Name: real-time measurement of epithelial barrier permeability in human intestinal organoids
Uploaded: 2017-12-18T15:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

著者は、organoid マイクロインジェクションの多くの有益な議論の夫妻ステファニー スポーンとバーゼルの Abuaita を感謝したいです。JRS は、腸管幹細胞コンソーシアム (U01DK103141)、国立研究所糖尿病・消化器・腎臓病 (NIDDK) と国立研究所のアレルギー ・感染症 (NIAID) によって資金を供給する共同研究プロジェクトによってサポートされます。JRS と VBY NIAID の小説、腸疾患 (NAMSED) コンソーシアム (U19AI116482) のための代替モデル システムによってサポートされます。DRH のサポート臨床および健康のためミシガン研究所国立研究所のアレルギーおよび感染症 (NIAID, T32AI007528) と臨床トランスレーショナル科学賞から微生物病因メカニズムの訓練グラント研究 (UL1TR000433)。
Https://github.com/hilldr/HIO_microinjection では、完全なデータ ファイルやこの原稿で使用されるデータ解析コードを楽しめます。

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	<li>Standring, S., Borley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gray's anatomy: The anatomical basis of clinical practice. , (2008).</li><li>Buckley, A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+tight+junction+barrier+regulation+and+mucosal+disease.">Cell biology of tight junction barrier regulation and mucosal disease.</a> Cold Spring Harb Perspect Biol. , (2017).</li><li>Clayburgh, D. R., Shen, L., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porous+defense:+The+leaky+epithelial+barrier+in+intestinal+disease.">A porous defense: The leaky epithelial barrier in intestinal disease.</a> Lab Invest. 84, 282-291 (2004).</li><li>Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+mucosal+barrier+function+in+health+and+disease.">Intestinal mucosal barrier function in health and disease.</a> Nat Rev Immunol. 9, 799-809 (2009).</li><li>Bischoff, S. C., 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=Intestinal+permeability-a+new+target+for+disease+prevention+and+therapy.">Intestinal permeability-a new target for disease prevention and therapy.</a> BMC Gastroenterol. 14, 189 (2014).</li><li>Odenwald, M. A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+epithelial+barrier:+A+therapeutic+target?.">The intestinal epithelial barrier: A therapeutic target?.</a> Nat Rev Gastroenterol Hepatol. 14, 9-21 (2017).</li><li>Krugliak, P., Hollander, D., Schlaepfer, C. C., Nguyen, H., Ma, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+sites+of+mannitol+permeability+of+small+and+large+intestine+in+the+rat.">Mechanisms and sites of mannitol permeability of small and large intestine in the rat.</a> Dig Dis Sci. 39, 796-801 (1994).</li><li>Johnston, S. D., Smye, M., Watson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+permeability+tests+in+coeliac+disease.">Intestinal permeability tests in coeliac disease.</a> Clin Lab. 47, 143-150 (2001).</li><li>Salles Teixeira, T. F., 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=Intestinal+permeability+measurements:+General+aspects+and+possible+pitfalls.">Intestinal permeability measurements: General aspects and possible pitfalls.</a> Nutr Hosp. 29, 269-281 (2014).</li><li>Wang, L., 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=Methods+to+determine+intestinal+permeability+and+bacterial+translocation+during+liver+disease.">Methods to determine intestinal permeability and bacterial translocation during liver disease.</a> J Immunol Methods. 421, 44-53 (2015).</li><li>Donato, R. P., 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=Studying+permeability+in+a+commonly+used+epithelial+cell+line:+T84+intestinal+epithelial+cells.">Studying permeability in a commonly used epithelial cell line: T84 intestinal epithelial cells.</a> Methods Mol Biol. 763, 115-137 (2011).</li><li>Balimane, P. V., Chong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture-based+models+for+intestinal+permeability:+A+critique.">Cell culture-based models for intestinal permeability: A critique.</a> Drug Discov Today. 10, 335-343 (2005).</li><li>Vidyasagar, S., MacGregor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ussing+chamber+technique+to+measure+intestinal+epithelial+permeability.">Ussing chamber technique to measure intestinal epithelial permeability.</a> Methods Mol Biol. 1422, 49-61 (2016).</li><li>Herrmann, J. R., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Ussing's+chambers:+contemporary+thoughts+on+integration+of+transepithelial+transport.">Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport.</a> Am J Physiol Cell Physiol. 310, C423-C431 (2016).</li><li>Sato, T., 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=Single+lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165, 1586-1597 (2016).</li><li>Drost, J., 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=Organoid+culture+systems+for+prostate+epithelial+and+cancer+tissue.">Organoid culture systems for prostate epithelial and cancer tissue.</a> Nat Protoc. 11, 347-358 (2016).</li><li>Rookmaaker, M. B., Schutgens, F., Verhaar, M. C., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+human+adult+stem+or+progenitor+cell+organoids.">Development and application of human adult stem or progenitor cell organoids.</a> Nat Rev Nephrol. 11, 546-554 (2015).</li><li>Spence, J. R., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470, 105-109 (2011).</li><li>Aurora, M., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPSC-derived+lung+and+intestinal+organoids+as+models+of+human+fetal+tissue.">HPSC-derived lung and intestinal organoids as models of human fetal tissue.</a> Dev Biol. 420, 230-238 (2016).</li><li>Dedhia, P. H., Bertaux-Skeirik, N., Zavros, Y., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+gastrointestinal+development+and+disease.">Organoid models of human gastrointestinal development and disease.</a> Gastroenterology. 150, 1098-1112 (2016).