この作品は、健康補助金NIH / NICHD＃HD051998（CAMまで）の米国国立研究所によってサポートされていました。

1。コラーゲンビーズの調製<ol><li>前の3次元細胞培養のためのロードEVTsに、一つはCytodex 3マイクロキャリアビーズを準備する必要があります。 <ol><li>実験に必要なCytodex - 3ビーズの適切な量を秤量。このプロトコルは、ビーズの0.05グラムが必要とされる、10ミリリットルRCCSの容器に適合されている。 50ミリリットルRCCSの容器の場合、スケールは調整する。 50mLのオートクレーブ可能な円錐管で、12mLダルベッコのリン酸との混合250mgのCytodex - 3ビーズが溶液（DPBS）をバッファ。この金額は、5 RCCS船で十分です。 </li></ol></li><li>オートクレーブ処理が蒸発につながるように十分なボリュームが、円錐管に存在していることを確認します。大まかに110℃で10分間チューブ、オートクレーブを締めくくる℃に</li><li>オートクレーブサイクルの完了時にコニカルチューブを外し、Cytodex - 3ビーズ溶液を冷却することができます。 </li><li>コニカルチューブは室温に冷却した後、1X DPを使用して12.5mLに総量をもたらすために無菌テクニックを使用してBS。 </li><li>キャップと室温で作製Cytodex - 3ビーズを格納する。準備ビーズが膨潤し、使用前に室温に冷却してください。 Cytodex - 3ビーズの上記製剤は、5つの10mLのRCCSの船のために提供します。我々は、コラーゲンが約1ヶ月後に不安定になるように室温でCytodex - 3ビーズの拡張ストレージは、パフォーマンスの低下につながることを観察した。 133から215μmからサイズのCytodex - 3ビーズの範囲。 </li></ol> 2。メディアの準備<ol><li>プラスサプリメント、60％L - 15リーボビッツのメディア（22から適応）、40％MEMアルファで構成されてGTSF - 2メディアを（22から適応）、最適化されたRCCSの1Lを準備します。最初に、補充したMEMアルファの合計量で400mlの準備： 21.2mM重炭酸ナトリウム 0.06％ペプトン 0.7mmのフルクトース 1.4mmのガラクトース 5.6mMグルコース 1％HEPES 1％L -グルタミン 0.5パーセントのITS 10％のFBS </li><li> ITSのストック溶液を準備するには、氷酢酸（約0.05mL）を添加することによって準備さ5mLの滅菌酸性化したH 2 Oに溶解する。解散する渦巻、45mL滅菌水で従ってください。 </li><li>組織培養グレードH 2 Oに溶解して十分なL - 15培地の600ミリリットルのためのリーボビッツの粉体を秤量</li><li> L - 15リーボビッツの培地を1Lに細胞培養液の合計量をもたらす。フィルター滅菌し、4℃で保存して暗所で。使用した培地の各アリコートに個別に1％ペニシリン - ストレプトマイシンを追加。 </li><li> GTSF - 2メディア以外の多くのメディアの配合に成功RCCSでテストされています。個々の研究室では実験が行われてのために最適である培地自分で決定する必要があります。 </li></ol> 3。細胞とビーズインキュベーション<ol><li> 〜80％コンフルエントにEVTsを伝播、トリプシンおよび認められた細胞培養のプラクティスを使用して数える。 </li><li>暖かいの4mlの中の1 × 10 6 EVTsを一時停止EDメディア。 </li><li>ゆっくりと準備Cytodex - 3ビーズをミックス。無菌テクニックを使用して、幅広い先端、10mLの血清ピペットおよび未使用の15mlのコニカルチューブへの転送を使用して、準備したビーズの2.5mLを削除します。彼らはピペットにアタッチするときにビーズの小さな損失があるかもしれないことに注意してください。 </li><li> Cytodex - 3ビーズをチューブの底に沈殿することができます。沈降した後、Cytodex - 3ビーズを乱すことなくDPBSの最上層を除去するピペッターを使用してください。 </li><li>準備Cytodex - 3ビーズをメディアで1 × 10 6準備EVTsを混ぜる。 </li><li> 30分間室温で細胞ビーズ混合物をインキュベートする。定期的に穏やかに混合する。 37℃、5％CO 2でさらに30分間インキュベートする。定期的に穏やかに混合する。 </li></ol> 4。 RWVのロード<ol><li>層流キャビネットでは、パッケージから10mLのRCCSを削除し、安定性のための滅菌、6ウェル培養プレートに入れてください。 RCのラベルが付いた図については図1を参照してくださいCS。 </li><li>港から大型ストッパーを外します。 </li><li>温めたメディアと10mLのためにインキュベート細胞ビーズ混合物の合計量をもたらす。 </li><li>大規模なポートを介してRCCSに細胞/ビーズ混合物をロードする。潜在的な気泡の除去を助けるためにロードするときRCCS（最大大きいポート）45 °の角度で傾斜させることが必要。ゆっくりと着実に容器を充填することにより、正圧のビルドアップを防ぐ。 </li><li>大規模なポートでストッパーを交換してください。 </li><li> 3 mLシリンジからピストンを外し、小さな港に空の注射器を置きます。各シリンジへのメディアの1 - 3ML追加。ゆっくりと2つの弁を開く。静かにシリンジで注射器のピストンを交換してください。すべての気泡が内側のチャンバーから除去されるまで1シリンジからメディアを追加します。 </li><li> RCCSをピックアップし、気泡を確認するために、目の前でそれを回転させながらそっと側をタップします。 