Name: in vitro culture of epicardial cells from mouse embryonic heart
Uploaded: 2016-04-27T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

この作品は、DUKE-NUS大学院医学部シンガポール、Manvendra K.シンへゴー基盤とシンガポールNRFの交わり（NRF-NRFF2016-01）からの資金によってサポートされていました。

すべての実験は、デューク-NUS大学院医学部制度動物実験委員会によって承認されました。 1.胚性心室を取得<ol><li>二酸化炭素ガスの供給または他の承認された安楽死の方法で安楽死チャンバーを用いて所望の胚段階（E11.5またはE12.5）で時限妊娠マウスを生け贄に捧げます。 </li><li>解剖台に背の上にマウスを置きます。 70％エタノールで女性の腹部を消毒します。両手で皮膚を保持し、腹壁を露出するように引き離した後、腹の上に皮膚を持ち上げて、刃で小切開（1-2センチ）を作ります。 </li><li>今、子宮角を露出させるために腹壁をカット。子宮角を持ち上げ、慎重に脂肪組織と子宮角に取り付けられた血管を切り取るために滅菌ピンセットを使用してください。子宮を取得するために、子宮頸部で切断。 </li><li>無菌の冷たい1×リン酸バフを含むペトリ皿に子宮角を配置<html> ered生理食塩水（PBS）と優しく洗い流します。氷上のペトリ皿を置きます。 </li><li>卵黄嚢の内部に残っていると、胎盤を通じて子宮壁に取り付けられた胚を露出させるために（胎盤が配置されているサイトとは反対側）の子宮角の正中線を切断するはさみを使用してください。 </li><li>滅菌ピンセットを用いて、胚を解放するために、胚の卵黄嚢を切り​​開い。 </li><li>滅菌冷たい1×PBSを含む新しいペトリ皿に胚を置きます。胚を刎ねると、その背中の上に置きます。心臓を露出するために胸壁を開いてカットします。 </li><li>心を持ち上げ、胸の壁から心を解放するためにその周りに船を離れてカットする滅菌ピンセットを使用してください。手術ツールで心臓の心外膜に損傷を与えないように注意を払ってください。 </li><li>流出路との両方の心房を切り落とします。冷たい1×PBSで別の皿に心室を転送します。氷の上の皿を置きます。 </li><li>繰り返して、すべての心室が検索されるまで、1.6から1.9を繰り返します。 </li></ol>ル &quot;&gt; 2。心外膜外植片培養注意：文化ガラスチャンバースライド上または下流のアプリケーションのためのコラーゲンゲル上のいずれかのこれらの心外膜の外植片。 <ol><li>ガラスチャンバースライド<ol><li> 、培地を調製したダルベッコ改変イーグル培地（DMEM）に10％ウシ胎児血清（FBS）、1％ペニシリン/ストレプトマイシンおよび2 ngの/ mlの組換え線維芽細胞増殖因子2（FGF2）を追加します。層流フード内のすべての手順を実行します。 </li><li> 8ウェルチャンバーの各ウェルに500μlの培地を追加します。 </li><li>ウェルの中央にそれぞれ切除した心室を置きます。東洋ウェルの底面を下に切開面を維持する脳室。 </li><li>穏やかに37℃、5％CO 2細胞培養インキュベーター中でプレートを置きます。 </li><li>外植片が付着することを可能にするために妨害を最小限にして文化を維持します。心外膜の単分子層の形成は、48〜72時間後に観察することができます。 </li><li> epicardの分化のために平滑筋細胞、培養をさらに6日間分化培地における心外膜単層細胞にIAL細胞。分化培地を調製するために、DMEMに10％FBS、1％ペニシリン/ストレプトマイシンおよび50 ngの/ mlの組換え型トランスフォーミング増殖因子β1（TGF-β1）を加えます。 </li><li>日おきに培地を変更します。 </li></ol></li><li>コラーゲンゲル<ol><li>製造業者のプロトコルに従って、商業用3Dコラーゲン培養キットを用いて96ウェルプレート中のコラーゲンゲルを準備します。 </li><li>コラーゲンゲルの所望のボリュームを準備するには、上下DMEMとピペットの5倍とコラーゲン溶液の適量を混ぜます。溶液は黄色に変わります。 </li><li>中和液の対応するボリュームを追加し、ピペッティングにより十分にすぐに混ぜます。解決策は、ピンク色に変わります。 </li><li>ピペット96ウェルプレートの各ウェルにこの混合物の100μlを。アルに30〜60分間、37℃、5％CO 2インキュベーターでプレートを置き低ゲルが重合します。 </li><li>各ウェルに、上記の培養培地を200μlを追加します。 </li><li>ウェルの中央にそれぞれ切除した心室を置きます。オリエントコラーゲンゲル上で解剖側を下に保つために心室（心室の頂点が実験者に直面している必要があります）。そっと背中細胞培養インキュベーターでプレートを置きます。 </li><li> 3日後、心室を削除します。別の2日間、細胞培養インキュベーターにバックプレートを置きます。 </li><li>日おきに培地を変更します。 </li></ol></li></ol> 3.固定と可視化注：心外膜細胞を、ガラスチャンバースライド、またはコラーゲンゲルのいずれかで可視化することができます。 <ol><li>培地アウト吸引し、細胞を洗浄し、滅菌1×等量のPBSを追加します。 </li><li>セルをカバーするだけの十分な4％パラホルムアルデヒド（PFA）を加えます。 4℃で10分間細胞を固定。 </li><li> PFAを削除し、で1×PBST（1×PBSで外植片を洗います細胞を透過するため、0.1％トリトンX-100）。 </li><li>所望の抗体（ 例えば 、アレクサフルオロ488ファロイジン; ZO-1、αチューブリンおよびα平滑筋アクチン）とイムノ7。 </li></ol>