</li><li>Dye, B. R., 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=In+vitro+generation+of+human+pluripotent+stem+cell+derived+lung+organoids.">In vitro generation of human pluripotent stem cell derived lung organoids.</a> Elife. 4, (2015).</li><li>Dye, B. R., Miller, A. J., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+grow+a+lung:+Applying+principles+of+developmental+biology+to+generate+lung+lineages+from+human+pluripotent+stem+cells.">How to grow a lung: Applying principles of developmental biology to generate lung lineages from human pluripotent stem cells.</a> Curr Pathobiol Rep. 4, 47-57 (2016).</li><li>McCracken, K. W., Howell, J. C., Wells, J. M., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+human+intestinal+tissue+from+pluripotent+stem+cells+in+vitro.">Generating human intestinal tissue from pluripotent stem cells in vitro.</a> Nat Protoc. 6, 1920-1928 (2011).</li><li>Leslie, J. L., 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=Persistence+and+toxin+production+by+clostridium+difficile+within+human+intestinal+organoids+result+in+disruption+of+epithelial+paracellular+barrier+function.">Persistence and toxin production by clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.</a> Infect Immun. 83, 138-145 (2015).</li><li>Leslie, J. L., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole+new+ball+game:+Stem+cell-derived+epithelia+in+the+study+of+host-microbe+interactions.">A whole new ball game: Stem cell-derived epithelia in the study of host-microbe interactions.</a> Anaerobe. 37, 25-28 (2016).</li><li>Zachos, N. C., 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=Human+enteroids/Colonoids+and+intestinal+organoids+functionally+recapitulate+normal+intestinal+physiology+and+pathophysiology.">Human enteroids/Colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.</a> J Biol Chem. 291, 3759-3766 (2016).</li><li>Hill, D. R., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+organoids:+Understanding+the+molecular+basis+of+the+host-microbe+interface.">Gastrointestinal organoids: Understanding the molecular basis of the host-microbe interface.</a> Cell Mol Gastroenterol Hepatol. 3, 138-149 (2017).</li><li>Miyoshi, H., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+and+genetic+modification+of+gastrointestinal+stem+cells+in+spheroid+culture.">In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.</a> Nat Protoc. 8, 2471-2482 (2013).</li><li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett's epithelium.</a> Gastroenterology. 141, 1762-1772 (2011).</li><li>Engevik, M. A., 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=Loss+of+nHE3+alters+gut+microbiota+composition+and+influences+bacteroides+thetaiotaomicron+growth.">Loss of nHE3 alters gut microbiota composition and influences bacteroides thetaiotaomicron growth.</a> Am J Physiol Gastrointest Liver Physiol. 305, G697-G711 (2013).</li><li>Forbester, J. L., 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=Interaction+of+salmonella+enterica+serovar+typhimurium+with+intestinal+organoids+derived+from+human+induced+pluripotent+stem+cells.">Interaction of salmonella enterica serovar typhimurium with intestinal organoids derived from human induced pluripotent stem cells.</a> Infect Immun. 83, 2926-2934 (2015).</li><li>Engevik, M. A., 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=Human+clostridium+difficile+infection:+Inhibition+of+nHE3+and+microbiota+profile.">Human clostridium difficile infection: Inhibition of nHE3 and microbiota profile.</a> Am J Physiol Gastrointest Liver Physiol. 308, G497-G509 (2015).</li><li>Bancroft, J., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histological techniques. , (2008).</li><li>Kennedy, S., Oswald, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR troubleshooting and optimization: The essential guide. , (2011).</li><li>Kurien, B., Scofield, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Western blotting: Methods and protocols. , (2015).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+image+to+imageJ:+25+years+of+image+analysis.">NIH image to imageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Schindelin, J., 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9, 676-682 (2012).</li><li>Winston, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microsoft excel data analysis and business modeling. , (2016).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+language+and+environment+for+statistical+computing.">A language and environment for statistical computing.</a> R Foundation for Statistical Computing. , (2017).</li><li>Rosenbaum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic pharmacokinetics and pharmacodynamics: An integrated textbook and computer simulations. , (2016).</li><li>Selden, L. A., Estes, J. E., Gershman, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tightly+bound+divalent+cation+regulates+actin+polymerization.">The tightly bound divalent cation regulates actin polymerization.</a> Biochem Biophys Res Commun. 116, 478-485 (1983).</li><li>Vojdani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+the+assessment+of+intestinal+permeability,+size+matters.">For the assessment of intestinal permeability, size matters.</a> Altern Ther Health Med. 19, 12-24 (2013).</li><li>Karve, S. S., Pradhan, S., Ward, D. V., Weiss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+organoids+model+human+responses+to+infection+by+commensal+and+shiga+toxin+producing+escherichia+coli.">Intestinal organoids model human responses to infection by commensal and shiga toxin producing escherichia coli.</a> PLoS One. 12, e0178966 (2017).</li></ol>

