Name: a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds
Uploaded: 2016-04-06T14:00:00
Description: Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

この研究は学際臨床ヴュルツブルクの大学病院の研究（IZKF、グラントBD247）及び（平家Wallesに付与された）バイエルンフィットプログラムのためのセンターが主催しました。

1.二次元（2D）細胞培養<ol><li>商業的に腫瘍細胞株HCC827（DSMZ）を得ました。培養RPMI-1640で肺腺癌細胞株HCC827（変異したEGFR、KRAS野生型）、20％FCSを補充しました。 3日間 - すべての2培地に変更します。週に2回、細胞を分割します。通路20に到達するまで細胞が使用されます。 </li><li>商業的に腫瘍細胞株A549（DSMZ）を得ました。培養物を10％FCSを補充したRPMI-1640中で肺癌細胞株A5...

<ol>
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我々は、バイオマーカー誘導の治療の予測のためのシリコ腫瘍試験システムのin vitro /で組み合わせ確立しています。 in vitroモデルは、また、 シリコ 17 でシミュレートすることができ、特定の突然変異背景に、腫瘍細胞の増殖およびアポトーシスの変化などの化合物のアクションの異なる重要な側面を評価します。ここでは、3D腫瘍モデルの生成およ...

製薬業界は、莫大なコスト1-5を引き起こす臨床段階における癌治療の分野で最大95％の高い離職率に直面しています。この赤字の1つの理由は、現在、潜在的な新規化合物の有効性は、癌細胞株の2D細胞培養でまたは動物モデルでの大規模スクリーニングで評価することです。動物モデルは、より高い複雑さを持っていますが、マウスと男性6,7間の決定的な違いがあります。 10年で、異なるアプローチを使用して3D癌モデルは、2D癌細胞株の培養およびインビボ腫瘍 6,8,9 における複合体との間のギャップを埋めるために生成されています。細胞分化にもシグナル伝達に対する3D環境の影響は数年前にいくつかの研究で示されている（ 例えば 、ミナザビッセルによって）10,11。今日では、多くの3D細胞培養モデルは、スフェロイド培養、ヒドロゲルまたはマイクロ流体チップ12-16として入手可能です。でもthesというかかわらずEモデルは、それらが主に腫瘍担持の効果と影響の薬効を有することが知られている組織の微小環境を欠いている、従来の2D培養システムと比較して複雑性を高めます。 この問題に対処するために、我々は、脱細胞化ブタ空腸に由来するSISmuc（ 小腸、粘膜下層+粘膜 ）と呼ばれる生物学的足場に基づいた3D腫瘍モデルを生成しました。これにより、組織構造や、異なるコラーゲンと同様に基底膜構造としてECMの重要な構成要素は、17を保持されます。このユニークな機能は、上皮から生じ、固形腫瘍の約80％を占める癌の腫瘍モデル生成のために重要です。さらに、我々の組織工学腫瘍モデルにおける増殖速度は、2D培養で達成され、人為的に高い速度に比べて低減されます。増殖が薬効を評価する上で重要なパラメータであるように、薬物検査は類似で我々のモデルで有効になっています17 in vivoでの腫瘍内への条件。 そのEGFRの -biomarker状態が異なる2つの異なる肺癌細胞株のための正常なバイオマーカー依存性薬物の有効性を予測するために我々のモデルの可能性を評価するために、ここで現在のデータ。この変異状態は、NSCLC患者に日常的に決定され始めています。例えば、白金ベースの化学療法18-21のものと比較して、活性化EGFR変異ショー優れた成果を保有する腫瘍に対するEGFR -inhibitorゲフィチニブなどのTKIとターゲットを絞った治療法。 我々は、化合物の有効性を評価するために関連するいくつかの読み出し技術を確立しました。さらに、TGF-β-1刺激後に、我々は、悪性形質転換22,23における重要なステップであると考えられ、薬物RESISTANに接続されたEMTプロセスを、開始した腫瘍細胞における化合物の作用を調査することができますCE 24。 3D腫瘍モデルは、良好なコントラストで標的治療、化学療法、または薬物の組み合わせに細胞特異的応答を監視することができます。さらに強化し、薬剤スクリーニングをスピードアップし、抵抗に遭遇するために、これはシリコシミュレーションにによって補完されます。いくつかの実験に基づいて、腫瘍反応は、薬物およびそれらの組み合わせの完全な範囲についての結果についてインシリコで予測することができます。

