筆者はLeif OxburghとDerek Adamsの知識共有、Leif Oxburghの原稿への有用なコメント、StefanWölflとUlrikeMüllerのテクニカルサポート、Saskia Schmitteckert、Julia Gobbert、Sascha Weyer、Viola Mayerに感謝します。ラボこの研究は、Development、The Biology of Company （CPへ）によって支持された。

マウスは、スウェーデンの規制およびEU法（2010/63 / EU）に従って維持された。すべての手続きはスウェーデン倫理委員会のガイドライン（C79 / 9、C248 / 11、C135 / 14の許可）に従って実施された。ハイデルベルク大学での動物被験者の手順は、ハイデルベルク大学のRegierungspräsidiumKarlsruheとAnimal Welfare Officersによって承認されています。 1.培養用試薬および材料の調製注：汚染を最小限に抑えるために、層流フードを使用してください。 <ol><li>解剖の日に、滅菌リン酸緩衝生理食塩水（PBS）中の1：5のモル比で抗ヒトFcヤギ抗体と混合することにより、ヒトFcに融合されたヒトFcのみまたはヒトFcに融合された組換えキメラエフリンタンパク質を前もってクラスター化する。 37℃で1時間インキュベートする。5 。 </li><li>ダルベッコ改変イーグル培地：栄養素を補充して培地を調製する1％ペニシリン/ストレプトマイシン（v / v）、1％グルタミン（v / v）、1％インシュリン - トランスフェリン - セレン（v / v）、50μg/ mlのヒトホロ - トランスフェリン、および1.5μg/ mLのアンホテリシンを含む。 注：16胚には培地10 mLで十分です。 </li><li> 20μg/ mLの最終濃度のクラスター化組換えエフリン溶液を培地に添加する。クラスター化されたヒトFcを対照として使用する。 </li><li>無菌のコーティングされていない平底のポリスチレン4ウェルプレートの各ウェルに培地500μLを加えて培養プレートを調製する。 </li><li>滅菌鉗子を使用して、培地の上に穴あたり5μmの孔径を有するポリカーボネート膜フィルターを注意深く1つ置きます。調製したプレートをインキュベーター（37℃/ 5％CO 2 ）に移す。フィルターが上面に濡れていて浮いていることを確認してください。 注：2つの腎臓の癌細胞には1つのフィルターで十分です。 </li><li>かみそりの刃を使用して、およそ30個のピペットチップ（容量：100μL）を切断して先端から1mmの位置で、より広い開口部をもたらす。チップをピペットチップラックに戻します。 </li></ol> 2. E11.5におけるメタネフリックルディメントの解体<ol><li> E11.5で妊娠しているマウスを頸椎脱臼により犠牲にする（または地元の倫理委員会で承認された方法を使用する）。 </li><li>ブラウンら 6 ）の記載に従って、子宮および胚を取り除く。この時点から、解剖顕微鏡下で作業する。 </li><li>第5時計メーカーの鉗子を使用して、前脚のすぐ下に胚を切断します。各胚のために新しいペトリ皿を使用してください。胚の脚と尾を外す。これを行うには、削除する組織を1組の5番時計メーカーの鉗子でつかみ、2番目のペアの先端を使用して切断します。 </li><li> 腹側器官の塊を取り除くには、一対の鉗子の先端を使用して、胚の体壁を吻側 - 尾側方向に横に開く。 <ol><li>表面と平行、tに平行彼は背の大動脈。血液充填された、したがって目に見える背側大動脈を方向付けに使用する。閉鎖した鉗子の先端を用いて体壁の腹側部分を背側部分から注意深く分離し、胚を裏返す。体側の壁を吻側から尾側に切断し、前側と同様に背側の大動脈を使用して向きを決めます。 </li></ol></li><li>腹側の身体の壁が背側の身体から分離されたら、1対の鉗子でそれをつかんで、それを臓器塊とともに慎重に引き抜く。 注：metanephric anlagenは、通常、背の体の壁に付着したままになります。それらは、後肢のレベルに位置する長さ約200μmの楕円形の構造である。 </li><li>腹側を上にして、1対の鉗子で背側の身体の壁を保持する。他の鉗子のペアを使用して、その吻側の端で背部の大動脈をつかんで背部の体の壁から背部の大動脈を注意深く剥がす。 注：両方のメタニュークリニック腎臓は、通常、背部大動脈に投与した。 </li><li> 1対の鉗子を用いて、大動脈を除去するために吻側に吻側に切断する。次に、慎重に鉗子の先端を使用して2つの腎臓のanlagenを分離します。 </li><li>慎重に鉗子の先端を使用して腎臓のanlagenから不要な組織を剥がす。間葉系に損傷を与えないよう注意してください。破損は、さらなる分析から外植片の成長不良および排除を招く可能性があるためです。 </li><li> 4ウェルプレートのポリカーボネートメンブランフィルターに10μLのPBSにマイクロタイターピペットとワイドオープンピペットチップを使用して、腎臓のanlagenを別々に移す。 注：表面張力のため、外植片は薄い培地層で覆われます。 2つの腎臓のマーカーには1つのフィルターで十分です。 <ol><li> 2つの腎臓のそれぞれをフィルターの各半分の中央に置きます。プレートを37℃/ 5％CO 2のインキュベーターに戻します。プレートはインキュベーターの中に放置してください次の胚からのイメントは、フィルター上に置く準備ができている。 </li></ol></li><li>各胚についてステップ2.3〜2.9.1を繰り返す。 注：いくつかの練習では、解剖の手順は、胚あたり約5分かかるでしょう。 </li><li> 37℃/ 5％CO 2で3日間、腎臓の蛍光物質をインキュベートする。 </li></ol> 3.固定化および染色のための試薬の調製<ol><li>カルシウムとマグネシウム（w / v）を含まないPBS中4％パラホルムアルデヒド（PFA）を調製するために、PFAを60℃でPBSに溶解し、連続的に撹拌する。室温まで冷却し、紙フィルターを使用して残りの粒子を除去する。分注し、すぐに使用する場合は4℃に保ち、長期間保存する場合は凍結してください。 注意：PFAは有毒です。現地の安全基準に規定されている個人用保護具を着用し、ヒュームフードで作業してください。施設ガイドラインと指定廃棄物容器を使用して廃棄物を廃棄してください。 </li><li>透過液を調製するために、希釈したTriton X-1000.3％（v / v）までのカルシウムおよびマグネシウムを含まないPBS中でインキュベートした。 </li><li>ブロッキング溶液を調製するために、5％（v / v）ヤギ血清および0.1％Triton X-100をカルシウムおよびマグネシウムを含まないPBSに加える。アジ化ナトリウムを0.02％（w / v）の最終濃度まで添加する。 4℃で保存する。 注意：アジ化ナトリウムは有毒である。現地の安全および環境保護規制に従ってナトリウムアジドを取り扱ってください。 注：染色のために抗体を使用する場合、二次抗体が由来する種からの5％血清を用いてブロッキング溶液を作製する。 </li><li>ビオチン化Dolichorus biflorusアグルチニンをブロッキング溶液で1：200希釈する。 Alexa488結合ストレプトアビジンをブロッキング溶液で1：200希釈する。 </li><li> すぐに使用できる包埋培地（材料の表を参照）を調製するために、0.1M Tris-HCl（pH 8.5）中の25％（w / v）グリセロールに0.1gの水溶性ポリビニルアルコール系粘膜接着剤/ 。連続的に攪拌しながら50℃に1時間加熱し、冷却し、1を用いてpHを8.0-8.5に調整する。M NaOH。より高いNaOH濃度を避けてください。 100μg/ mLのN-プロピルガレートおよびチメロサールを0.02％（w / v）の最終濃度に加える。室温で30分間撹拌する。 注意：チメロサールは有毒です。現地の安全および環境保護規制に従って取り扱ってください。 <ol><li> 50 mLのチューブに包埋培地を満たし、ローターをバランスさせ、3,200 xgで室温で10分間遠心する。透明な溶液を15 mLチューブに移し、ペレットを捨てる。分注し、数週間-20℃で保存します。解凍後、溶液を4℃に保ちます。使用直前に約30℃まで暖めます。 </li></ol></li><li>外植片を取り付けるためのフィルターをガラススライドとして準備します。このためには、インスタント接着剤を使用してスライドガラスの両側に小さなカバースリップ（18 x 18 mm）を2つ貼り付けます。間に15 mmのスペースを残してください。 </li></ol> 4.固定および染色<ol><li> 3の培養期間後in vitroでインキュベートし （div）、インキュベーターからプレートを取り出し、マイクロピペットを用いて各ウェルから培地を注意深く吸引する。フィルターに触れないで、先端開口部を井戸の壁の方に保つようにしてください。 </li><li> 500μLの4％PFA / PBSを各ウェルに加える。フィルターは固定液に浮かぶでしょう。マイクロピペットを使用して、注意深くPFA固定剤を滴下して、フィルターを水没させる。室温で30分間インキュベートする。 注記：以降のすべての手順では、外植片をフィルターから洗い流さないように注意しなければなりません。井戸の壁に向かってピペットチップの開口部を保つ。 </li><li> PFAを除去し、500μLのPBSで2回洗浄し、フィルターを浸します。 500μLの透過処理溶液を各ウェルに加え、室温で1時間インキュベートする。 </li><li> 500μLのPBSで2回すすぎ、500μLのブロッキング溶液を各ウェルに加えます。室温で1時間インキュベートする。 </li><li>ブロッキング溶液を250μLのビオチン化Dolichorus biflorus凝集素をブロッキング溶液で1：200に希釈し、4℃で24時間インキュベートする。 </li><li> Dolichorus biflorusアグルチニン溶液を除去し、1ウェルあたり500μLのPBSで2回リンスする。 </li><li>ブロッキング溶液で1：200に希釈したAlexa488結合ストレプトアビジン250μLを添加し、4℃で24時間インキュベートする。あるいは、室温で2時間インキュベートする。 注：4℃でインキュベートすると、より良いシグナル対ノイズ比が得られます。 </li><li> 1ウェルあたり500μLのPBSで2回すすぐ。鉗子を使用して、外植片を上にして、準備したガラススライドにフィルターを移す。 〜50〜100μLの包埋培地でマウントする。長さ60mmの1.5号カバースリップで覆う。包埋媒体溶液を暗所で室温で2時間固化させる。イメージングに進むか、フォイルに包まれたスライドを4°Cで画像化するまで保存します。 </li><li>広視野落射顕微鏡7 &lt;（励起帯域、460〜495nm;二色性鏡、505nm、および発光バンドパス、510〜550nm）および20X Plan-Apochromatレンズ、0.75数値絞り。 </li></ol>

