A new inject-embed 3D culture method enables the spheroid and aggregate formation from single or dual liver cancer cell types.

BACKGROUND: Three-dimensional (3D) cell culture techniques have emerged as a bridge between traditional two-dimensional (2D) cell culture and the complex 3D architecture of living organisms. To overcome the limitations of existing 3D culture methods, we aimed to develop a new 3D culture platform that provides a liquid-matrix interface for cells, enabling both cell-cell and cell-matrix interactions. METHODS: We generated an inject-embed 3D culture platform with a standardized procedure for operation and quantification. This platform was evaluated using seven cell lines: six liver cancer cell lines including four hepatocellular carcinoma (HCC) lines and two cholangiocarcinoma (CCA) lines, and LX2 hepatic stellate cells representing the liver cancer microenvironment. Cells were cultured in both mono-culture and co-culture setups to assess spheroid and aggregate formation, proliferation, assembly, and their 3D architecture. We also evaluated its utilization in assays of stemness-related gene expression, chemo-resistance, and the tumor malignancy phenotypes. RESULTS: In mono-culture, spheroids from all seven cell lines exhibited varying sizes and shapes. In co-culture setup, HCC cells with LX2 predominantly formed mixed LX2-HCC hybrid aggregates, while CCA cells with LX2 formed well-organized CCA-centered/LX2-surrounded aggregates. In both mono-culture and co-culture systems, this inject-embed method supported significant cell proliferation, spheroid/aggregate aggregation, and cell-cell communication. Compared with conventional 2D culture, cells in this 3D system altered gene expression profiles, enhanced stemness-associated gene expression and increased cell resistance to chemotherapeutic agents. Moreover, a known HCC malignancy regulator altered spheroid size and number in this method, demonstrating its suitability for functional studies in cancer field. CONCLUSIONS: This newly established inject-embed 3D culture method is highly reproducible, effectively promotes spheroid/aggregate growth and assembly, and enables the investigation of cell malignancy features under 3D microenvironment conditions. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12935-026-04250-1.