この作品は、(社) にドイツ研究振興協会 SFB688 によって支えられました。

マウス繁殖は Regierung Unterfranken によって承認された、すべてのドイツ語、欧州連合の法律、実験動物の管理と使用に関する規制に従い、すべての解析を行った。
1. コーティングのマイクロタイター プレート α4β1 インテグリンとEx 子宮尿膜培養
<ol><li>200 μ g/mL 滅菌リン酸緩衝生理食塩水 (PBS) で凍結乾燥させたマウス α4β1 インテグリンを再構成します。予め温めておいた (37 ° C) PBS で 10 μ G/ml の最終濃度に希釈します。ピペット希釈 α4β1 インテグリン溶液 20 μ L/ウェルのマイクロ プレート (1 尿膜/井戸) の井戸の必要な数に。 注: は、マイクロ プレートのウェルをコーティングする際、気泡の形成を避けるため。</li>
	<li>加湿の組織文化のインキュベーターでそれらを配置することによって 37 ° C で 60 分のマイクロタイター プレートを孵化させなさい。</li>
	<li>ピペットで上澄みを慎重に吸引します。穏やかな吸引を使用し、ピペット チップをウェル底面に触れることを避けるためにプレートを傾斜します。予め温めておいた (37 ° C) 培養液などダルベッコ変更イーグル培地 (DMEM)、50 μ L を追加することで 2 回洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>重要なステップ: 無料サイトを 50 μ L/ウェル 0.1% (w/v) ウシ血清アルブミン (BSA) を追加することでバインド ブロック/DMEM 加湿組織文化のインキュベーターでマイクロタイター プレートを配置することによって ≥ 37 ° C で 30 分間インキュベートし、。 注: 尿膜外植体血清の存在下での様々 な面にアタッチできます。非特異的結合部位をブロックする必要したがって、コーティング後、インテグリンとの切り裂かれた尿膜を追加する前に井戸は、血清を含む文化を外植体中 (手順 4.4 を参照)。</li>
	<li>4.4 の手順で必要になるまでの 37 ° C でブロッキング液でコーティングされた井戸を維持します。</li>
</ol>2. 子宮解剖 Dpc 8 で妊娠マウスから
注: は、重量ゲイン差別15によって偽陽性妊娠率を減らします。
<ol><li>頚部転位によってマウスを犠牲に。腹部のコートを 70% (v/v) エタノールを噴霧して消毒します。 注: は、マウスを処理するとき、手袋を着用します。麻酔なしの頚部転位による安楽死は、尿膜添付ファイルを妨げる可能性があります尿膜の化学汚染を防ぎます。</li>
	<li>ハサミで正中線で小切開を行うことで腹部の皮膚を開きます。上と下の切開、皮膚を保持するために手袋をはめた指を使って、胸やマウスの尻尾に向かって皮膚を引き離します。</li>
	<li>鉗子で腹膜を把握します。もう片方の手を前方から V 字切開は後方に向かって、はさみを使用して、離れて腹腔内を公開する組織を移動します。子宮へのアクセスを得るために 1 つの側面に腸をプッシュするのに鉗子を使用します。</li>
	<li>2 つの子宮角を探します。鉗子で子宮頸部 (尾側のセグメント) を押しながら高級ハサミで靭帯をカットします。その後、はさみで卵管を切断することによって両方の子宮角をデタッチします。</li>
	<li>部屋の温度 (RT) PBS 滅菌、滅菌 10 cm 皿含むに子宮を転送する鉗子を使用します。脂肪組織、血管、神経周囲の高級ピンセット、はさみ (図 1 a) と子宮角を削除します。</li>
</ol>3. 胚解離
注: 8:1 ズーム範囲を備えた顕微鏡の下で次の手順に従います。
<ol><li>はさみで、着床間に切断することによって胚を区切ります。鉗子で子宮芽の筋肉子宮内膜をつかんでそれを分解、脱落膜 (図 1 b) を公開します。</li>
	<li>RT PBS 滅菌、含んでいる新しい 10 cm 皿に子宮芽を転送鉗子を使用して、します。</li>
	<li>鉗子の先端で胚の周り脱落膜を固定します。</li>
	<li>微細鉗子 (図 1)、脱落膜の抗 mesometrial 側に切開をして、胚 (図 1) を公開するための鉗子で、脱落膜をはがします。 注: mesometrial ポール脱落膜の高い血管柄付きし、胚が含まれますされ、淡い反 mesometrial 部分より根元が広くできます。</li>
	<li>鉗子 (図 1E, F) で胎児を解剖します。</li>
	<li>マイクロ スプーンを使用して、滅菌 3.5 cm 皿に含まれている滅菌 PBS のドロップに胚を転送します。</li>
	<li>マイクロ スプーンを使用して同じ 3.5 cm 皿の PBS の新鮮なドロップに胚を転送そして鉗子 (図 1, H) による卵黄嚢から胎児を解剖します。</li>
