Abstract: BACKGROUND: Compared with two-dimensional culture techniques, three-dimensional culture technology can more accurately simulate the in vivo environment of natural cell growth, which becomes a key focus in the fields of basic scientific research and translational medicine.
OBJECTIVE: To construct prevascularized osteogenic organoids through three-dimensional culturing techniques.
METHODS: Stromal vascular fraction (SVF) and adipose mesenchymal stem cells were isolated and extracted from the same human adipose tissue, and the cell components were identified by flow cytometry. The SVF and adipose mesenchymal stem cells were inoculated into 96-well U-plates. After 3 days of culture, the SVF was self-assembled and the adipose mesenchymal stem cells were formed into spheres. On the 14th day of culture, the internal structure of the spheres and cell morphology were observed by hematoxylin-eosin staining and cytoskeletal staining. Viability of the cells was detected by Presto Blue assay on days 0, 7, 14, and 21 of culture. Osteogenic, chondrogenic, and lipogenic differentiation were induced after 3 days of culture. After 14 days of induction, alizarin red staining was used to observe osteogenic differentiation; alizarin blue staining to observe chondrogenic differentiation; oil red O staining to observe lipogenic differentiation; immunofluorescence staining to co-localize CD31 (endothelial cell marker), RUNX2 (early osteogenic marker), CD44 (mesenchymal stem cell marker), type I collagen (late-stage osteogenic marker), and peroxisome proliferator-activated receptor γ (lipogenic markers); and qRT-PCR to detect the mRNA expression of RUNX2, osteopontin, type I collagen, Sp7 transcription factor, CD31, and vascular endothelial growth factor.
RESULTS AND CONCLUSION: (1) Flow cytometry results revealed that the SVF contained various cell types including adipose mesenchymal stem cells and endothelial cells, whereas adipose mesenchymal stem cell spheroids were primarily composed of adipose mesenchymal stem cells. (2) Hematoxylin-eosin staining with cytoskeleton staining indicated that compared with adipose mesenchymal stem cell spheroids, SVF organoids more closely mimicked natural tissue structures with orderly arranged cells and richer extracellular matrix. Cell viability tests showed that adipose mesenchymal stem cell spheroids had higher activity on day 7, but SVF organoids surpassed the cells by day 21 (P < 0.05), exhibiting longer-lasting cell viability. (3) In tri-lineage differentiation experiments, SVF organoids demonstrated superior osteogenic and chondrogenic potential compared with adipose mesenchymal stem cell spheroids. Multicolor immunofluorescence colocalization revealed rich vascular networks and pronounced osteogenic marker expression in SVF organoids, with complex cell-to-cell and cell-to-matrix interactions, whereas adipose mesenchymal stem cell spheroids showed a simpler structure with a lack of endothelial gene expression. qRT-PCR results indicated significantly higher expression of RUNX2, osteopontin, type I collagen, Sp7 transcription factor, CD31, and vascular endothelial growth factor in SVF organoids compared with adipose mesenchymal stem cell spheroids (P < 0.05). Overall, the findings suggest that SVF organoids possess complex physiological structures with enhanced capabilities in vascular and bone formation, as well as sustained cell viability.

Key words: three-dimensional culture, stromal vascular fraction, adipose mesenchymal stem cells, endothelial cells, organoids, osteogenic differentiation, angiogenesis, engineered materials