Page 122 - IJB-9-1

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

1 μL propidium iodide (PI) was added to each well and 2.9. Statistical analysis
incubated at 37°C with 5% CO for 15–20 min. The live/ All data were presented as mean ± standard deviation
2
dead staining images of cell-scaffolds were observed under (SD). One-way analysis of variance (ANOVA) was used
single-photon laser confocal microscopy (IX83, Olympus, for analyzing significant difference of data in each group,
Japan). and P<0.05 indicated that there was significant difference
The proliferation of L929 fibroblasts on the scaffolds between groups. All statistical analyses were performed
was assessed by CCK-8 assay. The medium was removed using Origin Pro 9.5 software (Origin Lab, MA, USA).
followed by addition of 500 μL CCK-8 solution (CCK: 3. Results and discussion
α-DMEM = 1:10) to the cell-scaffolds cultured for 1 d, 4
d, and 7 d in each well, and placed in an incubator at 37°C 3.1. Evaluation of decellularization efficiency
with 5% CO for 3 h. The optical density (OD) values were If the process of decellularization is too intense, the
2
tested with an enzyme-linked immunoassay at 450 nm. The microstructure in tissues may be destroyed, and some
results were averaged in each group by performing three bioactive components, such as proteins, glycoproteins, and
parallel experiments. Moreover, the morphology of the other chemicals that are important for cell proliferation,
cells cultured on the scaffolds for 1 d and 7 d were observed may be lost in large quantities, which could inhibit cell
by SEM. The procedure was similar to that described in attachment and migration . If the decellularization
[40]
section 2.3. process is too mild, the original cells in the tissue may not
be completely removed, so the growth and proliferation of
2.8. Construction of breast tumor model subsequent inoculated cells may also be inhibited as a result
Briefly, a 4T1 cell suspension with the concentration of immune activity . Therefore, it is necessary to evaluate
[41]
of 1×10 cells/mL was prepared and 10 μL of the cell the efficiency of decellularization. Figure 1A showed the
6
suspension was inoculated on one side of the scaffold. After macroscopic morphology of porcine liver tissues before
0.5 h, 300 μL medium was added and the plate was put into and after decellularization. It was obvious that fresh porcine
a 37°C incubator with 5% CO . After 3 h, the same amount liver tissue was red before decellularization, while pale and
2
of 4T1 were inoculated on the other side of the scaffold and soft after being washed by distilled water. dECM obtained
supplemented with 200 μL of medium. 2D control group after decellularization was white, slightly transparent, and
was directly inoculated with 20 μL of the cell suspension softer, with visible pore structures on the surface. After
supplemented with 1 mL medium on the well plate. freeze-drying, the dECM turned into white slices, and the
The scaffolds inoculated with the cells for 1 d, 4 d, and internal tissue was found to be white floccules. This result
7 d, respectively, were taken out for fluorescence staining. was identical to the dECM of pig liver obtained by Sellaro
Briefly, 300 μL staining solution, which was prepared by et al. In order to confirm that the decellularization
[42]
mixing 1 mL PBS with 2 μL Calcein-AM, 1 μL PI and 5 μL process was complete and the original structure of the
Hoechst 33342, was added to each well and incubated at tissue was not damaged, SEM (Figure 1B), H&E staining
37°C with 5% CO for 15–20 min. The staining images (Figure 1C), and Masson staining (Figure 1D) were used to
2
of cell-scaffolds were observed under single-photon laser observe the tissues before and after decellularization, and
confocal microscopy. The diameters of tumor spheres the results were similar to those obtained by Saleh et al.
[43]
formed on 4 d and 7 d were determined by IPP software and Wu et al. SEM results showed that there were no cells
[44]
and sorted into a column graph. 4T1 grown on 2D plates in dECM and its surface showed irregular pore structure.
were observed by inverted microscope and pictures were In addition, a large number of fiber networks could be
also taken. seen in dECM under high magnification, which may be
collagen or other components in extracellular matrix that
The proliferation of L929 fibroblasts on the scaffolds provided sites for cells to attach and grow. A large number
was assessed by CCK-8 assay. The medium was removed of cells could be seen in H&E staining and Masson staining
followed by addition of 500 μL CCK-8 solution (CCK: images before decellularization, and actin was more than
α-DMEM = 1:10) to the cell-scaffolds cultured for 1 d, collagen in porcine liver tissue. ECM and collagen in liver
4 d, and 7 d in each well, and placed in an incubator at tissue were preserved in dECM, while actin was removed.
37°C with 5% CO for 3 h. The OD values were tested with The complex network structure was not damaged, which
2
an enzyme-linked immunoassay at 450 nm. The results was consistent with the results of SEM.
were averaged in each group by performing three parallel
experiments. Moreover, the morphology of the cells To further evaluate the efficiency of decellularization,
cultured on the scaffolds for 1 d, 4 d, and 7 d were observed collagen, GAGs, and DNA in the tissues before and after
by SEM. The procedure was similar to that described in decellularization were quantitatively detected. GAGs are
section 2.3. important component of ECM. Due to their hydrophilic

Volume 9 Issue 1 (2023) 114 https://doi.org/10.18063/ijb.v9i1.630