	<li>必要に応じて、必要な場合 (例えば、ポリメラーゼ連鎖反応による検出)、転送卵黄嚢生殖不能の管にそれを吸引によって ~ 大形オリフィス 200 μ L ピペット チップに 5 μ L の PBS。-20 ° C にてストア 注: 卵黄嚢準備を汚染する可能性が母体の組織 (すなわち、頭頂部の卵黄嚢ライヘルトの膜と胎盤のコーンなどを含む) は、誤ったジェノタイピングの結果につながります。これらの組織の除去は、卵黄嚢から胚解離の前に最も簡単です。</li>
</ol>4. 尿膜解離と固定化 α4β1 インテグリンのEx 子宮文化
<ol><li>微細鉗子 (図 1 H) による胚の後部の端にその基底挿入部位から尿をカットします。</li>
	<li>必要に応じて、発達のステージングが必要な場合は、段階の対照の光学と、5 ×、10 × 対物顕微鏡を用いた目視による胚の体節のペアをカウントします。 注: 胎児は体節を数えるための鉗子で拡張できます。漿の添付ファイルとの融合は ≥ 6 体節のペアを持つ胚で E 8.0 と 9.0 E の間発生します。6 8 体節のペアを持つ胚で回転を開始します。</li>
	<li>10% (v/v) ウシ胎児血清、L-グルタミン、プレコート マイクロタイター プレート井戸 (セクション 1 を参照) の必要な数の抗生物質と DMEM のピペット 40 μ L/ウェル。</li>
	<li>それを吸引によってフローティング尿を収集 〜 200 μ L ピペット チップの大形オリフィスに 5 μ L の PBS。1 尿膜/ウェルを転送します。</li>
	<li>例えば、加湿の組織文化のインキュベーター (37 ° C、5% CO2) でマイクロタイター プレートを配置することによって、allantoides をインキュベート 6、12、24 h。尿膜の添付ファイルとの干渉を防ぐためにこれらの時間ポイントの間にマイクロタイター プレートを移動しないでください。</li>
	<li>各時点でスコア尿膜添付ファイル (例えば、ない添付ファイル/フローティング尿膜部分的または完全にアタッチされた尿膜; 完全に広がり尿膜) 位相コントラストや微分干渉コントラスト光学系を搭載した顕微鏡を使用して、5 ×、10 × 目的 (図 2)。 注: 新鮮単離と完全に接続されている allantoides の代表的な例は図 2のとおりです。子宮 ex、野生型 c57bl/6 j マウスから分離した allantoides の遠位先端アタッチ (尿の部分的な添付ファイルとして呼ばれます) 4-6 時間、以内、全体の尿膜壁完全添付ファイルが可能です 〜 文化の 12 h。18-24 h 外植体が平坦化、CD31 陽性血管叢9,16(完全に広がった尿膜に呼ばれる) 円形の形状を想定します。</li>
</ol>5 血管内皮細胞マーカー CD31 Explanted 尿膜壁験染色
<ol><li>完全に広がり外植片の培養培地を吸引し、4% (w/v) パラホルムアルデヒド/PBS の 40 μ L/ウェルを追加してセルを修正します。 注: パラホルムアルデヒドは有毒です。手袋、皮膚と眼との接触を避けるために安全ゴーグルを着用します。</li>
	<li>常温では、10 分間インキュベートし、上清を吸引します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>セルを permeabilize し PBS の 40 μ L/ウェルを追加することによって二度 0.1% (v/v) した洗浄で 30 分の PBS でトリトン X-100/0.1% (w/v) BSA/10% (v/v) ヤギ血清の 40 μ L/ウェルを追加することで非特異的結合部位をブロックし、穏やかな吸引によって洗浄媒体を削除します。 注: プロトコルを一時停止できる一晩この時点で。</li>
	<li>4 ° C で一晩に 20 μ L/ウェル主アンチ CD31 抗体の希釈 1:50 0.1% (w/v) BSA/PBS で追加することによって細胞を染色します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>暗い室内で 0.1% (w/v) BSA/PBS アルミホイルで包装することによって配置の光から保護したマイクロタイター プレートで 1 時間で蛍光標識した二次ヤギ抗ラット抗体希釈 1: 200 の 20 μ L/ウェルを追加することでバインドされた一次抗体を検出します。PBS の 40 μ L/ウェルを追加して 3 回を洗浄し、穏やかな吸引によって洗浄媒体を削除します。</li>
	<li>メディアをマウントの 20 μ L/ウェルを追加することによって細胞を埋め込みます。インキュベート 〜 RT 取付培地が固化するまでで 1 時間。マイクロタイター プレート常温または 4 ° c、光から保護を格納します。</li>
	<li>5 ×、10 × 客観的かつ適切な蛍光フィルターを搭載した蛍光顕微鏡下で外植体画像尿膜壁血管叢 注: 完全普及尿膜の CD31 陽性血管叢の代表的なイメージは図 3のとおりです。Flk-1、Flt、に対して指示される抗体などの血管叢の他のマーカーとしてよく16ネクタイ 1 またはネクタイ 2 を使用できます。</li>
</ol>

