Name: an intestinal gut organ culture system for analyzing host-microbiota interactions
Uploaded: 2021-06-30T15:00:00
Description: Watch this Scientific Journal Video about An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions at JoVE.com

我们感谢Yissachar实验室的过去和现在的成员在优化肠道器官培养系统协议方面做出的宝贵贡献。我们感谢耶尔·劳尔对手稿进行批判性编辑。这项工作得到了以色列科学基金会（第3114831号赠款）、以色列科学基金会-博德研究所联合方案（第8165162号赠款）和以色列加斯纳医学研究基金的支持。

本议定书遵循动物福利伦理委员会批准的动物护理准则。
1. 实验准备
<ol><li>肠道器官培养装置的制造（3天）<ol>
<li>使用 3D 打印机，打印可重复使用的塑料模具用于器官培养装置（设备有 6 口井，有 24 个大大小小的孔，以及设备盖盖）（附加 3D 文件）。 注：这些塑料模具可用于制造许多设备。</li>
		<li>将钝端针头（22 G + 18 G）插入设备模具内的适当位置，为一组设备和?...

<ol>
	<li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nature Reviews Immunology. 14 (10), 667-685 (2014).</li><li>Pearce, 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+in+vitro+and+ex+vivo+Models+to+Study+Host-Microbiome+Interactions+and+Acute+Stressors.">Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors.</a> Frontiers in Physiology. 9 (1584), (2018).</li><li>Hooper, L. 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=Molecular+analysis+of+commensal+host-microbial+relationships+in+the+intestine.">Molecular analysis of commensal host-microbial relationships in the intestine.</a> Science. 291 (5505), 881-884 (2001).</li><li>Haller, D., 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=Non-pathogenic+bacteria+elicit+a+differential+cytokine+response+by+intestinal+epithelial+cell/leucocyte+co-cultures.">Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.</a> Gut. 47 (1), 79-87 (2000).</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>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>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. 61 (7), 1007-1015 (2012).</li><li>Gazzaniga, F. 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=Harnessing+Colon+Chip+Technology+to+Identify+Commensal+Bacteria+That+Promote+Host+Tolerance+to+Infection.">Harnessing Colon Chip Technology to Identify Commensal Bacteria That Promote Host Tolerance to Infection.</a> Frontiers in Cellular and Infection Microbiology. 11, 638014 (2021).</li><li>Yissachar, N., 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=An+Intestinal+Organ+Culture+System+Uncovers+a+Role+for+the+Nervous+System+in+Microbe-Immune+Crosstalk.">An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.</a> Cell. 168 (6), 1135-1148 (2017).</li><li>Duscha, 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=Propionic+Acid+Shapes+the+Multiple+Sclerosis+Disease+Course+by+an+Immunomodulatory+Mechanism.">Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.</a> Cell. 180 (6), 1067-1080 (2020).</li><li>Grosheva, 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=High-Throughput+Screen+Identifies+Host+and+Microbiota+Regulators+of+Intestinal+Barrier+Function.">High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.</a> Gastroenterology. 159 (5), 1807-1823 (2020).</li><li>Blaize, J. 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本文描述了Yissachar等人最近开发的一种针对前体内肠道器官培养物的优化协议（已发布9个和未发表的数据）。肠道器官培养系统支持完整肠道片段的多重培养，同时保持发光流动。它提供完全控制内部和外发光环境（包括刺激剂量，暴露时间和流速），并保持天真的肠道组织结构及其细胞复杂性9。
协议中的关键步骤包括以下步骤。...

肠道是一种高度复杂的器官，包含多种细胞类型（上皮细胞、免疫系统细胞、神经元等），组织在特定结构中，允许细胞相互作用和交流，并具有发光量（微生物群、食物等）1.目前，可用于分析宿主-微生物群相互作用的研究工具箱包括体外细胞培养和体内动物模型2。在体内动物模型提供了一个生理组织结构3，但实验控制不力，操纵实验条件的能力有限。另一方面，体外培养系统使用原细胞或细胞系，这些细胞线可以补充微生物4，对实验参数提供严格控制，但缺乏细胞复杂性和组织结构。现代体外测定允许高级使用健康和病理的人体组织样本，如从小鼠或人类来源5，6，和模拟粘膜微环境7的上皮器官。另一个例子是"芯片上的古特"检测，其中包括人类结肠上皮细胞系（Caco2）、细胞外基质和微流体通道，以模拟肠道不变体8的生理状况。然而，尽管体外样本可能先进和创新，它们不能维持正常的组织结构或天真的细胞组成。
为了解决这个问题，Yissachar等人最近开发了一种前体内器官培养系统9（图1），它能保持完整的肠道片段，同时受益于体内和体外模型的优势。这个前体内肠道器官培养系统基于一个定制的文化装置，支持六个结肠组织的多路复用培养物，允许在可比条件下检查实验输入，同时控制系统的输入和输出。最近的研究表明，该系统对于分析肠道对个体肠道细菌的反应很有价值，9个，全人类微生物群样本10个，微生物代谢物11个。该系统首次允许研究这些早期宿主-微生物群相互作用，对宿主、微生物和环境成分进行高度控制。此外，它允许在整个实验中实时监控和操纵系统。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62779/62779fig01.jpg"> 图1：肠道培养装置的原理图。 整个肠道组织片段连接到腔室的输出和输入端口（顶部），泵调节流明内和外部中室的中流。整个设备（底部）包含 6 个此类室。这个数字已经从2017年Yissachar等人修改。 <a href="https://www.jove.com/files/ftp_upload/62779/62779fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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an intestinal gut organ culture system for analyzing host-microbiota interactions