著者を開示するない競合する金銭的な利益やその他の利害の関係があります。

このプロトコルは、リアルタイムで hPSC 派生 HIOs と組織由来腸オルガノイドのマイクロインジェクションと上皮関門の透過性の測定のための一般的な方法を確立します。これらのメソッドを使用して生成されたデータの分析と解釈へのアプローチもわかってきました。腸オルガノイドの成長の採用モデル システムを与えられた16,20,21,28と生理関連として腸管バリア透過性, 機能の長年の興味結果3,4,5,6、我々 はこの分野で働く人は適用し、これらのメソッドに基づいて構築することができることを見込んでいます。
この手法の適用に不可欠ないくつかの手順があります。高品質 hPSC - または派生 HIO 組織はマイクロインジェクションの広範な実験の前にすべき組織にアクセスできます。HIO マクロ組織は、組織アイデンティティおよび細胞形態が再現性の高い、この HIOs24を生成する確立された方法論を活用した異種サイズや形のバリエーションを持つ可能性があります。単一の半透明な内腔から成ると直径約 1 mm の球形 HIOs はマイクロインジェクションとリアルタイムで内腔の蛍光の測定に最適です。いくつかのケースで、HIO の崩壊や注入材の明らかな漏れのインジェクションは失敗します。失敗した HIOs は、標準的なマイクロ ピペットを使用してユーザーの裁量でよく文化から削除できます。マイクロインジェクションとイメージングの HIOs を選択するときは、イメージング プラットフォームで使用可能な対物レンズを検討してください。一般に、2-4 倍の対物レンズは完全な HIO 蛍光信号をキャプチャするために理想的な対物レンズ 10 倍は低消費電力のレンズが利用できない場合、または使用可能な HIOs が使用できますが &#60; に直径 1 mm。イメージング ソフトウェア定義されたポイントで蛍光画像の自動キャプチャ時間をかけて考慮しなければなりません。
このプロトコルのいくつかの変更は、実験の条件に適するために可能です。障壁関数テストの結果が使用43化合物の分子の大きさに依存して可能性がありますなど、さまざまな分子量デキストラン製剤をテストする適切なことがあります。また、組織25の全体的構造健全性の指標としての蛍光イメージングに加え明視野イメージングを実行可能性があります。生きたバクテリア25,28,31,32,33,44のマイクロインジェクションを実行すると、ペニシリンを追加する必要があり、ストレプトマイシンまたは前または後マイクロインジェクション HIO 文化メディアにゲンタマイシン。細菌培養懸濁液充填時、マイクロキャピ ラリーの外側が汚染になるし、これは HIO メディアに転送することがあります。また、マイクロインジェクションは HIOs 媒体なしで、細胞外基質 (例えば、マトリゲル) で中断、マイクロインジェクションが完了した後にメディアを追加することで実行できます。これは、細胞外マトリックスと、HIO の外部面への汚染を制限があります。微生物の増殖の試金を計画するときは、減速または microinjected の生物の成長を防止するを避けるために 1-2 時間後メディアで抗生物質を削除する必要があります。
最後に、撮影した画像をこのプロトコル蛍光データを収集するために記載されている手順を適用できることを指摘することが重要ですすべての研究者が体外イメージングに適した顕微鏡機器へアクセスできることを認識し、で自動化された画像のキャプチャや環境制御なし標準 epifluorescent 顕微鏡を用いた固定の縦長。このアプローチの例がレスリーと黄らレポート25、腸オルガノイドの hPSC から派生したと Karve と Pradanら c. ディフィシル毒素活性を調べた44、ライブ大腸菌と microinjected と同様 hPSC 由来腸 organoids 上皮関門の透過性を調べた。大きい変化と蛍光信号の正規化の難しさの画像装置の手動操作があります。HIOs を注入するデキストラン投与により手動イメージングを実行する固定倍率、蛍光励起強度および蛍光強度測定の歪みを防ぐため実験全体の露光時間を維持するために不可欠です。

腸上皮は内腔間の他の粒子の非特異的な拡散を介した exchange を最小限に抑えながら栄養素、H2O、イオン、および廃棄物の方向の輸送を仲介する選択的なバリアを形成し、間葉系組織または血液供給1,2。腸管の上皮バリアの非特異的な透磁率は長く健康と病気3,4,5,6の割合を反映する重要な機能パラメーターを考慮されました。傍細胞スペースを介して上皮小分子の拡散。上皮関門の透過性の測定を行なえる動物モデル7と消化管とその後のコレクションで特定のトランスポーターの無い、ラクツロースの摂取を介して人間の患者8ラクツロース末梢血中濃度の測定。蛍光に分類された炭水化物などバリア機能の代替摂取マーカー使用可能な9,10しています。このアプローチは、腸管上皮細胞文化 Transwell サポート11より実験的に制御できますが、 in vivoの貧しい予言者として批判されても単純化されたアプローチの栽培に適応されています。透水性上皮分化のサブタイプと組織構造12の不在のため。チャンバーを使用してもう一つのアプローチを表し、腸粘膜ex vivo13上皮バリア機能の測定を可能にします。この技法の応用頻繁、組織の可用性と条件13,14によって制限されます。従って生理学的な関連性と再現性とスループットのバランスを調整する新しい方法が必要です。
器官形成の in vitroにおける最近の動向は、複雑な組織15,16,17 のダイナミクスの概説のため高度なプラットフォームとして 3次元組織培養モデル システムの採用につながっています。 ,18,19,20,21,22,23。特に、ひと多能性幹細胞 (hPSC) に由来ひと腸 organoids (HIOs)19,24が宿主と細菌の相互作用の研究の再現性と実験的に解き得るモデル システムとして浮上していると上皮バリア ダイナミクス25,26,27,28。同様に、ヒト組織由来 organoids (として知られている enteroids) は単純な生検の手順から派生できるし、人間の生理と疾患15,29,30の勉強を扱いやすいシステムとして使用することができます。人間の腸オルガノイドのマイクロインジェクション実験化合物25を配信できますまたは微生物25,31,32,33頂端に上皮をライブorganoid 内腔の表面。レスリーと黄ら25最近 HIOs かに (FITC) デキストラン細菌毒素への暴露を次のラベルの microinjected の関門の透過性を測定するこの手法に適応。
このプロトコルは、hPSC 由来 HIOs と蛍光顕微鏡を用いた組織由来 HIOs 上皮関門の透過性の測定のためのガイドとして使用されます。マイナーな変更と実験的化合物と HIOs のマイクロインジェクションの一般的なプライマーとしても使えます。ユーザーは、マイクロインジェクション後追加または代替のダウン ストリーム アプリケーションでこのプロトコルを補う可能性があります。