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プロの接着細胞外マトリックス分子フィブロネクチンなどの豊富な源である血清の存在下で尿膜外植体は容易に様々 なプラスチックやガラス、フィルター表面9,16にアタッチされます。したがって、実行される分析の目的は、遺伝子組み換えの効果を具体的に検討する場合、尿膜の添付ファイルに他の処理固定化 α4β1 は、非特異的結合部位 (例?...

胎盤が子宮環境の胚の成長と開発のために不可欠です。それは内分泌と免疫器官としての酸素代謝物、胎児と母体の循環とも機能間の栄養交換のためのインターフェイスを構成します。胎盤発育を損なう内因性または外因性の侮辱は、子宮内胎児発育遅延、胎児の損失、または妊娠合併症、マウスと人間1の両方につながることができます。対象となるマウスを異常な胎盤の生理を引き起こす突然変異の数が 219 遺伝子多型のマウスのゲノム情報学 (MGI) ウェブサイトに現在表示されている2を増やす続けます (http://www.informatics.jax.org/vocab/ mp_ontology/MP:0010038)、これらの表現型の多くはよく機械論的視点から理解されていません。に加えて遺伝子変異、小分子薬、モノクローナル抗体、毒素、病原体、余分な代謝物や様々 な環境要因や影響を与える胎盤開発関数3,4,5,6,7します。 まだ、簡単な生体外モデル胎盤の開発で重要な手順が不足している試金。
(臍帯発達前駆体) 尿膜が絨毛膜8に向かって育つ芽として胚の後部の端から現れるとき、初期の経産婦でマウス胎盤の発育が開始されます。尿の芽の外側の層の Chorioadhesive セルは、添付ファイルおよび絨毛膜 (漿「融合」) の表面に尿膜の広がりを仲介します。絨毛膜はその後に尿膜壁血管成長迷路、栄養素と酸素が密接に並置母体血と胎児血管9 間交換される胎盤内層を形成する絨毛に折る ,10。
絨毛膜へ尿の付着迷宮形成の初期および重要なステップであり、このプロセスの欠陥は midgestation1胚性致死性の最も一般的な原因のうち。マウス突然変異体の数が、漿添付ファイル10が発生するが失敗し、MGI データベース現在異常な漿融合 (http:// によって特徴付けられる 108 マウスの突然変異体の一覧を記載されてwww.informatics.jax.org/vocab/mp_ontology/MP:0002824)、血管細胞接着分子 1 (VCAM 1、尿中胚葉に発現) と (は絨毛の中皮に表される α4β1 インテグリンの細胞間相互作用初期胚胚体外胚葉由来) 漿添付ファイルと融合11,12,13のため不可欠であることが表示されます。(E) 7.5 日約 VCAM 1 が尿の茎の遠位 3 分の 2 以内単位があることを示している全体マウント野生型胚の免疫組織化学的染色13 (1-4 体節期)、およびその発現状態のまま高漿の添付ファイルと - 融合11,12。絨毛膜尿愛着の障害は、通常、子宮の芽の組織学的解析による検出します。ただし、尿膜サイズはの間と同じ発達段階14リットル内でも高い生理学的変数であり、尿は、物理的な接触をするのに十分な大きさに達したときに絨毛膜にのみ添付できます。この自然の変動を反映して、漿添付が行われる間のいつか 〜 E 8.0 と 9.0 E子宮内、および組織によって、このプロセスの統計的信頼性の高い評価は大きい数の解析に依存したがって淘汰適切な発達段階で十分な検体を採取します。
ここでは、尿の添付ファイル子宮 ex少ない尿膜のサイズに依存している評価方法について述べる。妊娠マウスの子宮からマウス胚とその allantoides の郭清を紹介 〜 8 日のポストの coitum 定め(dpc; dpc 0.5 が膣にプラグインの検出を示す)、固定化 α4β1 の外植体尿膜の後続の文化とインテグリンです。このメソッドは、添付ファイルおよび複数並列で、異なる連続した時点で尿膜外植体の挙動を広がりの急速な機能評価できます。プロトコルを使用することがあります尿膜添付ファイル前のヴィヴォに及ぼすターゲット変異、薬、または様々 な環境要因の画面に。

