Name: a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay
Uploaded: 2018-04-12T14:15:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

この作品は、上海科学委員会、技術 (15411951800、15410723200) からの助成金によって支えられました。著者教授 YuMei 温とその技術支援復旦の病原微生物部大学教授・ チャオ趙を感謝したいです。

このメソッドは、承認されたガイドラインや研究倫理委員会の華山病院、復旦大学の規則に従い行った。対応する標準的な安全対策は、各ステップで続いていた。図式、図 1を参照してください。
1. 細胞培養とプラスミド構築
<ol><li>定期的に管理ダルベッコでひと臍帯静脈内皮細胞 (株) のイーグル培地 (DMEM) 10% 牛胎児血清 (FBS)、100 単位/ml ペニシリン、ストレプトマイシン加湿の 5% の 100 μ g/mL と補われる、変更された CO2インキュベーター37 ° C で</li>
	<li>ShRNA フラグメントの合成の ApaI と EcoRI PLKO.1 プラスミドを消化します。VHL shRNA ベクターのオリゴヌクレオチドのシーケンスが CCGGGATCTGGAAGACCACCCAAATCTCGAGA TTTGGGTGGTCTTCCAGATCTTTTTG14であることを確認します。</li>
	<li>オリゴ、逆オリゴの 5 μ L、NEB バッファー x 10 の 5 μ L、ddH2O 一緒に (総容積は 50 μ L) の 35 μ L の 5 μ L 前方で、20 の µΜ システムを混ぜます。数時間で 4 分、ゆっくりと室温まで冷却 98 ° C で混合物を加熱します。</li>
	<li>一晩焼なまし oligos と一緒に 4 ° C で T4 リガーゼ PLKO.1 プラスミドを縛る。16 時間後、25 μ L の有能な DH5α セルに結紮ミックスの 5 μ L を追加します。正常に結紮プラスミドをスクリーンし、シーケンスによって挿入されたフラグメントを確認します。</li>
</ol>2. レンチ ウイルス パッケージと感染症
<ol><li>合計では、PLKO.1 shVHL または PLKO.1 shScramble のベクトルの 10 μ g、プラスミド psPAX2 のパッキングの 7.5 μ g、500 μ L の 25 分のためのウシ胎児血清 (FBS) なし DMEM メディアで封筒プラスミド pMD2.G の 2.5 μ g を混ぜます。</li>
	<li>FBS なし 7 mLDMEM を上記準備ソリューションに追加します。</li>
	<li>直径 10 cm のティッシュの培養皿で FBS なし DMEM 293 FT 細胞。293 FT 細胞数が 1 × 107セルのカウンターを使用してであることを確認します。</li>
	<li>293 FT 細胞の培地に transfection のため上記準備溶液 1 mL を追加します。 注:293 FT 細胞ラインは、293 f 細胞ラインから派生し、安定 SV40 大きい T の抗原を表現します。そのため、レンチ ウイルスの生産のための適したホストです。</li>
	<li>10% を含む DMEM 培に置き換える 6 h 後 FBS。</li>
	<li>トランスフェクション後 48 h でピペットを使用してメディアを収集します。 注:10% を含む DMEM にウイルスが存在する政府短期証券。死んだ細胞は、培地を浮遊します。0.45 μ m の無菌膜を使用すると、死んだ細胞や不純物をフィルター処理します。一般的に、感染症 (MOI) の多様性をチェックする必要はありません。これは、安定した細胞ラインを確立することです。最後のステップで成功の感染なしすべての生きている細胞は、ピューロマイシンによって死んでしまいます。ShScramble グループと血管内皮細胞は、コントロール グループです。すべての血管内皮細胞が 72 h で使用されるピューロマイシン濃度と死ぬことを確認します。同じ数のセルをすべてきちんと備えています。</li>
	<li>72 h のレンチ ウイルスのメディアで血管内皮細胞を培養します。ピューロマイシンを対照群として治療なし 24 時間使用血管内皮細胞の 2 μ G/ml の濃度で血管内皮細胞のメディアに追加します。この時点でセルは死に、コントロールを確認します。 注:生きている細胞は、VHL ノックダウン細胞と shSramble 細胞の安定したセルラインを表します。メッキ密度で 5,000/ウェルの 96 ウェル プレートであります。</li>
</ol>3. 血管内皮細胞スフェロイドの世代
<ol><li>Huvec 細胞を trypsinize し、10 %dmem 培地で再懸濁します FBS。10 cm 培養皿の細胞の中程度で、typsin EDTA の 1 mL を追加します。</li>
	<li>1 × 103細胞/ウェルの細胞密度で、3 D の丸底 96 ウェル プレートで細胞を播きます。まあ回転楕円体を停止する 0.50 μ L の回転楕円体を収容することができますそれを使用します。製造元の指示に従って細胞カウンター システムを用いた細胞を数えます。</li>
	<li>37 ° C で 5% CO2連続 72 時間のセルを孵化させなさい。これらの条件の下で浮遊細胞は自動的に細胞スフェロイドを形成することを確認します。36 時間後培地の半分を交換してください。</li>
</ol>4.体外血管新生アッセイ
<ol><li>4 ° C でゲル溶液を解凍し、減らされた血清培地で希釈比が 1:5 で割りますと。</li>
	<li>マイクロ ピペットを DMEM 培地から回転楕円体を吸います。減少血清培地 5 mL の回転楕円体を洗浄した後希釈のゲルで、ゆっくりと慎重に、回転楕円体を中止すること。空気の泡がないことを確認します。</li>
	<li>15 ウェル プレートに 300 μ L の混合液を埋め込みます。37 ° C と 1 h の 5% CO2で潜伏後 400 μ L 減少血清培地を各ウェルに追加します。滅菌水を蒸発を妨げる加湿環境を生成する井戸の周りをご記入ください。</li>
	<li>その後、文化で 37 ° C 1 時間の湿度 100% で 5% CO2プレート。</li>
	<li>慎重に古い培地を吸引し、各ウェルで内皮細胞成長サプリメントで 1% 減少血清中の 600 μ L で置き換えます。</li>
	<li>倒立顕微鏡下で撮影後 12 h、24 h (40 *)。</li>
</ol>5. データの解析
<ol><li>スプラウト長さデータ (資材表) を取得するオンライン画像解析プラットフォームに画像をアップロードします。</li>
	<li>Web ページ、メールでアカウントを作成します。</li>
	<li>[アップロード] ボタンをクリックして画像をアップロードします。</li>
	<li>ダウンロード ボタンをクリックして結果をダウンロードします。 注:ソフトウェアは、処理後の画像とデータが生成されます。データに 5 つの部分が含まれている: 累積的な芽の長さ、平均スプラウト長さスプラウト長さ、スプラウト領域と回転楕円体領域の標準偏差。処理後の画像の識別を容易にする別の色では、処理後の画像の各パラメーターの輪郭が表示されたら: 赤い表すもやしスケルトン、芽数を黄色、青い芽構造、白芽エンドとオレンジ回転楕円体。</li>
</ol>