いずれかの気泡がある場合は空気の気泡が形成Oを妨害するため、お客様は、チャンバーからそれらを得る必要がありますfは、セルの集合体と剪断力を紹介。気泡が小さいポートの下になるまで、RCCSを回転させて気泡を取り除きます。その後そっとチャンバーのポートと外に泡を強制的に反対側に注射器を押し下げます。 </li><li>片側のポートを閉じます。ゆっくりと形成される気泡を防ぐ容器、中に正圧の少量を導入する他の注射器のピストンを押し下げます。第二バルブを閉じます。 </li><li>ローターにRCCSをロードします。 37℃CO 2インキュベーターで19rpmで回転を開始します。 </li></ol> 5。メディアを変更する<ol><li>毎日その後も、最初の3日間は一日おきにメディアを変更します。 </li><li>ローターの電源を切り、RCCSを取り外します。 、いくつかの吸引を作成し、各小ポートからシリンジを削除し、ビーズが大きいポートの反対を解決するような角度でRCCSを配置するピストンを引き上げます。 </li><li>ビーズは、すべて決着した後に、小弁の1つを開き、メディアは、RCCの、廃棄物の容器に流出することができます。この方法でメディアの2 / 3を取り外します。ビーズを邪魔しないため、任意の集合体を破棄しないようにしてください。 </li><li>小さなバルブを閉じて、大規模なポートを開きます。バック大きいポートを介してRCCSにメディアを追加します。大規模なポートでストッパーを交換してください。 </li><li> 4.9 - 手順4.6を繰​​り返します。 </li><li>凝集体のサイズが大きくなるに従い、お客様は最適な速度で小さな循環パターンに理想的に、常時懸濁液中にそれらを保つために回転速度を増加する必要があります。凝集体が目に見えて成長し始めると、一般的に、それぞれの給餌後に0.3から0.7 rpmの間に速度が向上します。 </li></ol> 6。伝播細胞を収集<ol><li>ローターからRCCSを削除します。それぞれの小さなポートからシリンジを取り外し、ビーズが大きいポートの反対側の沈殿ができるように角度でRCCSを置きます。 </li><li>ビーズは、すべて決着した後に、小弁の1つを開いて、メディアが流出することができますRCCSの、廃棄物の容器に。この方法でメディアの1 / 3を取り外します。 </li><li>小さなバルブを閉じて、大規模なポートを開きます。優しくスワール容器をバックソリューションに集約して分散させる。滅菌50ミリリットルコニカルチューブに空の液体混合物。収集された凝集体は完全に凝集体の回復を最大化するために培養容器を洗浄するために上清を使用する前に、コニカルチューブに定住することができます。集計は、このようなフローサイトメトリー、浸潤アッセイ、免疫蛍光などとして、すぐにダウンストリームアッセイに用いることができる。 </li></ol> 7。代表的な結果 Cytodex - 3ビーズでRCCSで育ったEVT様細胞（SGHPL - 4栄養膜細胞株）の例を図2に示されています。 EVTのような細胞株は、離れてメインクラスタから拡張し、近隣のクラスタに接続する突起が表示されます。ビーズの多くは、完全に伝播する細胞で覆われている。一度RCCSとPLAから削除Tedは、細胞外マトリックスに、EVT -のような3次元培養した細胞は積極的におよび/ ​​または移動を（図3）に侵入。 RT - PCRのデータは、従来の細胞培養単層（図4）とは対照的に、3次元凝集体に見られるようにMMPの発現増加を確認する。興味深いことに、侵略に関連付けられていない遺伝子もRCCSにアップレギュレートされている（表1）とトロホブラストの細胞に侵入すると免疫の相互作用などの分野を線引きで助けることができる。 <img alt="図1" src="/files/ftp_upload/3367/3367fig1.jpg" /> 図1。回転細胞培養システム（RCCS）の漫画の描写。 <img alt="図2" src="/files/ftp_upload/3367/3367fig2.jpg" /> 図2。RCCSの5日間の成長後の代表的な集合体の位相コントラスト顕微鏡写真。矢印は浸潤細胞を示し、*はCytodex 3マイクロキャリアビーズを表します。 <imgALT =&quot;図3&quot;SRC =&quot;/ files/ftp_upload/3367/3367fig3.jpg&quot;/&gt; 図3。RCCS成長凝集体は、フィブリンゲルに包埋した。フィブリンゲルを介して浸潤は（A）24時間後にフィブリンの埋め込みには早くも観察され、（B）48時間を通して継続した。矢印は浸潤細胞を示し、*はCytodex 3マイクロキャリアビーズを（参考文献から、許可を得て、脚色7）を表します。 <img alt="図4" src="/files/ftp_upload/3367/3367fig4.jpg" /> 図4単層とRCCSの比較ゼラチンザイモグラムはSGHPL - 4 EVT様細胞を伝播。 MMP - 2及びMMP - 9のプロと、アクティブなフォームは、RCCS栽培栄養膜細胞（文献[7]から、許可を得て、適応）によって分泌された。 <img alt="表1" src="/files/ftp_upload/3367/3367table1.jpg" /> 表1。RCCSで育ったSGHPL - 4細胞（文献[7]から、許可を得て、適応）からマイクロアレイの結果のまとめ。 <ol class="lalpha"><li正規化された3 - D遺伝子の値/正規monlayerの遺伝子の値：3 - D集合体で&gt;誘導倍率は、次のように計算された。 </li><li>単分子膜における検出限界以下であったの遺伝子を示している。 </li></ol>

