我々は酸素室を提供するための優れた技術サポートと博士はアントニオ·ディ·サバティーノのためエリカMiletiに感謝します。 資金調達 ：MRへとFondazione Cariploとマリーキュリー国際トレーニングモビリティネットワーク（クロストーク、グラント契約番号：21553から2）による支援へ：この作品は、第7回EUフレームワークプログラム（Dendroworld IBDase、ERC）からの補助金によって支えられてKT。

1。入手とティッシュの準備<ol><li>組織（健康またはIBD粘膜）が手術中に得られる。一度試料を4℃または氷上でペニシリン/ストレプトマイシンを補充したのCa + + / Mg + +を含まフリーHBSSバッファにそれを維持、できるだけ早く研究室に転送を切除されています。 </li></ol> *試料の大きさは、組織の可用性に依存します。得られた組織の一部は、診断のために必要とされておらず、健康でIBDの被験者間で大きく異なることがあります何厳密です。健康な組織は、大腸癌の手術を受けた患者から切除されています。 <ol start="2"><li>そっとティッシュを洗って、すべての腸間膜の材料でそれをきれいに。ペトリ皿（必ずしも漬せず）内にHBSSバッファで組織を維持しながら、滅菌ハサミとピンセットを用いて粘膜から粘膜層を分離する。できるだけ少ないようにピンセットで粘膜表面をタッチします。 </li><li>少なくとも1 cがストライプ状に粘膜をカットメートル幅とできるだけ長く。あなたは食品を切断されたかのように多く、2滅菌メスを使用してください。 </li><li> HBSSに優しくクリーンな粘膜を洗浄し、上向きに直面している頂端側をシャーレの上に広げる。 </li></ol> 2。ティッシュの取り付け<ol><li>新鮮な培地（tisDMEM）を準備。 DMEMはグルタミン（2 mM）をとNaPyr（1mMの）を補充し使用し、FBS - Naは15％を追加（ウシ胎児血清 - 北米）、1％ITS-Xおよび使用時の200 ngの/ mlのEGF。 </li><li> FBS-ナ（金属グリッドが完全に水没されるように約30mlを使用してください）​​と水没金属グリッドの残りの部分と10センチメートルシャーレを埋める。プレートを開いたままにし、そのタップのプラスチック製のシリンダーをサポートしています。 </li></ol> *金属グリッドは、鉄で作られている。メッシュは0.5mmの辺を有する正方形である。 <ol start="3"><li> 10または20μlのピペットを用いて外科的接着剤の3μLを取る。 fは、それを保持しながら、プラスチックシリンダーの境界のいずれかに慎重にそれを適用します鉗子でIRM。 </li><li>静かにできるだけ近いストリップの境界のいずれかに、粘膜の頂端側にシリンダーを配置。 </li><li>しっかりと組織上のシリンダーを接続するために接着剤の時間を与えるために、センターウェル器官培養プレートを準備：よく中央に培地850μL-1ミリリットルを分注し、ウェルプレートの中央の端に金属グリッドをサポートしています。 </li><li>前のように2つの滅菌メスを使用してシリンダーの境界線から余分な組織を切り取る。 </li><li>優しく添付組織とシリンダーを持ち上げ、プレートの中央に金属グリッドに基底側に置く。 </li></ol> 3。刺激<ol><li>お好みの濃度でお好みの刺激を使用してください。 </li><li>ピペットチップでシリンダや粘膜表面を乱すことなく、プラスチックシリンダーの中心に適切な音量を適用します。選択したボリュームが均一にシリンダ内表面を覆っていることを確認してください。でdicatedボリューム： 大型プラスチックシリンダー：200μlの ミディアムプラスチックシリンダー：100μlの 小さなプラスチック製のシリンダー：50μlの</li><li>アップ従来のインキュベーター内で3時間までインキュベートする。 </li></ol> *あなたが長い時点インキュベートしたい場合は、（3.5を参照）を使用すると、刺激のはるかに小さい音量（10〜20μL）を使用していることを確認し、直接、1気圧の圧力では100％O 2雰囲気中で組織を配置。 <ol start="4"><li>頂端面上の媒体の厚い層がガス交換ができない場合、組織が​​2 O十分に取得していないので、組織の先端面からの刺激と培地を取り、新鮮な培地と交換しないでください。 プレートをカバーしています。 </li><li> 1気圧の圧力では100％O 2雰囲気中で組織を置きます。 </li></ol> 4。収穫<ol><li>総培養時間（±2.5時間）の24時間後、注意深く番目からシリンダーを取り外すメス優しくこするグルーオフの助けを借りて、電子ティッシュ、それは希望の解析（RNAの精製および遺伝子発現プロファイリングやタンパク質精製およびウェスタンブロットアッセイのための液体窒素中で免疫組織化学、および/または免疫蛍光アッセイの修正、またはスナップ凍結に応じて保管）。 </li><li>サイトカイン分泌プロファイリングのための基底培地を収穫。ペレット破片〜7分間8,000 rpmで遠心分離し、エッペンドルフチュー​​ブ（あるいはアリコート）で上清を転送し、直ちに-20℃で保存し</li></ol> *あなたが長い上清を維持したい場合には、-80℃で保管し℃まで

