这项研究是由赠款RO1 CA 135561，R01 CA151494，R01 CA174432和P3013330来自美国国立卫生研究院的支持。 我们要感谢米歇尔·休斯顿，艾琳娜Dhima和安娜Velcich博士开发的地下室隔离协议提出宝贵的意见。 我们也感谢糖尿病的培训和医学用NIH P60DK20541支持的阿尔伯特爱因斯坦医学院的研究中心，以及迈克尔·布朗利博士和博士学梁笃，直接和运营海马工厂，分别是谁。

按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行研究。该方案经委员会医学爱因斯坦医学院动物实验的伦理。 1.地穴的分离与培养<ol><li>隔离小肠隐窝: <ol><li>从任何感兴趣的小鼠模型中分离出肠腺。安乐死的小鼠用CO 2，随后通过颈脱位。 </li><li>打开腹部纵向和补小肠（SI）的用冰冷的磷酸盐缓冲盐水，PBS（-Ca 2+ -Mg 2+）用2×抗生素-抗真菌剂（防反）。快速隔离小肠。 </li><li>深入剖析免费使用手术刀肠系膜脂肪。要小心，不要穿透组织 - 在任何时候都保持组织湿润冰冷的PBS。打开INTE斯坦纵向和用冰冷的PBS彻底清洗。 </li><li>切小肠分成两部分，并拉平用湿式棉尖，轻轻用预冷却的滑动刮去绒毛。在冰冷的PBS几次大力清洗。 </li><li>孵育于20ml的1×PBS每3毫乙二胺四乙酸（EDTA）/0.5毫二硫苏糖醇（DTT），3分钟为组织的非酶分解。 </li><li>切割组织成使用于预冷的滑动刀片的小块（2-4毫米）。传输块放入50ml管含有20ml冰冷的PBS中。 </li><li>移液器上下轻轻地用10倍的有10毫升无菌一次性吸管。让组织碎片沉淀在重力作用。用移液管去除上清。重复附加三次;确保上清液是明确的。 </li><li>加入20毫升的PBS每2mM EDTA的。漩涡孵育在4℃下进行30分钟，并轻轻摇动。 </li><li>让组织中沉积，弃上清。加入15ml冰冷的PBS和吸管上下5倍，用10毫升吸管。让组织碎片沉淀，收集上清液作为馏分1（F1）。重复采集F2-F5，单独维护每一个部分。 </li><li>添加15ml冰冷的PBS及此时抖动用手剧烈持续15秒。收集F6和重复F7，F8。如果组织块开始浮动，挖掘，帮助他们解决。 </li><li>检查在显微镜下每个级分的等分试样。普尔那些含有隐窝分数。 </li><li>通过70微米尼龙细胞过滤器的汇集级分，收集隐窝在50ml管中。 </li><li>离心机在100×g离心5分钟，在4℃，弃上清，重悬沉淀于10ml冰冷的PBS用2×防反和转印隐窝到15毫升管。重复洗一次。 </li><li> 2个防反 - 用10毫升冰冷的自动进稿器，先进的DMEM / F-12（贝科"改良Eagle培养基/火腿"S F-12）洗一次隐窝。 </li><li>洗一次隐窝用10mlDF - 1×防反。 </li><li>计数使用血球隐窝。调整隐窝溶液体积并转移到1.5ml试管中，使悬浮在最终隐窝浓度将每50μl胶状蛋白质混合物（基质胶）100-500隐窝。离心机在100×g离心5分钟，在4℃下重悬隐窝中的胶状蛋白质混合物。 </li><li>板50微升隐窝-每孔胶状蛋白质混合物的悬浮液在24孔板中（生长面积:2平方厘米 ），小心地放置在每一个的中心的悬浮液一滴良好。保持所述板在CO 2，37℃培养箱中培养约30分钟，直到悬浮液凝固。 </li><li>加入500微升冰冷的完全培养基（先进的DMEM / F12，带有1反 - 反，10mM的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），1个含有GlutaMAX，1×B27的补充液，1×N2补充液，1毫N-乙酰基-L-半胱氨酸（NAC），50纳克/毫升的表皮生长因子（EGF），100ng / ml的脑袋，500毫微克/毫升的R-脊椎）。 <ol><li>重组生长因子在0.1％牛血清白蛋白（BSA）的PBS中。 </li></ol></li></ol></li><li>地穴化培养和传代: <ol><li>通道隐窝组织体14-21天接种后（或根据需要），如下所示: <ol><li>变化的媒体，每星期五和星期一。上周三，用新鲜培养基替代一半的媒体。 </li></ol></li><li>除去培养基。以5分钟的间隔用500μl的PBS洗两次的样品。 </li><li>除去PBS，轻轻向上突破用无菌P1000微枪头胶状蛋白质混合物。 </li><li>加入1 ml完全培养基，以单井，重悬在1ml媒体的组织体。 </li><li>轻轻地破坏通过上下吹打20-30x使用P1000的组织体（检查显微镜进行适当的组织体离解以良好产率隐窝直到一致的技术下被显影）。 </li><li>转让隐窝到1.5 ml离心管中;离心机在100×g离心在4℃，5分钟。 </li><li>用1毫升完全培养基两次洗涤沉淀。 </li><li>裂以1:3的比例，或1:6​​根据需要，再悬浮隐窝中每孔加入50μl基质胶。继续上面的（ 部分1.1.16-18）的说明。 </li></ol></li><li>地穴类器官冷冻: <ol><li>除去培养基。洗500微升PBS样品。 </li><li>分散使用的是P1000的枪头的胶状蛋白质混合物。 </li><li>加入1毫升媒体单井重悬在1ml媒体的组织体。 </li><li>轻轻打散使用P1000的组织体吹打向上和向下20-30x。 </li><li>转移的组织体到1.5ml管中。离心机在100×g离心在4℃，5分钟。 </li><li>用1毫升完全培养基两次洗涤沉淀。 </li><li>隐窝重悬在500μl冷冻介质。 </li><li>转移隐窝到冻存管，将管在冷冻容器，存放于-80℃冰箱overniGHT。 </li><li>隐窝转移到液态氮罐。 <ol><li>通过在37℃水浴中快速解冻冷冻复苏隐窝类器官。用500微升，在100 XG，5分钟，悬浮在胶状蛋白质混合物，文化如上所述洗完整的培养基一次，离心机。为更好的恢复，ROCK抑制剂（Y-27632）加入到冷冻介质。 </li></ol></li></ol></li></ol> 2.地穴类器官代谢分析<ol><li> 24孔板准备: <ol><li>隔离并根据协议（见隐窝隔离和1.2节中的地穴通道第1.1节 ）数量隐窝。 </li><li>在胶状蛋白质混合物悬浮隐窝（每20微升100-200隐窝）。 </li><li>在24孔测定板中板隐窝胶状蛋白质混合物的悬浮液（确保有至少一式三份对各样品），并允许该悬浮液在37℃下固化，在CO 2孵化器;再加入500μl完整的培养基。 </li><li>文化隐窝（如1.1节所述），并在显微镜下观察到隐窝成长为全面发展类器官。 </li></ol></li><li>外流量分析: <ol><li>水合物盒过夜在非CO 2（0％CO 2）中 ，37℃培养箱中培养。 </li><li>除去培养基，并用500μl的DMEM洗两次组织体（无:葡萄糖，L-谷氨酰胺，丙酮酸钠和碳酸氢钠;以及与:酚红）。等待5分钟。 </li><li>添加675微升每孔的测定培养基（DMEM有2mM L-谷氨酰胺和5mM D-葡萄糖）到每个孔中。 </li><li>检查隐窝组织体的微观形貌，以确保组织体和凝胶状蛋白混合物的洗涤后完好无损。孵育1小时的非CO 2，37℃培养箱中培养。 </li><li>制备10μM的可注射的化合物（寡霉素，羰基氰化物对 - 三氟甲氧基苯基酮腙（FCCP）中，rotenone）在测定培养基中。 </li><li>建立墨盒通过加载75微升10微米的化合物注射进顺序墨盒的端口:端口A - 寡; B口 - FCCP;和C口 - 鱼藤酮（在试验过程中的最终浓度为1微米）。 </li><li>培养盒30分钟-在非CO 2培养箱中1小时，在37℃。 </li><li>同时，打开XF分析仪，并创建分析方案模板。 </li><li>将墨盒和公用事业板块成为XF分析和运行"校准"墨盒。 </li><li>检查地穴组织体显微镜，以确保它们连接到胶状蛋白质混合物。 </li><li>负载细胞培养板为工具和运行试验的协议。 </li></ol></li></ol>

