这项工作得到了美国国立卫生研究院拨款R01DK120986（KPM）的支持。

肠道炎症对已建立的小鼠结肠样的上皮影响

<ol>
	<li>Martini, E., Krug, S. M., Siegmund, B., Neurath, M. F., Becker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mend+your+fences:+The+epithelial+barrier+and+its+relationship+with+mucosal+immunity+in+inflammatory+bowel+disease.">Mend your fences: The epithelial barrier and its relationship with mucosal immunity in inflammatory bowel disease.</a> Cellular and Molecular Gastroenterology and Hepatology. 4 (1), 33-46 (2017).</li><li>McGuckin, M. A., Rajaraman, E., Simms, L. A., Florin, T. H. J., Radford-Smith, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+barrier+dysfunction+in+inflammatory+bowel+diseases.">Intestinal barrier dysfunction in inflammatory bowel diseases.</a> Inflammatory Bowel Diseases. 15 (1), 100-113 (2009).</li><li>Joshi, A., Soni, A., Acharya, 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+models+and+ex+vivo+systems+used+in+inflammatory+bowel+disease.">In vitro models and ex vivo systems used in inflammatory bowel disease.</a> In Vitro Models. 1 (3), 213-227 (2022).</li><li>Goyal, N., Rana, A., Ahlawat, A., Bijjem, K. R., Kumar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+inflammatory+bowel+disease:+A+review.">Animal models of inflammatory bowel disease: A review.</a> Inflammopharmacology. 22 (4), 219-233 (2014).</li><li>Ostanin, D. V., 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=T+cell+transfer+model+of+chronic+colitis:+Concepts,+considerations,+and+tricks+of+the+trade.">T cell transfer model of chronic colitis: Concepts, considerations, and tricks of the trade.</a> American Journal of Physiology. Gastrointestinal and Liver Physiology. 296 (2), 135-146 (2009).</li><li>Bhinder, 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=The+Citrobacter+rodentium+mouse+model:+Studying+pathogen+and+host+contributions+to+infectious+colitis.">The Citrobacter rodentium mouse model: Studying pathogen and host contributions to infectious colitis.</a> Journal of Visualized Experiments. (72), e50222 (2013).</li><li>Bhan, A. K., Mizoguchi, E., Smith, R. N., Mizoguchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colitis+in+transgenic+and+knockout+animals+as+models+of+human+inflammatory+bowel+disease.">Colitis in transgenic and knockout animals as models of human inflammatory bowel disease.</a> Immunological Reviews. 169, 195-207 (1999).</li><li>Okayasu, I., 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=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Dotti, I., Salas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+use+of+human+stem+cell-derived+intestinal+organoids+to+study+inflammatory+bowel+diseases.">Potential use of human stem cell-derived intestinal organoids to study inflammatory bowel diseases.</a> Inflammatory Bowel Diseases. 24 (12), 2501-2509 (2018).</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 (7244), 262-265 (2009).</li><li>Santos, A. J. M., Lo, Y. H., Mah, A. T., Kuo, C. J. <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+stem+cell+niche:+Homeostasis+and+adaptations.">The intestinal stem cell niche: Homeostasis and adaptations.</a> Trends in Cell Biology. 28 (12), 1062-1078 (2018).</li><li>Yin, X., 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=Niche-independent+high-purity+cultures+of+Lgr5++intestinal+stem+cells+and+their+progeny.">Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.</a> Nature Methods. 11 (1), 106-112 (2014).</li><li>Wilson, S. S., 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=Optimized+culture+conditions+for+improved+growth+and+functional+differentiation+of+mouse+and+human+colon+organoids.">Optimized culture conditions for improved growth and functional differentiation of mouse and human colon organoids.