Name: real-time measurement of epithelial barrier permeability in human intestinal organoids
Uploaded: 2017-12-18T15:00:00
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. 24</td>
		</tr>
		<tr>
			<td>B27 supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Component of ENR media; see McCraken et al. 24</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. 24</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td>Component of ENR media; see McCraken et al. 24</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 C. difficile Toxin A/TcdA Protein</td>
			<td>R&#38;D Systems</td>
			<td>8619-GT-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>R-spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>4645-RS</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>6057-NG</td>
			<td>Component of ENR media; see McCraken et al. 24</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

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