Page 120 - IJB-9-1

International Journal of Bioprinting Fabrication of 3D breast tumor model for drug screening

staining kit and Masson staining kit and observed under solution containing 10 mg pepsin at room temperature for
optical microscope (IX83, OLYMPUS, Japan) to evaluate 3 d until dECM pieces were completely dissolved. Then, the
the decellularization efficiency. pH of dECM solution was adjusted to 7 with 1 M NaOH

The decellularization efficiency was further evaluated to solution. The gelatin/sodium alginate/dECM (Gel/SA/
determine the DNA, GAGs, and collagen contents of dECM dECM) bioinks were prepared by mixing the dECM with
compared to the native tissue [33–36] . For quantification, different weight ratio of Gel and SA. The concentrations of
1 mg/mL of lyophilized dECM was digested in a papain gelatin were 4%, 5%, 6%, and 7%, while the concentrations
solution (125 μg/mL papain in 0.1 M Na PO with 5 mM of sodium alginate were 2% and 3%. The prepared bioinks
3
4
Na EDTA and 5 mM cysteine at pH 6.5) for 16 h at 60°C. were placed in 4°C refrigerator for later use.
2
Native tissue of similar weight was also digested in a same The rheological properties of bioinks were tested by
manner as the control. The DNA content was determined a rheometer (Anton-Paar, Austria) . To determine the
[37]
using Hoechst 33324 assay. Briefly, 200 μL sample solution viscoelasticity of the bioinks, the measuring position was
and 10 μL Hoechst dye were added to a 96-well plate, set to 0.1 mm, and the angular frequency range was 0.01–
and incubated away from light for 30 min to measure the 100 rad·s . During the test, the temperature was set as
−1
fluorescence intensity (excitation wavelength: 360 nm, 20°C, and the shear strain remained at 1%. The temperature
emission wavelength: 450 nm). The standard curve for sensitivity (“G’-Temp” model) of the bioinks was tested by
DNA was generated using calf thymus DNA and used for serious reduction of temperature from 40 to 10°C (5°C /
quantifying the DNA in samples. The GAGs content was min), and the frequency was set to 1 Hz. To measure the
estimated via quantifying the amount of sulphated GAGs steady viscosity, the relationship between shear rate and
using 1, 9-dimethylmethylene blue (DMMB) solution. viscosity was tested at the shear rate ranged of 0.01 to 1000
Briefly, 20 μL sample solution and 200 μL DMMB were (1∙s ) at 20°C, and then the test was performed in the same
−1
added to a 96-well plate, and incubated for 4–6 min to manner again at the shear rate range of 1000 to 0.01 s to
−1
measure the absorbance at wavelength of 520 nm. The determine thixotropy of the boinks. The thixotropic curves
standard curve was made using chondroitin sulphate A were graphed by fitting these two inverse shear rate-shear
and used for estimating the sulphated GAGs in samples. stress curves.
The collagen content was determined via a conventional
hydroxyproline assay. Briefly, 1 mL 0.01mol/L CuSO 2.5. Manufacture of Gel/SA/dECM hybrid scaffold
4
solution, 1 mL 2.5 M NaOH solution, and 0.2 mL 3.6% The bioinks were pre-printed to select printable inks for
H O were added to 1 mL sample solution and shaken for 5 the construction of 3D hybrid scaffolds by a 3D bioprinter
2
2
min. Then, it was left to stand for 30 min, bathed in water (Pro, Regenovo, Hangzhou). Bioinks with appropriate
at 30°C for 10 min, and shaken violently again for 5 min. concentrations were selected to construct 3D hybrid
4 mL H SO and 2 mL 5% p-dimethylaminobenzaldehyde scaffolds, and the printed scaffolds were crosslinked with
2
4
(P-DMAB) solution were added after water bath at 65°C CaCl solution and EDC/NHS successively. Briefly, the
2
for 20 min. Finally, 100 μL solution was added to a 96-well hybrid scaffolds were soaked in 3% CaCl solution for 2 h
2
plate to measure the absorbance at 560 nm and quantified and washed with distilled water for 3 times to remove the
by referring to a standard curve made with hydroxyproline. residual crosslinking agent. Then, the hybrid scaffolds were
immersed in EDC/NHS crosslinking agent (50 mM EDC,
The surface of specimen was gently smoothed with 50mM NHS, 50mM MES, 60% ethyl alcohol) for 24 h and
a blade and the water contact angle of specimen was were washed three times with distilled water (0.5 h each
investigated by a contact angle meter with a high-speed time) to remove the residual crosslinking agent. The Gel/
camera (OCAH200, Data Physics, Germany). The videos SA/dECM (GSd) scaffolds were obtained after lyophilizing
were recorded from the moment the droplets touched the for 24 h.
materials, until the droplets completely penetrated the
scaffolds or became stable on the scaffolds. The photos of 2.6. Scaffolds characterization
droplet at 0 s, 0.5 s, and 1 s were captured, and the water 2.6.1. Macro- and micro-structure of scaffolds
contact angles were measured by Image Pro Plus 6.0 (IPP, The 3D-printed scaffolds were cut into the size of 5 mm ×
Media Cybernetics, USA). The results were averaged in 5 mm × 1 mm and were pasted on the conductive adhesive
each group by performing three parallel experiments. of the sample table. The surfaces of specimens were gently
blown with nitrogen, and specimens were placed on the
2.4. Preparation and characterization of bioinks metal spraying instrument and sprayed with metal evenly
The dECM solution was prepared by dissolving the for 10 min. Then, the microstructure of the scaffold was
lyophilized dECM pieces with pepsin. Briefly, 1 g dECM observed by SEM. The pore size data of the scaffolds
pieces were added and stirred in 100 mL 0.5 M acetic acid determined by IPP software were sorted into pore size

Volume 9 Issue 1 (2023) 112 https://doi.org/10.18063/ijb.v9i1.630