この研究は、鈴木マンペイ糖尿病財団(YT)、ボストン小児病院OFD/BTREC/CTREC教員キャリア開発助成金、ボストン小児病院眼科学財団、BCHパイロット賞、BCHマントンセンターフェローシップ、マントンセンターフェローシップの助成金によって支援されました。 そしてリトルキリン財団(ZF)、ドイツ研究財団(DFG;紀元前[CA1940/1-1])、NIH R24EY024868、EY017017、R01EY01717-13S1、EY030904-01、BCH IDDRC(1U54HD09025)、マサチューセッツ州ライオンズ財団(1U54HD09025)、マサチューセッツ州ライオンズ財団(1U54HD09025)、LEHS財団(

記載されているすべての動物実験は、ボストン小児病院の施設動物ケアおよび使用委員会(ARCHプロトコル番号19-04-3913R)によって承認されました。
1. 準備
<ol><li>ペニシリン/ストレプトマイシン(10000 U/mL)5 mLと5mLと10mLの市販サプリメントを、完全な古典的な培地の500 mLに血清で加えます。初期の培地のアリコート50mL。 注:汚染を避けるために、在庫に媒体を戻さないようにしてください。</li>
	<li>氷の上に完全な古典的な媒体のアリコートを置きます。</li>
	<li>70%エタノールを使用して、解剖顕微鏡、鉗子、ハサミを洗浄してください。</li>
	<li>解剖顕微鏡に1つ、氷の上に1つ、2つの細胞培養皿(10 cm)を準備します。各皿に完全な古典的な媒体の10 mLを入れてください。</li>
</ol>2. 実験ステップ (図 1)
<ol><li>75-100 mg/kgケタミンを使用した出生後(P)20の周りのC57BL/6Jマウスを犠牲にし、7.5-10 mg/kgキシラジンを腹腔内注射する。解剖する前に氷の上に完全な古典的な媒体で目を保ちます。</li>
	<li>結合組織(筋肉と脂肪組織)と眼の視神経を取り除きます。</li>
	<li>マイクロハサミを使用して角膜のリンブに0.5mm後部を円周的に切断します。角膜/虹彩複合体、ガラス状、レンズを取り除きます。</li>
	<li>1mm切開を視神経に向かって切り取りエッジに垂直にし、幅1mmの円周バンドを切断します。複合体の中央領域と周辺領域を分離します。鉗子を使用して、RPE/脈絡/強膜複合体からレチナを剥がします。</li>
	<li>氷の上に完全な古典的な媒体で末梢脈絡膜バンドを保ちます。もう一方の目を分離し、2番目のバンドを切断するプロセスを繰り返します。</li>
	<li>円形のバンドを6~等しい正方形(〜1mm×1mm)に切ります。 注意:どのエッジにも触れないでください。</li>
	<li>製造指図ごとに基底膜抽出物(BME)を解凍します。BMEの30 μL/wellを24ウェル組織培養プレートの各ウェルの中央に加えます。BMEの液滴が、エッジに触れることなくプレートの底部に凸型ドームを形成していることを確認してください。 注:冷蔵庫で一晩BMEを解凍してください。BMEは解凍後いつでも氷の上にあるべきです。</li>
	<li>BMEの真ん中に組織を置きます。 注意:脈絡膜の外植を平らにしないでください。一般に、組織をBME内で拡大させる。組織の向き(強膜側の上下)は、実験結果に影響を与えません。</li>
	<li>37°Cでプレートを10分間インキュベートし、ゲルを固化させます。</li>
	<li>完全な古典的な媒体/井戸の500 μLを加える。</li>
	<li>クラシックメディアを1日おきに変更します(500 μL)。脈絡芽は顕微鏡で3日後に観察することができる。 注:成長因子治療のために、4時間組織を飢えます。増殖因子還元培地で試験化合物を希釈する(1:50の代わりに1:200ブースト)。</li>
</ol>3.SWIFT-コロイドコンピュータ化定量方法17 (図2)
注:成長する船舶がカバーする面積を測定するためのコンピュータ化された方法が使用されました。ImageJ ソフトウェアへのマクロプラグインは、定量化の前に必要です (詳細については 補足情報 を参照してください)。
<ol><li>イメージJでチョロイドの発芽画像を開き、「画像|タイプ|グレースケールの 8 ビット "</li>
	<li>「 イメージ |に移動する|を調整する明るさ/コントラスト(Ctrl/シフト/C)を使用し、コントラストを最適化します。</li>
	<li>魔法の杖機能を使用して、新芽の中心に存在する脈絡膜組織を画像から外し、(ショートカットキーを使用してF1")(図2A,B)を使用します。 注:魔法の杖の許容率を20〜30%に設定します。</li>
	<li>自由選択ツール (図 2C)で画像の背景を削除します。「 イメージ |に移動する|を調整するしきい値 (Ctrl/シフト/T)"。閾値関数を使用して、背景および周辺に対する微小血管芽を定義する(図2D)。</li>
	<li>[F2]をクリックすると、概要が表示されます。[保存] をクリックして、選択した領域の画像を保存します。今後の参照のために、元の画像と同じフォルダに保存します。</li>
	<li>サンプルのグループを測定した後、データ分析のために記録されたデータをコピーします。 注:また、「分析|で面積(μm²)を測定することも可能です。スケールバー付きのイメージを使用してスケール " を設定します。</li>
</ol>

