Name: mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development
Uploaded: 2014-09-04T14:00:00.000+00:00
Duration: 6 min 46 s
Description: Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

注：すべてのマウスを人道的に実験動物医学ミシガン大学医学部号機大学によって承認されたプロトコルを使用し、動物の使用およびケアに関する研究委員会のガイドラインに従って取り扱った。 1全臓器培養システム<ol><li>培地および培養プレートの調製<ol><li>組織培養フードでは、ストックボトルからBGJbメディアの5ミリリットルを除去し、ペニシリン/ストレプトマイシンを5ml加える。 （ペニシリン/ストレプトマイシンで）BGJb培地49 mlに5 mg / mlのアスコルビン酸の1ミリリットルを加えることで、50ミリリットルコニカルチューブ内のメディアの作業ストックを準備します。 </li><li>トランスウェルのそれぞれの下に1ミリリットルを追加することによって、6ウェル培養プレートを準備します。 注記：すべての6トランスウェルを使用しない場合は、後で使用するために、新鮮な無菌6ウェルプレートに余分なトランスウェル移動します。 3 10センチメートル無菌ペトリ皿のそれぞれに作業BGJbメディアの3ミリリットルを追加します。 </li></ol></li><li>解剖の準備<ol><li> 70％のETHAに解剖ツールを浸すとNOL作業中にきれいな道具を保管してください。 </li><li>大きな氷​​のバケツに解剖エリアを設定し、氷上でBGJbメディアを6ウェル培養プレートから10cmペトリ皿を置きます。解剖と文化のための腸の準備中に、できるだけ多くの氷の上で作業。 </li><li>胎児の腸の収穫のために頸椎脱臼により安楽死の前にイソフルラン（100μL）をチャンバー内での成体雌マウスを麻酔。 </li><li>母親から胎児を収穫した後、タイラーステージングチャート13に従って慎重にそれぞれの胎児を上演。日常的に、単一のゴミ内で観察された発生段階で最大24時間の変動などの各胎児の年齢を決定するために、後の性交日に依存しないでください。 </li></ol></li><li>胎児の腸の解剖<ol><li>前腸の除去に断頭によって胎児を安楽死させる。 </li><li> 1×ダルベッコのリン酸緩衝生理食塩水（DPBS）で各腸を分析した後、ナンプラーBGJbメディアを作業した10センチメートルペトリ皿にそれをCE。 </li><li>慎重に胃および上部十二指腸から胃や膵臓から脾臓を除去します。 </li><li>静かに腸がまっすぐにさせることが可能とだけ十分な接続を分離する限り添付大網を残すように注意しながら大網を分離。 注：E14.5より古い腸以上24時間未満培養されたもののために、静かに管腔内容が腸を通って流れるようにし、切断端から排出されるために可能にするために3等分腸を分離する。培養はE13.5前に始まっただし、漿膜が正常に腸の長さにわたって成長を可能にするために、腸の3つのセグメントを分離する前に、培養24時間後まで待ちます。 </li><li>培養プレート（1.1.2）のトランスウェル上に腸の部分を転送するために口のピペットを使用してください。 </li><li>彼らはtranswel渡って真っ直ぐになるように、必要に応じて静かに腸の位置を変更lである。 注：腸管を通って管腔内容物を移動させ始めると、腸がねじれは、腸へのバックアップや損傷の原因となります。 </li></ol></li><li>文化と治療<ol><li>トランスウェルの下に（追加された組換えタンパク質または薬理学的阻害剤と作動媒体）、トランスウェルの下から培地を除去処理培地700μlのを追加します。 </li><li>腸の上に処理媒体の滴ずつ300μlのを追加し、それがトランスウェルを通して浸すことができます。必要に応じて腸の位置を変更。 </li><li>処理媒体は、少なくとも一日一回またはより頻繁に治療試薬の半減期に応じて変化する。 </li></ol></li><li>治療試薬の局在化のためのタンパク質源浸したアガロースビーズの調製<ol><li>彼らの貯蔵容器にアガロースビーズを再懸濁し、1.5mlマイクロチューブにアガロースビーズを100μlを移す。 </li><li>無菌1×pHのチューブの残りの容量を埋めるosphate-緩衝生理食塩水（PBS）と、それらを洗浄するためにビーズを混ぜて。ビーズが底に沈殿した後、ビーズの上から、PBSをオフにピペッティングすることを可能にするために数分間ラックにマイクロチューブを置きます。