Fig 1.
Schematic illustrations of the thiol-ene based microfluidic chip for intestinal transport studies.
(A) Cross-sectional view of human intestinal microvilli. (B) Microarchitecture of one microchamber on the thiol-ene microfluidic chip consisting of upper and lower channel cell culture chambers separated by an ECM coated Teflon membrane. (C) Co-administration of an absorption enhancer to increase Caco-2 monolayer permeability.
Fig 2.
Fabrication process of the microfluidic device for Caco-2 culture.
(A) Schematic process of fabricating the different layers of the thiol-ene chip (i) process of fabricating upper and lower fluidic layers; (ii) process of fabricating the thiol-ene coated Teflon membrane. (B) Exploded view of the high throughput multi-layer thiol-ene microchip for cell culture (Dimension of microchip: 76 mm x 52 mm x 2.7 mm). Thickness of the modified membrane is 0.3 mm. The membrane is coated with a thiol-ene mixture on both sides to ensure good bonding between the chip layers. Fluids were pumped in the upper and lower layers. (C) SEM images of Teflon membrane (Top view). Surface morphology was changed significantly after coating a layer of thiol-ene. The surface of the membrane has become very smooth after coating and curing a layer of thiol-ene (as indicated by red arrow). (D) Expanded view of Teflon membrane that was masked off and rinsed with methanol, maintained its porous structure in these areas.
Fig 3.
Biocompatibility of thiol-ene.
(A) Metabolic activity of Caco-2 cells (AlamarBlue® assay) over day 0 to day 21 of in vitro cell culture. (n = 3; mean ± SEM; scale bar = 50μm) (B) Microscopic images of Caco-2 cells cultured in thiol-ene microchip (i) Phase contrast image of Caco-2 cells cultured on day 1 of in vitro cell culture; (ii) Day 11 of in vitro cell culture; (iii) live/dead cell staining of Caco-2 cells on day 11 of in vitro cell culture.
Fig 4.
Morphology of Caco-2 cells cultured in the microfluidic device.
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).
Fig 5.
Barrier functions of Caco-2 cell monolayers in the microfluidic device or Transwell system.
(A) TEER measurements of Caco-2 cells cultured in thiol-ene microchip and Transwell inserts for the same cell concentration of 2.55 x 105 cells/cm2. (Here, number of measurements per data point n = 12 for microfluidic device; and n = 5 for Transwell inserts). (B) Effect of test compounds alone or with TDM on Caco-2 TEER in the Transwell or microfluidic system, immediately after the experiment or following 24 h recovery in medium. (C) Rh 123 accumulation profile in the basolateral and apical chambers across Caco-2 monolayers in microfluidic device. Data points represent mean ± SEM (n = 3). Where ns = not significant and *** p < 0.001. (D) Immunofluorescence staining of P-gp on Caco-2 cells cultured in microfluidic device (nucleus in magenta, P-gp in cyan). Magnification 20x; scale bar = 50 μm.
Fig 6.
Permeability studies of Caco-2 layers with different compounds.
(A) Schematic drawing of membrane enhancer and drugs flowed across the Caco-2 cells cultured in the microfluidic device. Insert is an enlarged schematic view of disrupted tight junctions upon co-administering the membrane enhancer TDM. (B) Comparison of permeability profiles of different compounds with or without TDM experimented on Caco-2 layers cultured in microfluidic device versus Transwell: (i) mannitol, (ii) FD4 and (iii) insulin. Data points represent mean ± SEM (n = 4; *p ≤ 0.02, **** p < 0.0001 and ns = not significant).