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有没有披露。

我们测试的耗氧率（OCR）和隐窝中分离出8个月大的小鼠并生长成组织体在体外的细胞外酸化速率（ECAR）的基础量的测定之后，隐窝代谢物通过增加寡霉素，羰基氰化物评价-p-trifluoromethoxyphenylhydrazone（FCCP）和鱼藤酮，按顺序。 基底OCR和基底ECAR记录从0 - 29分钟（ 图2A和2B）。在第 29 届分钟，寡霉素（来自端口A）注射到每...

结直肠癌（CRC）是癌症相关死亡在美国的第三大原因。散发性大肠癌- 即后来的生活产生（&gt; 50岁），并没有明确的遗传易感因素-占〜80％ ​​的情况下，随着发病率的长期膳食模式1,2的强烈影响。这些肿瘤中表现出对依赖性代谢移对氧化糖酵解，被称为Warburg效应，这可能部分地使较高浓度的蜂窝构件和能量可用（通过谷氨酰胺），以允许和也许驱动的肿瘤细胞的增殖3-5率高。结肠癌的研究以及其他胃肠道癌症，包括小肠肿瘤提供重要的洞察肿瘤形成的原因。调查胃肠器官系统，可协助DET正常的，有利于肿瘤发生和肿瘤发生的国家之间的代谢差异发芽对肿瘤发展的相对风险，以及早期发现肿瘤的。此外，了解生物能量代谢涉及到线粒体呼吸和糖酵解提供基本的深入了解细胞生理学，老化和疾病状态扰乱肠道内环境稳定。生物能学分析技术，细胞外流量分析，可以利用在细胞生长在培养实时6,7同时评估线粒体呼吸和糖酵解的速度。 直到最近， 在体外研究小肠被限制在由良性或恶性肿瘤8,9的细胞系，并没有代表正常的肠道上皮细胞的生理和他们所在的微环境的影响。在2009年，Sato 等人 10导入离体培养体系生长的三维（3D）的小鼠的肠上皮组织体，或epithelial"微型胆"，适用于实验，诊断和治疗的研究10,11。此外，隐窝从calorically限制小鼠中分离维持其改变的生长特性类器官在这样的文化12。相比于转化的细胞系，隐窝类器官培养物可以被用于产生生理相关数据呈现出好得多的模型，以了解在体内的状态。 我们调整生物能量学分析技术测定肠隐窝组织体的能量代谢。小鼠小肠隐窝类器官培养体外发展提出的墓穴类器官的能量代谢研究。耗氧率（OCR）和隐窝组织体的细胞外酸化速率（ECAR）测定在不存在和两种不同的代谢抑制剂（寡霉素，鱼藤酮），和离子载体（羰基氰 - 对 - trifluoromethoxyphenylhydrazone）存在下进行。地穴奥尔加这些化学物质NOID代谢反应，通过改变ECAR和OCR值成功体现。 细胞生物能的研究将阐明代谢状态和疾病的风险和表型之间的相互交流的癌症，肥胖症，糖尿病，代谢紊乱和线粒体疾病，并有助于与转化医学直接影响预先筛选方法。在这里，我们描述了一个详细的协议来隔离小肠隐窝和文化隐窝类器官。此外，我们引入了一种新的方法来使用地穴类器官培养的代谢试验。

<table><tbody><tr><td>BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td><td>Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Advanced DMEM/F-12 (1x)</td><td>Life Technologies</td><td>12634-028</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Modified Eagle&amp;prime;s Medium w/o glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate</td><td>Sigma-Aldrich</td><td>D5030</td><td></td></tr><tr><td>Phenol red sodium salt</td><td>Sigma-Aldrich</td><td>P4758</td><td>Final Concentration 15 mg/l in DMEM (D5030) - &lt;strong&gt;step 2.2.2&lt;/strong&gt;</td></tr><tr><td>Antibiotic-Antimycotic, 100x, 100 ml</td><td>Life Technologies</td><td>15240-062</td><td>Final concentration 1x or 2x</td></tr><tr><td>Penicilin-Streptomycin, liquid</td><td>Life Technologies</td><td>15140-122</td><td>Final concentration 1x</td></tr><tr><td>Gibco&amp;reg; GlutaMAX&amp;trade; supplement</td><td>Life Technologies</td><td>35050061</td><td>Final concentration 1x</td></tr><tr><td>Gibco&amp;reg; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td><td>Life Technologies</td><td>15630-080</td><td>Final concentration 10 mM</td></tr><tr><td>N-acetyl-L-cysteine, 25 g</td><td>Sigma-Aldrich</td><td>A9165-25G</td><td>Final concentration 1 mM</td></tr><tr><td>100x N-2 supplement, liquid</td><td>Invitrogen</td><td>17502-048</td><td>Final concentration 1x</td></tr><tr><td>50x B-27&amp;reg; supplement minus Vitamin A, liquid</td><td>Invitrogen</td><td>12587-010</td><td>Final concentration 1x</td></tr><tr><td>Recombinant Mouse R-Spondin 1, CF, 50 &amp;mu;g</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>Final concentration 500 ng/ml</td></tr><tr><td>Recombinant Murine EGF, 100 &amp;mu;g</td><td>Peprotech</td><td>315-09</td><td>Final concentration 50 ng/ml</td></tr><tr><td>Recombinant Murine Noggin, 20 &amp;mu;g</td><td>Peprotech</td><td>250-38</td><td>Final concentration 100 ng/ml</td></tr><tr><td>Gibco&amp;reg; L-glutamine, 200 mM</td><td>Life Technologies</td><td>25030-081</td><td>Final concentration 2 mM</td></tr><tr><td>Gibco&amp;reg; glucose powder</td><td>Life Technologies</td><td>15023-021</td><td>Final concentration 5 mM</td></tr><tr><td>Ambion&amp;reg; 0.5 M EDTA (ethylenediaminetetraacetic acid), pH 8.0</td><td>Life Technologies</td><td>AM9260G</td><td>Final concentration 3 mM for &lt;strong&gt;step 1.1.5&lt;/strong&gt;; 2 mM for &lt;strong&gt;step 1.1.8&lt;/strong&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>DTT (dithiothreitol), 1M</td><td>Life Technologies</td><td>P2325</td><td>Final concentration 3 mM</td></tr><tr><td>Albumin from bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>A2058</td><td>0.1% in PBS</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td>1% in PBS</td></tr><tr><td>Recovery&amp;trade; Cell Culture Freezing Medium</td><td>Life Technologies</td><td>12648-010</td><td></td></tr><tr><td>ROCK inhibitor (Y-27632)</td><td>Sigma-Aldrich</td><td>Y0503</td><td>Final concentration 10 &amp;mu;M</td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>O4876</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>Final concentration 0.1 N in PBS</td></tr><tr><td>XF24 Extracellular Flux Analyzer (XF Analyzer)</td><td>Seahorse Bioscience</td><td></td><td></td></tr></tbody></table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

