APSは、デブラ·インターナショナルと英国のスキン財団によってサポートされています。ダンディーパートナーシップ博士号プログラムの大学 - YZNはA * STARはサポートされています。

本研究は、ヘルシンキ原則宣言に従って実施し、適切な倫理委員会によって承認された。 1。培地および試薬の調製<ol><li> L-アスコルビン酸2 - リン酸原液の200倍の調製<ol><li>ダルベッコ改変イーグル培地（DMEM）溶液5mlあたりL-アスコルビン酸2 - リン酸を29mg溶解し、0.22μmのメンブランフィルターを通して濾過する。ストア-20℃の中0.25ミリリットルの滅菌アリコートとして</li><li> Lの0.1 mMの最終濃度のために、必要な日には、線維芽細胞培地（1％L-グルタミンおよび10％ウシ胎児血清を含むDMEM）毎に50mlに200倍のL-アスコルビン酸-2 - リン酸原液0.25mlのアリコートを追加-アスコルビン酸2 - リン酸。 </li></ol></li><li>ケラチノサイト増殖培地の調製<ol><li>原発SCCケラチノサイトの単離および培養は、以前に21に記載されている。ケラチノサイト増殖培地の調製が記載されているそこにも。 </li><li>以下のように簡単に説明すると、メディアを準備します。 300mlのDMEMおよび10％FBSを補充したHamのF-12を100ml 0.4 mg / mlのヒドロコルチゾン 5 mg / mlのインスリン 10 ng / mlのEGF 5 mg / mlのトランスフェリン 8.4 ng / mlのコレラ毒素 13 ng / mlのチロニン 1×ペニシリン - ストレプトマイシン溶液</li></ol></li></ol> 2。L-アスコルビン酸2リン酸添加培地での線維芽細胞由来のネイティブマトリックスのインビトロ建設に <ol><li> L-アスコルビン酸2 -リン酸を補充した線維芽細胞培地中の種子6ウェルプレート中のウェル200,000線維芽細胞（20,000細胞/ cm 2）。メディア2-5 mlで2〜3日ごとに再給。 （注：再給頻度とボリュームは、異なるセルの個々のニーズに合わせて調整することができる「ディスカッション」を参照してください）​​。 </li><li>細胞外マトリックスに埋め込まれた細胞の厚い層は、（肉眼で見える、6週間の終わりに形成する<stroNG&gt;図1）。組織培養プレートから、この層を解放するために、穏やかに1ミリリットルマイクロピペットチップを用いてマトリックスの周囲をこすり、次いでウェルの中心に向かって行列の縁を押してください。この細胞の層とマトリックス（天然のマトリックス）をプレート表面から容易に着脱可能であるべきであり、今媒体中に浮遊する。それはそれ自体の上に折り畳む避けるために、媒体中でのネイティブ行列を広げる。 </li><li>ネイティブ行列フロートを聞かせて2〜3日ごとにメディアを変更し、5日間、改造。による引張強度及びマトリクス内の内因性リモデリングは、マトリックスは急激に収縮し、より小さな、より厚い天然のマトリックスに還元なり、浸潤アッセイのために利用される準備ができているであろう。 </li></ol> 3。腫瘍SCCケラチノサイトと浸潤アッセイ<ol><li>鈍ピンセットでそっとネイティブ行列をピックアップし、ナイロンネットに転送します。一度ナイロンネット上で、1ミリリットルマイクロピペットを用いて可能な限り平坦に位置するように優しく行列を広げる先端と鈍鉗子。 </li><li>一端に無菌ワセリンを少量塗りつけによって無菌クローン性シリンダーを準備します。 </li><li>ワセリン側を下にして、ネイティブのマトリックス上に、クローンシリンダーを配置します。これは、ネイティブの行列と、クローンリング間の緊密なシールを確保することである。 </li><li>クローン性シリンダー（ケラチノサイト増殖培地100μl中250,000細胞）にCSCCセルを追加します。 </li><li> CSCC細胞が本来のマトリックス上に落ち着いたときに6時間後に、クローンシリンダーを取り外します。 </li><li>曲がったステンレス製のワイヤメッシュ支持体上に気液界面へのネイティブ行列とCSCC細胞とナイロンネットを持ち上げます。 </li><li>メディアレベルは、天然のマトリックスの底部に接触するまでのアスコルビン酸を補充したケラチノサイト増殖培地を加える。 </li><li> CSCC細胞の播種後7日および14日に2〜3日ごとに、収穫のメディアを変更します。 </li></ol> 4。 3D培養物の収穫と組織学のための準備<ol><li> 4％のパーで修正室温で一晩aformaldehyde。 </li><li>サンプルを二等分し、切断面が外側に向けた状態で、ホルマリン固定パラフィン包埋ブロックのためのワックスに埋め込む。 </li><li>代わりに、10月に二等分サンプルを埋め込み、スナップ凍結を直ちに液体窒素中で新鮮凍結組織ブロックのために。 </li><li>ミクロトームと脱ワックス（必要な場合）に4μmの切片を切った。標準ヘマトキシリン​​およびエオシンを使用して染色する。 CSCC細胞を可視化するために、ケラチン14に対するマウスモノクローナル抗体（インハウスLL001は）免疫組織染色に使用することができる。 </li></ol>

