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.