Scaffold-Free Self-Assembled Cell Ring Organoids Enable Efficient Tumor Modeling with High Engraftment Rates, Superior Tumor Uniformity, and Reduced Animal Consumption

Background: Conventional tumor cell suspension xenografts suffer from low engraftment efficiency, prolonged latency, and poor tumor uniformity, with high animal consumption conflicting with the 3R principles (Replacement, Reduction, Refinement). This study aimed to develop a scaffold-free self-assembled cell ring organoid platform to address these bottlenecks. Methods: Tumor cells and fibroblasts were mixed at optimized ratios and seeded into microwell molds, spontaneously self-assembling into ring-shaped organoids within 24–48 h without exogenous matrices or hydrogels. In vitro characterization included live/dead staining, histology, immunofluorescence, flow cytometry, Western blotting, and transcriptomic sequencing. In vivo performance was evaluated by orthotopic transplantation into the lateral femoral condyle of immunodeficient or immunocompetent mice, with longitudinal monitoring by bioluminescence imaging and micro-CT. Results: Cell rings maintained high viability during in vitro culture and were compatible with both cell lines and clinical primary sarcoma cells. Immunofluorescence and Western blotting revealed that co-cultured fibroblasts underwent cancer-associated fibroblast (CAF)-like phenotypic transformation (α-SMA upregulation) within the cell ring organoids. Transcriptomic analysis further demonstrated that the gene expression profiles of cell ring organoids closely resembled those of original tumors, whereas 2D-cultured cells showed the greatest number and magnitude of differentially expressed genes. In vivo, cell ring organoids achieved higher engraftment rates, better tumor uniformity, and stronger bone invasion capacity compared with cell suspensions. Conclusion: The scaffold-free self-assembled cell ring organoids platform provides an efficient, uniform, and 3R-compliant strategy for animal tumor modeling. Without requiring exogenous matrices, this technology achieves significantly higher engraftment rates and tumor uniformity compared with conventional methods, effectively reduces animal consumption, and offers a standardized platform for preclinical drug screening and tumor microenvironment (TME) research.