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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4-&#956;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&#160;</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;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&#160;</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&#39;s phosphate-buffered saline without Calcium and Magnesium</td>
			<td>Gibco&#160;</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&#160;</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 &#181;g/&#181;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 &#956;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&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Stock Concentration (1000 ng/&#181;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hIL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hTNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</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 &#181;g/&#181;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 &#181;g/&#181;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&#160;</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/&#181;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&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration (0.25 &#181;g/&#181;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&#160;</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Y-27632 dihyddrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td>Stock Concentration (10 mM); Final Concentration (10 &#181;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。

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

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  

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

Through our research, we aim to explore how murine colonoids could be modified from a conventional colonoid culture system, to study certain aspects of intestinal physiology and their alteration in the presence of an inflamed disease state. Studying barrier function and intestinal physiology in disease states like inflammatory bowel disease is limited when using a traditional murine colonoid culture system that reflects only stem cell physiology. Our protocol is advantageous in that it provides the reader with multiple techniques to study how inflammatory mediators affect the intestinal epithelium, by either terminally differentiating them, or creating a 2D monolayer system.In the future, our laboratory will concentrate on comprehending how the human colonoid culture system can be altered to investigate different aspects of intestinal physiology and diseases, particularly inflammatory bowel disease.

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

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。

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

738 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  

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

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

科研

生物学

Intestinal Epithelial Monolayers Derived from Established Murine Colonoids

Begin by preparing all the reagents prior to the experiment and thaw the basement membrane matrix on ice. Then prepare the murine monolayer medium. Dilute the thawed basement membrane one to 20 with the cold murine monolayer medium, and keep it on ice.Place a 0.4 micrometer transparent cell culture insert, using sterile forceps, into a single well of a 24 well tissue culture plate. Grab a corner prong of the insert and transfer it into the well. Repeat until the appropriate number of cell culture inserts are available for culture.To cover the apical surface of each cell culture insert with diluted basement membrane matrix, place the tip of the P 200 pipette in the center of the insert and expel 150 microliters matrix. Then, transfer the plate to a sterile 37 degrees incubator with 5%carbon dioxide, for at least one hour. At the end of the incubation, rotate the 24 well plate at a 45 degree angle.Place a single finger on the corner of the prong to stabilize the insert. Gently place the P 200 pipette tip along the bottom side of the insert and remove the basement matrix. Remove any excess residue by washing each cell culture insert with 150 microliters of sterile DPBS without calcium or magnesium ions and allow them to dry in a biological safety cabinet for a minimum of 10 minutes.Once the inserts are dried, add 400 microliters of the murine monolayer medium to the bottom, and 75 microliters on top of the cell culture insert. Repeat until all the cell culture inserts are immersed. Leave the plate in the biological safety cabinet or the incubator until needed.Then remove the 24 well plate of mature intestinal colonoids from the incubator and place it under the biological safety cabinet. Aspirate the medium using sterile tips and wash each well with one milliliter of sterile DPBS at room temperature. Using sterile tips, remove the DPBS without calcium or magnesium ions.Digest each plug of the basement membrane matrix by adding 500 microliters of enzymatic dissociation reagent in each well to be passaged. To break up the plug, rotate the 24 well plate at a 45 degree angle. Place the tip of the pipette at the edge of the plug and gently dislodge it by pipetting each plug approximately six to 10 times.Place the 24 well plate back in a sterile incubator at 37 degrees with 5%carbon dioxide, for three to four minutes. After incubation, pipette the trypsin up and down five to seven times for each well under a biological safety cabinet. Transfer the dissociated colonoids from each well into a sterile 15 milliliter conical tube and bring up to a volume of 10 milliliters with an ice cold mouse wash medium.Next, centrifuge the partially digested colonoids at 300 G for five minutes at four degrees Celsius in a swinging bucket centrifuge. Under the biological safety cabinet, remove the supernatant from the pellet using a 10 milliliter pipette. Also, remove any remaining medium using a P 200 pipette.Then, re-suspend the colonoid pellet by adding 75 microliters of murine monolayer medium. Dispense 75 microliters of the colonoid suspension to the center of each cell culture insert. Gently pipette the suspension once or twice before adding it to the well to ensure uniform platting.Place the 24 well plate with cell culture inserts on a rotating platform for 10 minutes to allow for uniform dispersion across the cell culture insert, before placing the plate in a sterile 37 degree Celsius in 5%carbon dioxide incubator. At the end of the incubation, dislodged the unattached colonoids fragments by placing the tip alongside the inside of the cell culture and pipetting the medium three to five times with a P 200 pipette. Avoid disrupting the attached intestinal cells.Remove the medium and colonoid mixture immediately. Repeat until all the cell culture inserts have been cleaned. Now add 150 microliters of pre-warmed murine monolayer medium to the apical side of each cell culture insert.Add 400 microliters of warmed murine monolayer medium to each well of a new 24 well plate. Transfer the cell culture insert to the new plate by grabbing its prong using sterile forceps. Change the medium every other day until the desired confluency is attained.The enzymatically disrupted colonoids plated on the cell culture inserts initially appeared in cellular clusters ranging from 15 to 150 cells. On day three, the cells flattened out and slowly covered the cell culture insert, generally reaching confluency. Over the next couple of days, monolayers continued to grow and formed a continuous barrier that can be quantitatively measured.