Formulation-dependent alginate hydrogels for ovarian cancer spheroid drug screening

Introduction: Three-dimensional (3D) tumor spheroid models provide a more physiologically relevant platform for cancer research and drug screening than conventional two-dimensional (2D) cultures. However, the development of cytocompatible hydrogel scaffolds that support reproducible spheroid formation and drug response evaluation remains challenging. This study evaluated formulation-dependent calcium-cross-linked sodium alginate (SA) hydrogels for ovarian cancer spheroid culture and comparative drug sensitivity assessment. Materials and methods: SA hydrogels were prepared using polymer concentrations of 2–3% (w/v) and calcium chloride (CaCl 2 ) concentrations of 102–103 mM. Scaffold microarchitecture was characterized by field-emission scanning electron microscopy, and Fourier transform infrared spectroscopy (FTIR) analysis was used to evaluate chemical interactions associated with ionic cross-linking. Cell viability and apoptosis profiles were assessed using Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry with PA-1 human ovarian teratocarcinoma cells. Comparative drug sensitivity to olaparib was evaluated in 2D monolayer and 3D spheroid cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The olaparib drug response assay was performed using three independent biological replicates ( n = 3). Results: Alginate concentration and cross-linking density influenced scaffold pore architecture and matrix compaction. Selected formulations enabled the formation of compact PA-1 spheroid-like cellular aggregates under the tested conditions, while viability observations were assessed qualitatively. Drug response analysis demonstrated dose-dependent cytotoxicity in both culture systems. The fitted half-maximal inhibitory concentration (IC 50 ) was higher in 3D spheroids (111.60 nM) than in 2D monolayer cultures (82.87 nM), indicating an apparent reduction in olaparib sensitivity under the examined conditions. Interpretation of the 2D–3D IC 50 comparison is limited by the different starting cell numbers (3 × 10 4 cells/well in 2D versus 5 × 10 4 cells/well in 3D), extrapolation of the 3D IC 50 beyond the highest tested concentration of 100 nM, and the absence of a direct inferential statistical comparison between the fitted IC 50 values. Conclusions: Calcium-cross-linked alginate hydrogels provide preliminary feasibility for ovarian cancer spheroid-like aggregate culture and comparative drug response evaluation under the tested conditions. Optimization of scaffold composition enables regulation of three-dimensional cellular organization and therapeutic response. Further optimization and quantitative validation are needed before alginate hydrogel-based spheroid models can be considered reliable platforms for cancer drug screening.