Page 403 - v11i4

International Journal of Bioprinting Bioprinted liver dECM/GelMA tumor model

solution for 14 h, followed by treatment with a DNase For morphology characterization, the bioinks with
solution of 100 U/mL for 6 h, and washed with sterile different ratios were cross-linked using ultraviolet (UV)
PBS for 3 days, with 5 mg ciprofloxacin added to prevent light and subsequently immersed in PBS for 12 h to reach
bacterial contamination. The resulting decellularized swelling equilibrium. After freeze-drying, the samples
liver matrix (DLM) was collected, washed with sterile were fractured at the midpoint and coated with gold for
PBS, freeze-dried, and stored at –20°C overnight. After 60 s using a conductive gel to secure them onto the holder.
72 h of freeze-drying, the DLM was then digested in a The fracture surface morphology of the hydrogels was
solution of hydrochloric acid and pepsin. The digestion then observed using scanning electron microscopy (SEM).
process took place at 37°C and 250 rpm for 6 h. The Additionally, the internal pore sizes of the hydrogels were
pepsin concentration was maintained at 1 mg/mL, with quantitatively analyzed using the Nano Measure software.
a hydrochloric acid concentration of 0.1 M and a ratio of For rheological measurements, all tests were conducted
1:10 between the weight of DLM and the amount of pepsin using a rotational parallel plate rheometer (MCR 302,
used. After digestion, the pH was adjusted to neutral using a Anton Paar, Austria). A volume of 5 mL of each bioink was
sodium hydroxide solution. The digested mixture was then pipetted onto the bottom plate of the stage, with the gap
subjected to dialysis with a 1000 D cutoff membrane for 3 distance set to 1 mm. Shear rate sweeps were conducted in
days, with water replenished every 4 h. Finally, the dialyzed the range of 0.1–800 s at room temperature. Temperature–
–1
dECM was collected and freeze-dried for further use. viscosity measurements were performed from 40 to 15°C.
Both native liver tissues and DLM were immersed in a The storage modulus (G’) and loss modulus (G’’) of the
4% paraformaldehyde solution for 24 h to fix the tissues. bioinks were evaluated by changing the temperature from
Following fixation, the tissues were embedded in the 40 to 5°C, with a cooling rate of 5°C/min.
paraffin. Paraffined sections were cut into thin slices of 5 2.4. Three-dimensional printing
μm for deparaffinization and rehydration. Cell component To bring the bioink to a pre-gel state, it was placed at 4°C
residues were then observed by hematoxylin and eosin for 20 min. Subsequently, the bioink cartridges were loaded
staining. In addition, 5-μm-thick sections of native and into a 3D bioprinter, with the nozzle temperature set to
decellularized liver tissues were stained using the nuclear 25°C and the print platform maintained at 4°C. During
stain, 4’,6-diamidino-2-phenylindole. The staining process the printing process, the extrusion pressure was set to 0.25
was performed at room temperature for 5 min. Collagen MPa and the fill spacing was 1.2 mm. A porous scaffold
I and III were also observed by immunohistochemical with dimensions of 20 mm × 20 mm × 3 mm (length ×
staining of tissue collagen. width × height) was printed. After extrusion, the printed

For DNA, protein, collagen, GAG content, and porous scaffold was exposed to UV light at an intensity of
2
proteomic analysis of dECM, a Tissue DNA extraction 30 mW/cm for 30 s to induce cross-linking. To evaluate
kit (Sigma-Aldrich, USA) was used to determine DNA the printability of the bioink and the print resolution of
content, a hydroxyproline assay kit (Aladdin Biotechnology the porous scaffold, the scaffold’s surface morphology was
44
Co., China) was employed to measure collagen content, analyzed using SEM, and the Pr value was calculated.
and a GAG enzyme-linked immunosorbent assay kit This value was determined based on the circularity of an
(Sinopharm Chemical Reagent Co., China) was used enclosed area, as shown in Equation (I):
to assess GAG content as an indicator of residual cells
in dECM. A microplate reader (Thermo Scientific™ c = 4ΠA/L^2 (I)
Multiskan™ GO, USA) was used to quantify the DNA,
GAG, and collagen content. where c is circularity, L is the perimeter, and A is the
area. A perfect circle yields a value of 1. For square shapes,
2.3. Preparation of three-dimensional printing the maximum circularity value is π/4. Therefore, the
bioinks and characterization Pr parameter for square shapes can be calculated using
Five different printing bioinks were prepared with varying Equation (II):
compositions and ratios as follows: (i) 10% (w/v) GelMA
(GM); (ii) 10% (w/v) GelMA and 5% (w/v) gelatin (GM/G);
2
and (iii–v) GM/G combined with dECM at concentrations Pr = Π / 4C L / 16A (II)
of 1%, 3%, and 5% (w/v), labeled as GM/G/d-1, GM/
G/d-3, and GM/G/d-5, respectively. The components To measure the swelling ability of scaffolds, the freeze-
were dissolved in a PBS solution containing 0.25% (w/v) dried scaffolds were first weighed and recorded as Wd.
lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The scaffolds were then immersed in PBS (pH 7.4) at

Volume 11 Issue 4 (2025) 395 doi: 10.36922/IJB025160142