Name: incorporating pericytes into an endothelial cell bead sprouting assay
Uploaded: 2018-02-16T15:00:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

有用な議論、夫妻ビクトリア Bautch とジョシュア ブーシェに感謝し、標準的なビーズの最適化に関するアドバイス発芽アッセイ条件と発芽の試金のプロトコルを汚します。S.H.A. だったから国立科学研究所の一般医療下での補助金によって一部にはサポート 5T32 GM007092 賞を受賞します。

<ol>
	<li>Hanahan, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+emerging+mechanisms+of+the+angiogenic+switch+during+tumorigenesis.">Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.</a> Cell. 86 (3), 353-364 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+ischemic+and+neoplastic+disorders.">Angiogenesis in ischemic and neoplastic disorders.</a> Annu Rev Med. 54, 17-28 (2003).</li><li>Zhang, Z. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+three-dimensional+in+vitro+model+for+the+analysis+of+angiogenesis.">An optimized three-dimensional in vitro model for the analysis of angiogenesis.</a> Methods Enzymol. 443, 65-82 (2008).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> Microvasc Res. 50 (3), 311-322 (1995).</li><li>Sims, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pericyte--a+review.">The pericyte--a review.</a> Tissue Cell. 18 (2), 153-174 (1986).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Tattersall, I. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+endothelial+interaction+with+macrophages+and+pericytes+demonstrates+Notch+signaling+function+in+the+vascular+microenvironment.">In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.</a> Angiogenesis. 19 (2), 201-215 (2016).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+an+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.</a> Science. 307 (5706), 58-62 (2005).</li><li>Tahergorabi, Z., Khazaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Popson, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-induced+transmembrane+protein+1+regulates+endothelial+lumen+formation+during+angiogenesis.">Interferon-induced transmembrane protein 1 regulates endothelial lumen formation during angiogenesis.</a> Arterioscler Thromb Vasc Biol. 34 (5), 1011-1019 (2014).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438 (7070), 937-945 (2005).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. 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著者が明らかに何もありません。

このプロトコルは、複雑なステージと徹底解明研究を実施する遺伝とイメージングの方法を採用する研究者を有効にすることによって血管新生を発芽の血液細胞間相互作用を特徴付けるための方法を示します。分析を実行すると、そのビーズの内皮塗着効率の間に起こるビーズ撹拌手順が不可欠です。貧しい内皮コーティングはビーズ ゲル注入前に次の朝のゴルフ ボールのような粗面を有?...

血管新生は適切な胚形成、創傷治癒に不可欠ながん進行1冠動脈疾患を含む多くの疾患で重要な役割を果たします。2,3は、小説、効果的な治療法の開発のために重要な血管新生がどのように通常の開発時に発生して、それは病理学的コンテキストの再アクティブ化方法のより良い理解を持っていることです。良い運転して血管新生の分子機構を特徴と新しい発見をする研究者を許可する重要な段階と生体内の血管新生に関与する細胞の種類を再現忠実の in vitroモデルが必要内皮の規則。
中津とヒューズは、血管新生を発芽の多くの知られていた段階を経るを示している発芽ビーズ アッセイを最適化しています。4,5ここで紹介した方法の目的は、アッセイに perictyes を組み込むことで中津とヒューズを最適化壁画発芽血管内皮細胞のパラクリンおよび juxtracrine の役割に組み込むことができるアッセイに構築するには新規血管新生研究。ペリサイトは、細胞を維持する彼らの血管基底膜に埋め込まれているため内皮細胞に物理的に接触の近くにその役割で定義されている壁画のセルです。6ペリサイトおよび内皮細胞の複雑なクロス話-従事ノッチを含む経路のシグナルを介したシグナリング、Ang Tie2、PDGFRβ、TGFRβ、および他の多くの。7,8マウス モデルこれらのシグナル伝達経路の欠乏を示す悪い血管リモデリングにつながる胚の血管と血管系の機能不全を開発の貧しい周報道。7また、病的血管新生におけるペリサイトの役割は重要ですが、しばしば感謝です。たとえば、腫瘍血管の特徴にある血管より未熟な漏れ、貧しい周報道による機能不全。9それが劇的にペリサイトの有無は、腫瘍の血管の表現型に影響を与えるし、抗血管新生および抗腫瘍療法への応答の重要な仲介者は、提案されています。9このように、生体外の試金のペリサイトの役割を含むは内皮規制の重要なメカニズムをより完全にキャプチャするためのキーです。
現在、血管新生の研究に採用多くの体外と体外の試金が、それぞれに考慮する欠点があります。内皮細胞増殖や血管内皮移動分析など、いくつかは過度に簡略化し、焦点組織培養プラスチックの分離設定では 1 つの血管内皮機能に。10他のアッセイが発生する詳細 3 次元 (3 D) の設定でなどマトリゲル管形成アッセイ、10しかし、これらの試はまだ単純化移行・ デ ・ ノボ血管を形成する内皮細胞の機能に重点既存の血管から発芽するのではなく、構造体。さらに、これらのアッセイのどれもは壁画のセルの種類を組み込みません。前のヴィヴォモデル リング大動脈アッセイなどペリサイト ホスト器官に存在を組み込むことがありますが、これらのモデルの遺伝子操作はのノックアウト、トランスジェニック マウス モデルの生成の必要性のためにより挑戦的、関心の経路。発芽アッセイ ビーズは、内皮発芽、増殖、移行、および 3 D マトリックスでも吻合と内腔形成をモデル化するので最適です。4アッセイは内皮細胞またはより制御された設定でペリサイトの直接遺伝的変更を許可しながら、発芽の多くの異なる段階の機構の評価のために忠実にできます。発芽のビーズを含むフィブリン血栓とが簡単固定、染色、発芽; のさまざまな段階でイメージこれらもやしは、発芽のリアルタイム イメージングを実行するライブ イメージングの商工会議所にも配置できます。ここで紹介する方法は、深さの表現型解析における血管新生の基本的なメカニズムと血管新生中に活性化経路を徹底的に分析研究に最適です。