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The authors declare that they have no competing financial interests.

心臓の開発と再生に心外膜の重要性が増しに応えるために心外膜の研究を容易にする技術を開発することが極めて重要です。心外膜培養系は、心外膜の研究のための重要な利点をもたらします。 心外膜細胞を単離する別の方法は、蛍光活性化細胞選別（FACS）を使用することです。この方法は、他の系統から心外膜細胞を分離するための心外膜マーカー（または蛍光タン?...

実験データの富は、心外膜は、心臓の開発における重要なステップに影響を与えることが示されています。開発中に、横中隔はproepicardium 1-4として知られている中皮細胞の塊を生じさせます。 proepicardiumからの細胞は、次に移動して、心外膜を形成する心筋を包みます。これに続いて、心外膜細胞のサブセットは、その後、心筋に侵入心外膜由来細胞（EPDCs）の遊走集団を生じるEMTを起こします。実験トレース遺伝子、ならびにレトロウイルス系統はEPDCsは、平滑筋細胞、線維芽細胞、内皮細胞および心筋細胞（もしあれば）を含む種々の系統に分化することを実証しました。したがってEPDCsは、冠血管系および心筋アーキテクチャ1,2,4-9の発展に大きく貢献しています。さらに、心外膜は、心室緻密層10-12の発展のために不可欠です。 EのXAMPLE Gittenbergerドはグルートら proepicardiumの伸長を阻害することが、このような薄い心筋、心臓や異常心室中隔形成の欠損ループとして、その結果、胚致死13等の欠陥の配列につながることを実証しました。胚性心外膜から分泌パラクリン要因は、心筋細胞の増殖および分化を調節します。これと一致して、レチノイン酸（RA）、線維芽細胞増殖因子（FGF）およびWnt /βカテニンシグナル伝達経路などの心外膜特異的欠失は、欠陥のある心筋の増殖および胚致死14-16もたらしました。 心外膜は、大人の心の中に静止状態であると考えられていたが、最近の研究では、発達プログラムが心外傷17,18以下の心外膜に再活性化されることが示されています。活性化されると、細胞はEPDCの形成をもたらす急速な増殖およびEMTを起こします。これらの細胞は、capacitを示しますyは、線維芽細胞や平滑筋細胞ではなく、心筋細胞または内皮細胞18に分化します。また、EPDCsは、損傷部位の血管新生を助ける、したがって梗塞サイズを減少させることにより改善された心機能を促進する血管新生促進因子を分泌します。これらの知見に、心外膜は、心血管の開発、病気と再生の研究に関心を集めています。 トランスジェニック技術は、21世紀の医学研究に革命をもたらしました。トランスジェニック技術の助けを借りて、代謝および病態生理学的に人間の条件を模倣する病気のマウスモデルが正常に開発されています。しかし、これらの変異体における心外膜細胞の挙動を研究すると、初期胚致死性が主な原因課題でした。心外膜は、心臓の開発と再生に果たしていることが重要な役割を考えると、我々は、マウスの噴門上部のためのin vitro培養系を確立していますL細胞。この方法は、心外膜細胞の長期培養を可能にし、心外膜の二つの重要な特性の詳細な検討が容易：移行と分化する能力を。マウスから切除した心室は、遊走アッセイを実施するために使用することができるコラーゲンゲル上で培養することができます。より良い心外膜下の層のコラーゲンが豊富な細胞外マトリックスを複製インビボの細胞生理学を再現する3Dマトリックスで培養されています。あるいは、それらは、次いで、下流の適用の種々のために使用することができる心外膜単層を確立するために、チャンバースライド上で培養することができます。この単分子膜は、移行のために重要であるEMTを受ける心外膜の能力に関する洞察を提供するタイトジャンクショ​​ンタンパク質を染色するために使用することができます。また、分化実験は、これらの細胞上で行うことができます。さらに、遺伝子発現プロファイルは、細胞からRNAを抽出することによって分析することができると定量的ポリメラーゼ連鎖反応（qPCR）を行います。最後に、単層はまた、潜在的な治療薬をテストするために、分子分析を行っ剤を用いて治療することができます。まとめると、この心外膜培養系は、視覚化し、心外膜の開発についての我々の理解をfurthers分子データを収集する機会を提供してくれます。 この方法の別の望ましい特徴は、それが簡単であり、いかなる精巧なセットアップを必要としないことです。簡単に言えば、胚は、心臓を摘出されたE11.5またはE12.5以下で収穫されています。心室はその後、コラーゲンゲルまたはチャンバースライドのいずれかで培養されます。その後、これらの細胞は、下流の実験を行うために使用することができます。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Life tech invitrogen&amp;nbsp;</td><td>11995065</td><td></td></tr><tr><td>Penicillin/streptomycin solution</td><td>Life tech invitrogen&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Life tech invitrogen&amp;nbsp;</td><td>10500064</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-5KG</td><td></td></tr><tr><td>Recombinant fibroblast growth