<ol>
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(50), e2555 (2011).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+interaction+in+the+development+of+the+mouse+metanephros.">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li><li>Sax&#233;n, L., Toivonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary Embryonic Induction. , (1962).</li><li>Sax&#233;n, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system.">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li><li>Dudley, A. 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著者は何も開示することはない。

この写本は、発達中のメタネフリカのanlagenをマウスの胚から単離し、器官を培養する方法を記述しています。この方法は、Grobstein 8とSaxén9,10によって開発された標準的な手法であり、多くの他者11,12によって適応され、変更されています。この方法の成功は、主に、外植片の生存および誘導電位の低下が切開時間の延長に伴う切開の持続時間に依存する。?...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table><tbody><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14040117</td><td>use for dissection</td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>T0665</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100x)</td><td>Thermo Fisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Amphotericin B solution</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Thimerosal</td><td>Sigma-Aldrich</td><td>T5125</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Biotinylated Dolichorus Biflorus Agglutinin</td><td>Vector Laboratories</td><td>B-1035</td><td></td></tr><tr><td>Alexa488 conjugated Streptavidin</td><td>Jackson Immuno Research</td><td>016-540-084</td><td></td></tr><tr><td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td><td>R&amp;amp;D Systems</td><td>496-EB</td><td></td></tr><tr><td>Recombinant Human IgG1 Fc, CF</td><td>R&amp;amp;D Systems</td><td>110-HG-100</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc Antibody</td><td>R&amp;amp;D Systems</td><td>G-102-C</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>use for fixation and immunostaining</td></tr><tr><td>Dumont #5, biologie&lt;br/&gt; tips, INOX, 11 cm</td><td>agnthos.se</td><td>0208-5-PS</td><td>2 pairs of forceps are needed</td></tr><tr><td>Iris scissors, straight, 12 cm</td><td>agnthos.se</td><td>03-320-120</td><td></td></tr><tr><td>Dressing Forceps,&lt;br/&gt; straight, delicate, 13 cm</td><td>agnthos.se</td><td>08-032-130</td><td></td></tr><tr><td>Petri dishes Nunclo Delta treated</td><td>Thermo Fisher Scientific</td><td>150679</td><td></td></tr><tr><td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &amp;micro;m, 13 mm, white, plain</td><td>MerckMillipore</td><td>TMTP01300</td><td></td></tr><tr><td>Nunclon Multidishes&lt;br/&gt; 4 wells, flat bottom</td><td>Sigma-Aldrich</td><td>D6789-1CS</td><td></td></tr><tr><td>Microscope cover glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm</td><td>nordicbiolabs</td><td>107222</td><td></td></tr><tr><td>Cover glasses No.1.5, 18 mm x 18 mm</td><td>nordicbiolabs</td><td>102032</td><td></td></tr><tr><td>Slides ~76 x 26 x 1, 1/2-w. ground plain</td><td>nordicbiolabs</td><td>1030418</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430828</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430055</td><td></td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td><td>Sigma-Aldrich</td><td>WHA1213125</td><td></td></tr><tr><td>Fixed stage research mircoscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td>Black 6 inbred mice, male, C57BL/6NTac</td><td>Taconic</td><td>B6-M</td><td></td></tr><tr><td>Black 6 inbred mice,female, C57BL/6NTac</td><td>Taconic</td><td>B6-F</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>Leica</td><td>Leica S6 E</td><td></td></tr></tbody></table>