</a> Frontiers in Immunology. 11, 547102 (2021).</li><li>Kim, J. J., Shajib, M. S., Manocha, M. M., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+intestinal+inflammation+in+DSS-induced+model+of+IBD.">Investigating intestinal inflammation in DSS-induced model of IBD.</a> Journal of Visualized Experiments. (60), e3678 (2012).</li><li>Cunningham, K. E., 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=Peroxisome+proliferator-activated+receptor-&#947;+coactivator+1-&#945;+(PGC1&#945;)+protects+against+experimental+murine+colitis.">Peroxisome proliferator-activated receptor-&#947; coactivator 1-&#945; (PGC1&#945;) protects against experimental murine colitis.</a> Journal of Biological Chemistry. 291 (19), 10184-10200 (2016).</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> Nature Protocols. 8 (12), 2471-2481 (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 (5), 1762-1772 (2011).</li><li>Julian, M. W., 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+epithelium+is+more+susceptible+to+cytopathic+injury+and+altered+permeability+than+the+lung+epithelium+in+the+context+of+acute+sepsis.">Intestinal epithelium is more susceptible to cytopathic injury and altered permeability than the lung epithelium in the context of acute sepsis.</a> International Journal of Experimental Pathology. 92 (5), 366-376 (2011).</li><li>Haberman, 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=Ulcerative+colitis+mucosal+transcriptomes+reveal+mitochondriopathy+and+personalized+mechanisms+underlying+disease+severity+and+treatment+response.">Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response.</a> Nature Communications. 10, 38 (2019).</li><li>Yang, S., 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=Mitochondrial+transcription+factor+A+plays+opposite+roles+in+the+initiation+and+progression+of+colitis-associated+cancer.">Mitochondrial transcription factor A plays opposite roles in the initiation and progression of colitis-associated cancer.</a> Cancer Communications. 41 (8), 695-714 (2021).</li><li>Maidji, E., Somsouk, M., Rivera, J. M., Hunt, P. W., Stoddart, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+CMV+in+the+gut+of+HIV-infected+individuals+and+epithelial+barrier+dysfunction.">Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction.</a> PLoS Pathogen. 13, 1006202 (2017).</li><li>Noel, 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=A+primary+human+macrophage-enteroid+co-culture+model+to+investigate+mucosal+gut+physiology+and+host-pathogen+interactions.">A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions.</a> Scientific Reports. 7, 452-470 (2017).</li><li>Grondin, J., Kwon, Y., Far, P., Haq, S., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+Intestinal+mucosal+defense+and+inflammation:+Learning+from+clinical+and+experimental+studies.">Mucins in Intestinal mucosal defense and inflammation: Learning from clinical and experimental studies.</a> Frontiers in Immunology. 11, 2054 (2020).</li><li>Metcalfe, C., Kljavin, N. M., Ybarra, R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5++stem+cells+are+indispensable+for+radiation-induced+intestinal+regeneration.">Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration.</a> Cell Stem Cell. 14 (2), 149-159 (2014).</li><li>Yui, S., 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=YAP/TAZ-Dependent+reprogramming+of+colonic+epithelium+links+ECM+remodeling+to+tissue+regeneration.">YAP/TAZ-Dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration.</a> Cell Stem Cell. 22 (1), 35-49 (2018).</li><li>Komiya, Y., Habas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signal+transduction+pathways.">Wnt signal transduction pathways.</a> Organogenesis. 4 (2), 68-75 (2008).</li></ol>