<table><tbody><tr><td>Sterile Pipette tips</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipettors</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Complete EGM2 Media Bullet Kit</td><td>Lonza</td><td>CC-3162</td><td>HUVEC Media</td></tr><tr><td>MEM</td><td>Gibco</td><td>11095114</td><td>10T1/2 Media</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965118</td><td>NHLF Media</td></tr><tr><td>Tissue culture-grade PBS</td><td>Gibco</td><td>14190-144</td><td>Magnesium and calcium free</td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>For lifting HUVEC</td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>15050065</td><td>For lifting 10T 1/2 and NHLF</td></tr><tr><td>Customs siRNAs</td><td>Sigma</td><td></td><td></td></tr><tr><td>Lipofectamine RNAiMax</td><td>Life Technologies</td><td>13778150</td><td></td></tr><tr><td>HUVEC</td><td>Lonza</td><td>C2517A</td><td></td></tr><tr><td>10T 1/2</td><td>ATCC</td><td></td><td></td></tr><tr><td>NHLF</td><td>ATCC</td><td></td><td></td></tr><tr><td>Cytodex 3 microcarrier beads</td><td>Sigma</td><td>C3275</td><td></td></tr><tr><td>Tissue culture-coated 6 and 10 cm plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Sigma</td><td>F8630</td><td></td></tr><tr><td>Thrombin</td><td>Sigma</td><td>t9549</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>a3428</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue culture incubator and hood</td><td></td><td></td><td></td></tr><tr><td>24-well glass bottom plates</td><td>MatTek</td><td>P24G1.513F</td><td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td></tr><tr><td>Sterile Filtration Device</td><td></td><td></td><td></td></tr></tbody></table>

incorporating pericytes into an endothelial cell bead sprouting assay

血管新生は既存の血管から新しい血管の成長を胚形成と開発、創傷治癒、腫瘍の成長と転移を含む多くの生物的プロセスと眼と心血管疾患の重要なコンポーネントです。要約血管新生学効果の in vitroモデルは、適切にこのプロセスを研究し、新しい治療戦略のための最終的にターゲットすることができます調節機構が必要です。ビーズ血管新生アッセイは、以前内皮発芽の複数の段階を要約する実証されている体外。しかし、この試金の制限が無い体内の血管内皮-キーを内皮細胞の分子と表現の規制である壁画細胞の相互作用,機能します。ここに与えられたプロトコル ビーズ血管新生アッセイに壁細胞の混入のための方法論を提示し、発芽の培養中に血管内皮細胞と血管の緊密な関連を示します。プロトコルでは、効果的なサイレンシング機構研究の内皮細胞で siRNA を用いた遺伝子のための方法論についても詳しく説明します。完全に、このプロトコルは提供の in vitroアッセイをより適切に発芽して、血管新生に関与するさまざまな細胞タイプをモデル化し、治療評価の新規発見より生理学的関連性の高いプラットフォームを提供します血管新生の調節機構。

このプロトコルは、2 つのセル型in vitro でのタイトな協会と、ペリサイトの存在が発芽 (図 1 a, B) の発生を補完します。プロトコルも効果的なサイレンシング (例えばRNA 干渉経由) (具体的には血管内皮細胞の VEGFA) またはペリサイトの PDGFRβ など7,12のセル型の興味の遺伝子の?...

Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

新規体外ビーズ アッセイをこのプロトコルより適切に体内の血管を組み込むことによって血管新生を萌芽の過程をモデル化します。この変更により、血管内皮細胞と血管新生に重要な壁画と有性の細胞間相互作用をより忠実に再現するビーズ アッセイ。

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 ...

incorporating-pericytes-into-an-endothelial-cell-bead-sprouting-assay

Research

JoVE Journal

Developmental Biology

7.9K ビュー.  University of North Carolina at Chapel Hill. 新規体外ビーズ アッセイをこのプロトコルより適切に体内の血管を組み込むことによって血管新生を萌芽の過程をモデル化します。この変更により、血管内皮細胞と血管新生に重要な壁画と有性の細胞間相互作用をより忠実に再現するビーズ アッセイ。

ビデオ: Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

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 ...

発生生物学

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

微細と3D微小血管の組立

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

生物工学

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 ...

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 ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

血液脳関門メッシュ様血管形成および崩壊を評価するためのインビトロアッセイ

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

医学

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3 D 細胞培養と非高血圧症全血を用いた血管内皮細胞の抗凝固および抗炎症作用の評価

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

マウスにおけるマトリックスゲルプラグアッセイを用いたヒト内皮-周皮細胞の相互作用のin vivo研究で

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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 ...

ペリサイトの発芽アッセイ内皮細胞ビーズを組み込む

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血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

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

微細と3D微小血管の組立

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

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

血液脳関門メッシュ様血管形成および崩壊を評価するためのインビトロアッセイ

3 D 細胞培養と非高血圧症全血を用いた血管内皮細胞の抗凝固および抗炎症作用の評価

マウスにおけるマトリックスゲルプラグアッセイを用いたヒト内皮-周皮細胞の相互作用のin vivo研究で

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

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