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BioVoxxel Toolbox (ImageJ / Fiji)</td>
			<td style="width:261px;">Jan Brocher, Thorsten Wagner, https://github.com/biovoxxel/BioVoxxel_Toolbox</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell crowns</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">for static 3D culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellDesigner</td>
			<td style="width:261px;">http://www.celldesigner.org/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for drawing the network.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">citrate buffer stock solution (10x)</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">42 g/l Citric acid monohydrate, 17.,6 g/l Sodium hydroxide pellets in deionized water, pH 6,.0, stored at RT.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">citrate buffer working solution</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">10 % Citrate buffer stock solution in demineralized water, stored at RT.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid monohydrate</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td style="width:119px;">1002441000</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slips</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td>631-1339</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">DAPI Fluoromount-GTM</td>
			<td style="width:261px;">SouthernBiotech, Birmingham (USA)</td>
			<td style="width:119px;">SBA-0100-20</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Databases such as KEGG, HPRD and QIAGEN (Genes &#38; Pathways)</td>
			<td style="width:261px;">http://www.genome.jp/kegg/pathway.html; http://www.hprd.org/; https://www.qiagen.com/de/geneglobe/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">Different known literature databases were used for generating the network topology.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female Luer Lug Style Tee</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">FTLT-1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Female Luer Thread Style with 5/16&#34; Hex to 1/4-28 UNF Thread</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">SFTLL-J1A&#160;</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:261px;">Bio&#38;SELL, Feucht (GER)</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td style="width:212px;">not heat-inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gefitinib</td>
			<td style="width:261px;">Absource Diagnostics GmbH, M&#252;nchen (GER)</td>
			<td style="width:119px;">S1025-100 mg</td>
			<td style="width:212px;">100 mM stock solution with DMSO</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glas flask (Schott, GER) provided with glas hose connection</td>
			<td style="width:261px;">Weckert, Kitzingen (GER)</td>
			<td style="width:119px;">custom made</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histofix 4 % (Paraformaldehyd)</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">P087.1</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hose coupling</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>CC-9</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Incubator for bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">M30 CytoDeathTM ELISA</td>
			<td style="width:261px;">Peviva, Bromma (SWE)</td>
			<td style="width:119px;">10900</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male Luer Integral Lock Ring</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">MTLL230-J1A</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moisture chamber</td>
			<td style="width:261px;">custom made</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mouse anti Pan-Cytokeratin</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">C2562-2ML</td>
			<td style="width:212px;">Clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2, used 1/100 for immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needlefree Swabable Valve Female Luer</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>NVFMLLPC</td>
			<td style="width:212px;">Bioreactor setup, for sampling, gamma-sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 10 red 37*3 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">21444</td>
			<td>O-ring large, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 70 red 27*2.5 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">19170</td>
			<td>O-ring small, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PAP pen</td>
			<td style="width:261px;">Dako, Hamburg (GER)</td>
			<td>S002</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">6642.6</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:261px;">Ismatec, Wertheim-Mondfeld (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">D8537-6x500ml</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pump tubing cassette</td>
			<td style="width:261px;">Ismatec, Wertheim (GER)</td>
			<td>IS 3710</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit anti Ki67</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab16667</td>
			<td style="width:212px;">Clone SP6, used for 1/100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti Vimentin</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab92547</td>
			<td style="width:212px;">used 1/100 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 medium</td>
			<td style="width:261px;">Life technologies, Darmstadt (GER)</td>
			<td style="width:119px;">61870-044</td>
			<td style="width:212px;">warm in 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicone tube</td>
			<td style="width:261px;">Carl Roth GmbH, Karlsruhe (GER)</td>
			<td style="width:119px;">HC66.1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Hydroxide</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td>30620-1KG-R</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SQUAD</td>
			<td style="width:261px;">http://sbos.eu/docu/docu/SQUAD/doku.php.htm</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for performing the semiquantitative simulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile air filter, pore size 0.2 &#181;m</td>
			<td style="width:261px;">Sartorius Stedium Biotech, G&#246;ttlingen (GER)</td>
			<td style="width:119px;">16596-HYK</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Luer Lok 5ml</td>
			<td>BD Biosciences, Heidelberg (GER)</td>
			<td>309649</td>
			<td style="width:212px;">for bioreactor sampling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture test plates: 6-,&#160;&#160;&#160;&#160;&#160; 12-, 24-, 96- well</td>
			<td style="width:261px;">TPP Techno Plastic Products AG, Trasadingen (GER)</td>
			<td style="width:119px;">92006, 92012, 92024, 92048&#160;</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transforming growth factor-beta 1 (TGF-&#946;1) with carrier</td>
			<td style="width:261px;">Cell Signaling, Frankfurt (GER)</td>
			<td style="width:119px;">8915LC</td>
			<td style="width:212px;">stock solution in sterile citrate buffer pH 3.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">X100-1L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween-20</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">P7949-500ml</td>
			<td style="width:212px;">for washing buffer of immunofluorescent staining</td>
		</tr>
	</tbody>
</table>

