Page 404 - v11i4

International Journal of Bioprinting Bioprinted liver dECM/GelMA tumor model

room temperature, and their mass wet weight (Ws) was of the hydrogel precursors were added to each scratch well.
measured at 0, 2, 4, 6, and 12 h. The water swelling ratio Images of the cells were captured at 0, 12, and 24 h using an
(Sr) was calculated as a percentage using the following Olympus microscope (n = 3). Finally, quantitative analysis
formula (Equation III): was performed using Image J software (National Institutes
of Health, Germany).
Sr = (Ws – Wd)/Wd × 100% (III) Liver-derived GelMA/dECM bioinks were utilized
to fabricate an in vitro 3D liver cancer tumor model.
To evaluate the degradation behavior of the composite Briefly, HepG2 cells were collected by centrifugation and
porous scaffold, it was first subjected to swelling suspended in the bioinks at a density of 1 × 10 cells/mL.
6
equilibrium, then freeze-dried and accurately weighed. The 3D printing procedure was performed as described
Subsequently, the scaffold was immersed in a culture previously. Finally, the bioprinted scaffold was transferred
medium and incubated in a 37°C cell culture incubator. to a six-well plate, rinsed with PBS, and incubated in the
After 3, 6, and 9 days, the scaffold was retrieved, freeze- culture medium.
dried again, and its mass was recorded. The degradation To assess the cell viability of the tumor model, 3D
rate of the hydrogel was calculated using the following scaffolds (3D control and 3D-dECM) cultured for 2, 4, 6,
formula (Equation IV):
and 8 days, along with 2D cultured cells, were removed and
washed twice with PBS to remove the culture medium. The
D = (Wc – Wd)/Wc × 100% (IV) cell proliferation was analyzed using CCK-8 for both 2D
and 3D cultures. Meanwhile, live/dead staining was used
Cell cytotoxicity of printed scaffolds was assessed to assess the survival state of cells.
using L929 cells. The cells were cultured in Roswell Park For cell morphology, the sizes of tumor spheroids were
Memorial Institute (RPMI) medium and maintained at monitored during 3D culture, with photographs taken for
37°C with 100% humidity and 5% carbon dioxide. The 2, 4, 6, and 8 days using an optical microscope. The sizes
cells were passaged every 2 days. The cytotoxicity of the of tumor spheroids were quantified using Image J software
scaffolds was assessed by extract assay. Briefly, using the (National Institutes of Health, Germany). Cells cultured in
culture medium as an extraction solvent, the freeze- 2D were observed using phalloidin staining.
dried scaffolds were soaked at a ratio of 0.2 g/mL for 24
h. A cell density of 8 × 10 was seeded into each well, and To assess liver function, culture supernatants were
3
100 μL of the corresponding scaffold extract was added. collected on day 8, and parameters including alanine
Three replicates were set for each group. After 24 and 48 aminotransferase (ALT), aspartate aminotransferase
h of culture, CCK-8 was added and viability was a 2-h (AST), albumin (ALB), and total bile acid (TBA) were
incubation; the absorbance at 562 nm was measured to analyzed using a multimode microplate reader.
calculate cell viability. Similarly, live/dead staining was
performed using calcein-acetoxymethyl/propidium iodide 2.6. Chemosensitivity assay
staining, where green indicates live cells and red indicates The chemosensitivity of the 3D and 2D culture samples
dead cells, to observe cell growth. was assessed by treating them with various concentrations
of different drugs. After 6 days of cultivation, the samples
2.5. Three-dimensional in vitro tumor were treated with PTX and amrubicin hydrochloride.
model construction Specifically, PTX was dissolved in 0.1% dimethyl sulfoxide
Human hepatocellular carcinoma (HepG2) cells were and diluted with the culture medium, while amrubicin
cultured in five types of gels at a density of 6 × 10 cells/ hydrochloride was dissolved in ultrapure water and
4
mL and were crosslinked for 30 s with a 405 nm UV light. similarly diluted. The drug concentrations used were 10,
An appropriate medium was added to wash the scaffold, 20, 30, 50, 100, and 200 µg/mL. Each well was filled with
and the liquid culture was then changed. After 1, 2, and the corresponding drug concentration, and cell viability
3 days of culture, the absorbance was measured using and survival rate were measured using the CCK-8 assay
the CCK-8 method. Simultaneously, the effect of the after 12 and 24 h. Live/dead staining was performed
above hydrogels on HepG2 migration was determined simultaneously to assess cell survival.
using scratch assays. First, approximately 1 × 10 cells
6
were seeded onto a six-well plate and incubated until 2.7. Statistical analysis
they attained 80% confluence. A sterile 200 μL pipette tip At least three independent experiments were performed,
was used to scratch the cell monolayer across the center, unless otherwise stated. One-way analysis of variance and
creating a cross-shaped wound in each well. Then, 500 μL t-tests were used to analyze the differences between the

Volume 11 Issue 4 (2025) 396 doi: 10.36922/IJB025160142