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我々は、開示することは何もない。

ここで紹介する培養技術は、高度に侵襲EVT様細胞との調査官が用意されています。それは今分化の損失が三次元（心尖部、基底、および横方向のセルの表面）10,13における化学と分子の手がかりに対する細胞応答の阻害に起因する単層で発生することが認識されている。この手法は、EVTの細胞を侵入に子宮内に記載されている特性を反映している。手順は、従来の単層組...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> Cytodexマイクロキャリアビーズ</td><td>シグマアルドリッチ</td><td> C3275 </td><td></td></tr><tr><td>回転細胞培養システム（RCCS） </td><td> Synthecon </td><td> RCCS - D </td><td>ローターベース、電源、4使い捨てRCCSユニットが含まれています</td></tr><tr><td> RCCS使い捨てユニット</td><td> Synthecon </td><td>お問い合わせSynthecon </td><td></td></tr><tr><td> 3ミリリットルルアーロックチップシリンジ</td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ミリリットルワイド先端血清用ピペット</td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEMアルファ</td><td>インビトロジェン</td><td> 12561-072 </td><td></td></tr><tr><td>リーボビッツのL - 15培地、粉</td><td>インビトロジェン</td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O、エンドトキシンフリー</td><td>フィッシャー</td><td> MT - 25 - 055 - CM </td><td></td></tr><tr><td>重炭酸ナトリウム</td><td>シグマアルドリッチ</td><td> S - 7795 </td><td></td></tr><tr><td>ペプトン</td><td>フィッシャーサイエンティフィック</td><td> BP1420 - 100 </td><td></td></tr><tr><td>果糖</td><td>シグマアルドリッチ</td><td> F3510 - 100 </td><td></td></tr><tr><td>ガラクトース</td><td>シグマアルドリッチ</td><td> G5388 - 100 </td><td></td></tr><tr><td>グルコース</td><td>シグマアルドリッチ</td><td> G7528 - 250 </td><td></td></tr><tr><td> HEPES </td><td>インビトロジェン</td><td> 15630-080 </td><td></td></tr><tr><td> L -グルタミン</td><td>インビトロジェン</td><td> 25030 </td><td></td></tr><tr><td>インスリン - トランスフェリン - 亜セレン酸ナトリウム（ITS） </td><td>シグマアルドリッチ</td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td>インビトロジェン</td><td> 10437 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>インビトロジェン</td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

