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2.10. RNA-sequencing (RNA-seq) kit (KGA9501-1000, KeyGEN BioTECH, China) following
To investigate the molecular mechanisms underlying the the manufacturer’s protocol. Briefly, the TBM was rinsed
−6
effects of the TBM embedded with bergamottin (B757586, with PBS and then incubated in 2 × 10 M Calcein AM and
−6
Aladdin, China) or miR-138-5p antagonist (RQCON 4 × 10 M PI for 30 min at room temperature in the dark.
Biological Technology Co., Ltd., China), mRNA-sequencing Stained microspheroids were visualized using a confocal
was performed on TBM-treated hMSCs. Total RNA was microscope (FV3000, Olympus, Japan) with 4 μm thick
extracted using TRIzol reagent (15596018, Invitrogen, slices.
USA), and RNA quantity and quality data were assessed 2.14. 5-Ethynyl-2’-deoxyuridine labeling cell
using the NanoDrop OneC spectrophotometer (Thermo proliferation assay for TBM organoids
Fisher Scientific, USA). After confirming RNA integrity,
RNA-seq was conducted by Annoroad Gene Technology To assess the effects of treatment on cell proliferation,
Co. Ltd. (China). Raw sequencing data were processed 5-ethynyl-2’-deoxyuridine (EdU; RQCON Biological
using the Fastp software (v.0.19.11; https://github.com/ Technology Co., Ltd., China) labeling was conducted using
OpenGene/fastp). Fold changes in differentially expressed the EdU labeling/detection kit (C10310, RiboBio, China).
RNAs were calculated as log2 values (normalized intensity The TBM-embedded primary ADSCs were cultured in
of the treatment group/normalized intensity of the control 24-well plates. Then, 50 μM of EdU was added to each
group). well, and the cells were further incubated for 12 h at
37°C. Following incubation, the TBM was fixed with 4%
2.11. Enrichment analysis paraformaldehyde (F1635, Sigma-Aldrich, USA/BL539A,
Gene ontology (GO) enrichment analysis and Kyoto BioSharp, China) for 30 min at 37°C and treated with 0.5%
Encyclopedia of Genes and Genomes (KEGG) pathway Triton X – 100 (T9284, Sigma-Aldrich, USA) for 30 min at
enrichment analysis were conducted to investigate the 37°C. After three washes with PBS, 100 μL of 1 × Apollo
differentially expressed genes (DEGs) between blank TBM reaction cocktail (C10310-3, RiboBio, China [in the EdU
and TBM loaded with bergamottin, as well as between labeling/detection kit]) was added to each well, and the
TBM loaded with an empty recombinant RNA (MSA) and cells were incubated for 45 min at 37°C. Then, the cells were
TBM loaded with recombinant miR-138-5p antagonist. stained with 100 μl of Hoechst 33,342 (C10310, RiboBio,
Gene set enrichment analysis was conducted using the China) for 30 min at 37°C. The cells were visualized
“clusterProfiler” package from the Bioconductor project using a confocal microscope (FV3000, Olympus, Japan).
(https://bioconductor.org/packages/release/bioc/html/ All cells were gamma-adjusted, merged with Hoechst
clusterProfiler.html). A p<0.05 was set as the significance staining, and analyzed using Image-Pro Plus 6.0 software
threshold. (Media Cybernetics, USA). The EdU incorporation rate
was calculated as the ratio of EdU-positive cells to total
2.12. Cell embedding in the TBM
Hoechst-positive cells. All experiments were performed in
A total of 25 8-month-old C57BL/6 male mice (37 ± 1.4 g) triplicate and repeated independently 3 times.
were used in the cell embedding study. The mice were
anesthetized using CO and inguinal fat tissue was collected 2.15. Immunofluorescence
2,
and washed. The tissue was cut into pieces and digested in The TBM samples (2 mm 3 sec tions) were subjected to
0.25% collagenase I (BS032B, Biosharp, China) at 37°C for immunofluorescence staining in 96-well plates. Samples
26
30 min. Primary ADSCs were collected by centrifugation were first incubated with primary antibodies at 4°C
and resuspended in DMEM. 25 overnight, followed by incubation with fluorescent-labeled
Cultured ADSCs were detached using trypsin and secondary antibodies at room temperature for 1.5 h. The
suspended in a culture medium. HAMA was dissolved primary antibodies used were ALP rabbit polyclonal
in the cell suspension with a density of 6 × 10 /cm , then antibody (pAb) (1:20; ET1601-21, HUABIO, China, RRID:
3
6
crosslinked and overlaid onto the Porous to construct an AB_3069604), runt-related transcription factor 2 (RUNX2)
ADSC-embedded TBM. These constructs were either rabbit pAb (1:25; ET1612-47, HUABIO, China, RRID:
cultured in a microfluidic system or subcutaneously AB_2924311), ACTIN rabbit pAb (1:40; BL005B, Biosharp,
implanted over the calvarial surface of mice, with each China), cluster of differentiation (CD)31 (1:20; AG2849,
mouse receiving an autologous ADSC-embedded TBM. Beyotime, China). A fluorescent-labeled secondary
The samples were then ready for CCK-8 assays. antibody CoraLite 488-conjugated goat anti-rabbit (1:100;
20000259, Proteintech, USA, RRID: AB_2797132) was
2.13. Cell viability assay for TBM organoids used for detection. Stained cells were visualized using laser
Cell viability within the TBM was qualitatively assessed scanning confocal microscopy (FV3000, Olympus, Japan)
using the Calcein AM/PI (live and dead cell staining) test with excitation wavelengths of 488 nm and 405 nm.

Volume 1 Issue 2 (2025) 4 doi: 10.36922/OR025040003