肠道组织的结构促进宿主和肠道微生物群之间的密切和相互作用。这些交叉会谈对于维持局部和系统性平衡至关重要：肠道微生物群成分（肌瘤）的变化与广泛的人类疾病有关。解剖宿主-微生物群相互作用的方法包括生理组织结构保存（ 在体内 动物模型中使用时）和控制实验因子（如简单的 体外 细胞培养系统）之间的固有权衡。为了解决这一权衡，Yissachar等人最近开发了肠道器官培养系统。该系统保留了天真的结肠组织结构和细胞机制，它也允许严格的实验控制，促进实验，不能轻易 在体内进行。它最适合解剖各种肠道成分（如上皮、免疫学和神经元元素）对发光扰动（包括厌氧或有氧微生物、小鼠或人类的整个微生物群样本、药物和代谢物）的短期反应。在这里，我们使用定制的肠道培养装置，详细描述了多个肠道片段的器官培养优化协议。通过组织部分或全安装组织片段的免疫荧光染色、荧光就地杂交（FISH）或延时成像，可以可视化主机对发光扰动的反应。该系统支持广泛的读数，包括下一代测序、流细胞学以及各种细胞和生化检测。总体而言，这种三维器官培养系统支持大型完整肠道组织培养，对局部肠道环境中宿主-微生物群相互作用的高分辨率分析和可视化具有广泛的应用。

肠道器官培养系统维持组织生存能力 。 在整个培养期对组织生存能力进行了评估。结肠组织片段在肠道器官培养系统中孵育，并在2/12/24h培养后固定。肠道上皮细胞（IEC）层的完整性通过使用E-卡德林和细胞角蛋白-18抗体的免疫荧光染色来验证。同样，如Ki-67染色（图2）所示，在结肠上皮和流明内粘液分泌物中检测到粘液填充的血小板细胞，并在结肠墓穴中增殖IEC。?...

Watch this Scientific Journal Video about An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions at JoVE.com

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

本文提出了一种独特的方法，利用一种新的肠道器官培养系统进行 前体内 实验来分析宿主-微生物群相互作用。

分析宿主-微生物群相互作用的肠道肠道器官培养系统

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

肠道器官培养系统结合了体外和体内测定的优势，在简单的体外测定与复杂的体内实验之间作为中间实验步骤。该技术的主要优点是将严密的实验控制与肠道生理学的保存相结合。该方法为分析肠道对特定刺激的反应提供了见解，对宿主、微生物和环境成分具有高度的控制力。演示这个程序的将是来自我实验室的硕士生阿隆·谢梅什和我们的实验室经理西万·阿米德罗博士。首先，将钝端针插入设备模具内的适当位置，为一组设备和盖子铸造约 20 克聚二甲基硅氧烷或 PDMS 混合物。将模具放在真空室中 30 分钟，从 PDMS 混合物中去除气泡，然后在 55 摄氏度的夜间孵育模具，以完成 PDMS 聚合。设置 PDMS 时，从模具中取出针头，小心地从塑料模具中释放培养装置和盖子。使用手术刀片从井轮廓中取出 PDMS 残留物。使用无毒硅胶粘剂将PDMS设备和设备盖板连接到75至50毫米微滑动的盖玻璃上，让部件过夜。将粘合胶粘在设备的光滑侧。为流明插入 12、22 个量表针和为井插入 12、18 个量表针。使用硅胶固定所有针头，让它在一夜之间固定。清除输入注射器，确保井介质从所有管子流出到废玻璃中，然后清除输入注射器，并确保所有管子中的刺激流出到废玻璃中，注意不污染不同的刺激。牺牲小鼠后，用锋利的剪刀和钳子解剖它，通过切割所有的脂肪和结缔组织，从胃到肛门取出消化道。切开结肠，放在一个新的盘子里。在轻轻地握住组织并仅在边缘时尽量减少接触。在解剖显微镜下执行结肠冲洗。用文本中描述的准备的 10 毫升注射器用无菌 IMDM 轻轻冲洗结肠含量。从肠道组织中取出粪便后，将结肠放入一个新的六井盘中，里面装满了0.5毫升的无菌IMDM。接下来，取结肠组织，并小心地将其连接到22仪表针，与两根线紧密相连。保持结肠对流明流的正确方向，使近端和基数分别等于输入和输出。重复结肠冲洗和针绑在所有组织。将输入和输出管连接到设备，然后以所需的速率启动泵开始实验。如KI67染色所示，在结肠上皮和流明内粘液分泌物中检测到充满粘液的血小板细胞，并在结肠墓穴中增殖IEC。结果表明，肠道培养系统维持肠道功能和结构前体内。经过两个小时的细分丝菌，或SFB，介绍，典型的SFB丝被发现与小肠病毒密切结合，使用荧光就地杂交。此外，传输电子显微镜在小肠上皮刷边的微米内呈现SFB。整个组织样本的基因表达特征是在与SFB输注两小时后以三足鼎鼎的三足鼎鼎的方式产生的。控制培养物被注入无细菌或细菌法吉利斯单色小鼠的粪便悬浮。与无细菌控制相比，SFB 所说服的这些变化幅度较小。结肠的方向非常重要。将结肠绑在针上时要格外小心。适当的领带将防止与流明含量的油井污染。广泛的读出技术可以遵循此协议，如下一代测序、成像、细胞排序等。此读物提供了对宿主微生物群在健康和疾病中的相互作用的新见解。

Research

JoVE Journal

Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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

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

该<em&gt;离体</em&gt;结肠器官培养及使用抗菌宿主防御研究

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

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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

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 ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Optimization of the Cuff Technique for Murine Heart Transplantation

穆林心脏移植袖口技术的优化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...