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。

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			<td>2139</td>
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			<td>Sigma&#160;</td>
			<td>A-2158</td>
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			<td>Boekel Scientific</td>
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			<td>DuPont Corporation&#160;</td>
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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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

医学

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

人間の皮膚生検における侵害受容性表皮内神経線維の三次元イメージング

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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

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

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

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

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

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

複雑な大動脈の異常の三次元印刷

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

3次元印刷ガイドテンプレートアシスト経皮椎形成術(PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...