<table><tbody><tr><td>EGTA 0.5 M sterile (pH 8.0)</td><td>Bioworld</td><td>405200081</td><td></td></tr><tr><td>Cell matrix solution (Matrigel)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Deltavision RT live cell imaging system</td><td>GE Life Sciences</td><td>29065728</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/deltavision/</td></tr><tr><td>Camera</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>softWoRx Imaging software</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Biosafety cabinet</td><td>Labconco</td><td>Cell Logic+</td><td>http://www.labconco.com/product/purifier-cell-logic-class-ii-type-a2-biosafety-cabinets-2/4262</td></tr><tr><td>1X PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Life Technologies</td><td>12634-010</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>B27 supplement (50X)</td><td>Life Technologies</td><td>17504044</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>L-glutamine (100X)</td><td>Life Technologies</td><td>25030-081</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>HEPES buffer</td><td>Life Technologies</td><td>15630080</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Manipulator</td><td>Narshge</td><td>UM-3C</td><td></td></tr><tr><td>Micromanipulator</td><td>Narshge</td><td>UM-1PF</td><td></td></tr><tr><td>Pipette Holder</td><td>Narshge</td><td>UP-1</td><td>Alternate to Xenoworks pipette holder</td></tr><tr><td>Magnetic stand</td><td>Narshge</td><td>GJ-1</td><td></td></tr><tr><td>Dissecting scope</td><td>Olympus</td><td>SX61</td><td>Recommended scope, although other models are likely compatible</td></tr><tr><td>Olympus IX71 Fluorescent microscope</td><td>Olympus</td><td>IX71</td><td>Included with Deltavision system</td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Recombinant &lt;em&gt;C. difficile&lt;/em&gt; Toxin A/TcdA Protein</td><td>R&amp;amp;D Systems</td><td>8619-GT-020</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>R-spondin 1</td><td>R&amp;amp;D Systems</td><td>4645-RS</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>FITC-dextran (4 kDa)</td><td>Sigma-Aldrich</td><td>46944</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Nunc Lab-Tek II Chamber Slides</td><td>ThermoFisher Scientific</td><td>154526PK</td><td></td></tr><tr><td>Glass filaments</td><td>WPI</td><td>TW100F-4</td><td></td></tr><tr><td>Micropipette holder</td><td>Xenoworks</td><td>BR-MH2</td><td>Preferred device</td></tr><tr><td>Analog Tubing kit</td><td>Xenoworks</td><td>BR-AT</td><td></td></tr><tr><td>1/16 in clear ferrule</td><td>Xenoworks</td><td>V001104</td><td></td></tr><tr><td>1-1.2 mm O-ring</td><td>Xenoworks</td><td>V300450</td><td></td></tr></tbody></table>

real-time measurement of epithelial barrier permeability in human intestinal organoids

