Materials and methods
Preparation and characterization of GO hydrogel The GO suspension was purchased from Suzhou Tanfeng Graphene Technology Co., Ltd,Suzhou, China, centration of 10 mg/mL and graphene diameter between 0.5 and 5 μm. FeCl3·6H2O (Macklin) was dissolved in deionized water to prepare a 0.5 mmol/L stock. GO suspension was vialed into 50-mL centrifuge tubes and concentrated up to 31 mg/mL by centrifuging at 9500 rpm for 30 min. After removal of the supernatant, the hydrogel was mixed with FeCl3 and stirred for 20 min. FeCl3 particles would appear at the beginning and dissolve gradually with stirring when the concentration of Fe3+ was ≤ 8 mmol/L in GO hydrogel. Rheology of the prepared GO hydrogel was tested by the rheometer MCR 302 (Anton Paar GmbH).
3D printing set up Optimized GO hydrogel was collected into 1-mL syringes with nozzles (inner diameter ranging from 0.11 μm to 0.42 μm) (fig. 2a). After assembling the GO-loaded syringe onto the desktop cell 3D printer ALPHA-CPD1 (SunP Biotech Co. Ltd.), the GO hydrogel was printed into woodpile structure, with a 20 mm × 20 mm × 4 mm cuboid dimension, at room temperature. The printability of the hydrogel was verified by printing with different filament distances, from 1 mm to 3 mm.
Lyophilizing process and porous structure control The well-controlled unidirectional temperature field from -20 ℃ to room temperature was established within a custom-made mold as previously described (Fang, Zhang, Zhang, Gong, & Sun, 2019). After freezing, all samples were then dried at -50 °C and 0.05 mbar for 48 h in a freeze drying machine Alpha 1-2 LDplus (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Afterward, Samples were observed under scanning electron microscope (SEM), and diameters were measured by Image Pro Plus 6.0 software.
Cell adhesion measurement The culture medium was composed of Dulbecco’s modified Eagle’s Medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 0.05% insulin, 5×10-5 mol/L hydrocortisone hemisuccinate, 1% penicillin-streptomycin, 1% Gluta Max™ supplement, and 1% MEM nonessential amino acid solution (NEAA). Printed samples with filament distance of 1 mm and uniform freezing condition of -80 ℃, were used for the cell experiments. For sterilization, GO samples were soaked in 70% ethanol for 15 min under UV light and then transferred to PBS on a rotary shaker for 45 min to elute the residual ethanol. For cell attachment experiments, collagen type I was coated on the GO structure to improve the biocompatibility and cell attachment. Briefly, the sterilized sample was incubated in 0.3 mg/mL collagen solution for 1 h and washed with PBS. The samples were moved to untreated 6-well plates with 2 mL of 1×106/mL HepaRG cell suspension in each well. After two days of co-culture, cell attachment and viability were evaluated by live/dead assay. The samples were carefully washed with PBS and then stained with calcein acetoxymethyl (calcein AM) and propidium iodide (PI) to identify the live-cell condition. The samples were subsequently observed under a confocal microscope.
Conflicts of Interest: The authors have no conflicts of interest to declare.