Page 370 - IJB-10-6

International Journal of Bioprinting 3D-printed NAFLD model

hemisuccinate, Nile red, and calcein-AM were obtained three times. After complete dissolution and cooling to
from Shanghai Aladdin Biochemical Technology Co., Ltd. approximately 37°C, the solution was mixed with the cell
(China). Fetal bovine serum was sourced from Lonsera suspension to prepare cell-laden bioinks with varying
(Uruguay). Trypsin-EDTA, PAS staining solution, and concentrations. The gelatin concentrations were set at
FAA fixative were obtained from Wuhan Servicebio 3, 5, and 7%, and the alginate concentrations were set
Technology Co., Ltd. (China). Anhydrous calcium at 0.5, 1, 1.5, and 2% (w/v) for subsequent experiments.
chloride, DAPI staining solution, propidium iodide (PI), Next, the fibrinogen and laminin powders were weighed
Triton X-100, xylene, absolute ethanol, palmitic acid, oleic and dissolved in phosphate-buffered saline (PBS) to
acid, and bovine serum albumin (BSA) were obtained prepare stock solutions with concentrations of 10 mg/
from Sangon Biotech (Shanghai) Co., Ltd. (China). mL for fibrinogen and 100 μg/mL for laminin. Different
Albumin and CYP3A4 primary antibodies were purchased concentrations of fibrinogen (0.5, 1, 3, and 5 mg/mL) and
from Proteintech Co., Ltd. (China). Secondary antibodies laminin (10, 30, 50, and 70 μg/mL) were then added to
Alexa Fluor 488 and 594 were from Invitrogen (USA). the optimized gelatin/alginate bioinks to create optimized
Commercial Tissue/Cell RNA Rapid Extraction Kits, bioinks suitable for liver tissue culture.
cDNA Synthesis Kits, and SYBR Green were obtained from
Mei5 Biotechnology, Co., Ltd. (China). 2.5. Printing and culturing of liver tissue
A HepaRG cell suspension with a density of 4 × 10 cells/
6
2.2. Cell culture mL was prepared and thoroughly mixed with bioink at a
HepaRG cells were cultured in DMEM supplemented 1:1 ratio to produce the cell-laden bioink. The cell-laden
with 1% penicillin-streptomycin, 5 μg/mL insulin, 2 mM bioink was injected into a sterile printing syringe and
glutamine, 50 μM hydrocortisone hemisuccinate, and 10% loaded into the temperature-controlled printhead of the
fetal bovine serum. The cultures were maintained at 37°C 3D bioprinter. The syringe temperature was set to 5°C,
in a humidified incubator with 5% CO₂. When the cells and the ambient temperature was maintained at 25°C. The
reached 90% confluence, they were passaged with 0.25% liver tissue was printed in a layered square grid structure
trypsin-EDTA. The culture medium was changed every at an extrusion pressure of 0.178 kPa and a speed of 5
2–3 days. mm/s. Upon completion of printing, the liver tissue was
immediately transferred to a 2% CaCl solution for 5 min
2
2.3. Design of the printing model to induce crosslinking. After crosslinking, the tissue was
The design of the liver tissue printing model was rinsed with PBS and then cultured. HepaRG is a human
developed via BIOCAD software (provided with the hepatic progenitor cell line that requires induction to
REGENHU/R-GEN 200). To ensure adequate nutrient differentiate into functional hepatocytes and biliary
supply and timely removal of metabolic waste, the epithelial cells. In this study, we used the conventional
printing model was designed in a grid pattern. The method of adding 2% dimethyl sulfoxide (DMSO) to the
overall structure is a 10 × 10 mm square, printed with culture medium for differentiation. 19
a 0.33 mm inner diameter needle and divided into four
layers. The printing method is extrusion-based, with a 2.6. Cell viability
line spacing of 1 mm (Figures 1A and S1A, Supporting Calcein-AM can permeate the cell membrane, where
Information). The printing direction of the first and intracellular esterases hydrolyze it to calcein, which
third layers is horizontal, whereas the second and fourth remains inside the cells and emits green fluorescence,
layers are printed vertically. This alternating printing thereby labeling live cells. PI is an ethidium bromide
direction forms a grid-like structure. analog that binds to double-stranded DNA, producing
red fluorescence under UV excitation. PI can only enter
2.4. Preparation of the bioink cells and stain the nucleus when the cells are dead and
Based on our previous experience, we further optimized their membranes are compromised. In the experiment,
29
the bioink formulation to enhance its printability, the working concentration of PI was 10 μg/mL, and the
mechanical properties, and biocompatibility, making it working concentration of calcein-AM was 5 μM. After
more suitable for liver tissue printing. A precise amount the cultured liver tissue was harvested, it was incubated
of gelatin and alginate powder was weighed and sterilized with the calcein-AM/PI working solution at 37°C for 30
by ultraviolet (UV) lamp irradiation for 1 h before use. min. Imaging was then performed via a laser light source
The powders were then dissolved in the culture medium dual spinning disk confocal high-content analysis system
and incubated in a water bath at 60°C for 1 h. To ensure (CQ1; YOKOGAWA, Japan). To ensure the accuracy and
thorough pasteurization, the solution was removed every reliability of calcein-AM/PI staining, we conducted three
hour and cooled for 30 min, and this process was repeated replicate stains for each experimental condition.

Volume 10 Issue 6 (2024) 362 doi: 10.36922/ijb.4312