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).