Morphology of Caco-2 cells cultured in the microfluidic device.

<p>Caco-2 cells cultured in microfluidic system (A-G). Caco-2 cells cultured in Transwell system (H-I). Phase contrast images of the Caco-2 cells cultured in the microchambers on the thiol-ene microchip over 10 days. (A) Day 1; (B) Day 2; (C) Day 5; (D) Day 8. Cells multiply and differentiate over the days of culture. Folds in the monolayer of Caco-2 cells start appearing from day 4 of cell culture. The folds in the Caco-2 monolayers are more prominent from day 5 onwards (indicated by red arrows). Dark ‘patches’ also start appearing on the Caco-2 monolayers from day 7 onwards. They become more prominent from day 8 of cell culture (indicated by blue arrow). (E) Immunostaining of zonula occludens-1 (ZO-1) (green) and nuclei (magenta) for Caco-2 cells cultured in microfluidic device. (F) Caco-2 cells cultured in Transwell stained for nucleus and ZO-1 (Nuclei in red and ZO-1 in green) (Day 21). (G) Vertical cross-section view of the Caco-2 monolayer (nuclei in magenta, ZO-1 in green). The Caco-2 cells are ≈ 40 μm– 50 μm in height on day 3 of cell culture in the thiol-ene microfluidic chip. (H) Vertical confocal image of Caco-2 cells in Transwell (Nuclei in red and tight junctions in green). Immunofluorescence staining of nucleus and mucus on Caco-2 cells cultured in: (I) Thiol-ene microchip on day 10 of cell culture (nucleus in red, mucoprotein 2 (MUC-2) in green). The fluorescent images of the cells demonstrate that the cells have polarised into columnar cells of about 100 μm in height and formed villous-like structures. (J) Cells in the Transwell inserts were stained for nucleus and mucoprotein 2 at day 21. Only the nuclei could be fluorescently imaged but not MUC-2. Height of cells were about 25–30 μm at day 21. Cells were photographed at 10 x magnification. (Scale bar = 50 μm) (K) Differentiation of Caco-2 cells cultured in Transwell inserts and thiol-ene microchip as indicated by the activity of the brush border enzyme aminopeptidase. (n = 3, mean ± SEM; * p < 0.03, *** p < 0.001, **** p < 0.0001).