腸粘膜の高度な体外モデル腸組織生検により得られたまたは、分化多能性幹細胞から生成される 3 D の文化の進歩をもたらしました。これらの新興国のモデル システムを活用ツールと 2次元培養系や動物のための技術の適応が必要になります。ここでは、リアルタイムで人間の腸 organoids 上皮関門の透過性を測定するための手法について述べる。これは、蛍光標識デキストランと epifluorescent フィルター装備倒立顕微鏡のイメージングのインジェクションで。腸オルガノイドの関門の透過性のリアルタイム計測を容易に人間の腸上皮組織の高分解能時系列データの生成この手法は固定 timepoint アプローチをイメージングにも適用できます。このプロトコルは生きている微生物や毒素、細菌の製品薬理学的薬剤への露出に続く上皮関門の透過性の測定のため容易に適応可能であります。マイナーな変更でこのプロトコルは腸オルガノイドのマイクロインジェクションの一般的な入門としても利用できます、ユーザー可能性があります次のマイクロインジェクション追加または代替のダウン ストリーム アプリケーションでこのプロトコルを補完するを選択します。

HIOs ひと多能性幹細胞から分化した培養細胞マトリックス ソリューションとして以前に説明した19,24.文化の 4 週間後、HIOs はマイクロインジェクションように十分に拡大していた。HIOs は、PBS またはクロストリジウム ・ ディフィシル毒素 TcdA を含む PBS で中断 4 kDa FITC 結合デキストランと microinjected いた。C. ディフィシルは HIOs25で毒素を介する上皮毒性を示す日和見消化管病原体です。肯定的な制御としてグリコールエーテルジアミン四酢酸は、PBS と FITC デキストラン注入 HIOs のサブセットの HIO 文化メディアに追加されました。グリコールエーテルジアミン四酢酸はアクチン細胞骨格42の急速な解消重合を引き起こすカルシウムのキレート剤です。FITC の蛍光は制御環境チャンバ ライブ イメージング顕微鏡上でリアルタイムに監視され、10 分間隔で撮影します。
画像データの非定型分析では、FITC の蛍光 (図 5) の保持の相当な相違を明らかにしました。PBS の注入 HIOs 保持ほぼすべてtでの蛍光信号のグリコールエーテルジアミン四酢酸で処理の TcdA を注入したまた HIOs によって 8 時間後 (図蛍光強度の相当な減少を展示したしかし 0 を =5 a). イメージ投射データの各実験条件 (図 5 b) に時間をかけて蛍光強度の相対的な変化を表す高解像度データセットを生成するすべての時点ですべて HIOs の定量を行った。各治療群 (表 1) の FITC の平均除去時間 (t1/2 ) を計算してt t 1/2グループ間の違いを比較することによって評価した上皮の透過性の違い学生のtを使用して-をテストします。制御処理 HIOs 保持 16 時間以上の FITC の蛍光信号の大半 (t1/2 = 17 ± 0.3 h)。グリコールエーテルジアミン四酢酸による治療が相対 HIOs FITC デキストラン除去時間を大幅に削減 (t1/2 = 2.6 ± 0.2 h;P = 10-8x 1.3)。以前に公開した結果25に一貫した、TcdA の大幅増加コントロール治療を基準にして上皮バリア透過性 (t1/2 = 7.6 ± 0.6 h;P = 10-4x 5.4)。したがって、外部 (グリコールエーテルジアミン四酢酸) と microinjected (TcdA) の化合物は、HIOs 上皮関門の透過性の重要な変化を誘発することができます。このアプローチを使用して上皮バリア機能に及ぼす薬理学的薬剤、代謝物質、細菌の製品、サイトカイン、成長因子および他の化合物の広い範囲を評価することがありますが示唆されました。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>治療</td>
			<td>半減期 (h)</td>
			<td>SEM (h)</td>
			<td>下限 95年 %ci (h)</td>
			<td>上 95年 %ci (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>コントロール</td>
			<td>17.11</td>
			<td>0.3</td>
			<td>16.45</td>