factor 2 (FGF2)</td><td>PeproTech</td><td>450-33</td><td></td></tr><tr><td>Recombinant transforming growth factor beta 1 (TGF-&amp;beta;1)</td><td>PeproTech</td><td>100-21</td><td></td></tr><tr><td>ZO-1 antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>40-2200</td><td></td></tr><tr><td>&amp;alpha;-Tubulin antibody</td><td>Sigma</td><td>T 6074</td><td></td></tr><tr><td>&amp;alpha;-smooth muscle actin (SMA) antibody</td><td>Sigma</td><td>A 2547</td><td></td></tr><tr><td>Phalloidin antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>A12379</td><td></td></tr><tr><td>3D Collagen Culture kit&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ECM 675</td><td></td></tr><tr><td>8-well chamber slide</td><td>Fisher Scientific</td><td>NNU 154534-PK</td><td></td></tr><tr><td>Trizol reagent</td><td>Life Technologies</td><td>15596-018</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Life Technologies</td><td>4453536</td><td></td></tr><tr><td>Superscript First Strand Synthesis kit</td><td>Life Technologies</td><td>11904-018</td><td></td></tr></tbody></table>

in vitro culture of epicardial cells from mouse embryonic heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

この培養プロトコルを使用して、主要な心外膜細胞は、下流アプリケーションのための高純度で単離することができます。培養した細胞は、EMTを起こし移行し、心外膜細胞が生体内で同じよう分化することができます。 R26 トム/ +胚;当社の主要な心外膜細胞培養物の純度を決定するため...

Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

マウス胚心臓から心外膜細胞の体外培養で

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

in-vitro-culture-of-epicardial-cells-from-mouse-embryonic-heart

Research

JoVE Journal

Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.3K ビュー.  Duke-NUS Graduate Medical School. The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. 

ビデオ: In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜の伸長文化検定と<em&gt; ex vivoで</em&gt;心外膜由来の細胞移動の評価

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

発生生物学

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

エレクトロポレーションに基づくマウス心臓細胞のトランスジェネシス

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

著者スポットライト:迅速な遺伝子スクリーニングのための心臓細胞トランスジェネシス

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

マウスの心臓から初代メラノサイト様細胞を単離する

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

生物学

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

マウス胚心臓から心外膜細胞の体外培養で

要約

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心外膜の伸長文化検定と

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

マウス心臓分野特異的心臓前駆細胞のインビトロ生成

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

マウスの心臓から初代メラノサイト様細胞を単離する

新生仔マウス心筋細胞の単離および培養

小説

胚心室内皮細胞の単離