Figure 8 Identification of HepG2 cells and HUVECs by immunostaining. (A)
HUVECs (green, stained with VEGF) attached on the surface of channel.
(B) HepG2 cells (red, stained with MRP-2) located in Gel-MA hydrogel.
Nuclei were stained blue by DAPI. White line was the borderline of
microchannel.
To visually depict the cellular organization within the chip, we
immunostained MRP-2 and VEGF to identify HepG2 cells and HUVECs,
respectively. MRP-2 is specifically expressed in hepatic cells that
mediates the drug transportation 36, while VEGF is
secreted by HUVECs but not HepG2 cells to regulate the endothelial
angiogenesis and permeability 37. As shown in Figure
8A, VEGF-positive HUVECs were located in the channel zone, while the
MRP-2 positive HepG2 cells were dispersed inside Gel-MA hydrogel.
Zoom-in images showed the specific expression of VEGF (green, Figure 8B)
and MRP-2 (red, Figure 8C) by HUVECs and HepG2 cells, respectively. The
HepG2 cells formed irregular aggregates in Gel-MA hydrogel (Figure 8C),
consistent with previous reports 38.
While the above example did not cover all the possible applications of
layer-by-layer adhesion in constructing hydrogel-based microfluidics, it
presented a novel and straightforward concept for fabricating complex
and precise 3D architectures. Moreover, another example was shown in
Figure S5, where a bilayer chip was constructed for coculture of HepG2
cells and fibroblasts. To replicate the physiology of organs, current
biofabrication techniques require the spatially-precise organization of
cells and extracellular matrix, mimicking physical cues in vivosuch as chemical components, topography, stiffness and shear stress2. Hence, the layer-by-layer adhesion could aid in the
rational design of in vivo -like tissues, as multilayered
hydrogels facilitate the mimicking of chemical and structural properties
of extracellular matrix in vivo . This paper demonstrated the
construction of liver-on-a-chip with vascular structure by organizing
liver and endothelial cells in the appropriate positions (Figure 7),
well mimicking the vasculature and sustaining cellular activity in the
liver 39. Similarly, the layer-by-layer adhesion could
also benefit the design of other organs-on-chips such as skin consisting
of various layers with different extracellular matrix and cells40,41.
4. Conclusion
We have introduced a new method called “layer-by-layer adhesion” for
constructing hydrogel-based microfluidic chips. Four types of hydrogels
were well stitched together using the adhesive properties of CS-MA,
which exhibited adhesion energy of 1.2-140 N/m. The CS-MA diffused into
the hydrogels and then crosslinked at the interface of two hydrogels to
create a density zone. Such adhesion maintained good stability even
after autoclaving, stretching and twisting. This method allowed for the
assembly of perfusable hydrogels with snail, spiral, vascular-like and
bilayer microchannels with high resolution. As an example of
application, we used this method to construct liver-on-a-chip based on
Gel-MA/F127-DA layers and coculture of HepG2 cells with HUVECs. Our
method of layer-by-layer adhesion offers a new way to design 3D
architectures in hydrogels and construct microfluidic organs-on-chipsin vitro .
5. Data availability
statement
The data that supports the findings of this study are available in the
supplementary material of this article.
6. Conflicts of interest
There are no conflicts of interest to declare.
7. Acknowledgement
We gratefully acknowledge the financial support of this study by NSFC
(National Natural Science Foundation of China, No 22078287 and
21978257).
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