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

作者想感谢 Drs. 斯蒂芬妮 Spohn 和巴塞尔 Abuaita 对化微注射的许多有益的讨论。JRS 由肠道干细胞财团 ( U01DK103141 ) 支持 , 这是一个由国家糖尿病、消化和肾脏疾病研究所 ( NIDDK ) 和国立反应和传染性疾病研究所 ( NIAID ) 资助的合作研究项目。JRS 和 VBY 支持的 NIAID 小说, 替代模型系统的肠道疾病 (NAMSED) 联合体 (U19AI116482)。DRH 被支持由国立反应和传染性疾病研究所 ( NIAID , T32AI007528 ) 的微生物病机培训补助金机制 , 以及密歇根临床与健康研究所的临床和转化科学奖研究 (UL1TR000433)。
本手稿中使用的完整数据文件和数据分析代码可在 https://github.com/hilldr/HIO_microinjection。

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作者没有相互竞争的金融利益或其他利益冲突的披露。

本协议建立了 general-purpose hPSC HIOs 和组织衍生肠 organoids 的显微注射方法, 并实时测量上皮屏障的通透性。我们还展示了我们对使用这些方法生成的数据进行分析和解释的方法。考虑到肠道 organoids 模型系统的越来越多的采用16,20,21,28和对肠道屏障通透性的长期兴趣作为生理相关功能结果3,4,5,6, 我们预计在此字段中工作的其他人将能够应用和构建这些方法。
有几个步骤是关键的应用这项技术。在广泛的显微注射试验之前, 应建立高质量的 hPSC 或组织衍生的 HIO 组织。HIO 的宏观结构可能是不均匀的, 在大小和形状上的变化, 虽然组织认同和细胞形态学是高度可再生的, 当使用建立的方法生成 HIOs24。球形 HIOs 由单一的半透明腔和测量直径约1毫米是理想的显微注射和测量腔荧光在 real-time。在某些情况下, 注射会失败, 导致 HIO 或明显泄漏的注射材料的崩溃。失败的 HIOs 可以使用标准微从用户的判断中删除。当选择 HIOs 进行显微注射和成像时, 请考虑成像平台上可用的物镜。一般来说, 2 4X 物镜是理想的捕捉完整的 HIO 荧光信号, 虽然一个10X 的目标可以使用, 如果低功耗镜头是不可用或如果可用的 HIOs 是和 #60; 1 毫米直径。成像软件必须允许自动捕获的荧光图像在定义点随着时间的推移。
为了适应实验要求, 可以对该协议进行若干修改。例如, 屏障功能测试的结果可能取决于使用43的化合物的分子大小, 并且可以测试不同分子量的葡聚糖制剂。此外, 除了荧光成像外, 明成像还可以作为组织整体结构完整性的指标25进行。当进行活菌的微注射时25,28,31,3233,44, 可能需要添加青霉素和链霉素或庆大霉素对 HIO 培养基在注射之前或之后。microcapillary 的外侧会在细菌培养悬浮液中被污染, 这可能被转移到 HIO 介质。另外, 微注射可以进行 HIOs 悬浮在细胞外基质 (如, 基质) 没有介质, 添加介质后, 注射完成。这可能会限制污染的细胞外基质和外部表面的 HIO。在规划微生物生长试验时, 可能有必要在 1-2 小时后清除培养基中的抗生素, 以避免减慢或防止微量生物体的生长。
最后, 认识到并非所有的研究人员都能获得适合于体外成像的显微设备, 重要的是要指出, 本协议中概述的收集荧光数据的程序可以应用到图像在固定 timepoints 使用标准的 epifluorescent 显微镜, 没有自动图像采集或环境控制。此方法的示例可以在莱斯利和黄et al.的报告中找到25, 在 hPSC 的肠道 organoids 中检查了 C. 难辨性毒素活动, Karve 和 Pradan et al.44, 在类似 hPSC 的肠道 organoids 微量与活的大肠杆菌检查上皮屏障通透性. 人工操作成像设备可能会导致更大的变化和困难的正常化的荧光信号。在执行手动成像 FITC-葡聚糖注射 HIOs, 必须保持固定放大倍数, 荧光激发强度, 和曝光时间在整个实验, 以避免扭曲的荧光强度测量。

