.9K ビュー.  Center for Research and Advanced Studies (CINVESTAV-IPN). Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

ビデオ: Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工学

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3次元自己組織化ペプチドハイドロゲル中のヒト神経前駆細胞の栽培

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

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 ...

がん研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D培養におけるがん細胞浸潤とT細胞毒性のモニタリング

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

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.

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 ...

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

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

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

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

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

医学

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

ヒアルロン酸を用いた神経膠芽腫患者由来細胞の 3次元培養ヒドロゲル

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

癌細胞の浸潤や衛星腫瘍形成を探検するための三次元培養アッセイ

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells

Source: Koss, K. M, et al. <a href="https://app.jove.com/v/56615">Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to establish a three-dimensional culture of glial cells. Rat-brain-derived glial cells are encapsulated within a photocrosslinkable polymer matrix containing extracellular matrix components. Upon incubating in a growth medium, the glial cells proliferate within the supporting matrix to create a three-dimensional culture.

ラット脳由来グリア細胞の三次元培養法の確立

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use ...