無菌1×PBSでマイクロチューブを補充し、さらに2回の洗浄工程を繰り返します。 </li><li> 2回の治療のために所望の濃度で目的のタンパク質を準備します。 </li><li> 2倍のタンパク質原液5μlの水で洗浄したアガロースビーズを5μlを混合し、静かに、タンパク質中のビーズを攪拌する。静かに10〜15分毎に混合、1時間チューブを氷上に置きます。 </li><li>腸にビーズを配置する前に、滅菌1×PBS中で簡単にビーズをすすぐ。 </li></ol></li><li>アガロースビーズの配置<ol><li>静かに引っ張らキャピラリー針で口のピペットを用いて腸の上にアガロースビーズを配置します。乱暴な取り扱いが腸を損傷し、損傷した領域に絨毛の発達を防ぐことができますように細心の注意を払ってビーズを配置します。 </li><li>腸蠕動運動や培養時間にわたる腸のオフにロールバックされます置かれたビーズの約半分を受けるので、分析に必要とされる2倍のビーズを配置します。 </li></ol></li><li>培養された腸の固定<ol><li>慎重にトランスウェルの下から処理媒体を除去し、3分間空気乾燥腸を可能にします。 </li><li>その後定着時間の残りのための固定液2mlで腸の上部をカバーする、2分間のトランスウェルの下の固定液1ミリリットルを追加します。 4℃でまたは室温で2時間、4％パラホルムアルデヒドのO / Nで固定してください。 </li></ol></li></ol>固定腸の2イメージング<ol><li>ホールマウントイメージング<ol><li> 1X DPBS100μlのスライド上で（内因性蛍光を含む）全体の腸を置きます。優しく腸を配置する鉗子を使用してください。 </li><li>組織MOを作るために慎重に70％グリセロール100μlのDPBSを削除し、すぐに追加するには、実験室の組織を使用して、透明な再およびイメージングの可能性の深さを増加させる。 注：腸のさらなる浸透が望まれる場合、代わりに70％グリセロール中に腸を取り付ける、10〜30分間、フォーカスクリア溶液の数滴で腸を浸す。ピペットでフォーカスクリアソリューションオフとマウント解除と腸をマウントします。 </li><li>粘土にカバースリップの四隅を浸し、腸を平坦からカバースリップを保つために、スライドとカバーガラスの間にスペーサーとして使用するために、粘土の少量を拾う。 </li><li>綿棒で塗布溶融しVALAPでスライド上にカバーガラスを密封。 </li><li>イメージ直立または倒立型共焦点顕微鏡のいずれかで、腸。詳細については、議論を参照してください。 </li></ol></li><li> （腸の構造の断面図のために）固定された腸のビブラトーム切片<ol><li>小さなプラスチック金型7％アガロース（10×10×15ミリメートル）に埋め込む腸とにアガロースブ​​ロックを許可する冷却して凝固。 </li><li>瞬間接着剤でビブラコレクションステージにプラスチック金型及びグルーアガロースブ​​ロックからアガロースブ​​ロックを削除します。 </li><li>氷冷1X DPBSで収集段階を埋める。 </li><li> 100〜150ミクロンの間の厚さでセクションをカット。厚い部分は、断面が深く可視化すること（セクション2.1.2で説明します）決済ソリューションを使用してください。 </li><li> 4℃での保存のために6ウェルプレートのウェル内に収集段階からビブラトーム切片を注ぐ。 </li></ol></li><li>固定、ビブラトーム切片の組織の免疫蛍光染色<ol><li> 1X DPBSで24ウェルプレートにウェル当たり5-12ビブラトームのセクションを配置します。 </li><li> 1X DPBSを削除し、ウェルあたり透過処理溶液500μlを加える。静かに、室温で25分間サンプルを揺する。 </li><li>透過処理した後、1×PBSでサンプルを3回洗浄する。 </li><li>ブロッキング溶液500μlに、少なくとも30分間のサンプルをブロックします。 </li><li>ブロックした後、電子メールブロッキング溶液で希釈した一次抗体を500μlとブロッキング溶液をXchangeは、4℃のCO / Nでサンプルを保つ。 注：凍結し、パラフィン切片上でうまく動作し、一次抗体希釈液は、通常、しかし、一般的に、このプロトコル（ 例えば、BrdU）のではうまく機能しない核抗原回復を必要とする抗体、あまりにビブラトーム切片上でうまく動作します。 </li><li>翌日、ブロッキング溶液で15分間、試料を毎回3回洗浄する。 </li><li>洗浄後、ブロッキング溶液中に希釈した二次抗体を500μlを適用します。光から蛍光団を保護し、室温で2時間に45分のためのサンプルを揺するようにアルミホイルで24ウェルプレートを包みます。 </li><li> 15分ごとに1×PBSでサンプルを3回洗浄し、2.4節で説明したようにスライド上のビブラトーム切片をマウントします。 </li></ol></li><li>取付ビブラトーム切片<ol><li>ビブラトームダブルSTIの薄切りを切断して取り付けるためのスライドを準備するCKテープとスライドの長辺に沿ってそれらを配置する。 </li><li>慎重に1×100μlのPBSの低下をスライド上に両面テープの間に5-10ビブラトームセクションを配置。 </li><li>すべてのセクションを展開するとスライド全体で単一層にそれらを広げた。 </li><li>フラットに座ってからカバースリップを妨げる余分なアガロースまたは不均一なビットを削除します。 </li><li>慎重にビブラトーム切片の周囲から過剰1X PBSを吸収するために実験室組織を使用しています。 </li><li>ビブラトーム切片の上部に水性封入剤のドロップを追加し、その後カバースリップ。 </li><li>綿棒で塗布溶融しVALAPでスライド上にカバーグラスを密封。 </li><li>イメージ直立または倒立型共焦点顕微鏡のいずれかでビブラトーム切片。イメージングシステムの選択についての詳細は、説明を参照してください。 </li></ol></li></ol>全腸の3。ライブイメージング腸fetuseから収穫E12.5後のsは、そのままに接続された大網で、成功した上記のシステムを用いて、培養中の絨毛を開発しています。したがって、このエキソビボシステムは、経時的な形態形成の三次元ビューを再構成することができる現像腸の生のz断面画像の捕捉を可能にする。 <ol><li>ライブイメージングの設定<ol><li>細かいメッシュのスクリーンに個別のポリカーボネート膜を接着する瞬間接着剤を使用してください。これは直立共焦点顕微鏡の浸漬レンズ下の場所に腸を保持するための支持足場を提供します。ライブイメージング顕微鏡の選択についての詳細については、議論のセクションを参照してください。 </li><li>中央ウェルの培養プレートにメッシュスクリーンを配置します。 </li><li>ポリカーボネート膜上に解剖し、腸を置き、両端に瞬間接着剤を一滴加える。腸に固定するだけの十分な接着剤を使用してくださいではなく、コート、それ！ </li><li>ポリカーボネート以下のフェノールレッドを含まない培養培地を追加します。培養プレートの中央ウェル中の膜。 </li><li>湿度を提供するために、培養プレートの外縁に水を加える。 </li><li>イメージングしながら、腸の動きを制御するために、メディアの3ミリリットルの1×PBS中のキシラジンの5溶液：胎児の腸は、培養中の収縮の蠕動様の波を起こしているので、1を100μlを追加。この投与量は、絨毛の発達を損なうことなく、約8〜10時間、腸の動きを制御します。 </li><li>腸の横断ビューの場合は3〜4％アガロース溶液中でのライブ腸を埋め込み、ビブラトーム上の厚い部分（150〜500μm）をカット。切断された腸の剛性とアガロースの剛性を合わせて、経験的に発達段階を決定した。一般的に、3％アガロース溶液はE14.5よりも若い腸に適していますし、4％溶液腸E15-E16に適しています。 </li><li> （3.1.1項のように）メッシュスクリーンに貼付されたポリカーボネート膜にビブラトーム切片を接着し、セクション3.1.2-3.1.6で説明したように文化。 </li><li>調整可能なテーブル付きのアップライトスタンドに二光子共焦点蛍光顕微鏡（690-1,040 nmの励起レーザー）を使用したライブイメージングを実施する。 </li><li> GFPシグナルのための最高の解像度と深い浸透を提供するから900nmに同調二光子レーザー。イメージングながら、組織への損傷を低減します。また、可能な限り低い発光レーザのパワーを設定する組織損傷を減少させる。典型的には、二光子レーザーに12％の電力を使用して、GFPの良好な励起を得る。 </li></ol></li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Fu, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtome-free+3-dimensional+confocal+imaging+method+for+visualization+of+mouse+intestine+with+subcellular-level+resolution.">Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution.</a> Gastroenterology. 137, 453-465 (2009).</li></ol>