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著者らは財務上の開示を行っていない。コンピュータ化された方法は、著者を通じて学術機関に無料で利用可能です。

脈絡膜発芽アッセイは、新血管AMD9、10、18、19、20の研究を助ける。脈絡膜外植は、マウスだけでなく、ラットおよびヒト17、21から単離することができる。脈絡膜外植は、IC、マクロファージ、およびペリサイト17<...

脈絡膜血管新生調節異常は、新血管加齢黄斑変性症(AMD)1と関連している。脈絡膜は、網膜色素上皮(RPE)の下に存在する微小血管床である。脈絡膜における血流の減少は、AMD2の進行と関連することが示されている。血管内皮、RPE、マクロファージ、包皮細胞および他の細胞との間の複雑な関係は、組織3、4、5の恒常性を担う。従って、再生可能なアッセイは、新血管AMDの研究に重要な脈絡膜微小環境をモデル化する。
Ex vivo血管新生アッセイおよびインビトロ内皮細胞培養は、生体内の微小血管挙動の研究、新薬の試験、および病原性の研究を補完することができる。ヒトの眼内小血管内皮細胞(HRMEC)、ヒトアンビリカル静脈内皮細胞(HUVEC)、単離された原発性動物の脳または眼内脳の脳などの内皮細胞は、眼血管新生研究6、7、8の体外研究でしばしば使用される。特にHRMECは、内皮増殖、移動、管形成、および血管漏れを評価することによってインビトロ脈絡膜新血管新生(CNV)9のモデルとして広く使用され、介入を評価する6,10。しかし、培養中のECは、脈絡膜に見られる他の細胞型との相互作用が欠如し、これらのアッセイで使用されるほとんどのECが脈絡膜に由来しないため、CNVのモデルとして制限されています。マウス脈絡膜電子株は培養中に分離し維持することが困難である。
大動脈環アッセイは、マクロ血管増殖のモデルとして広く用いられている。大動脈外植植物からの血管芽は、IC、ペリサイトおよびマクロファージ11を含む。大動脈環アッセイは、大血管血管新生ウェル12、13、14をモデル化する。しかし、大動脈環としての脈絡膜新血管化のモデルとしての限界があり、特徴的な脈絡膜微小血管環境を欠いた大血管組織であり、大血管からの芽は微小血管病理に関与する毛細血管ネットワークとは異なる可能性がある。最近、グループは、元生体のレティナルアッセイ15、16を発表しました。しかし、それは、疾患性新血管疾患に適しているが、それはAMDに見られるほど脈絡膜新血管化に適していない。
マウスRPE、脈絡膜、および硬化分離組織を用いた脈絡膜発芽アッセイは、より良いモデルCNVのために開発された。組織はマウス(または他の種)の目17から容易に単離することができる。このアッセイは、薬理学的化合物のプロおよび抗血管新生電位の再現性評価及び遺伝子組換えマウスおよび対照18からの組織を用いた脈絡膜新生血管形成における特定の経路の役割の評価を可能にする。この脈絡膜発芽アッセイは、その後の多くの出版物9、10、18、19、20で参照されています。ここでは、このアッセイの使用に関与する方法が示される。

<table><tbody><tr><td>AnaSed (Xylazine)</td><td>AKORN</td><td>59339-110-20</td><td></td></tr><tr><td>Basal membrane extract (BME) Matrigel</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Cell culture dish</td><td>NEST</td><td>704001</td><td>10cm</td></tr><tr><td>Complete classic medium with serum and CultureBoost</td><td>Cell systems</td><td>4Z0-500</td><td></td></tr><tr><td>Ethyl alcohol 200 Proof</td><td>Pharmco</td><td>111000200</td><td>use for 70%</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>06-666</td><td></td></tr><tr><td>Microscope</td><td>ZEISS</td><td>Axio Observer Z1</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>GIBCO</td><td>15140</td><td>10000 U/mL</td></tr><tr><td>Tissue culture plate (24-well)</td><td>Olympus</td><td>25-107</td><td></td></tr><tr><td>VetaKet CIII (Ketamine)</td><td>AKORN</td><td>59399-114-10</td><td></td></tr></tbody></table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

