仕事は医学研究評議会からのコア資金によってサポートされていました。我々は技術的な図面を製造するためのクレイグニコルに感謝しています。我々は彼らのイメージングをサポートするためのマシュー·ピアソン、ポール·ペリーに感謝しています。

全ての動物作業は、英国内務省（プロジェクトライセンス番号PPL 3785分の60および4424分の60）によってライセンスの下で施設のガイドラインに準じて行った。 1。準備<ol><li> CREB 8とのWnt1 :: Creを 14は、Cre発現ラインとして成功裏に使用されている:: CREA、Tyrの:: Tyrの蛍光レポーターマウス系統との適切なのCreリコンビナーゼを発現するマウス系統を組み合わせて開発した皮膚におけるメラノブラストにラベルを付け、リポーターラインとしてR26YFPR 9。 </li><li>層流フード内の培養液を準備します。 （1％v / vのペニシリン/ストレプトマイシン）を含むダ​​ルベッコ改変イーグル培地（DMEM）を補う、10％v / vのウシ胎児血清および0.584グラム/ LのL-グルタミン。 </li><li>ライブイメージング装置は70％エタノールで拭くことにより、撮像チャンバ·ベースを滅菌し、図1に概説されている。 OVを浸漬することによって挿入、撮像クリップ、およびO-リングを滅菌70％エタノール中ernight。チャンバーの蓋として6ウェルプレートから無菌の蓋を使用してください。 </li><li>撮像チャンバーの底面は、O-リング（ 図1）によって適所に保持不器用な人膜からなる。 35ミリメートルのガス透過性のろまディッシュで始まります。イメージングチャンバーの上の皿を置き、片刃かみそりの刃で気体透過膜をカット。イメージング室に固定した膜の上にOリングを押してください。 </li><li>画像クリップ（合計6）が所定の位置に胚性皮膚使用の前に、彼らは1％アガロースで満たされなければならない（ 図1）をクランプします。 60℃に電子レンジと冷却することにより、無菌のdH 2 Oでアガロースの2％溶液を準備しますを10mlの培地との混合1:1（ステップ2を参照）を60℃に予熱滅菌した6ウェルプレート中で6撮像クリップスタンド。微細pastetteを使用して、各クリップの中央に1％アガロース溶液を垂らし冷却する。 αを防止するために各ウェルに1ミリリットル冷滅菌PBSを追加乾燥からgarose。撮像クリップは、PBS中で数時間保存することができる。 </li><li>胚皮膚の繊細な分離を実施することとする代わりに竹のケバブ串を使用できる箸を使用するのでは、柏木ら 3で説明したように解剖し、組織が ​​「ヘアスティック方式&#39;を使用処理する。片刃カミソリの刃を使用して串の平らな端の小さなスリットを作り、毛柔らかい歯ブラシを挿入します。それは、長さが15cm程度になるように串をトリミング。 </li><li>空気中のCO 2 5％を提供する完全な環境室やステージトップ室と共焦点顕微鏡を使用してください。撮像前に、少なくとも1時間、37℃に顕微鏡ステージを予熱し。これは、ウォームアップ、ステージ構成要素の膨張によるサンプルのドリフトを防ぐことができます。 </li><li> 1は、迅速かつ容易にexperimenを設定するために、各文化の真ん中を中心にできるように、顕微鏡の制御ソフトウェアを使用してマルチポジションセットを準備しますT。 </li></ol> 2。手順<ol><li>マウスを嵌合させ、日E0.5として初日ポスト交尾を指定します。日E14.5で、頚椎脱臼により女性を間引くと、小さな正中切開を作る頭と尾に向けて皮を引き離すと、基になる腹膜を切断し、後身頃の両側に剥がすことで体腔を公開します。ピンセットでしっかりと保持し、外科はさみを使用して子宮間膜に沿って切り取ることで冷滅菌PBSに子宮を解剖。必要になるまで氷上で子宮を保つ。詳細については、シェイら （2007）15を参照してください。 </li><li>第一の微細鉗子二対でそれらを引き離すことによって脱落膜を解放するために、外科用ハサミで子宮壁の長さに切断し、次いで、脱落膜から胚を解剖することによって子宮から胚を解剖する。蛍光レポーター発現（必要な場合）のための画面が表示されます。 </li><li>解剖の前に断頭により胚を間引く。 SUBSequently尾を削除し、手足や片刃カミソリの刃を使用して、その後前肢後肢。必要に応じて、遺伝子型判定のための組織を保持します。 </li><li>吻側 - 尾側方向に正中線に近いventrumに切開する。ステップ1.6で説明した髪スティックを使用して、慎重に同時に胚を回転下にある結合組織から肌を離れていじめる。完全に皮膚を除去ではなく、最初の切開部から腹側のエッジ遠い沿って取り付け、それを放置しないでください。 </li><li>それはそれからマウントすることは非常に困難であるようになって肌がめちゃくちゃにさせないことが重要です。これを防ぐには、乾いたシャーレの表面に毛が付いています（真皮側を上にして）を使用して、肌を平らにしてから片刃カミソリの刃を使用して、離れて胚幹から切る。皮膚が除去される前に真皮側を切開し、組織および胚との間の接合部の注意深い観察により表皮側から区別することができる。切開した組織の領域である約0.5mm 2 E14.5胚を解剖。 </li><li>アガロースが組織の真皮側に触れているように、皮膚の上に画像クリップを配置します。 60秒間放置した後、慎重にクリップの側面まで組織の縁をいじめることができます。クリップを反転し、毛が肌を平らにし、極力（画像化される領域に）組織の表面に触れるように注意しながら、任意の気泡を除去するためにスティックを使用します。 </li><li>彼らは締めとクリップの上部に近い溝に皮膚を引っ張るように、イメージング·クリップの両側に二つの簡単な親指の結び目を用いた撮像クリップに肌を結ぶために絹の縫合糸を使用しています。クリップを反転、画像挿入とベース内の場所の中に押し込みます。 （ステップ1.2を参照）の培地〜5ミリリットルで挿入を埋める。 図1は、撮影室のアセンブリの概要を説明します。 </li><li>残りの5個のサンプルのプロトコルを繰り返します。 </li></ol> 3。ライブイメージング<ol><li>撮像チャンバは、標準的な6ウェルプレートと同じ寸法を有する。共焦点顕微鏡のステージ上のチャンバを取り付けるために、適切なプレートインサートを使用してください。 </li><li>自動化されたステージは、並行して培養物をイメージするために必要です。共焦点ソフトウェアイメージへの6つの位置を定義します。正確な設定は、使用される顕微鏡の能力に依存します。典型的には、小さなzスタックは、（3-5 z位置は、任意のステージのドリフトを考慮するため）6位のそれぞれで使用され、zスタックは、18〜24時間、2分毎に捕捉される。ライブイメージング用途のためには、レーザー出力と必要なz軸部の数を減らすために光学的に最適なピンホールよりも広く設定を使用することが一般に有利である。 </li></ol>

