2. Materials and Methods
2.1 Experimental procedure of hydrogel
adhesion
CS-MA was synthesized according to the previously reported method21. Briefly, 1.2 ml methacrylic anhydride was added
slowly to 1% (w/v) chitosan (CS, MW 30,000 Da) in acetic acid solution
(400 ml) and reacted for 4 h at 50 °C. The solution was neutralized by
10% (w/v) sodium bicarbonate solution and dialyzed against deionized
water for 3 days before lyophilization. The CS-MA was characterized by1H NMR with D2O as solvent and FTIR by
the KBr pellets method. The CS-MA adhesive solution was prepared by
dissolving the CS-MA powder at 4 wt% and VA-086 at 0.2 wt% into PBS
solution at pH 7.
Five representative hydrogels in tissue engineering were prepared
according to the methods outlined in the Supporting Information. The
prepared CS-MA solution was directly and uniformly applied to the
surface of two pieces of hydrogels. After 5 to 120 min of incubation,
one piece of hydrogel was placed on top of the other, and the two pieces
were integrated via slight compression using a glass slide for 5
to 120 min. Subsequently, the two pieces of hydrogels were exposed to
ultraviolet (UV) light for 30 s to crosslink the CS-MA monomer.
2.2 Characterization of bonded hydrogels
The adhesion energy of hydrogels was measured by T-peeling tests
(Supporting Information), while the mechanical properties were measured
by elongation and compressive testing using an Instron Series IX
Automated Materials Testing System (Zwick/Roell Z020)22. Moreover, the bonded surface was observed using a
field-emission scanning electron microscopy (FE-SEM). To prepare the
samples, the hydrogels were cut into slices perpendicular to the bonding
interface, and the slices were then transferred to a vacuum freeze dryer
for 24 h of dehydration and were sputter-coated with Pt before SEM
observation.
2.3 Construction of hydrogel-based microfluidic
chips
Firstly, a set of molds with diverse forms of microgroove forms were
designed with Solidworks software and manufactured through 3D printing
with white resin (Deed 3D Corporation, Guangzhou, China). Next, PDMS
stamps were prepared by casting prepolymerized PDMS on the 3D printed
molds and cured at 80°C for 3 h. After removing the 3D printed mold, the
monomer solution for different hydrogels was poured onto the PDMS stamp
and polymerized using the methods described in Supporting Information.
After discarding the PDMS stamp, the hydrogel layer with open channels
was coated with the CS-MA solution for 60 min, and the residue CS-MA
solution on the surfaces was subsequently removed before covered by a
flat gel sheet for 60 min and UV irritation for 10 min. For perfusion,
silicone tubes (1 mm×1.5 mm) connected with stainless steel tubes were
taped to the access holes on the chip and a red dye of Rho B was
injected into channels for dynamic perfusion.
2.4 Fabrication of liver-on-a-chip
Liver hepatocellular carcinoma HepG2 were from American Type Culture
Collection (ATCC) and cultured in DMEM with 10% FBS. Human umbilical
vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville,
MD, USA) and cultured in EGM-2 medium supplemented with 100 U/ml
penicillin and 100 mg/L streptomycin at 37°C and 5%
CO2.
To encapsulate the HepG2 cells inside the hydrogel, 10 wt% Gel-MA was
dissolved in phosphate buffer solution (PBS) at pH 7.4 and then
autoclaved at 121 °C for 30 min. The HepG2 cell suspension was mixed
with the Gel-MA solution which was supplemented with 0.2 wt% VA-086 to
obtain a final density at 106 cells/ml. The cell-laden
hydrogel was formed by pouring the solution into a sterilized PDMS
stamp, followed by a 30 s exposure to a UV light at 395 nm.
The Gel-MA and F127-DA sheets were coated with the CS-MA solution for 30
min in a sterilized glass mold. Then, the F127-DA layer was placed onto
the Gel-MA layer for a UV light exposure at 395 nm for 30 s to assemble
the chip. The hydrogel chip was then taken out and incubated in DMEM
medium with 10% FBS for 48 h at 37°C. Next, the HUVECs suspension in
EGM-2 medium at a density of 2×105 cells/ml was
injected into the channel of the chip. The chip was perfused by EGM-2
medium for another 2 days culture.
2.5 Cell activity analysis and
immunostaining
The cell viability of the chip was tested using a cell LIVE/DEAD assay
kit. After being rinsed with PBS, the chips were stained with Calcein AM
and PI solution at concentrations of 10 µM and 4 µM, respectively. After
incubation with the Calcein AM/PI solution for 40 min, the chips were
washed with PBS and observed under a fluorescence microscope (OLYMPUS
Ix70). Moreover, the MTT reduction was used to evaluate the cell
viability of HepG2 and HUVECs in the chips. Briefly, the chips were
rinsed by PBS before being immersed in 5 ml of the MTT-PBS at 1.15
mg/ml. After being incubated at 37°C for 3 h, the chips were washed by
PBS and then added by 5 ml of acidified isopropanol. After agitation for
3 h, the extraction was measured at absorbance of 570 nm on a
spectrophotometer.
The chips were immunostained by VEGF and MRP-2 to show the HUVECs and
HepG2 cells respectively. Briefly, the chip was blocked using 1.5% fish
skin gelatin in PBS containing 0.025% Trion-X 100 for 90 min at room
temperature and incubated with 1:100 diluted primary antibodies (Rabbit
Anti-VEGF and Mouse Anti-MRP-2) overnight at 4°C. After washing with PBS
and incubating with secondary antibody (DyLight 488-Goat Anti Rabbit IgG
and DyLight 594-Goat Anti Mouse IgG) for 1 h at room temperature, the
sample was stained by DAPI for 5 min and imaged by a fluorescent
microscope (OLYMPUS Ix70).
2.6 Statistical analysis
All data from cell experiments were analyzed by means ± SD from three
independent experiments. Comparisons between multiple groups were
performed with the ANOVA test by SPSS, or results from two different
groups were tested with the unpaired Student t-test. P-values less than
0.05 were considered statistically significant.
3. Results and discussion
3.1 Synthesis of hydrosoluble and photo-crosslinkable
CS-MA
Natural CS cannot be dissolved in a neutral pH solution due to the
hydrogen bonds between its amino and hydroxyl groups21. When the pH of CS solution (4 wt% CS dissolved in
0.1% acetic acid) was adjusted to 7, the pH-responsive CS precipitated
from the solution (Figure 2A). However, the pH-sensitivity of CS was
significantly attenuated after grafting of acrylate groups for the
reduced hydrogen bonds, resulting in the formation of a hydrosoluble
CS-MA in solution at pH 7 (Figure 2A).