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著者が明らかに何もありません。

最近では、血管生物学の研究の複数のフィールドが血管内皮15の研究によって刺激されました。この記事での新規候補分子を識別するために操作の血管内皮細胞における機能の VHL 遺伝子の損失に起因する血管の形成を研究する実験モデルとして技術を発芽内皮細胞スフェロイドを開発した、血管新生のカスケード。我々 の知る限り、これは天体の変更された血管内皮細胞?...

血管芽腫 (HBs) は、人間の中枢神経系 (CNS) にのみ見られる血管腫です。フォン ・ ヒッペル-リンダウ (VHL) 病や散発的な病変の患者で開発します。VHL HBs は頻繁に再発のため外科的治療によって治癒が困難とこれに起因する遺伝的多発障害1。VHL 癌抑制遺伝子の不活化は、VHL HBs の腫瘍の根本的な原因と考えられている、(新生血管を含む) 細胞の起源と HBs の進化プロセスのまま大きく物議を醸す2です。したがって、HB 新生血管生物学的メカニズムの理解は、VHL HBs の最も有望な抗血管戦略に有用な洞察を提供可能性があります。
最近の研究では、HB 新生血管が発生学的血管3,4、5と似ていることを示唆しています。古典的な血管内皮増殖因子 (VEGF)-を介した血管新生血管の内皮細胞に由来する、関数の VHL の損失によって牽引されて増殖し、新生血管形成されている6に挑戦します。1965 年 Cancilla とジマーマンは、HBs の由来内皮7電子顕微鏡を使用してください。その後、間質細胞由来 vasoformative エレメント8それが見つかりました。1982 年、Jurcoらは間質細胞が内皮細胞起源9あることを発見しました。したがって、我々 の仮説ひと血管内皮細胞が HB 血管新生10の元の細胞であります。VHL 患者手術から派生した HB 細胞の初代培養を使用することをお勧め、HB からプライマリ カルチャが、安定しない、細胞株の確立された3できませんでした私たちの以前の研究が示されます。また、HB 血管成分10,11の前駆細胞が含まれているために、3 D 環境で初代培養は HB 新生血管の細胞起源を識別できませんでした。したがって、プリミティブと古典的な血管内皮細胞のモデルとしてひと血管内皮細胞 (株) は、HBs の細胞の代替モデルとして役立つことができます。
回転楕円体の発芽アッセイは、組織工学12,13の新しいモデルです。本稿で、3 D コラーゲン ベース共システムの in vitroアッセイを発芽回転楕円体を使用して、開発された HBs に於いての役割と同様、HBs、古典的な腫瘍血管新生が存在するかどうかを評価するための全体的な目標を持つ。