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著者らは、開示することは何もありません。

これにより、実験の性質のために、完了のために必要な総時間は2ヶ月までとすることができる。この時間を通して、細心の注意と、無菌組織培養の実践は、微生物汚染を防止するために使用しなければならない。 脇コラーゲンのヒドロキシプロリンとヒドロキシリシンの合成における補因子としての役割から、アスコルビン酸は、線維芽細胞22のコラーゲン特異的なmRNA発現を刺激する。ここで選択されたアスコルビン酸はより安定なL-アスコルビン酸2 -リン酸23である。再給紙は、週に三回をお勧めしますが、これは常に研究された細胞に依存することになる。週間通過する初期の線維芽細胞培養は、より多くの栄養素を消費して、より大きなメディアボリュームとより頻繁に再給紙が賢明であるかもしれません。それは、変化するメディアやケアはネイティブのマトリックス産生の間に細胞層への障害を最小限に抑えるよう注意する必要があるとき、穏やかなことが重要です。 行列は、特によくの周囲に、通常は2週間後に目に見えるようになるはずです。まれに、行列は任意の機械的破壊せずに自分自身を解放します。これは内側に行列を引っ張るマトリックス、引張強さの本質的な改造を反映したものであるだけでなく、ネイティブの行列の周囲は、メディアの変更の際に邪魔されたこと徴候である可能性があります。 解放し、プレートからマトリックスセル層を解放すると、1はマトリックスを通して穴を突くしないように注意しなければなりません。小鈍細胞スクレーパーを1mlマイクロピペットチップの代わりにも使用することができる。ナイロン上に放出ネイティブ行列の配置（フラットネイティブ行列を築くために確認してから）最初のメッシュがはるかに容易に取り扱いや気液界面の後に持ち上げることができます。 このプロトコルは、最初に記載しているより正確かつ生理学的に関連するデータを得るために、腫瘍細胞アッセイにおいて微小環境をモデル化する真皮基質として天然繊維芽細胞マトリックスの使用。