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著者らは、開示することは何もありません。

私たちは、倒立型共焦点顕微鏡をONに生細胞イメージング従順特殊性である文化、胚、皮膚への方法を記載している。この方法は、6文化が並行して画像化し、マトリゲルと元のメソッド6のヌクレポアメンブレンへの依存を削除することを可能にする最近の改善が含まれています。類似の技術からの重要な技術的違いは、空気液体界面を確立するとともに、カバースリップとして作用?...

伝統的に、胚皮膚は、マウスの胚から解剖し、ポリカーボネートヌクレポア膜上に搭載することによって培養されています。次いで、膜をこのように発達中の組織3,4の表面にわたって空気-液体界面を維持し、培養培地に浮遊される。この技術は、組織を固定し、マーカ7のようにβ-ガラクトシダーゼを用いたメラノブラスト分布を評価することによってメラノブラスト挙動をアッセイするために使用されている。私たちは、ex vivoで皮膚培養6における蛍光標識されたメラノブラストのライブイメージングを可能にする方法を開発した。ここでは、共焦点イメージングを生きるために、セットアップに、解剖から詳細に方法を説明し、いくつかの最近の改善が含まれています。 メラノブラストは、メラノサイトの胚性前駆体、髪や肌に色素を産生する細胞である。メラノブラストは、発達中のマウス（e）で胎生9（E9）付近で神経管に隣接した神経堤で発生mbryo。その後彼らは、外胚葉と発展体節間の背側の経路に沿って移動する。 E12.5で彼らは増殖し、その移行を続行表​​皮に真皮から移動する。主な毛包のパターンはE14.5で表皮に形成し始めるとE15.5のメラノブラストすることにより、これらの卵胞にローカライズされています。メラノブラスト/メラノサイト開発のレビューのためにトーマス·エリクソン（2008年）1を参照してください。メラノブラストの人口を標識するために、我々は、Tyrを組み合わせている::条件付きでROSA26遺伝子座 9から黄色蛍光タンパク質（YFP）を発現R26YFPR動物とマウスチロシナーゼプロモーター8によって駆動のCre-リコンビナーゼを表現CREB動物。 私たちは、倒立型共焦点顕微鏡を用いて培養、胚、皮膚や撮影画像にする方法を説明します。それは、モートらに記載の元の方法。（2010年）6形に構成されている。現在この方法は、6 - ウェルフォーマットで画像化を可能にし、ヌクレポア膜上や文化を支援するマトリゲルへの依存を削除します。代わりに、胚の皮膚を安定させるために1％アガロースの小ブロックを用いた。マトリゲルへの依存を削除しても、特に水溶性の成長因子に対する胚の皮膚の応答が研究の焦点である状況では重要である。当社独自の方法では、すでにメラノブラスト開発10月13日に新たな洞察を得るために使用されており、我々は我々がここで説明する改善が複数の並列文化が要件で、特に実験手法として、それがより強力になると予想している。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM high glucose</td>
			<td style="width:132px;">Biochrom AG</td>
			<td style="width:119px;">F0475</td>
			<td style="width:167px;">without phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:132px;">Hyclone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live imaging chamber</td>
			<td style="width:132px;">Custom made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lummox dishes</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-well plate</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single edged razor blade</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">1244-3170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:132px;">Biogene</td>
			<td style="width:119px;">300-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine pastette</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kebab skewers</td>
			<td style="width:132px;">Waitrose</td>
			<td style="width:119px;"></td>
			<td>Bamboo BBQ skewers 30cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothbrush</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture thread</td>
			<td style="width:132px;">Look</td>
			<td style="width:119px;">SP115</td>
			<td>Black silk suture thread</td>
		</tr>
	</tbody>
</table>

ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration

メラノブラストは、メラニン細胞の神経堤に由来する前駆体である;皮膚及び毛髪における顔料の製造に関与する細胞である。メラノブラストは、彼らはその後、発展途上毛包1,2にコロニーを形成する胚の表皮を通って移動。神経堤細胞の移動が盛んにin vitroで検討されているが、in vivo法でまだよく、特に哺乳動物系では、開発されていない。一つの選択肢は、ex vivoでの器官培養3-6を使用することです。マウス胎児の皮膚の培養組織3,6の表面を横切って気液界面（ALI）のメンテナンスを必要とします。マウス胎児の皮膚の高解像度ライブイメージングは​​、このALIを維持だけでなく、文化が反転し、したがって、短い作動距離の対物レンズと、ほとんどの共焦点顕微鏡と互換性があることを可能にするだけでなく、良い方法がないことによって妨げられてきた。この記事では、メタへの最近の改善について説明しますこれらの問題を克服し、ex vivoで培養中の胚の皮膚6の高分解能共焦点イメージングを可能にするガス透過性膜を使用して外径。 R26YFPRレポーターラインと組み合わせるメラノブラスト特定のCreリコンビナーゼ発現するマウスラインを使用して、我々は、蛍光これらの皮膚の培養内メラノブラストの人口にラベルを付けることができます。技術はメラノブラスト、彼らが開発している組織と彼らの行動や相互作用の観察のライブイメージングを可能にする。代表的な結果を並列に6の文化をイメージ生きる能力を実証するために含まれる。

図2は、Tyrからの胚性スキンを使用して時間経過の実験からの代表的な結果を示しています:: CREBは E14.5でR26YFPR胚をX。 6胚性皮膚培養物を18時間ごとに2分、共焦点顕微鏡で画像化した。フリーウェアの画像解析ソフトウェアパッケージImageJの6映画のYFP-標識メラノ​​ブラストの挙動を分析した。メラノブラストは自動的に（イェスパーSøndergaardペダー?...

Watch this Scientific Journal Video about Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration at JoVE.com

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

我々は、マウス胚の皮膚の切開およびex vivoでの文化について説明します。培養系は、組織表面を横切って気液界面を維持し、倒立顕微鏡でイメージングを可能にする。メラノブラスト、現像皮膚の成分は、それらの蛍光挙動は、共焦点顕微鏡を用いて観察することを可能に標識される。

ex vivoで培養マウス胚の皮膚およびメラノブラストの移行のライブイメージング

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

ex-vivo-culture-mouse-embryonic-skin-live-imaging-melanoblast

Research

JoVE Journal

Biology

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

12.5K ビュー.  University of Edinburgh. 我々は、マウス胚の皮膚の切開およびex vivoでの文化について説明します。培養系は、組織表面を横切って気液界面を維持し、倒立顕微鏡でイメージングを可能にする。メラノブラスト、現像皮膚の成分は、それらの蛍光挙動は、共焦点顕微鏡を用いて観察することを可能に標識される。 

ビデオ: Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

ショウジョウバエの眼成虫ディスクに付属してグリア細胞の移行のイメージングを生きる

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

のライブイメージングキイロショウジョウバエ胚性血球移行

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

初代ヒト卵管上皮細胞のex vivoでの文化

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

ex vivoで培養マウス胚の皮膚およびメラノブラストの移行のライブイメージング

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