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., 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=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., 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=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., 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=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

利害の衝突は宣言されていない。

ヒト腸粘膜の治療が正当に試験することができるれた生理学的に関連するモデルの必要性が強調されている長い。多くの研究者がその目的のためにこれまで使用されるセル5及び6マウスモデルの両方で潜在的なアーティファクトや欠陥を同定した。有望な前臨床データは、多くの場合、取得されていても実際、これらはほとんど重要な臨床的利益のために翻訳しません。 <p...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>10X HBSS</td><td>Eurocalne</td><td>ECM4006XL</td><td></td></tr><tr><td>Peni/Strepto</td><td>Lonza</td><td>DE17602E</td><td></td></tr><tr><td>Gentamycin</td><td>Gibco</td><td>15750-037</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>BE12614</td><td></td></tr><tr><td>FBS-Na</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>100X ITS-X</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>EGF</td><td>Tebu-bio</td><td>100-15</td><td></td></tr><tr><td>L-Glutamine</td><td>Lonza</td><td>BE17605E</td><td></td></tr><tr><td>Non essential aminoacids 100X</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>NaPyr</td><td>Gibco</td><td>11360-039</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Cloning cylinders 6x8 mm</td><td>BellCo</td><td>2090-00608</td><td></td></tr><tr><td>Cloning cylinders 8x8 mm</td><td>BellCo</td><td>2090-0080</td><td></td></tr><tr><td>Cloning cylinders 10x10 mm</td><td>BellCo</td><td>2090-01010</td><td></td></tr><tr><td>Metal grids</td><td>Home made</td><td></td><td></td></tr><tr><td>Centre well organ culture plate</td><td>BD Falcon</td><td>353037</td><td></td></tr><tr><td>Surgical glue (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Oxygen Jars</td><td>Home made</td><td></td><td></td></tr><tr><td>Oxygen tanks</td><td>Air Liquid Sanit&amp;#224;</td><td></td><td></td></tr></tbody></table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