肠上皮形成一个有选择性的屏障, 介导的养分定向运输, H2O, 离子和废物, 同时尽量减少非特异性扩散介导的其他粒子之间的交换管腔和间充质组织或血液供应1,2。小肠上皮屏障的非特异性通透性一直被认为是健康和疾病的关键功能参数3,4,5,6, 它反映了通过细胞空间扩散小分子穿过上皮细胞。上皮屏障通透性的测量可以进行动物模型7和人类患者的8通过摄入果糖, 其中没有特定的运输在胃肠道, 并随后收集外周血中果糖浓度的测定。交替摄入的屏障功能标记, 如荧光标记碳水化合物也可用9,10。这种方法已经适应了肠道上皮细胞培养生长在 Transwell 支持11, 一种简化的方法, 允许更大的实验控制, 但也被批评为一个可怜的预测者的在体内渗透性由于缺乏分化的上皮亚型和组织结构12。使用室代表还另一种方法, 并允许测量的上皮屏障功能在整个肠道黏膜前体内13。此技术的应用通常受组织可用性和条件13,14的限制。因此, 新的方法, 平衡重复性和吞吐量与生理相关性是必要的。
最近的发展, 在体外器官分化已导致采用3D 组织培养模型系统作为一个复杂的平台, 以综述的动力学复杂性组织15,16,17 ,18,19,20,21,22,23。特别是, 人类多能干细胞 (hPSC) 衍生的人类肠道 organoids (HIOs)19,24已成为一个可重现和实验性的模型系统, 用于研究宿主-微生物相互作用和上皮屏障动力学25,26,27,28。同样, 人体组织衍生的 organoids (也称为 enteroids) 可以从一个简单的活检程序, 并可作为一个听话的系统来研究人体生理和疾病15,29,30。注射人体肠道 organoids 允许交付实验化合物25或活微生物25,31,32,33至顶端上皮化腔的表面。莱斯利和黄et al.25最近采用了这种技术来测量 HIOs 微量与荧光素异硫氰酸酯 (FITC) 标记为葡聚糖后暴露于细菌毒素的屏障渗透性。
本议定书的目的是作为一个指南的测量上皮屏障通透性 hPSC 衍生 HIOs 和组织衍生 HIOs 使用荧光显微镜。稍加修改, 它也可以作为一个一般的底漆, 注射 HIOs 与实验化合物。使用者可以在注射后附加或替代的下游应用补充本协议。

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

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

通过定向分化, 通过活检或从多能干细胞产生的肠道组织的3D 培养取得了进展, 导致了成熟的肠道黏膜的体外模型。利用这些新兴的模型系统将需要适应为2D 文化系统和动物开发的工具和技术。在这里, 我们描述了一项技术, 测量上皮屏障通透性的人肠道 organoids 在 real-time。这是通过注射荧光标记的葡聚糖和成像在倒置显微镜, 装有 epifluorescent 过滤器完成。肠 organoids 屏障通透性的实时测量有助于人类肠道上皮组织中的高分辨率时间数据的生成, 虽然这种技术也可以应用于固定的 timepoint 成像方法。这项协议是很容易适应的测量上皮屏障渗透性后, 接触到药理剂, 细菌产品或毒素, 或活微生物。在稍加修改的情况下, 本协议还可以作为肠道 organoids 注射的一般入门读物, 使用者可以选择在注射后附加或替代的下游应用补充本协议。

