由于疾病引起或藥物毒性導致的上皮屏障功能障礙可能危及生命。上皮屏障破壞主要表現(xiàn)為上皮細胞的細胞通透性增加。上皮屏障的細胞通透性的體外測試通常通過在剛性膜上培養(yǎng)細胞來實現(xiàn)，這種傳統(tǒng)的Transwell系統(tǒng)不適合高分辨率動力學測量，不能像3D培養(yǎng)一樣支撐更多的模擬和研究。微流控技術在體外模擬領域迅速發(fā)展。微流控技術固有的小尺寸特點使其恰好能與管狀上皮連接，以便通過灌注提供剪切應力和連續(xù)培養(yǎng)基。典型的微流體溶液利用人造膜進入上皮細胞而不通過ECM，ECM是參與分化和上皮-間充質轉化的細胞信號轉導中的關鍵參數(shù)。 

基于此，荷蘭Mimetas公司聯(lián)合創(chuàng)始人兼總經理Paul Vulto博士及其團隊開發(fā)了一種腸上皮屏障功能檢測系統(tǒng)來培養(yǎng)灌流的ECM支持的上皮細胞并以無膜方式測試它們的屏障功能。作為一個例子，研究者們開發(fā)了一種顯示細胞極化、緊密連接形成和關鍵受體表達的腸道上皮細胞模型。 40個腸道模型在器官芯片平臺（OrganoPlate）中以管狀形式生長，可從頂端和基底兩側進入。 

圖1展示了OrganoPlate平臺，其包含嵌入標準384孔微量滴定板格式的40個微流體細胞培養(yǎng)結構（圖1 a，b）。每個微流體通道結構由連接到相應孔的三條泳道組成的微孔板，其作為進口和出口以進入微流體培養(yǎng)基。圖1 c-j示出了微流體結構中心的垂直和水平橫截面以及生長出管狀結構的方法。首先，在中央通道引入ECM凝膠（圖1c，d）。ECM凝膠化后，將上皮細胞接種在一個側道中，通過將滴定板置于垂直位置，即站立在一側（圖1e-h），使它們直接沉積在ECM凝膠上。在細胞貼壁后，將該板水平放置在間隔搖桿上，該搖桿通過儲液器之間的相互調平引起流動。細胞增殖并開始鋪滿灌注通道的所有表面，形成匯合的管狀結構（圖1 i，j）。上皮的基底緊貼ECM凝膠并且可以與ECM凝膠層另一邊的灌注腔相通。為了模擬腸屏障，使用人結直腸腺癌細胞系（Caco-2）。圖1 l-p分別顯示了在第0,1,4,7和11天的管形成的相位對比圖。在第0天，將細胞接種到ECM上并開始在玻璃壁上增殖以形成融合管（圖1 n-p）。灌注對于管的形成至關重要。 3天內形成管并在第4天發(fā)現(xiàn)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.

圖2a展示了腸道管的共焦熒光顯微照片的三維重建。該管具有清晰的內腔并排列于凝膠和灌注腔的周邊。如ZO-1和Ezrin的免疫熒光染色所示，管中的Caco-2細胞顯示有緊密連接和刷狀緣形成（圖2a-d，g）。另外，細胞圓頂化說明流體運輸正常、上皮屏障功能完整（圖2f）。圖2h-j顯示了對于Glut-2，MRP2，ErbB1和ErBb2染色的管的最大強度投影圖像。與ECM接觸的細胞顯示轉運蛋白Glut-2，MRP2的表達強烈增加，并且ErbB1和ErbB2受體的表達較弱。這些染色結果說明了ECM在細胞分化和蛋白表達中起關鍵作用。此外，ECM凝膠表面的特性，如其（生物）化學成分和機械特性，使其可形成在體內觀察到的組織結構。

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管的屏障功能通過在管腔中灌注有熒光探針的培養(yǎng)基來評估，接著測定基底凝膠區(qū)域中的熒光水平。將高分子量熒光探針（150 kDa FITC-葡聚糖）和較低分子量探針（4.4 kDa TRITC-葡聚糖）都加入到通過管腔灌注的培養(yǎng)基中。在沒有完整管狀結構的情況下，熒光探針會滲入凝膠和基底側灌注通道（圖3a，d，g），而對于完全完整的屏障，熒光探針保留在管腔中圖（3b，e，h）。一旦（部分）喪失屏障功能，例如通過藥物誘導的毒性，熒光探針從管腔向基底側泄漏，在ECM中產生更高的信號（圖3c，f，i）。使用HCI系統(tǒng)測量屏障完整性，可并行監(jiān)測40個管。在達到0.4的熒光值時，認為管的屏障完整性丟失。在培養(yǎng)的第4,7和11天追蹤24管的屏障完整性，發(fā)現(xiàn)在第4天，所有管都是密封的，而在第7天和第11天，分別有三個和七個管泄漏。因此，在培養(yǎng)4天時進行屏障完整性測量。

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. 

本研究由荷蘭Mimetas公司聯(lián)合創(chuàng)始人兼總經理PaulVulto博士及其團隊完成，于2017年8月發(fā)表于Nature Communications。

論文信息：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.

論文鏈接：https://www.nature.com/articles/s41467-017-00259-3