The authors have no financial conflicts to disclose.

動的な性質と途上腸の複雑な組織の相互作用は、これらの形態形成イベントの完全な理解を持つように3次元可視化を必要とします。イメージング技術の進化により、能力が/開発の改善とそれに、器官形成時の空間的なコミュニケーションと相互作用の理解が大幅に強化されている詳細に絨毛の形態形成を調べた。 全体腸を培養するための別の方法も試験されているが、...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table><tbody><tr><td>Fine dissecting forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;11254-20</td><td>2 pairs</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>1x sterile Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Gibco&amp;nbsp;</td><td>14040-133</td><td>500 ml</td></tr><tr><td>6 well plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>24 well plates</td><td>Costar&amp;nbsp;</td><td>3524</td><td></td></tr><tr><td>60 x 15 mm petri dishes</td><td>Falcon</td><td>&amp;nbsp;451007</td><td></td></tr><tr><td>Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td><td>Corning Costar&amp;nbsp;</td><td>3428</td><td>6 inserts per plate</td></tr><tr><td>BGJb media</td><td>Invitrogen&amp;nbsp;</td><td>12591-038</td><td>500 ml</td></tr><tr><td>PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td><td>Gibco</td><td>&amp;nbsp;15140</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma&amp;nbsp;</td><td>A0278</td><td>make 5 mg/ml stock, filter, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td><td>Fisher</td><td>&amp;nbsp;21-180-10</td><td>remove the aspirator assembly and replace it with a 1,000 &amp;micro;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td></tr><tr><td>6 inch glass pasteur pipets</td><td></td><td></td><td></td></tr><tr><td>Capillary Tubes</td><td>World Precision Instruments&amp;nbsp;</td><td>TW100F-4</td><td>pull to needles</td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td>made in 1 x PBS, pH to 7.3</td></tr><tr><td>Culture plates</td><td>Falcon&amp;nbsp;</td><td>353037</td><td></td></tr><tr><td>Fine mesh stainless steel screen</td><td>purchase at hardware store</td><td></td><td></td></tr><tr><td>Polycarbonate membranes</td><td>Thomas scientific&amp;nbsp;</td><td>4663H25</td><td>alternatively, cut Corning Costar 3428 membranes off of transwell supports</td></tr><tr><td>Instant glue</td><td>purchase at hardware store</td><td>gel based preferrably</td><td></td></tr><tr><td>35 x 10 mm plates</td><td>Falcon&amp;nbsp;</td><td>351008</td><td></td></tr><tr><td>7% agarose</td><td>Sigma</td><td>&amp;nbsp;A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>minutien pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Phenol red free media (DMEM)</td><td>Gibco&amp;nbsp;</td><td>21063-029</td><td></td></tr><tr><td>Xylazine (100 mg/ml)</td><td>AnaSed&amp;nbsp;</td><td>139-236</td><td></td></tr><tr><td>Matrigel</td><td>BD</td><td>356231</td><td>basement membrane matrix, growth factor reduced, phenol red-free</td></tr><tr><td>3-4% agarose</td><td>Sigma&amp;nbsp;</td><td>A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>Imaging of fixed intestines</td><td></td><td></td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>vaseline</td><td>purchase at pharmacy</td><td></td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>lanolin</td><td>Sigma&amp;nbsp;</td><td>L7387</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>paraffin</td><td>Surgipath</td><td>39601006</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>70% glycerol in 1 x PBS</td><td></td><td></td><td></td></tr><tr><td>Focus clear and Mount Clear</td><td>CelExplorer Labs Co.</td><td>&amp;nbsp;F101-KIT</td><td></td></tr><tr><td>Modeling clay</td><td>purchase at art supply store</td><td></td><td></td></tr><tr><td>double stick tape</td><td></td><td></td><td></td></tr><tr><td>cotton applicator swabs</td><td></td><td></td><td></td></tr><tr><td>plastic molds, 10mm x 10mm x 5 mm)</td><td>Tissue Tek&amp;nbsp;</td><td>4565</td><td></td></tr><tr><td>slides</td><td></td><td></td><td></td></tr><tr><td>coverslips</td><td></td><td></td><td></td></tr><tr><td>lab wipe</td><td>Kimberly Clark</td><td>34155</td><td>lint free delicate task wipe</td></tr><tr><td>Theiler staging chart&amp;nbsp;</td><td></td><td></td><td>&lt;a href=&quot;http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf&quot;&gt;http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf&lt;/a&gt;</td></tr><tr><td>Leica SP5X confocal microscope&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Leica DMI 6000 stand&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Aqueous mounting medium (Prolong Gold)</td><td>Molecular Probes&amp;nbsp;</td><td>P36930</td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>24 well plate</td><td>Costar</td><td>&amp;nbsp;3524</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T-8787</td><td>used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td></tr><tr><td>Goat serum</td><td></td><td></td><td>used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td></tr><tr><td>Tween20&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;P9416</td><td></td></tr></tbody></table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