			<td>1,778万</td>
			<td>11</td>
		</tr>
		<tr>
			<td>グリコールエーテルジアミン四酢酸</td>
			<td>2.58</td>
			<td>0.19</td>
			<td>1.78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TcdA</td>
			<td>7.56</td>
			<td>0.63</td>
			<td>5.93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
表 1: 除去時間を意味する (t1/2) HIOs の FITC デキストラン グリコールエーテルジアミン四酢酸または TcdA で治療したため。単位は、時間後のインジェクションです。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
図 1: マイクロインジェクターと HIO のインジェクション用マイクロマニピュレーターの基本的なレイアウトします。この画像は、HIOs のマイクロインジェクションを実行するために使用される機器の完全な補完を示します。主要なコンポーネントのラベルが付いています、材料表に注文情報を見つけることができます。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
図 2: マイクロ ピペット引き手。加熱コイルhc、上側クランプc1、下部クランプ (c2)、銅の引き手の腕 (pa)、熱の選択の切り替え (ht) は、矢印によって識別されます。熱 1 とプルの正しい設定は赤のテキストで表示されます。はめ込みは、暖房の準備が正しくマウントされているガラス フィラメントを示しています。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
図 3: FITC デキストラン注入組織由来の HIO の代表的なイメージです。(A) イメージは HIO とマイクロインジェクション挿入直前ガラス マイクロキャピ ラリーの位置を示します。(B) FITC デキストランの顕微注入後の組織由来、人間の腸オルガノイドの明視野イメージ。蛍光信号が特定のフィルターのセットの使用がなくても明らかであることに注意してください。この配色機能は、マイクロインジェクション精度を支援します。3 倍の倍率<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
図 4: 画像処理ワークフローのライブします。(A) スクリーン ショット顕微鏡パラメーターを設定、スライド上の HIOs を見つけるし、イメージを作成する各の HIO のための段階の位置を保存するための手順を示します。それぞれの位置で定期的に FITC チャンネルをキャプチャするため (B) 事前イメージング設定が、DV フォーマット データの A. (C) イメージ作成後輸出時点あたり HIO 1 つの TIFF イメージを持つ TIFF イメージとしてのファイルを指定します。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">この図の拡大版を表示するのには、ここをクリックしてください。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
図 5: 代表結果。(A) 幹細胞由来 2 mg/ml の FITC デキストラン microinjected 人間の腸 organoids (HIO) 20 時間 (4 kDa) のイメージを作成します。HIOs いた PBS (コントロール) とクロストリジウム ・ ディフィシル毒素 TcdA microinjected も (12.8 ng/μ L) か、外部の文化メディアに追加されますグリコールエーテルジアミン四酢酸 2 mM。4 倍の倍率。HIOs で時間の経過と共に平均正規化 FITC 強度 (B) プロットは PBS (コントロール)、TcdA、またはグリコールエーテルジアミン四酢酸で処理。誤差範囲を表す S.E.M. とn = 11 HIOs (コントロール)、3 HIOs (グリコールエーテルジアミン四酢酸)、6 HIOs (TcdA)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