小肠粘膜表现出组织成两个基本结构的重复架构:绒毛，伸入肠腔和成熟肠细胞，杯状细胞和肠内分泌细胞组成;和隐窝，驻留近端粘膜下层和肌层，窝藏成体干细胞和祖细胞和成熟的帕内特细胞，以及基质和隐窝微环境的免疫细胞。直到最近几年， 在小肠体外研究仅限于从良性或恶性的肿瘤衍生的细胞系，并没有表示的正常肠上皮细胞的生理机能和在它们所在的微环境的影响。在这里，我们展示了从培养C57BL / 6小鼠的原代小鼠小肠隐窝类器官改编自佐藤等人的方法。（2009）。此外，我们目前使用的crypt类器官培养法检测进行实时测量地穴新陈代谢换货基础耗氧量，糖酵解速率，ATP的产生和呼吸的能力。类器官维持他们的源代码中定义的属性，并保留其代谢改编耗氧量和胞外酸化率反映的各个方面。在这个地穴化培养系统实时代谢研究是一个强大的工具来研究隐窝类器官的能量代谢，以及如何通过营养和药物因素的调节。

地穴类器官是8月龄C57BL / 6小鼠饲喂建立纯化营养76A（AIN76A）的啮齿动物饲料美国学院。肠隐窝组织体可以生长在培养物从单个隐窝（ 图1A，单红色箭头）长时间。组织体生长出隐窝状结构在18-20天后在培养物（ 图1B，红色箭头）。隐窝传代，每3周和组织体有效回收下列各通道。 海马生物能量的仪器可以在细胞生长在培养的实时同步​​评估线粒体呼...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

小肠隐窝类器官培养离体提供的组织培养系统，该系统概括隐窝依赖于干细胞和它们的生态位的增长。我们建立了一个方法，以测定在实时在原代小​​鼠隐窝类器官的代谢分布图。我们发现组织体保持了其源定义的生理特性。

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

16.2K 次观看.  Albert Einstein College of Medicine. 小肠隐窝类器官培养离体提供的组织培养系统，该系统概括隐窝依赖于干细胞和它们的生态位的增长。我们建立了一个方法，以测定在实时在原代小​​鼠隐窝类器官的代谢分布图。我们发现组织体保持了其源定义的生理特性。 

视频: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

科研

发育生物学

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

癌症研究

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

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

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

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

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 ...

免疫与感染

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

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

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 ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

生物学

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

肠内增殖细胞和死细胞的定量

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

使用活性氧敏感荧光探针分析小鼠肠道类器官中的氧化应激

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

小鼠肠隐窝和单干细胞类器官的 3D 培养

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

作者聚焦：来自小鼠肠隐窝的类器官的 3D 培养和用于类器官研究的单个干细胞  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

代谢谱的实时分析

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使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

该

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