dissection and culture of mouse embryonic kidney

このプロトコールの目的は、マウスのメタネフリカの基礎の解剖、単離および培養のための方法を記述することである。 哺乳動物の腎臓発達中、2つの前駆組織、尿管芽および間葉間葉は、最終的に腎臓の収集系およびネフロンを形成するために、細胞機構を伝達し相互に誘導する。哺乳動物の胚が子宮内で増殖し、したがって観察者が接近できないので、器官培養が開発されている。この方法により、腎臓器官形成の間に上皮間葉相互作用および細胞の挙動を研究することが可能である。さらに、先天性腎臓および泌尿生殖路奇形の起源を調べることができる。慎重な解剖の後、metanephricの手先は、培地上に浮遊するフィルター上に移され、数日間細胞培養インキュベーターに保持することができます。しかし、条件が人工的であり、組織内の代謝に影響を及ぼす可能性がある。また、外植片に存在する細胞外基質および基底膜のために、試験物質の浸透が制限される可能性がある。 器官培養の1つの主な利点は、実験者が器官に直接アクセスできることである。この技術は安価で簡単であり、生物学的に活性な物質の添加、遺伝子変異の研究、高度なイメージング技術の適用など、多数の改変が可能である。

Metanephric kidney anlagenは、E11.5で妊娠したBlack-6近交系マウスに由来し、培養した。 3日後、尿管芽は5回まで分岐し、最初はT字型の尿管芽が分岐した。各外植片を撮影し、分節世代を決定し、分枝あたりのエンドポイントの数を計算するために、セグメントおよびエンドポイントの数を定量化した（ 図1 ）。 ImageJ（ rd世代;第4世代は、移植外植片の?...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

このプロトコルは、マウス胚からのmetanephric基盤を単離および培養するための方法を記載する。

マウス胎児腎臓の解剖および培養

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

11.9K ビュー.  Uppsala University. このプロトコルは、マウス胚からのmetanephric基盤を単離および培養するための方法を記載する。 

ビデオ: Dissection and Culture of Mouse Embryonic Kidney

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

マウス全胚培養における神経管閉鎖

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

神経科学

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培養とマウスウォルフダクトの全マウント免疫蛍光染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

発生生物学

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

血管化、拡張開発、および顕微鏡イメージングの改善のための腎オルガノイドおよびオルガノイド培養の最適化

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

生物学

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

生物工学

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 ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

マウス萌芽期の神経幹細胞の分離と培養

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

無血清全胚培養系で、マウス胚発生

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

上頭頂皮細胞活性化に関与する経路を解析するエクスビボアッセイとしての糸球体成長

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

マウス胎児腎臓の解剖および培養

要約

さらに動画を探す

この動画の章

マウス全胚培養における神経管閉鎖

器官培養とマウスウォルフダクトの全マウント免疫蛍光染色

血管化、拡張開発、および顕微鏡イメージングの改善のための腎オルガノイドおよびオルガノイド培養の最適化

マウス胎児肺の文化、分岐の分子機構を評価するシステム

腎組織のプライマリセグメントと新規スキャフォールドフリー構造技術と定款の

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

マウス萌芽期の神経幹細胞の分離と培養

無血清全胚培養系で、マウス胚発生

上頭頂皮細胞活性化に関与する経路を解析するエクスビボアッセイとしての糸球体成長

一次マウス網膜色素上皮細胞の効率的解離と培養

マウス胎児腎臓の解剖および培養

要約

さらに動画を探す

この動画の章

マウス全胚培養における神経管閉鎖

器官培養とマウスウォルフダクトの全マウント免疫蛍光染色

血管化、拡張開発、および顕微鏡イメージングの改善のための腎オルガノイドおよびオルガノイド培養の最適化

マウス胎児肺の文化、分岐の分子機構を評価するシステム

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

マウス萌芽期の神経幹細胞の分離と培養

無血清全胚培養系で、マウス胚発生

上頭頂皮細胞活性化に関与する経路を解析するエクスビボアッセイとしての糸球体成長

一次マウス網膜色素上皮細胞の効率的解離と培養

腎組織のプライマリセグメントと新規スキャフォールドフリー構造技術と定款の