A pilot evaluation of a 3D bioprinted tumor model for assessment of electroporation-based therapies

Head and neck squamous cell carcinomas (HNSCCs) are aggressive malignancies with poor prognosis and limited therapeutic options. Electrochemotherapy (ECT), combining short electric pulses with chemotherapeutic agents to enhance intracellular drug uptake, has shown clinical potential but still requires physiologically relevant in vitro models for protocol optimization and mechanistic studies. Here, we introduce a three-dimensional 3D bioprinted in vitro HNSCC model specifically designed for the assessment of electroporation. Structures were fabricated using a composite hydrogel composed of 8% sodium alginate and 4% gelatin (w/w), crosslinked with calcium chloride at concentrations of 0.5%, 1%, and 2%. Uniaxial compression testing confirmed elastic moduli spanning the physiological tumor stiffness range, with the 1% calcium chloride formulation providing optimal mechanical and handling characteristics (42.96 ± 19.89 kPa). Hypopharyngeal carcinoma FaDu cells (5×106/mL) embedded in three-layer structures (thickness: 1.05 mm) maintained 75–80% viability for up to 21 days and formed tumor-like spheroids (mean diameter: 303 ± 113 μm), reflecting native tumor architecture. Electroporation with eight pulses at 200 V for 100 μs efficiently permeabilized the cell membrane, as evidenced by the internalization of propidium iodide, while maintaining high cell viability as confirmed by live/dead analysis. Programmed death-ligand 1 expression was preserved and upregulated in 3D spheroids compared to two-dimensional (2D) controls, supporting the platform’s relevance for immuno-oncology studies. Compared to other 3D HNSCC models, our system integrates mechanical tuning, electroporation compatibility, and immune-related biomarker expression, enabling functional validation of electric field-mediated intracellular delivery. This proof-of-concept platform demonstrates structural fidelity, long-term cell viability, and high reproducibility, offering a scalable, human-relevant tool for preclinical optimization of ECT and other electrically based therapies, bridging the gap between conventional 2D cultures and complex in vivo models.