<table><tbody><tr><td>C57BL/6J wildtype mice</td><td>The Jackson Laboratory</td><td>Stock No:&amp;nbsp;000664</td><td></td></tr><tr><td>70 % (v/v) Ethanol</td><td>VWR International</td><td>930031006</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Forceps used to open the abdominal cavity.</td></tr><tr><td>Micro dissecting forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Dumont # 5 medical biology forcepts with fine, sharp tip.&amp;nbsp;</td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14094-11</td><td>Straight blades.</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15012-12</td><td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td></tr><tr><td>Microspoon</td><td>Carl Roth</td><td>AT18.1</td><td>5 mm Spoon diameter.</td></tr><tr><td>Microtiter plates</td><td>ibidi</td><td>81501</td><td>We use uncoated, sterile angiogenesis slides (internal volume 50 &amp;micro;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-&amp;#945;4&amp;#946;1-mediated binding to plastic.</td></tr><tr><td>10 cm dish</td><td>Nunc</td><td>150350</td><td>Sterile tissue culture dishes.</td></tr><tr><td>3.5 cm dish</td><td>Nunc</td><td>150288</td><td>Sterile tissue culture dishes.</td></tr><tr><td>Large orifice pipette tips&amp;nbsp;</td><td>Biozym</td><td>VT0140X</td><td>Low binding pipette tips, 200 &amp;micro;L.</td></tr><tr><td>&amp;#945;4&amp;#946;1 integrin</td><td>R&amp;amp;D Systems</td><td>6054-A4</td><td>Murine &amp;#945;4&amp;#946;1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>PAN Biotech</td><td>P04-03600</td><td>The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO&lt;sub&gt;3&lt;/sub&gt; but no L-glutamine, which is added separately.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (ThermoFisher Scientific)</td><td>15140-122</td><td>100 x (10,000 U/mL Penicillin and 10,000 &amp;micro;g/mL streptomycin) stock solution.&amp;nbsp;Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>L-Glutamine</td><td>PAN Biotech</td><td>P04-80100</td><td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S 0115</td><td>We use heat-inactivated FBS (heated to 56 &amp;deg;C for 30 min with mixing to inactivate complement).</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A7030</td><td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &amp;micro;m pore size and low protein-binding membrane, and store aliquots at -20 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;para&lt;/em&gt;-Formaldehyde</td><td>Carl Roth</td><td>0335.2</td><td>To make a formaldehyde solution in PBS, weigh &lt;em&gt;para&lt;/em&gt;-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &amp;deg;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &amp;deg;C for approximately four weeks.&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td></tr><tr><td>Normal goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td>To block non-specific binding of antibodies in immunofluorescence analyses.</td></tr><tr><td>&amp;#945;-CD31 Antibodies</td><td>BD Biosciences</td><td>550274</td><td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td></tr><tr><td>Secondary goat anti-rat antibodies</td><td>Thermo Fisher</td><td>A-11006</td><td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td>Mounting medium for cytochemistry. MW ~31,000</td></tr><tr><td>Labovert&amp;nbsp;</td><td>Leitz</td><td>Labovert&amp;nbsp;</td><td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>M80</td><td>Leica Microsystems</td><td>M80</td><td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td></tr><tr><td>DM4000B</td><td>Leica Microsystems</td><td>DM4000B</td><td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&amp;nbsp;</td></tr><tr><td>Digital camera</td><td>JVC</td><td>KY-F75U</td><td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>SP5</td><td>Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td></tr></tbody></table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