<table><tbody><tr><td>human umbilical vein endothelial cell</td><td>Fudan IBS Cell Center</td><td>FDCC-HXN180</td><td></td></tr><tr><td>dulbecco&amp;rsquo;s modified eagle&amp;rsquo;s medium&amp;nbsp;</td><td>Gibco</td><td>11995040</td><td></td></tr><tr><td>fetal bovine serum</td><td>Gibco</td><td>&amp;nbsp;26400044</td><td></td></tr><tr><td>PLKO.1-puro vector</td><td>Addgene</td><td>#8453</td><td></td></tr><tr><td>packing plasmid psPAX2&amp;nbsp;</td><td>Addgene</td><td>#12260</td><td></td></tr><tr><td>envelope plasmid pMD2.G</td><td>Addgene</td><td>#12259</td><td></td></tr><tr><td>3D round-bottom 96-well plates</td><td>S-Bio</td><td>MS-9096M</td><td></td></tr><tr><td>matrigel</td><td>BD Biosciences</td><td>354234</td><td></td></tr><tr><td>Opti-MEM medium</td><td>Gibco</td><td>31985-070</td><td>reduced serum medium&amp;nbsp;</td></tr><tr><td>15-well plate</td><td>Ibidi</td><td>81501</td><td>Air bubbles in the gel can be reduced by equilibrating the &amp;mu;&amp;ndash;Slide angiogenesis before usage inside the incubator overnight</td></tr><tr><td>endothelial cell growth supplements</td><td>Sciencell</td><td>#1052</td><td></td></tr><tr><td>10-cm culture dish</td><td>Corning</td><td>Scipu000813</td><td></td></tr><tr><td>Puromycin</td><td>Gibco</td><td>A1113802</td><td></td></tr><tr><td>typsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Automated Cell Counter System &amp;nbsp;</td><td>BioTech</td><td></td><td></td></tr><tr><td>Image Analysis software&amp;nbsp;</td><td>Winmasis</td><td></td><td>http://mywim.wimasis.com&amp;nbsp;</td></tr></tbody></table>

a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay

・ フォン ・ ヒッペル-リンダウ (VHL) 癌抑制遺伝子の不活化は、人間の中枢神経系 (CNS) 内血管芽腫 (HBs) の開発で重要な役割を果たしています。しかし、細胞の起源と HBs (新生血管を含む) の進化の過程の両方残る物議を醸す、VHL HBs、古典的な HB 新生に基づいての抗血管新生が臨床試験で残念な結果を生成しました。抗血管内治療の臨床成功の翻訳 1 つの主要な障害は、この血管豊富な腫瘍血管新生の徹底的な理解の欠如です。この記事では体外でHBs に於いての役割と同様、HBs、古典的な腫瘍血管新生が存在するかどうかを評価する包括的な手順を提案する.この手順で研究者は正確に HB 血管新生の複雑さを理解し、HBs で血管新生のこの一般的な形式の関数を識別できます。これらのプロトコルは、腫瘍の治療のため、または HBs 抗血管新生治療の最適化で将来の翻訳を支援する並進可能性が高い腫瘍については、最も有望な抗血管治療の評価に使用できます。結果は、HB の血管新生の複雑さを強調表示し、この一般的なフォームの血管新生が HB 血管新生の補完機構のみであることを示唆します。

元の画像は倒立顕微鏡で撮影しました。コントロール グループと VHL グループの典型的なイメージは、図 2に示す-A1および図 2-A2。コントロール グループの発芽の長さは VHL グループのより短いです。
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Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

発芽アッセイ回転楕円体を使用して推定される芽新生体外パフォーマンスを評価する包括的な手順

In vitro血管芽腫 (HBs) と HBs におけるその役割の古典的な腫瘍血管新生が存在するかどうかを評価する包括的な手順を提案します。結果は、HB 血管新生の複雑さを強調表示し、血管新生のこの共通の形態の HB 血管新生における補完機構のみが示唆されました。

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

a-comprehensive-procedure-to-evaluate-vitro-performance-putative

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

7.9K ビュー.  Fudan University. In vitro血管芽腫 (HBs) と HBs におけるその役割の古典的な腫瘍血管新生が存在するかどうかを評価する包括的な手順を提案します。結果は、HB 血管新生の複雑さを強調表示し、血管新生のこの共通の形態の HB 血管新生における補完機構のみが示唆されました。

ビデオ: A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

がん研究

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

神経膠芽腫の3次元スフェロイドモデル

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

ペリサイトの発芽アッセイ内皮細胞ビーズを組み込む

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

発生生物学

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

内皮コロニー形成細胞と間葉系幹細胞によって形成された共培養スフェロイドを用いた3次元血管新生アッセイ系

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

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 ...

医学

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

生物工学

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

iPSC由来内皮細胞の浸透した3D血管新生発芽をインビトロで研究するための標準化およびスケーラブルなアッセイ

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼小血管血管新生のエキビボロイド発芽アッセイ

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

生物学

スクラッチ創傷移動とスフェロイドスプラウティングアッセイを用いた血管新生の研究

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

著者スポットライト:アッセイ開発における課題と革新による血管新生の調査

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Scratch Wound Migration Assay と Spheroid Sprouting Assay を活用した機能的および分子レベルでの血管新生の調査

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...