いくつかのモデルは、現在現実的に相互作用の様々な場所を取る複雑な人間の腸内の微小環境をシミュレートするために存在します。誤って食物と一緒に摂取した病原性微生物に対して効果的な免疫応答をマウントすると同時に、彼らは食物抗原に対する全体の免疫応答誘導耐性を形作るように適切な恒常性を直接、これらの相互作用に依存する。 腸の恒常性は粘液のアタッチメント/劣化を規制する微生物（食品関連の有益な細菌株を含む）とホストの間の様々な複雑な相互作用を介しても保存され、上皮バリアによって抗菌ペプチドの産生、および &quot;教育&quot;集団 &#39;ユニーク、腸常駐リンパ系細胞の免疫寛容や免疫表現型を制御する&#39;を上皮細胞。これらの相互作用は、in vitroアッセイを再現するこれまでのところ非常に困難であった培養された細胞系または末梢血単核細胞のいずれかを使用して。また、マウスモデルは、ヒトの腸に関して腸粘膜（粘液層の組織、共生細菌のコミュニティ）の構成成分で大きく異なる。したがって、このような過敏性腸症候群、炎症性腸疾患または結腸直腸癌などの重要なストレス関連または病理学的状態のために診療所に持ち込まれる治療の様々な研究が行うことが困難であった。 これらの問題に対処するために、我々は我々が分極方法で、ホストの細菌叢と免疫応答によりその場コンディショニングで自分を維持する人間の腸粘膜の外植片を刺激することができた新規のシステムを開発しました。偏尖刺激の結果のために非常に重要である免疫応答を誘発した。これを繰り返し、それらが腸エピの先端面をバイパスするときに、同じ刺激​​が全く異なる応答を生成できることが示されている乳房、免疫原性への寛容原性の表現型をスイッチング領域に不要な過剰な炎症を引き起こし、基底外側上皮細胞を刺激または粘膜固有層コンポーネントと直接接触する。 私たちは、接着することで、プロトコルに記載されるように粘膜の頂端面上に区切られた刺激の面積洞窟シリンダーを分極刺激を達成しました。我々は他の人、三つの異なる乳酸菌株の差の影響の中で、検討するために、このモデルを使用していました。我々はこのモデルシステムは、健康と病気の条件でプロバイオティクスの免疫調節特性を評価することは非常に強力であることを示している。

私たちは、組織の健康を維持し、少なくとも24時間、文化の中で、健康でIBD人間の腸粘膜を維持することに成功しました。組織は、従来のインキュベータで24時間（ 図2）が生存しなかったとして、培養時間の大部分（全体の少なくとも85％）は、O 2で行われた場合、以前の観測値1によれば、これは可能であった。また、外植片の異なる応答を刺激（頂端対側底）?...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

私たちは、少なくとも24時間以上の刺激の様々なタイプに対応しての文化やモニタリングにおけるヒト腸粘膜の維持のための新規な方法をご紹介します。我々の方法では、組織の極性が尖経路で生理的刺激を可能にし、維持されている。

人間の腸粘膜の外植片の文化と偏刺激する新規な方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

a-novel-method-for-culture-polarized-stimulation-human-intestinal

Research

JoVE Journal

Biology

13.0K ビュー.  European Institute of Oncology. 私たちは、少なくとも24時間以上の刺激の様々なタイプに対応しての文化やモニタリングにおけるヒト腸粘膜の維持のための新規な方法をご紹介します。我々の方法では、組織の極性が尖経路で生理的刺激を可能にし、維持されている。

ビデオ: A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

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

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

医学

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

ザ<em&gt; ex vivoで</em&gt;コロン器官培養および抗菌生体防御研究におけるその使用

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

病原体、共生生物、およびホストの腸上皮間の相互作用を研究する人間 Colonoid 単分子膜

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

ヒト幹細胞由来腸腸単層におけるM細胞様細胞の誘導分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

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 ...

生物学

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

腸内ビリ上皮によるオルガノイドの3D培養

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

腸オルガノイドモデルを用いたアプリカル特異的相互作用を研究する培養法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

凍結保存された上皮陰窩からの子豚腸3Dオルガノイドの培養と細胞単分子膜の樹立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

確立されたマウスコロノイドに対する腸の炎症の上皮的影響

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

著者スポットライト:確立されたマウスコロノイドに対するIn vitroでの腸の炎症の上皮的影響の研究  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

確立されたマウスコロノイドに対する腸の炎症の上皮効果を in vitro で研究する

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...