胎盤は、成長と哺乳動物の胎児の開発のために不可欠です。このため、多数の遺伝子変異と胎盤の開発または機能を妨げる可能性が高いのも環境の侮辱が、マウスとヒトの妊娠初期の損失にあります。それにもかかわらず、画面の胎盤形成に対する潜在的な影響に簡単な生体外の試金が不足しています。ここでは、モデリングにおける胎盤絨毛膜に尿膜壁添付ファイルから成っている最初の重要なステップに焦点を当てます。Chorio 擬態の基板となる固定化 α4β1 インテグリンの尿外植片の添付ファイルを迅速に評価する方法について述べる。この体外アプローチにより、添付ファイルと異なる連続した時点で複数の尿膜外植体の挙動を拡散の定性的な評価です。プロトコルは、ターゲットを絞ったマウス突然変異、薬、または妊娠合併症や尿膜添付ファイル前のヴィヴォの胎児の損失にリンクされているさまざまな環境要因の効果を調べるために使えます。

このプロトコルを特定しマウス allantoides前のヴィヴォ、外植体について述べるし、体内漿融合中 α4β1 インテグリンに尿膜添付ファイル、重要なプロセスの定性的な評価の詳細。図 1 a-Hに示す代表的な結果は、妊娠子宮から始まって尿膜分離の連続した手順を示します。脂肪組織と血管 (図 1...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

モデル漿添付ファイル、胎盤形成の最初のステップの in vitroアッセイについて述べる。プロトコルは、固定化 α4β1 インテグリンのマウス allantoides の郭清と植の文化を示しています。尿膜の添付ファイルは、あらかじめ決められた時点で病理組織学的に評価されます。

郭清と尿の添付ファイルのIn Vitro解析マウス尿膜の培養

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

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Research

JoVE Journal

Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K ビュー.  University of Würzburg. モデル漿添付ファイル、胎盤形成の最初のステップの in vitroアッセイについて述べる。プロトコルは、固定化 α4β1 インテグリンのマウス allantoides の郭清と植の文化を示しています。尿膜の添付ファイルは、あらかじめ決められた時点で病理組織学的に評価されます。

ビデオ: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

体内と組織や細胞植アプローチを使用して形態形成と胚および周産期の大動脈の病因を研究するには

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

発生生物学

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

造血の発達における大動脈生殖巣中植文化システムのアプリケーション

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

免疫・感染学

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

ザ<em&gt; ex vivoで</emマウス腸オルガノイドの&gt;文化とパターン認識受容体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

マウス卵管の微小解剖と解離:個体セグメントの同定と単一細胞単離

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

頭蓋間葉の細胞挙動を評価するための外植片アッセイ

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

生物学

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

マウス小腸上皮オルガノイドと先天性リンパ球細胞との共培養

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

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.

マウス胎児腸全体培養システム<em&gt;生体外</em&gt;シグナル伝達経路の操作と絨毛開発の3次元ライブイメージング

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016).
This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

ヒト母体‐胎児インターフェースのex vivoモデルとしての絨毛および脱落器官培養の確立

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...