三次元組織培養における生体材料の使用は、2D接着性プラスチック基板とで要約のものよりインビボ環境により類似の生理学的条件下で、実験室における細胞の挙動を研究する研究者を有効にしている。特に、長足の進歩が前方気液界面1-4における3次元培養法を採用することで重層上皮のモデル化がなされている。このような技術は忠実にケラチノサイトの分化およびこれらのプロセスを研究する研究者のための柔軟性と忠実度を可能にする腫瘍細胞の浸潤を模倣する。間質環境を模倣する生体材料の基板の選択は、主にI型コラーゲン、Engelbreth-Holm-Swarmマウス肉腫行列およびデepidermized真皮の使用を伴っています。例えば、癌関連線維芽細胞が癌の浸潤5に寄与することが示されている、開始、および進行間質上皮相互作用を介して、6,7 WHENこのような基板で栽培。 皮膚内の間質環境を模倣するためのゴールドスタンダードは、このような技術を用いて、最大かつ最も広く研究されている重層上皮は、人間の真皮（DED）を解除epidermizedされるように考えられている。 DEDの調製は、人間の死体皮膚3,4からトリプシン処理または物理的解離を介した表皮の除去を含む。しかしながら、このような皮膚へのアクセスは、医療機関に関連付けられていない研究室のために非常に困難であることができ、病気の真皮を得ることが近くに不可能である。別の方法として、研究室は頻繁にI型コラーゲン（ラット尾から分離）および/またはEngelbreth-Holm-Swarmマウス肉腫マトリックスタイプを組み合わせて使用​​します。 コラーゲンは容易酢酸および塩沈殿を用いて単離することができることNageotte 8 1927年の発見後、組織培養への適用は、その後Huzelによって開拓されたLAや同僚9。コラーゲンコーティングはアマンとをゲイ9によって問い合わせなど29株および組織外植片の細胞培養用のガラスよりも優れていることが判明した。現在、組織培養に用いられるコラーゲンの主要なタイプは、ラット尾の腱から単離され、通常の商業的供給源から購入される。しかし、忠実な基板要約のための欠点は、ラット尾部コラーゲンI型およびIII型コラーゲンは、主要な成分として存在し、単離されたラット尾部コラーゲンは、常に断片化されたヒトコラーゲン、またはヒト真皮、同一ではないということである。 エンゲルブレス-ホルム-スウォームマウス肉腫マトリックスは、培養されたエンゲルブレス-ホルム-スウォームマウス肉腫細胞から分泌される10ゼラチン状のタンパク質混合物である。主要成分は、ラミニン、IV型コラーゲン、ヘパリン硫酸プロテオグリカン、エンタクチンおよびニドゲンであり、これらのタンパク質の正確な比率は、バッチからバッチに変化します。さておき、構造プロからteins、このマトリックスはまた、増殖因子β、上皮成長因子、インスリン様成長因子1、ウシ線維芽細胞成長因子、および細胞挙動11,12を変化させるであろう血小板由来成長因子の形質転換のような成長因子の有意なレベルを含有する。エンゲルブレス-ホルム-スウォームマウス肉腫行列の複雑性の方を向いて、1851のタンパク質の総量は、最近のプロテオミクス研究13で同定された。解釈し、その11を利用して様々な実験を比較するとき、この行列の豊かで複雑 ​​な性質に照らして、十分に注意が知らされていた。 私たちの研究室では、遺伝性皮膚疾患、皮膚扁平上皮癌（CSCC）14を発症する素因を持つ特に強い関心を持っている。 COL7A1遺伝子に劣性栄養障害性表皮水疱症（RDEB）、生殖細胞変異を伴う重度の水疱性疾患の場合<s&gt; 15〜17まで、我々は、これらの患者における皮膚微小環境18を促進する腫瘍であることを決定した。この研究の過程で、我々は、コラーゲン内のI / Engelbreth-Holm-Swarmマウス肉腫行列を組み込み、細胞自身の、ネイティブの行列を評価する方法を検討した皮膚線維芽細胞の性質を促進する腫瘍を評価することができませんでした。これを達成するために、我々はヒト皮膚同等物19,20に取り組んでルーシー·ジェルマンの研究室から以前の手法を変更しました。サンジェルマンの技術は、合成または死体足場がない状態で初代ヒトケラチノサイトおよび線維芽細胞培養を用いてよく組織基底膜をヒトの皮膚を再構築することができました。 本研究では、皮膚の腫瘍間質微小環境（ネイティブ行列）反復するために使用する手順は、in vitroでの一次間質性線維芽細胞に直接由来は18に記載されている。ネイティブの行列をP線維芽細胞の長期培養によりroducedはCSCC細胞浸潤についてアッセイするために真皮同等物として使用した。私たちは、RDEB線維芽細胞（VII型コラーゲン（C7）が欠損した）が分泌する細胞外マトリックスのいずれかから、またはレトロウイルス腫瘍上の単一のコラーゲンの絶大な効果を構築し、実証する表現VII型コラーゲンを用いて形質導入RDEB線維芽細胞から誘導されたネイティブの行列を使用してデータを提示細胞浸潤。

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			<td class="xl63" height="64" style="height:48.0pt;width:66pt" width="88">L-Ascorbic acid 2-phosphate</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">A8960</td>
			<td class="xl64" style="width:66pt" width="88"></td>
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		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="height:31.5pt;width:66pt" width="88">DMEM with l-glutamine,</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">Life Technologies</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">11995-073</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
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		<tr height="106" style="height:79.5pt">
			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl63" height="85" style="height:63.75pt;width:66pt" width="88">100x Penicillin-Streptomycin</td>
			<td class="xl63" style="width:66pt" width="88">Life Technologies</td>
			<td class="xl63" style="width:66pt" width="88">15070</td>
			<td class="xl64" style="width:66pt" width="88"></td>
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			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Vaseline</td>
			<td class="xl63" style="width:66pt" width="88">VWR</td>
			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
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		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">Z370789</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="148" style="height:111.0pt">
			<td class="xl63" height="148" style="height:111.0pt;width:66pt" width="88">Nylon Net Filter Disc Hydrophilic 100um 25um diameter 100/pk</td>
			<td class="xl63" style="width:66pt" width="88">Millipore</td>
			<td class="xl63" style="width:66pt" width="88">NY1H02500</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="127" style="height:95.25pt">
			<td class="xl63" height="127" style="height:95.25pt;width:66pt" width="88">Bent stainless steel wire mesh support</td>
			<td class="xl63" style="width:66pt" width="88">Made in house</td>
			<td class="xl63" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88">Dimensions were made so that the mesh would fit into 6-well plates</td>
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tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