加齢黄斑変性症の顕著な特徴である病的脈絡膜血管新生は、視力障害および失明を引き起こしうる。ヒトのレチナル微小血管内皮細胞(HRMEC)または単離された一次性の腎細胞を用いた内皮細胞(EC)増殖アッセイは、腎血管新生を研究するために、インビトロモデルで広く使用されている。しかし、純粋なマウスのレチナル内皮細胞を単離することは技術的に困難であり、RETINAL ECは脈絡膜内皮細胞および異なる細胞/細胞相互作用とは異なる増殖応答を有する可能性がある。脈絡膜微小血管増殖のモデルとして、再現性の高いex vivo脈絡膜発芽アッセイが開発された。このモデルには、脈絡膜血管系(EC、マクロファージ、ペリサイト)と網膜色素上皮(RPE)の相互作用が含まれます。マウス RPE/脈絡膜/鎖状外植体は、成長因子減少基底膜抽出物(BME)(0日目)で単離され、インキュベートされる。培地は1日おきに変化し、6日目に脈絡芽が定量される。個々のチョロイド外植の画像は反転した位相顕微鏡で撮影され、発芽領域は、本研究室で開発されたImageJソフトウェアへの半自動マクロプラグインを使用して定量化されます。この再現性ex vivo脈絡膜発芽アッセイは、潜在的な治療のための化合物を評価し、微小血管疾患研究のために、野生型および遺伝子組み換えマウス組織を用いた脈絡膜微小血管増殖に関与する経路を評価するために使用することができる。

1日あたりの脈絡芽成長の比較
BMEに埋め込まれた鎖骨で脈絡膜を解剖し、6日間培養した(図1)。C57BL/6Jマウスで3日目から6日目までの脈絡芽を顕微鏡で調べ、ImageJでの半自動定量法をSWIFT-チョロイドで定量した。代表的な場合、発脈芽領域(外植から伸びる血管は、外植自体を除く)は、3日目(図3A)、4日目(図3B)で1.47mm2?...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

このプロトコルは、微小血管増殖の元生体モデルである脈絡芽発芽アッセイを提示する。このアッセイは、脈絡膜微小血管の増殖に関与する経路を評価し、野生型および遺伝子組み換えマウス組織を用いた薬物治療を評価するために使用することができる。

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 ...

an-ex-vivo-choroid-sprouting-assay-ocular-microvascular

Research

JoVE Journal

Biology

5.5K ビュー.  Boston Children's Hospital, Harvard Medical School. このプロトコルは、微小血管増殖の元生体モデルである脈絡芽発芽アッセイを提示する。このアッセイは、脈絡膜微小血管の増殖に関与する経路を評価し、野生型および遺伝子組み換えマウス組織を用いた薬物治療を評価するために使用することができる。

ビデオ: An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

血管新生研究のための角膜ポケットアッセイのマウスモデル

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

医学

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

アン<em&gt; ex vivoで</em&gt;メソッド培養ラット腸間膜微小血管ネットワークのタイムラプスイメージングのための

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

発生生物学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

神経科学

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜マイクロアッセイ：マウス眼における血管新生のモデル

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

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 ...

生物工学

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

網膜神経血管疾患研究のための ex vivo モデルとしての成体マウス網膜網膜の外植片

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

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 ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

インビトロ 血管疾患モデリングおよび薬物検査のためのマウス胚性幹細胞を用いた血管新生の三次元発芽アッセイ

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

眼小血管血管新生のエキビボロイド発芽アッセイ

要約

さらに動画を探す

この動画の章

血管新生研究のための角膜ポケットアッセイのマウスモデル

アン

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色

角膜マイクロアッセイ：マウス眼における血管新生のモデル

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

網膜神経血管疾患研究のための ex vivo モデルとしての成体マウス網膜網膜の外植片

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

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

インビトロ血管疾患モデリングおよび薬物検査のためのマウス胚性幹細胞を用いた血管新生の三次元発芽アッセイ

眼小血管血管新生のエキビボロイド発芽アッセイ

要約

さらに動画を探す

この動画の章

血管新生研究のための角膜ポケットアッセイのマウスモデル

アン

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色

角膜マイクロアッセイ：マウス眼における血管新生のモデル

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

網膜神経血管疾患研究のための ex vivo モデルとしての成体マウス網膜網膜の外植片

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

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

インビトロ 血管疾患モデリングおよび薬物検査のためのマウス胚性幹細胞を用いた血管新生の三次元発芽アッセイ

インビトロ血管疾患モデリングおよび薬物検査のためのマウス胚性幹細胞を用いた血管新生の三次元発芽アッセイ