Result
Design
and operation of the generic organ-on-a-chip platform
As shown in Fig. 1A , each chip consists of 32 chip units, with
each chip unit consisting of three horizontally arranged wells. The
holes at both ends are connected by the passageway of the lower channel,
where culture the medium was perfused with a rocker. The middle hole is
designated for cell culture and is separated from the lower layer by a
transparent PET porous membrane. The specifications of the porous
membrane can be chosen according to specific requirements. This design
not only reduces the consumption of culture medium and other materials
but also facilitates pipetting operations and characterizations with
enhanced convenience.
To validate the usability of the chip, we constructed a simple
gut-on-a-chip model. In the specific
experiments,
Caco-2 and HT-29 cells were seeded
in a 9:1 ratio, totaling about 50,000 cells, in the central well of the
chip unit to form a compact layer of intestinal epithelial cells on the
membrane (Fig. 1B) . Then the chip was placed on a rocker at a
speed of 10 rpm and an angle of ± 8° to induce basal fluid flow. To
further investigate the fluid dynamics within the chip, computational
simulations were performed using COMSOL software.
The results indicate that
the fluid shear stress
(~0.4
dyn/cm2) at the center of the membrane was relatively
lower compared to the surrounding regions (0.8~2
dyn/cm2) (Fig. 1C) . Additionally, the fluid
shear stress beneath the membrane remained stable at around 0.4 dyn/cm2(Fig. S3) . These observed shear force values align well with
physiological
data.[26]
It should be noted that although we
focus on seeding cells on the upper side of the membrane in this work,
the chip design (Fig. 1B) allows for easy cell seeding on the
opposite side of the membrane by simply seeding cells in the bottom
channel and inverting the chip. This unique feature enables the
co-culture of multiple cell types, thus highlighting the scalability of
our system and its potential for diverse
applications. For instance, a
multicellular model of inflammatory bowel disease can be established on
this platform by seeding intestinal epithelial cells in the central
well, inoculating intestinal endothelial cells on the opposite side of
the membrane, and introducing immune cells into the bottom channel.
Additionally, the versatility of the system extends further with the
ability to control the cultivation conditions. By adjusting the angle
and speed of the rocker, different shear forces can be easily applied to
achieve distinct cultivation conditions for various experimental needs.
Characterization of the
intestinal epithelium on theorgan-on-a-chip platform
Previous
studies have elucidated the crucial role of basal-side fluid flow in
promoting the morphogenesis of Caco-2 cells.[27,
28] This effect is attributed to the accumulation of Wnt signaling
pathway inhibitors, such as DKK-1, beneath the membrane under static
conditions, leading to hindered cell proliferation and impaired
morphogenesis of the Caco-2 cell layer. The introduce of fluid flow can
effectively eliminate the accumulation of these inhibitory molecules. In
our research, a rocker with adjustable angle and speed was employed to
induce reciprocating fluid flow. This dynamic flow not only dilutes the
inhibitory molecules but also facilitates the occurrence of
intestinal-like morphogenesis that closely mimics in vivo conditions.
In our chip system, a prominent distinction was observed between the
perfused and static conditions. Under perfusion conditions, the
intestinal cell layer cultured on the chip developed 3D villous
structures, while cells without perfusion predominantly remained in a
single-layer state. After three days of culture with fluid, noticeable
disparities were observed under an optical microscope (Fig.
2A) . The intestinal cells under flow conditions formed convex and
stacked structures, appearing as dark arc curves or circular structures
with well-defined boundaries. Similar to transwell or petri dish
culture, intestinal cells can be cultured on the chip for up to 30 days,
but after the sixth day, there is no significant change in the
morphology of the cells under the optical microscope (Fig. S4) .
Consequently, cells after six days of fluid introduction were taken for
characterization.
Live/dead staining images revealed that most of the cells exhibited
normal growth after six days of perfused culture on the chip(Fig. 2D) . Additionally, a CCK-8 assay was performed to assess
cell viability under static and perfused conditions, demonstrating that
the viability of cells cultured on the chip was comparable to or even
higher than that of cells cultured in a static state (Fig. 2E) .
Furthermore, differences in cell layer height and microvillus morphology
were observed through H&E staining (Fig. 2B) and SEM(Fig. 2C) , respectively. These results highlight the successful
establishment of an intestinal-like morphogenesis on our chip system
under perfusion conditions.
Improved functionality of
intestinal epithelium under flow condition
To assess the integrity of the cell
layer, we evaluated the permeability of the intestinal barrier by
measuring the fluorescence intensity of FITC-dextran with varying
molecular weights that permeated the cell layer within a specific time
frame (Fig. 2F) . Notably, regardless of molecular weight, the
Papp of dextran in the perfused chip was significantly
lower than that observed in the static culture. In the 1-hour
experiment, the Papp of 4 kDa-dextran was as low as 5 x
10-7cm/s, indicating a substantial enhancement in the
integrity of the intestinal barrier in the perfusion environment.
To further assess the integrity of the intestinal barrier, we measured
the TEER using a commercially available instrument, EVOM3, which was
facilitated by our chip design (Fig. S3) . Interestingly, there
was minimal disparity between the TEER values of the perfused and static
cultures (Fig. 2G) ,
deviating from previous studies. Both groups displayed an upward trend
in TEER over the six-day culture period, eventually reaching an
approximate value of 100 Ω·cm2. While this value may
seem relatively low compared to other reports, it is essential to note
that co-culturing Caco-2 and HT-29 cells typically leads to lower TEER
values.[29, 30] Consequently, the observed TEER
value aligns more closely with physiological conditions in humans.
Immunofluorescence staining of ZO-1 (Fig. 3C) revealed the
presence of well-formed tight junctions and brush border formation in
the perfusion environment, indicating a more robust barrier compared to
static culture. Moreover, the detection of Villin (Fig. 3F) in
apical cells provided additional evidence for the presence of intestinal
villi in the perfusion environment, which was further supported by SEM
images showing well-defined microvilli (Fig. 2C) . Furthermore,
by analyzing the distribution of nuclei in images at different levels,
we could clearly visualize the hollow structure of intestinal villi(Fig. 3A) . The expression of MUC-2 was also detected. As shown
in Fig. 3D , MUC-2 was evenly distributed on the intestinal
villus, and superposition with the bright field image provided a clear
view of the villi’s shape. Side-view confocal 3D images allowed us to
calculate the height of the villi, and the results indicated that the
average villi height in static culture was about 33 μm (Fig.
3E) , while the average villi height on the chip was close to 90 μm.
Additionally, the expression of Ki67 (Fig. 3G) indicated active
proliferation of intestinal cells, suggesting the potential for further
growth into higher intestinal villi.