このプロトコルは、蛍光顕微鏡を用いたヒトの腸オルガノイドのリアルタイム次の薬理学的治療における上皮バリア透過性の測定について説明し、細胞顕微鏡検査をライブします。

人間の腸 Organoids 上皮関門の透過性のリアルタイム計測

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

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14.1K ビュー.  University of Michigan. このプロトコルは、蛍光顕微鏡を用いたヒトの腸オルガノイドのリアルタイム次の薬理学的治療における上皮バリア透過性の測定について説明し、細胞顕微鏡検査をライブします。

ビデオ: Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

医学

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

ヒトオルガノイドと臓器オンチップ技術を組み合わせて腸領域特異的機能をモデル化

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

生物工学

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

腸透過性と上皮細胞流れ性能を評価する生体内顕微鏡ベースのアプローチ

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

免疫・感染学

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

根尖腸管モデルにおけるルシファーイエローを用いた腸透過性の決定

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

生きているオルガノイドにおける腸のバリア破壊の調査

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

胎児組織由来エンテロイドの上皮透過性の試験

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

生物学

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

オルガノイド由来の上皮単分子膜:腸管バリア機能のための臨床的に関連するインビトロモデル

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

ウシ腸オルガノイドからの2D単層培養物の開発

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

著者スポットライト:病原体相互作用研究のためのウシオルガノイドからの2D腸単層モデルの開発と特性評価

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

小腸および大腸単分子膜界面を用いたウシオルガノイド技術の進歩

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...