基于微流控技术的肠上皮屏障功能检测系统

由于疾病引起或药物毒性导致的上皮屏障功能障碍可能危及生命。上皮屏障破坏主要表现为上皮细胞的细胞通透性增加。上皮屏障的细胞通透性的体外测试通常通过在刚性膜上培养细胞来实现，这种传统的Transwell系统不适合高分辨率动力学测量，不能像3D培养一样支撑更多的模拟和研究。微流控技术在体外模拟领域迅速发展。微流控技术固有的小尺寸特点使其恰好能与管状上皮连接，以便通过灌注提供剪切应力和连续培养基。典型的微流体溶液利用人造膜进入上皮细胞而不通过ECM，ECM是参与分化和上皮-间充质转化的细胞信号转导中的关键参数。 

基于此，荷兰Mimetas公司联合创始人兼总经理Paul Vulto博士及其团队开发了一种肠上皮屏障功能检测系统来培养灌流的ECM支持的上皮细胞并以无膜方式测试它们的屏障功能。作为一个例子，研究者们开发了一种显示细胞极化、紧密连接形成和关键受体表达的肠道上皮细胞模型。 40个肠道模型在器官芯片平台（OrganoPlate）中以管状形式生长，可从顶端和基底两侧进入。 

图1展示了OrganoPlate平台，其包含嵌入标准384孔微量滴定板格式的40个微流体细胞培养结构（图1 a，b）。每个微流体通道结构由连接到相应孔的三条泳道组成的微孔板，其作为进口和出口以进入微流体培养基。图1 c-j示出了微流体结构中心的垂直和水平横截面以及生长出管状结构的方法。首先，在中央通道引入ECM凝胶（图1c，d）。ECM凝胶化后，将上皮细胞接种在一个侧道中，通过将滴定板置于垂直位置，即站立在一侧（图1e-h），使它们直接沉积在ECM凝胶上。在细胞贴壁后，将该板水平放置在间隔摇杆上，该摇杆通过储液器之间的相互调平引起流动。细胞增殖并开始铺满灌注通道的所有表面，形成汇合的管状结构（图1 i，j）。上皮的基底紧贴ECM凝胶并且可以与ECM凝胶层另一边的灌注腔相通。为了模拟肠屏障，使用人结直肠腺癌细胞系（Caco-2）。图1 l-p分别显示了在第0,1,4,7和11天的管形成的相位对比图。在第0天，将细胞接种到ECM上并开始在玻璃壁上增殖以形成融合管（图1 n-p）。灌注对于管的形成至关重要。 3天内形成管并在第4天发现最佳屏障功能。

Fig. 1 Overview of the method for modeling intestinal tubules in the OrganoPlate platform. a Photograph of the bottom of an OrganoPlate showing 40 microfluidic channel networks with inlay showing thetop view of the 384-well plate format device; b Zoom-in on a single microfluidic channel network comprising three channels that join in the center. c, e, g, i Horizontal projection and d, f, h, j vertical cross section ofcenter region for subsequent steps in establishing the gut model. c, d Anextracellular matrix gel (light gray) is patterned by two phaseguides (darkgray), e, f culture medium is introduced in the two lanes adjacent to the ECMgel, one of which comprises cells. g, h Cells are allowed to settle against the ECM gel surface by placing the plate on its side. i, j Upon application of flow, cells form a confluent layer lining the channel and gel surfaces, resulting in a tubular shape. k 3D artist impression of the center of a chip comprising a tubule, an extra cellular matrix gel and a perfusion lane; two phaseguides (white bars) are present that define the three distinct lanes inthe central channel. The tubule has a lumen at its apical side that isperfused. l–p Phase-contrast images of the formation of the tubular structure at day 0, 1, 4, 7, and 11, respectively. Scale bars are 100 μm.

图2a展示了肠道管的共焦荧光显微照片的三维重建。该管具有清晰的内腔并排列于凝胶和灌注腔的周边。如ZO-1和Ezrin的免疫荧光染色所示，管中的Caco-2细胞显示有紧密连接和刷状缘形成（图2a-d，g）。另外，细胞圆顶化说明流体运输正常、上皮屏障功能完整（图2f）。图2h-j显示了对于Glut-2，MRP2，ErbB1和ErBb2染色的管的最大强度投影图像。与ECM接触的细胞显示转运蛋白Glut-2，MRP2的表达强烈增加，并且ErbB1和ErbB2受体的表达较弱。这些染色结果说明了ECM在细胞分化和蛋白表达中起关键作用。此外，ECM凝胶表面的特性，如其（生物）化学成分和机械特性，使其可形成在体内观察到的组织结构。