人間の栄養膜研究の分野では、胎盤形成の間に確立された複雑な環境を理解する上で役立ちます。これらの研究の性質上、in vivoの実験で 、人間は不可能です。初代培養、外植培養と栄養膜細胞株1の組み合わせは、子宮壁2の侵略と妊娠の成功確立のために必要とされるextravillous栄養膜細胞（EVTs）による子宮螺旋動脈3,4の改造の我々の理解をサポートしています。このようなモデルから収集した豊富な知識にもかかわらず、それは彼らのin vivoでの対応で 5,6と比較した場合、EVTのような細胞株を用いた in vitro細胞培養モデルにおいて細胞のプロパティを変更し表示することが受け入れられている。回転細胞培養システム（RCCS）で培養した細胞は、より密接にUで差別化を模倣することEVTのような細胞株の、形態学的表現型、および機能的特性を表示する侵略（例：マトリックスメタロプロテアーゼ（MMP））と栄養膜の分化7,8,9を仲介する遺伝子の発現増加とテロササナEVTs、。サンジョルジュ病院胎盤細胞ライン- 4（SGHPL - 4）（親切に先生ガイウィットリーと博士ジュディスカートライトによる寄贈）RCCSでテストするために使用されたEVT様細胞株です。 RCCSの培養容器のデザインは、臓器および組織の3次元（3 - D）の環境で機能するという原則に基づいている。生理学的に適切なせん断の条件を含む容器内の動的な培養条件、のために、三次元的に培養した細胞は、天然の細胞の親和性に基づいて、凝集体を形成し、アセンブリ10,11,12器官のような組織に分化する。流体の軌道の維持は、 生体内に存在する条件と同様の低せん断、低乱流の環境を提供します。培養細胞の沈降は、回転を調整することによって打ち消された細胞の一定の自由落下を確実にするためにRCCSの速度。ガス交換は、バイオリアクターの背面に透過性の疎水性膜を介して行われます。それらが多孔質マイクロキャリアビーズの表面上で培養されているため、 生体内での彼らの親の組織と同様に、RCCSに成長した細胞は三次元（それらの、頂端基底、および側面即ち）で化学と分子勾配に対応することができます。プラスチックのような不浸透性の表面上の2次元単層として増殖させた場合、細胞はその基底面では、この重要なコミュニケーションを奪われている。そのため、環境によって課された空間の制約が深くこうして3次元環境13の重要な役割を示唆し、周囲の微小環境からどのように細胞の感覚とデコード信号に影響を与えます。 我々は、7,14,15,16、様々な人間の上皮組織の生物学的に意味のある3 - Dモデルを設計するRCCSを使用している。実際、多くの過去の報告では、DEMデータを持っているRCCSで培養された細胞は、他のモデル10,17-21では実現されていない生理学的に関連する表現型を担うことができることonstrated。要約すると、RCCSの文化は、実験操作の様々な従順な分化した細胞を大量に提供する簡単に、再現性、高スループットのプラットフォームを表します。次のプロトコルでは、例としてEVTsを使用して、我々ははっきりとRCCSに3次元的に培養接着細胞に必要な手順を説明します。

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

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

伝統的な、二次元細胞培養技術は、しばしば、分化マーカー、サイトカインおよび増殖因子に関する変更特性になる。 extravillous栄養膜細胞株を以下に示すように回転する細胞培養系における三次元細胞培養（RCCS）は、これらの要因の多くの表現を再確立します。

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

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

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Research

JoVE Journal

Bioengineering

16.4K ビュー.  Tulane University Medical School. 伝統的な、二次元細胞培養技術は、しばしば、分化マーカー、サイトカインおよび増殖因子に関する変更特性になる。 extravillous栄養膜細胞株を以下に示すように回転する細胞培養系における三次元細胞培養（RCCS）は、これらの要因の多くの表現を再確立します。

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

異なる系統にヒト胚性幹細胞（hESCの）の伝播との差別化のための3Dプラットフォームとしてのアルギン酸マイクロカプセル

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

腫瘍細胞の浸潤を間質液流れに及ぼす影響を測定するための三次元細胞培養モデル

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

ミササイトメトリー：CyTOFマスメーターで毎日のチューニングと実行した細胞サンプルのためのプロトコル

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

水性二相系を用いた細胞の共培養パターニング

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

有毒吸入化学試験のためのin vitroでの細胞培養モデルにおける

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

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

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...