HIOs 被区分了从人多能干细胞和培养在细胞矩阵解答前面描述了19,24。经过4周的培养, HIOs 已经扩展到足以允许微注射。HIOs 是微量与 4 kDa FITC 共轭葡聚糖悬浮在 pbs 或 pbs 含有难辨梭菌毒素 TcdA。梭菌是一种机会性胃肠病原体, 在 HIOs25中显示毒素介导的上皮毒性。作为一个积极的控制, EGTA 被添加到 HIO 培养基中的一个子集的 HIOs 注射 PBS 和 FITC 葡聚糖。EGTA 是一个钙合剂, 导致快速 de-polymerization 的肌动蛋白细胞骨架42。FITC 荧光监测实时成像显微镜在一个受控环境室和图像被抓获的10分钟间隔时间。
对成像数据的后期分析显示, FITC 荧光的保留有很大的差异 (图 5)。HIOs 注射 PBS 保留了几乎所有的荧光信号, 目前在t = 0, 但 HIOs 也注射 TcdA 治疗与 EGTA 表现出大幅下降的荧光强度8小时 post-microinjection (图5A.). 所有时间点的所有 HIOs 都量化了成像数据, 以生成一个高分辨率数据集, 表示每个实验条件下荧光强度的相对变化 (图 5B)。通过计算每个治疗组 (表 1) 中 FITC 的平均清除时间 (t1/2 ) 并比较各组之间在t1/2 之间的差异, 来评估上皮通透性的差异使用学生的t测试。控制处理 HIOs 保留了大多数 FITC 荧光信号超过16小时 (t1/2 = 17 ± 0.3 h)。与对照 HIOs (t1/2 = 2.6 ± 0.2 h 相比, EGTA 治疗可显著降低 FITC-葡聚糖的清除时间;P = 1.3 x 10-8)。与先前发布的结果25一致, 微注射 TcdA 明显增加了与对照治疗相关的上皮屏障通透性 (t1/2 = 7.6 ± 0.6 h;P = 5.4 x 10-4)。因此, 外 (EGTA) 和微量 (TcdA) 化合物都能诱发 HIOs 上皮屏障通透性的显著改变。这些结果表明, 广泛的药理作用, 代谢物, 细菌产品, 细胞因子, 生长因子, 和其他化合物的上皮屏障功能, 可以评估使用这种方法。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>治疗</td>
			<td>半生命 (h)</td>
			<td>扫描电镜 (h)</td>
			<td>下 95% CI (h)</td>
			<td>上 95% CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>控制</td>
			<td>17.11</td>
			<td>0。3</td>
			<td>16.45</td>
			<td>17.78</td>
			<td>11</td>
		</tr>
		<tr>
			<td>egta</td>
			<td>2.58</td>
			<td>0.19</td>
			<td>1.78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TcdA</td>
			<td>7.56</td>
			<td>0.63</td>
			<td>5.93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
表 1: 平均消除时间 (t1/2) 中的 FITC 葡聚糖在 HIOs 处理 EGTA 或 TcdA。单位是小时 post-microinjection。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
图 1: 用于 HIO 显微注射的微量和微的基本布局.此图显示了用于执行微注射 HIOs 的全套设备。关键组件被标记, 订购信息可以在材料表中找到。<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
图 2: 微拉拔器.铜加热线圈hc, 顶部钳位c1, 底部钳 (c2), 拉拔臂 (pa), 热选择开关 (ht) 由箭头标识。热1和拉的正确设置用红色文本表示。该插页显示了一个正确安装的玻璃灯丝准备加热。<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
图 3: 用 FITC 葡聚糖注射的组织衍生 HIO 的代表性图像(A) 图像, 演示在插入到 HIO 和微注射之前, 玻璃 microcapillary 的位置。(B) 明注射 FITC 葡聚糖后组织衍生的人肠道 organoids 的图像。注意, 即使不使用特定的过滤器集, 荧光信号也很明显。这种着色辅助显微注射精度。3X 放大倍数<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
图 4: 实时图像处理工作流(A) 屏幕截图, 说明设置显微镜参数所需的步骤, 在幻灯片上查找 HIOs, 并保存每个 HIO 的舞台位置。(B) 前成像设置, 用于在 A. (C) 中指定的每个位置定期捕获 FITC 通道. 将 DV 格式数据文件的后向输出导出为 tiff 图像, 每个时间点每 HIO 一个 tiff 图像。<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
图 5: 代表性结果.(A) 干细胞衍生的人肠道 organoids (HIO) 微量与2毫克/毫升 FITC 葡聚糖 (4 kDa) 成像20小时。HIOs 也微量与 PBS (控制) 或梭菌毒杆菌 TcdA (12.8 ng/µL) 或治疗2毫米 EGTA 添加到外部培养基培养基。4X 放大倍数。(B) 在使用 PBS (控制)、TcdA 或 EGTA 处理的 HIOs 中, 随着时间的推移, 平均归一化 FITC 强度的图。误差线表示 S.E.M. 和n = 11 HIOs (控制)、3 HIOs (EGTA)、6 HIOs (TcdA)。<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

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

本协议描述了在人体肠道 organoids 中采用荧光显微镜和活细胞显微镜进行药物治疗后 real-time 上皮屏障通透性的测量。

人肠道 Organoids 上皮屏障通透性的实时测量

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

real-time-measurement-epithelial-barrier-permeability-human

Research

JoVE Journal

Developmental Biology

14.1K 次观看.  University of Michigan. 本协议描述了在人体肠道 organoids 中采用荧光显微镜和活细胞显微镜进行药物治疗后 real-time 上皮屏障通透性的测量。

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

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

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

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

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

体外肠囊评估黏膜通透性在胃肠道疾病模型

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

科研

医学

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

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

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

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

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

生物工程

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. ...

免疫与感染

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

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

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

在根尖肠模型中使用路西法黄测定肠通透性

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

Investigating Intestinal Barrier Breakdown in Living Organoids

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

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

研究活体器官的肠道屏障分解

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

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

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

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

检测胎儿组织来源肠的上皮通透性

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

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

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

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

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

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 ...

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 ...

从牛肠道类器官开发 2D 单层培养物

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

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

作者聚焦:用于病原体相互作用研究的牛类器官 2D 肠道单层模型的开发和表征

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

使用小肠和大肠单层界面的牛类器官技术的进步

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