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.