贡献作者没有什么可透露的。

类器官的发展彻底改变了科学界在 体外 研究器官系统的方式，能够在培养皿中部分概括动物或人类的细胞结构和功能。此外，源自患有疾病的人类的类器官系统为个性化医疗提供了一种有前途的工具，可以指导治疗决策。在这里，我们描述了一种运行良好的加密隔离协议，并介绍了允许在电镀前清理隔离中多余碎屑的关键步骤。此外，我们还解释了一个在这个过程中经常被忽视的关键细?...

炎症性肠病 （IBD） 是一种慢性、缓解和复发性疾病，其特征是炎症发作和临床静止。IBD 的病因是多因素的，但该疾病的关键特征包括屏障功能缺陷和肠上皮通透性增加，此外还有上皮区间内激活的促炎信号级联反应 1,2。几种体外和体内模型已被用于概括IBD期间的上皮反应，包括炎症的细胞培养和小鼠模型3。然而，所有这些系统都有重要的缺点，限制了它们在IBD4期间概括上皮变化的能力。大多数用于研究IBD的细胞系都是转化的，具有形成单层的能力，并且可以分化3种，但本质上的繁殖方式与宿主中未转化的肠上皮细胞不同。几种不同的小鼠炎症模型用于研究IBD，其中一些包括敲除模型，感染模型，化学炎症模型和T细胞转移模型5,6,7,8。虽然每个人都可以研究IBD的某些病因方面，例如遗传易感性，屏障功能障碍，免疫失调和微生物组，但他们研究疾病的多因素性质的能力有限。
肠道类器官，包括类肠类和结肠类，在过去十年中已被建立为一种有用的体外模型，不仅可以研究肠道干细胞的动力学，还可以研究它们在肠道稳态和疾病中的屏障完整性和功能的作用。这些实体为我们理解IBD9的发病机制做出了重大贡献，并为个性化医疗开辟了新的机会。结肠样或干细胞衍生的自组织组织培养物已经从小鼠和人体组织发展而来，其过程允许位于肠隐窝内的干细胞繁殖并无限期维持10。体内干细胞生态位依赖于细胞外因子来支持其生长，特别是典型的Wnt信号传导和骨形态发生蛋白信号通路11。这些因素的添加促进了结肠样细胞的健康和寿命，但也推动培养物走向干细胞样状态，这种状态不反映体内上皮细胞结构，该结构由自我更新和终末分化的细胞组成12,13。虽然肠上皮的功能取决于干细胞区室和分化细胞之间的持续串扰，但在结肠样培养系统中同时具有两者的能力相当有限。尽管存在这些限制，类器官培养系统仍然是研究体外上皮内在特性的金标准。尽管如此，可能需要考虑替代文化策略来回答手头的科学问题。
已经表明，连续7天葡聚糖硫酸钠（DSS）方案的小鼠发生上皮炎症和屏障功能障碍14。此外，线粒体生物发生失败和肠上皮内的代谢重编程，已被证明在人类IBD中很明显，也已捕获在结肠炎15的DSS模型中。然而，我们的初步数据表明，线粒体生物发生失败的特征并未保留在源自DSS处理动物隐窝的结肠中（补充图1）。因此，在检查小鼠肠道炎症期间炎症如何驱动上皮变化时，必须使用替代培养方法。在这里，我们概述了我们开发的一个协议，该协议描述了1）如何从整个结肠组织中分离隐窝以建立鼠结肠样，2）如何最终分化该细胞群以反映 体内的细胞群，以及3）如何在此 体外 模型中诱导炎症。为了研究发炎上皮内的药物相互作用，我们开发了一种方案，从鼠结肠样体中建立2-二脉（2D）单层，可以用炎症介质进行基础治疗，并用药物治疗进行顶端治疗。