a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds

本研究では、我々は特定の突然変異背景に基づいて、薬物応答の予測を最適化するために、 シリコモデルで用いたin vitro 3D肺腫瘍モデルを組み合わせます。モデルは、基底膜を含む細胞外マトリックス組成物およびアーキテクチャに関する組織特異的特性を再現脱細胞化ブタの足場上に生成されます。私たちは、薬物治療の3日を含め14日以内に人工的な腫瘍組織の生成を可能にするプロトコルを標準化しました。私たちの記事では、読み出しの有効性を評価するための便利なツールである増殖指数Ki67染色の、M30-ELISAによって上清からアポトーシスと間葉転換 （EMT） に上皮の評価の決意のようなスクリーニング技術を、3Dのいくつかの詳細な説明を提供します治療用化合物。我々は、2D培養物にrelatある我々の3D腫瘍モデルにおける増殖の減少を比較し示すことができ臨床状況にエド。このより低い増殖のにもかかわらず、このモデルは、HCC827肺癌細胞株の比較によって示されるように正確にバイオマーカーの状態に応じてEGFR -targeted薬物応答を予測（EGFRは -mutated、KRAS野生型）およびA549（EGFR野生型KRAS -変異） チロシンキナーゼ阻害剤 （TKI）ゲフィチニブで処理しました。ビメンチン/パン - サイトケラチン免疫蛍光染色によって評価されるように、より高度の腫瘍細胞の薬物応答を調べるために、我々は、TGF-β-1との長期治療によりEMTを誘導しました。フローバイオリアクターは、組織の生成を改善し、生理的条件下に培養物を調整するために使用しました。アポトーシス、増殖指数とEMT - -ブール値へのシリコモデルではさらに、我々は、ゲフィチニブ治療またはTGF-β-1刺激の際に薬剤応答の統合を示しています。さらに、当社は、特定の突然変異背景とカウントを有する腫瘍細胞のどの薬物応答を説明します抵抗に対するerstrategiesを予測することができます。我々は、特にそのインシリコ拡大を用いたin vitroのアプローチで私たちの 3Dは2D細胞培養におけるよりもより現実的な条件での前臨床薬物検査のための付加価値を提供することを確信しています。

SISmuc足場（Cに図2A）に基づき、我々は3D腫瘍試験システム（ 図2D）の生成、刺激及び治療 ​​のための標準化された操作手順を確立しました。それぞれ、 図1および図3に示すように、このモデルは、増殖指数及びM30-ELISAを使用して、アポトーシスの定量化の決意を可能にします。3に示すA549およびHCC827モ...

Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

操作された複合三次元組織<em&gt;インビトロ</em&gt; /<em&gt;インシリコ</em具体的な突然変異背景に薬物の有効性を予測するための&gt;肺腫瘍モデル

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Research

JoVE Journal

Bioengineering

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

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

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

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 ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

心臓医薬品安全性スクリーニングのための人間エンジニア心臓組織の自動収縮分析

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

化学のエピジェネティックな修飾剤の細胞機械装置をリダイレクトすることによって遺伝子の転写を抑制

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

シードとマウス モデルにおける合成組織設計気管移植片の移植

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

脳癌と神経変性疾患を研究するためのヒト神経オルガノイド

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

ラットにおける円周食道再建のための組織工学型移植片

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...