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Abstract Background Recently, increasing attention has been drawn to the impact of the tumor microenvironment (TME) on the occurrence and progression of malignant tumors. A variety of 3D culture techniques have been used to simulate TME in vitro. The purpose of this study was to reveal the differences in transcriptional and metabolic levels between osteosarcoma (OS) 2D cells, 3D cells, 3D cell-printed tissue, isolated tissue, and transplanted tumor tissue in vivo. Methods We cultured the OS Saos-2 cell line under different culture methods as 2D cells, 3D cells, 3D cell-printed tissue and isolated tissue for 14 days and transplanted tumors in vivo as a control group. Through transcriptomic and metabonomic analyses, we determined the changes in gene expression and metabolites in OS tissues under different culture methods. Results At the transcriptional level, 166 differentially expressed genes were found, including the SMAD family, ID family, BMP family and other related genes, and they were enriched in the TGF-β signaling pathway, complement and coagulation cascades, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ferroptosis, cGMP-PKG signaling pathway and other pathways. At the metabolic level, 362 metabolites were significantly changed and enriched in metabolic pathways such as the Fc Epsilon RI signaling pathway, histidine metabolism, primary bile acid biosynthesis, steroid biosynthesis, protein digestion and absorption, ferroptosis, and arachidonic acid metabolism. After integrating the transcriptome and metabolomics data, it was found that 44 metabolic pathways were changed, and the significantly enriched pathways were ferroptosis and pyrimidine metabolism. Conclusion Different culture methods affect the gene expression and metabolite generation of OS Saos-2 cells. Moreover, the cell and tissue culture method in vitro cannot completely simulate TME in vivo, and the ferroptosis and pyrimidine metabolism pathways mediate the functional changes of OS Saos-2 cells in different microenvironments.