Fig. 2 Tubule characterization by immunofluorescent staining. a 3D reconstruction of a confocal z-stack showing tubular morphology with a lumen. White arrows indicate the apical (A) and basal (B) sides. The tube is stained for tight junctions (ZO-1 in red) and brush borders (ezrin in green). b Max projection and c vertical cross-section of the tubular structure in a; d, e zoom of the epithelial layer at the bottom of the tube exhibiting d tight junctions (ZO-1 in red) and brush borders (ezrin ingreen), and e acetylated tubulin (green) and occluding (red). f Phase-contrastimage showing dome formation. g Zoom of a z-slice of the tube in a of the celllayer on top of the phaseguide showing apical positioning of ezrin, indicating polarization of the tube (white arrow indicates basal side B). h Expression of glucose and MRP2 transporters, respectively stained with Glut-2 in red and MRP2 stain in green. Both Glut-2 and MRP2 show significantly higher signal againstthe collagen gel compared to the regions that are not exposed to the collagen, indicating increased expression levels. Both stains clearly stain the apicalside of the tube. For z-slices above the phaseguide at a higher magnificationsee Supplementary Fig. 2b. i ErbB1 (red) and acetylated tubulin (green) expression. ErbB1 expression levels appear higher against the collagen. j Co-staining of Glut-2 transporter and ErbB2 receptor; both stains show highersignal levels against the collagen gel. ErbB2 is primarily expressed pericellularly (see also Supplementary Fig. 2d for a zoom)). All tubes arefixed after 4 days in culture. Nuclei are stained blue with Draq5 (a–c, g–j) and DAPI (d, e). Scale bars in white are 100 μm with the exception of d, e, f,and g, where they are 50 μm. Z-slices just above the phaseguide at higher magnification of the images g–j are available in Supplementary Fig. 2. All imagesare representative of at least three biological and at least three technical replicates.

Caco-2管的屏障功能通过在管腔中灌注有荧光探针的培养基来评估，接着测定基底凝胶区域中的荧光水平。将高分子量荧光探针（150 kDa FITC-葡聚糖）和较低分子量探针（4.4 kDa TRITC-葡聚糖）都加入到通过管腔灌注的培养基中。在没有完整管状结构的情况下，荧光探针会渗入凝胶和基底侧灌注通道（图3a，d，g），而对于完全完整的屏障，荧光探针保留在管腔中图（3b，e，h）。一旦（部分）丧失屏障功能，例如通过药物诱导的毒性，荧光探针从管腔向基底侧泄漏，在ECM中产生更高的信号（图3c，f，i）。使用HCI系统测量屏障完整性，可并行监测40个管。在达到0.4的荧光值时，认为管的屏障完整性丢失。在培养的第4,7和11天追踪24管的屏障完整性，发现在第4天，所有管都是密封的，而在第7天和第11天，分别有三个和七个管泄漏。因此，在培养4天时进行屏障完整性测量。

Fig. 3 Barrier integrity assay in OrganoPlate. A fluorescent dye is inserted in the channel comprising the tube. Integrity of the tube barrier is quantified by measuring the amount of dye thatis leaking out of the tube into the adjacent gel channel. a–c Sketch invertical cross section showing fluorescence distribution: a in absence of atube, b for the case of a leak-tight tube and c for a leaky tube. d–i Fluorescent images of microfluidic chips perfused with fluorescent molecules show experimental results for: gel only (d, g), leak-tight tube (e, h), andleaky tube (f–i) using both 150 kDa FITC-dextran and 4.4 kDa TRITC-Dextranduring the same experiment. 

本研究由荷兰Mimetas公司联合创始人兼总经理PaulVulto博士及其团队完成，于2017年8月发表于Nature Communications。

论文信息：Sebastiaan J. Trietsch, Elena Naumovska, DorotaKurek, Meily C. Setyawati, Marianne K. Vormann, Karlijn J. Wilschut, Henri?tteL. Lanz, Arnaud Nicolas, Chee Ping Ng, Jos Joore, Stefan Kustermann, AdrianRoth, Thomas Hankemeier, Annie Moisan & Paul Vulto*. Membrane-free cultureand real-time barrier integrity assessment of perfused intestinal epitheliumtubes. Nature Communications 2017, 8(1):262.

论文链接：https://www.nature.com/articles/s41467-017-00259-3