間葉系細胞が埋め込まれたマトリクス上の上皮細胞の3次元器官型培養物は、広く上皮細胞分化および浸潤を研究するために使用される。 Engelbreth-Holm-Swarmマウス肉腫細胞由来のラット尾I型コラーゲンおよび/またはマトリックスは、伝統的に、間葉細胞（通常、線維芽細胞）が移入されその中にマトリックスまたは間質微小環境をモデル化するために基板として用いられてきた。このようなマトリックスを用いた実験は非常に有益ですが、それはその原因なマウス由来の行列のように、単一のタンパク質（たとえば、I型コラーゲンのように）か、基底膜成分と成長因子の含有量が高い（最優先の存在に主張することができます肉腫細胞）、これらの基板は、最高の間質細胞自身によって行われたマトリックス組成への貢献度を反映していない。腫瘍を起こしやすい、遺伝水疱性障害（劣性ジストロフィーの患者から単離された初代皮膚線維芽細胞によって産生さネイティブ行列を研究するために、表皮水疱症）、我々は、腫瘍細胞の浸潤を研究するために、既存のネイティブ行列プロトコルを適応している。線維芽細胞は、培養中の長期間独自のマトリックスを産生するように誘導される。この天然のマトリックスは次いで、培養皿から剥離され、全体の共培養は、空気 - 液体界面まで上昇させる前に、上皮細胞はその上に播種する。細胞分化および/または浸潤、次いで経時的に評価することができる。この技術は、唯一の欠点は、天然のマトリックスを生成するために必要な長期間である、合成または外来マトリックスを必要とせずに、3次元設定における上皮 - 間葉細胞の相互作用を評価する能力を提供する。ここでは、腫瘍細胞浸潤を阻害するために、VII型コラーゲン、線維芽細胞によって発現される単一分子の能力を評価するためにこの技術の適用を記載している。

<html>この技術は、異なる次元間質環境下で腫瘍細胞（この場合はCSCC）の侵襲的挙動を調べ、比較する可能性を開く。この技術を使用して、RDEBの皮膚環境をで要約C7-欠損ネイティブ行列だけではなく、生成するだけでなく、遺伝的に過剰発現するC 7 18ように操作された追加的な行列することができます。 図2に見られるように、CSCC RDEBケラチノサイトの浸潤は、C7-欠損RDEB対照と比較して過剰発現するC7-ネイティブマトリックス中に有意に遅らせた。この侵攻は、ゲルは、固定パラフィンブロックに包埋した後、切断して免疫染色した標準的な組織学的な方法を介して可視化される。染色のために使用することが示唆抗体がCSCC細胞と線維芽細胞のための抗ビメンチン（V9）のための抗ケラチン14（LL001）である。 p_upload/51321/51321fig1highres.jpg &quot;幅=&quot; 500 &quot;/&gt; 図1。線維芽細胞由来のネイティブマトリックスと腫瘍の浸潤アッセイの世代のワークフロー 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51321/51321fig1highres.jpg" target="_blank">拡大画像を表示するにはここをクリックしてください。</a> <img alt="図2" fo:content-width="6in" src="/files/ftp_upload/51321/51321fig2highres.jpg" width="500" /> 図2。 C7の中の線維芽細胞を欠損制御RDEBから派生したネイティブマトリックスへのCSCC侵入のケラチン14（LL001）染色（AI＆BI）、またはRDEB線維芽細胞で過剰発現する完全長のC7式（AII及びBII）を、明確に実証する再発現C7の細胞外マトリックスにCSCCケラチノサイト侵入を遅らせることができます。核BiとBII中（緑色）ビメンチンと（青）DAPIおよび線維芽細胞を用いて染色した。

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

組織操作された線維芽細胞由来の天然のマトリックスは、上皮細胞の増殖および分化を支持する間質基質を生成するための新興のツールである。ここでは、腫瘍細胞の生物学上の異なる間質細胞タイプの影響を評価するためにこの方法を適用するプロトコルが提示される。

癌細胞浸潤への応用による腫瘍間質微小環境の組織工学

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

tissue-engineering-tumor-stromal-microenvironment-with-application-to

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.7K ビュー.  University of Dundee. 組織操作された線維芽細胞由来の天然のマトリックスは、上皮細胞の増殖および分化を支持する間質基質を生成するための新興のツールである。ここでは、腫瘍細胞の生物学上の異なる間質細胞タイプの影響を評価するためにこの方法を適用するプロトコルが提示される。 

ビデオ: Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

腫瘍微小環境を再作成するためのマイクロ流体デバイス<em&gt;体外で</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

生物工学

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

生体力学的解析と組織工学のための細胞由来の組織のリングを製作する監督セルラーセルフアセンブリ

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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 ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

電気刺激のマウス筋芽細胞前駆細胞およびアプリケーションからの工学骨格筋組織

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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

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

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

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

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

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

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

エンジニアリングフィブリンベースの組織の筋線維芽細胞と制約の適用からの構築と細胞とコラーゲン（再）組織を誘導するためにひずみ

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

落射蛍光による組織マトリックスのコンポーネントと二光子顕微鏡の長期生体内免疫蛍光イメージング

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心円状のゲルシステムは、3D細胞移動に対するマトリックスの微小環境の生物物理役割を研究する

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...