Evaluation of a Simple 3D Bioengineered Model: A Step Closer to Bridging the In Vitro–In Vivo Gap in Osteosarcoma Research

Standard in vitro models for cancer research and anticancer drug screening are primarily based on two-dimensional (2D) cell cultures. However, these models fail to accurately replicate the complex biology of cancer, leading to significant differences in drug efficacy between in vitro and in vivo studies. Therefore, the aim of this study was to develop and evaluate a simplified, practical three-dimensional (3D) osteosarcoma cell culture model that supports tissue-like organization, based on alginate microfibers with or without embedded hydroxyapatite (HAP) to effectively bridge the in vitro–in vivo gap in osteosarcoma research and anticancer drug screening. Alginate and HAP/alginate microfibers (500–600 µm in diameter) with uniformly immobilized murine osteosarcoma K7M2-wt cells (4 × 10⁶ cells cm⁻³) were produced by manual extrusion. The majority of cells remained viable, metabolically active, and retained their cancer cell morphology after immobilization, as well as after 21 days of cultivation. In addition, immobilized cells within alginate microfibers exhibited a trend of increased SOX9 expression during cultivation compared to 2D cell cultures. During the 21-day cultivation, cells in both microfiber types spontaneously formed aggregates (15–210 µm in diameter) and produced collagen and reticular fibers, components of the osteosarcoma extracellular matrix. Evaluation of the 3D cell culture models for anticancer drug screening revealed that the cells in microfibers treated with doxorubicin exhibited up to ~ 6-fold higher half-maximal inhibitory concentration as compared to those in 2D cultures (2–3 µg cm⁻³ vs. 0.5 µg cm⁻³). This increased resistance is consistent with the chemoresistance commonly observed in patients with osteosarcoma. Overall, these findings highlight the potential of alginate-based microfibers as practical 3D osteosarcoma models for relevant research and anticancer drug screening.