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International Journal of Bioprinting 3D-bioprinted meningioma model

the supernatant from each well of the 6-well plate was at 20–37°C. The slides were then incubated with a blocking
transferred to a blank 96-well plate, and the optical density solution containing Triton X-100 (Beyotime, China) at
(OD) values at 570 nm and 630 nm were measured using room temperature for 60 min. After overnight incubation at
an enzyme-linked immunosorbent assay reader (Epoch2, 4°C with the primary antibody, the slides were washed twice
BioTek, USA). The remaining Alamar Blue in the 6-well with 1× PBS and incubated with a fluorescent secondary
plate was replaced with fresh culture medium after three antibody. The fluorescent images of tumor markers were
washes with 1× PBS. The OD values for each well were captured using a fluorescence microscope (Eclipse Ti2-U,
calculated according to the instructions and measured Nikon, Japan) after staining with a fluorescent quenching
every 2 days. agent containing DAPI for 10 min.
2.6. Scanning electron microscopy analysis 2.9. Transwell invasion assay
Scanning electron microscopy (SEM) was performed to Eight-micrometer-pore Transwell chambers (Corning,
better observe the surface of the outer shell and inner core. USA) were placed in a 24-well plate. The extracellular
The 3D-bioprinted constructs were removed from the matrix (ECM) gel and serum-free medium were mixed
culture medium and washed with 1× PBS. Next, they were at a ratio of 1:7.5 on ice to prepare the working solution.
fixed overnight in 4% (w/v) paraformaldehyde (PFA) at Subsequently, 40 μl of the ECM gel working solution was
4°C, dehydrated using a graded series of ethanol solutions evenly spread on the bottom of the chamber to mimic the
(70%, 80%, 90%, 95%, and 100%) (v/v) for 10 min each, in vivo ECM layer. The chamber was then incubated at
and then frozen at -80°C overnight. Subsequently, the 37°C until the gel solidified. Next, 200 μl of cell suspension
constructs were freeze-dried using a freeze dryer, and with a density of 2.5 × 10 cells/ml was added to the upper
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sharp blades were used to cut open the hydrogel structure chamber, while 600 μl of serum-containing medium was
to obtain views from various angles. The constructs were added to the lower chamber. After 24 h, the cells on the
then sputter-coated with gold, and images were captured upper surface of the membrane were removed using
using SEM. a cotton swab. The Transwell chamber was then fixed
in 4% (w/v) PFA for 30 min and stained with Crystal
2.7. Real-time quantitative PCR (RT-qPCR) and Violet Staining Solution (Beyotime, China) for 15 min.
Western blotting analysis After 1× PBS washes, the samples were observed under a
For collection of cells from the inner core of 3D-bioprinted microscope, and 5 fields of view were randomly selected
structures, the 3D constructs were soaked in a 0.9% (w/v) for statistical analysis.
NaCl solution containing 55 mM sodium citrate and 20
mM ethylenediaminetetraacetic acid (EDTA; Sigma- 2.10. Nude mouse tumorigenesis experiment
Aldrich, USA) for 5 min to dissolve the sodium alginate. Twelve nude mice (4–6 weeks old) were obtained from
Subsequently, the resulting mixture was centrifuged at 1000 Nanjing Biomedical Research Institute of Nanjing
rpm, and the cell pellet obtained was used for mRNA and University (Nanjing, China) and divided into two groups,
protein extraction. Total RNA was extracted using RNA 2D and 3D, with six mice in each group. IOMM-Lee cells
Isolater Total RNA Extraction Reagent (BioFlux, China), cultured under 2D and 3D conditions were collected and
and reverse transcription and real-time PCR procedures resuspended in serum-free medium to a concentration of
were performed as previously described. For the Western 5 × 10 cells/ml. Then, 100 μl of matrix gel was added to
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blot assay, the following antibodies were used: anti- 100 μl of cell suspension, thoroughly mixed, and injected
actin antibody (Abcam, USA; 1:1000), anti-N-cadherin subcutaneously into the armpit of each mouse in each
antibody (Proteintech, China; 1:1000), anti-E-cadherin group. Tumor length and diameter were measured using
antibody (Proteintech, China; 1:1000), and anti-MMP9 a Vernier caliper every 5 days after injection, and tumor
antibody (Proteintech, China; 1:1000). Western blot assays volume was calculated to generate tumor growth curves.
were performed as previously reported. After 40 days, mice were euthanized by cervical dislocation,
and tumors were harvested for subsequent analyses.
2.8. Immunofluorescence analysis
To detect the expression level of meningioma-related 2.11. Statistical analysis
biomarkers in 3D-bioprinted constructs, we performed The data were analyzed using the statistical software SPSS
immunofluorescence staining analysis. The bioprinted 23.0, and the graphs were created using GraphPad Prism 5.
constructs were fixed in 4% (w/v) paraformaldehyde (PFA) All data are presented as the means ± standard deviations
at 4°C overnight, dehydrated with 30% (w/v) sucrose, and of three independent experiments. Differences between
then fixed in optimal cutting temperature (OCT) compound two groups were evaluated by t-test, while one-way analysis
at -80°C for 2 h. The sample was quickly cut into 10-μm- of variance (ANOVA) was used for comparisons between
thick slices using a cryostat and adsorbed onto glass slides multiple groups. A p-value of less than 0.05 was considered

Volume 10 Issue 1 (2024) 315 https://doi.org/10.36922/ijb.1342