List of Figures
Figure 1. Microfluidic device characterization. (a) Schematic
showing the geometry of the liver-on-a-chip used to develop the
NAFLD-on-a-chip model and for testing the effects of natural
polyphenols. In (a) top view and section, with magnified details, of the
chip designed as a human hepatic sinusoid, showing the device
microarchitecture with a grid of closely spaced and parallel
microchannels that simulate the endothelial-like barrier. (b)
Microscopic 3D image showing a cord of tightly packed HepG2 cells
(purple), injected through the cell loading channel (cell inlet),
surrounded by the mass transport channel (perfusion channel), which is
bordered by the endothelial-like barrier (microchannel barrier) for
diffusive transport. Dimensions are expressed in μm.
Figure 2. HCA of the effect of Quercetin and Hydroxytyrosol on hepatic
steatosis in the Liver-on-a-Chip microfluidic device. AdipoRed assay (a)
for the analysis of intracellular triglyceride accumulation after the
treatment of on-chip HepG2 cultures with different mixtures of FFAs (1mM
final concentration) for 48h, and representative confocal micrographs of
the lipid overload (green cells in b). In (a) the histograms show the
MFI of the exogenous FFA overload in HepG2 cells referenced to the
controls. AdipoRed assay following the addition of Quercetin (c and
representative confocal micrographs in d) or Hydroxytyrosol (e and
representative confocal micrographs in f), at 10 μM for 48h, to the same
combinations of FFAs shown in (a and b). The effect of Quercetin (red
bars) and Hydroxytyrosol (green bars), in combination with the different
mixtures of FFAs, on intracellular triglyceride accumulation (AdipoRed)
is compared to that of the corresponding FFA supplementation.
Micrographs in (b, d and f) are maximum intensity projection images in
Z-axis; ROIs of the microchambers occupied by the cells are shown.
Values are reported as mean ± SEM; n = 3 at least; * p<0.05,
** p<0.01. Scale bars: 50 μm in all images. Legend: Ctrl
(Control); Que (Quercetin); HT (Hydroxytyrosol).
Figure 3. HCA of the effect of Quercetin and Hydroxytyrosol on cell
viability under conditions of steatosis in the Liver-on-a-Chip
microfluidic device. HCA of cell viability following the different
treatments with FFAs (a), at 1mM for 48h, and in combination with
Quercetin (c and d) or Hydroxytyrosol (e and f) at 10 μM for 48h. In
(a), (c) and (e) the histograms show the percentage of live cells for
Ctrl (white bars) and treated groups (colored bars, indicated as w/ Que
or w/ HT in the legends). Representative confocal micrographs (b, d and
f) of the ROIs of the microchambers occupied by the cells, showing
nuclei of dead cells (PI dye in red) vs. total nuclei (Hoechst
33258 dye in blue). The effect of Quercetin (c in red) and
Hydroxytyrosol (e in green), in combination with the different mixtures
of FFAs, on cell viability (PI/Hoechst 33258) is compared to that of the
FFAs alone (w/o Que and w/o HT, respectively). Values are reported as
mean ± SEM; n = 3 at least; * p<0.05, ** p<0.01, ***
p<0.001, **** p<0.0001. Scale bars: 50 μm in all
images. Legend: Ctrl (Control); Que (Quercetin); HT (Hydroxytyrosol).
Figure 4. HCA of the effect of Quercetin and Hydroxytyrosol on oxidative
stress under conditions of steatosis in the Liver-on-a-Chip microfluidic
device. HCA of total ROS/RNS and Superoxide production following the
different treatments with FFAs (a, b and representative confocal
micrographs in c), at 1mM for 48h, and in combination with Que (d, e) or
HT (g, h) at 10 μM for 48h. Representative confocal micrographs of the
ROIs of the microchambers occupied by the cells treated with the FFAs in
(c), FFAs w/ Que in (f) and FFAs w/ HT in (i) showing the ROS/RNS
production (using the ROS/RNS detection probe, green cells) and
Superoxide production (using the Superoxide detection probe, red cells).
In (a) and (b) the histograms show the MFI expressed as the ratio
between FFA-treated cells and controls; the ROS inducer (Pyocyanin) at
500 μM was used as a positive control. The effect of Quercetin (d, e, w/
Que, red bars) and Hydroxytyrosol (g, h, w/ HT, green bars), in
combination with the different mixtures of FFAs, on ROS/RNS production
(d and g) and Superoxide production (e and h) is compared to that of the
FFAs alone (white bars) in each graph with histograms showing MFI values
expressed as in (a, b). Micrographs in (c, f and i) are maximum
intensity projection images in Z-axis. Values are reported as mean ±
SEM; n = 4 at least; * p<0.05, ** p<0.01, ***
p<0.001. Scale bars in (c, f and j): 50 μm in all images.
Legend: Ctrl (Control); Que (Quercetin); HT (Hydroxytyrosol).
Figure 1S. Log2-fold change (log2 F.C.) analysis of intracellular lipid
accumulation and oxidative stress following Quercetin and Hydroxytyrosol
addition to the different FFA mixtures. The fold decrease in
intracellular triglyceride accumulation (via the AdipoRed assay)
due to Que (a) and HT (b) administration is reported; the fold decrease
in oxidative stress (ROS/RNS in c and d, and Superoxide species in e and
f, respectively) due to Que (c and e) and HT (d and f) administration is
reported. All values are reported as mean ± SEM, n = 3 at least, and
plotted as the log2 of the ratio between the different mixtures of FFAs
w/ Que or HT and the FFAs alone normalized vs. blank control
(i.e., no effect); statistical significance was evaluated by one-way
ANOVA, * p<0.05, ** p<0.01, *** p<0.001,
**** p<0.0001. Legend: Ctrl (Control); Que (Quercetin); HT
(Hydroxytyrosol).