Fig 1.
Human Gut-on-a-Chip microfluidic culture device.
Photograph (A) and schematic diagram (B) of the human Gut-on-a-Chip device. (C) Phase contrast micrograph of human Caco-2 intestinal epithelial cells cultured for 6 days in a Gut-on-a-Chip under apical flow (30μl/hr; 0.02 dyne.cm-2) and cyclic mechanical strain (10% at 0.15 Hz); bar, 100 μm. (D) Apparent permeability (Papp) of the epithelium assessed by adding fluorescent inulin-FITC daily to the upper channel for 6 days after seeding (n = 3 chips). Note that an ECM-coated Gut-on-a-Chip without cells used as a control exhibited a very high permeability. (E) Confocal immunofluorescence micrograph of human villus intestinal epithelium formed inside the Gut-on-a-Chip and stained for villin (yellow) to visualize the apical brush border and nuclei (blue); bar, 10 μm. (F) Scanning electron micrograph of the apical surface of the villus epithelium cultured for 6 days in the Gut-on-a-Chip under flow and mechanical strain. Note the microvilli at the surface of the cells (bar, 10 μm).
Fig 2.
Coxsackie virus B1 readily infects the human Gut-on-a-Chip.
(A) Phase contrast micrographs of the villus epithelium within the Gut-on-a-Chip apically infected with CVB1 at 6, 24 and 48h post infection (hpi); bar, 100 μm. (B) Confocal immunofluorescence micrographs of uninfected and apically infected villus epithelium 24hpi that were stained for CVB1 (green), F-actin (red) and nuclei (blue). Note the destruction of villi in infected samples; bar, 100 μm. (C) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after apical infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi).
Fig 3.
CVB1 release is polarized towards the epithelial lumen.
(A) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after basal infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi). (B) Confocal fluorescence micrographs of apically and basally infected gut chips at 6 and 24 hpi, showing horizontal sections at the base, middle and top of the villi (left to right columns). Infected chips were stained for CVB1 (green) and nuclei (blue); bar, 100 μm.
Fig 4.
Apical fluid flow generates a gradient of CPE.
Phase contrast micrographs of apically and basally CVB1-infected Gut-on-a-Chips at 24hpi. The flow direction (from the inlet to the outlet) is from left to right (the arrow). Note the increasing CPE intensity towards the downstream outlet; bar, 100 μm.
Fig 5.
Cytokine secretion and caspase-3 expression in infected gut chips.
IP-10 (A) and IL-8 (B) were quantified in effluents from the apical (gray bars) and basal (white bars) microchannels in gut chips that were either uninfected or infected apically or basally (n = 4 chips/condition; *p < 0.05, **p < 0.001) at 24 hpi. (C) Epithelium lining gut chips that were uninfected (controls C1 and C2), or infected apically (A1, A2, A3) or basally (B1, B2, B3, B4), or infected apically or basally, were lysed and pro-caspase-3 and cleaved caspase-3 levels were visualized on the gel as indicated.