<table><tbody><tr><td>0.4-&amp;mu;M transparent transwell, 24-well</td><td>Greiner Bio-one</td><td>662-641</td><td></td></tr><tr><td>15-mL conical tubes</td><td>Thermo Fisher&amp;nbsp;</td><td>12-565-269</td><td></td></tr><tr><td>50-mL conical tubes</td><td>Thermo Fisher&amp;nbsp;</td><td>12-565-271</td><td></td></tr><tr><td>70-&amp;mu;M cell strainer</td><td>VWR</td><td>76327-100</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Invitrogen</td><td>12634-010</td><td>Stock Concentration (1x); Final Concentration (1x)</td></tr><tr><td>B-27 supplement&amp;nbsp;</td><td>Invitrogen</td><td>12587-010</td><td>Stock Concentration (50x); Final Concentration (1x)</td></tr><tr><td>Chopsticks Electrode Set for EVO</td><td>World Precision Instruments</td><td>STX2</td><td></td></tr><tr><td>Corning Matrigel GFR Membrane Mix</td><td>Corning</td><td>354-230</td><td>Stock Concentration (100%); Final Concentration (100%)</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D0632-5G</td><td>Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)</td></tr><tr><td>DMEM high glucose</td><td>Thermo Fisher</td><td>11960-069</td><td>Stock Concentration (1x); Final Concentration (1x)</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline without Calcium and Magnesium</td><td>Gibco&amp;nbsp;</td><td>14190-144</td><td>Stock Concentration (1x); Final Concentration (1x)</td></tr><tr><td>Ethylenediaminetetraacetic acid (ETDA)</td><td>Sigma-Aldrich</td><td>E7889</td><td>Stock Concentration (0.5 M); Final Concentration (30 mM)</td></tr><tr><td>Fetal Bovine Serum</td><td>Bio-Techne</td><td>S11150H</td><td>Stock Concentration (100%); Final Concentration (1%)</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm</td><td>Thermo Fisher&amp;nbsp;</td><td>12-550-15</td><td></td></tr><tr><td>G418</td><td>InvivoGen</td><td>ant-ga-1</td><td>Final Concentration (400 &amp;micro;g/&amp;micro;L)</td></tr><tr><td>Gentamicin Reagent</td><td>Gibco/Fisher</td><td>15750-060</td><td>Stock Concentration (50 mg/mL); Final Concentration (250 &amp;mu;g/mL)</td></tr><tr><td>GlutaMAX-1</td><td>Fisher Scientific</td><td>35050-061</td><td>Stock Concentration (100x); Final Concentration (1x)</td></tr><tr><td>HEPES 1 M</td><td>Gibco</td><td>15630-080</td><td>Stock Concentration (1 M); Final Concentration (10 mM)</td></tr><tr><td>hIFN&amp;gamma;</td><td>R&amp;amp;D Systems</td><td>285-IF</td><td>Stock Concentration (1000 ng/&amp;micro;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td></tr><tr><td>hIL-1&amp;beta;</td><td>R&amp;amp;D Systems</td><td>201-LB</td><td>Stock Concentration (10 ng/&amp;micro;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td></tr><tr><td>hTNF&amp;alpha;</td><td>R&amp;amp;D Systems</td><td>210-TA</td><td>Stock Concentration (10 ng/&amp;micro;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td></tr><tr><td>Hydrogen Peroxide&amp;nbsp;</td><td>Sigma</td><td>H1009</td><td>Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)</td></tr><tr><td>Hygromycin B Gold</td><td>InvivoGen</td><td>ant-hg-1</td><td>Final Concentration (400 &amp;micro;g/&amp;micro;L)</td></tr><tr><td>L-WRN Cell Line</td><td>ATCC</td><td>CRL-3276</td><td></td></tr><tr><td>mEGF</td><td>Novus</td><td>NBP2-35176</td><td>Stock Concentration (0.5 &amp;micro;g/&amp;micro;L); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)</td></tr><tr><td>N-2 supplement</td><td>Invitrogen</td><td>17502-048</td><td>Stock Concentration (100x); Final Concentration (1x)</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma&amp;nbsp;</td><td>A9165-5G</td><td>Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)</td></tr><tr><td>Noggin</td><td>Peprotech</td><td>250-38</td><td>Stock Concentration (0.1 ng/&amp;micro;L); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher</td><td>15140-122</td><td>Stock Concentration (100x); Final Concentration (1x)</td></tr><tr><td>Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>Polystyrene Microplates, 24 well tissue culture treated, sterile</td><td>Greiner Bio-one</td><td>5666-2160</td><td></td></tr><tr><td>R-Spondin</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>Stock Concentration (0.25 &amp;micro;g/&amp;micro;L); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)</td></tr><tr><td>Tryp LE Express</td><td>Thermo Fisher</td><td>12604-013</td><td>Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)</td></tr><tr><td>UltraPure Water&amp;nbsp;</td><td>Invitrogen</td><td>10977-023</td><td>Stock Concentration (1x); Final Concentration (1x)</td></tr><tr><td>Y-27632 dihyddrochloride&amp;nbsp;</td><td>Abcam</td><td>ab120129</td><td>Stock Concentration (10 mM); Final Concentration (10 &amp;micro;M); Solvent (UltraPure Water)</td></tr></tbody></table>

studying the epithelial effects of intestinal inflammation in vitro on established murine colonoids