胎児の腸内のほとんどの形態形成のプロセスは、発達段階的な変化のスナップショットを提供し、固定した組織の薄い部分から推論されてきた。薄連続切片から三次元情報が原因完全連続切片を再構築し、連続切片上の組織の適切な向きを維持することの難しさを解釈するために挑戦することができます。グロッセらによる最近の知見。、2011腸1の現像絨毛の形態形成を理解する上で三次元情報の重要性を強調している。単独でラベルされた腸細胞の三次元再構成は、腸管上皮細胞の大部分が頂端および基底表面の両方に接触することを実証した。さらに、上皮の頂端表面でのアクチン細胞骨格の三次元再構成は、腸管腔が連続しており、二次管腔は、複数のアーチファクトであることが示されectioning。これら2点は、腸管上皮におけるinterkinetic核移動の実証とともに、以前に1思った層別化多列上皮としないように開発する腸上皮を定義しました。上皮三次元的に観察する能力は、この点を実証し、胎児の腸上皮形態形成を再定義する精液だった。多光子イメージング技術と三次元再構築ソフトウェアの進化により、無傷の、現像器官を視覚化する能力が急速に向上している。二光子励起は、高解像度の組織への損傷が少ない深い浸透を可能にする。二光子イメージングとウォルトンら全体の胎児のマウスの腸の3D再構成は、2012年には絨毛伸長2のパターンを定義することができました。ここでは、許可する絨毛およびその培養系の拡張のex vivoで開発を可能にする全器官培養系を記述する腸を三次元的にその開発中に撮像される。

このシステムは、薬理学的試薬または組換えタンパク質とシグナル伝達の操作を可能として、胎児腸の全体の外植片の培養は、腸の開発を調整シグナル伝達分子の位置、分布、および期間の分析が可能になります。トランスウェル培養系（ 図1Aは 、ウォルトンらから再生される。2012）2が組織上に薬物またはタンパク質浸したアガロースビーズを配置するこ...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

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 ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

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

14.9K ビュー.  University of Michigan. 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. 

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

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

使い方<em&gt;エクスビボ</em&gt;全体の胎児器官の直立飛沫培養物をマウス器官中に発生過程を研究します

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

発生生物学

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

条件を使用して、マウス腸の再生成にパネート細胞の役割を分析するためのプロトコル<em&gt;のCre-LOX</em&gt;マウスモデル

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

生物工学

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

腸幹細胞機能をex vivoで研究するためのマウス結腸陰窩の三次元培養

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

がん研究

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

ガットオンチップマイクロ流体デバイスにおけるマイクロ加工ヒト腸絨毛と生活Microbiomeの共培養

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

胎児腸オルガノイドを用いたインビトロで吸引から破乳移行を再現する

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

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 ...

免疫・感染学

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 ...

生物学

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

腸ホルモン分泌の研究と腸内分泌細胞の生細胞イメージングを対象ネズミ小腸の混合初代培養

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

ラット腸陰窩の分離と腸オルガノイドの生成

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

著者スポットライト:ラット腸オルガノイドの生成と操作  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

マウス胎児腸全体培養システム

要約

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使い方

条件を使用して、マウス腸の再生成にパネート細胞の役割を分析するためのプロトコル

マウスモデルにおける腸のティッシュエンジニアリング

腸幹細胞機能をex vivoで研究するためのマウス結腸陰窩の三次元培養

ガットオンチップマイクロ流体デバイスにおけるマイクロ加工ヒト腸絨毛と生活Microbiomeの共培養

胎児腸オルガノイドを用いたインビトロで吸引から破乳移行を再現する

ザ

のためのシステム

腸ホルモン分泌の研究と腸内分泌細胞の生細胞イメージングを対象ネズミ小腸の混合初代培養

ラット腸管オルガノイドの作製と操作