肠上皮在人体健康中起着至关重要的作用，在宿主和外部环境之间提供了屏障。这种高度动态的细胞层提供了微生物和免疫群体之间的第一道防线，并有助于调节肠道免疫反应。上皮屏障的破坏是炎症性肠病（IBD）的标志，对于治疗靶向具有重要意义。三维结肠培养系统是研究IBD发病机制中肠道干细胞动力学和上皮细胞生理学的非常有用的 体外 模型。理想情况下，从动物发炎的上皮组织中建立结肠样体对于评估遗传和分子对疾病的影响最有益。然而，我们已经表明， 体内 上皮变化不一定保留在由急性炎症小鼠建立的结肠样中。为了解决这一限制，我们开发了一种方案，用通常在IBD期间升高的炎症介质混合物来治疗结肠样。虽然该系统可以普遍应用于各种培养条件，但该方案强调对分化结肠和源自已建立的结肠的二维单层的治疗。在传统培养环境中，结肠富含肠道干细胞，为研究干细胞生态位提供了理想的环境。然而，该系统不允许分析肠道生理学的特征，例如屏障功能。此外，传统的结肠样不能提供研究终末分化上皮细胞对促炎刺激的细胞反应的机会。这里介绍的方法提供了一个替代的实验框架来解决这些限制。二维单层培养系统也为 离体治疗药物筛选提供了机会。这种极化细胞层可以用细胞基底侧的炎症介质处理，并伴有假定的治疗方法，以确定它们在IBD治疗中的效用。

3D肠结肠样培养系统是研究上皮对肠粘膜稳态的内在贡献的宝贵工具。所描述的协议提供了有关如何在8周龄时从C57BL / 6J（WT）小鼠中分离隐窝并建立可用于多个下游应用的长期结肠样培养系统的详细说明。在基底膜基质中分离和电镀隐窝后，当通过明场显微镜观察时，隐窝在结构上显得致密和多细胞。它们可以是球形的，也可以是细长的圆柱形，类似于通常在 体内 肠道结构中观察到的单?...

Watch this Scientific Journal Video about Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids at JoVE.com

作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

我们描述了一个协议，详细说明了小鼠结肠隐窝的分离以开发三维结肠。然后，可以在接受炎症激发或被引导建立上皮单层之前对已建立的结肠样蛋白进行终末分化以反映宿主上皮的细胞组成。

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

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

通过我们的研究，我们的目标是探索如何从传统的类结肠培养系统修饰小鼠结肠，以研究肠道生理学的某些方面及其在发炎疾病状态下的改变。当使用仅反映干细胞生理学的传统鼠结肠样培养系统时，研究炎症性肠病等疾病状态下的屏障功能和肠道生理学是有限的。我们的协议是有利的，因为它为读者提供了多种技术来研究炎症介质如何影响肠上皮，通过最终分化它们或创建2D单层系统。未来，我们的实验室将专注于理解如何改变人类结肠培养系统，以研究肠道生理和疾病的不同方面，特别是炎症性肠病。

studying-epithelial-effects-intestinal-inflammation-vitro-on

Research

JoVE Journal

Biology

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

914 次观看.  UPMC Children’s Hospital of Pittsburgh. 通过我们的研究，我们的目标是探索如何从传统的类结肠培养系统修饰小鼠结肠，以研究肠道生理学的某些方面及其在发炎疾病状态下的改变。当使用仅反映干细胞生理学的传统鼠结肠样培养系统时，研究炎症性肠病等疾病状态下的屏障功能和肠道生理学是有限的。我们的协议是有利的，因为它为读者提供了多种技术来研究炎症介质如何影响肠上皮，通过最终分化它们或创?...

视频: 作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

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.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

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

科研

医学

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

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.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

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 ...

免疫与感染

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.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

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

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

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

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

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

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

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

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.

肠上皮细胞通透性的体外及体内测定方法

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.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

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

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

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 ...

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响

副本

摘要

探索更多视频

此视频中的章节

从整体组织和活检建立人类上皮Enteroids和Colonoids的

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

肠上皮细胞通透性的体外及体内测定方法

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

来自肠道密码的穆林原结肠上皮单层的生成

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

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

副本

摘要

探索更多视频

此视频中的章节

从整体组织和活检建立人类上皮Enteroids和Colonoids的

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

肠上皮细胞通透性的体外及体内测定方法

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

来自肠道密码的穆林原结肠上皮单层的生成

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

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响