Genipin-Crosslinked, Silane-Anchored 3D Tumor–Stroma Microtissues for High-Content On-Chip Drug Testing

Abstract Physiologically relevant 3D tumor models incorporating extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) are essential for studying tumor progression and drug resistance yet often suffer from hydrogel contraction and instability – especially in microfluidic formats, where ECM deformation hampers long-term culture and quantitative imaging. Here, we present a microfluidic tumor-stroma co-culture platform for head and neck squamous cell carcinoma (HNSCC) that overcomes these limitations through a dual-material strategy: APTES-mediated surface silanization anchors the ECM to the chip, while Genipin-based crosslinking enhances matrix stiffness without compromising cell viability. This approach stabilizes collagen-rich hydrogels for over 10 days, preserving 3D architecture, sustaining >85% viability, and supporting active proliferation. Fourier-transform infrared spectroscopy (FTIR) confirmed successful collagen crosslinking, combining covalent modification of biomaterials with improved mechanical performance. The platform further integrates AI-assisted, high-content imaging to quantify dynamic phenotypic drug responses at both single-cell and higher multicellular/tissue level resolution. Drug chemosensitivity assays, including the co-culture of tumor cells with patient-derived CAFs, demonstrated the quantitative assessment of clinically relevant chemoprotective effects. By combining biomaterial engineering with functional microfluidic design, this system enables reproducible, physiologically relevant modeling of tumor-stroma interactions, offering a scalable tool for preclinical drug screening and personalized medicine or precision oncology applications. Competing Interest Statement The authors have declared no competing interest. Footnotes Funding This research was funded by the Polish National Science Centre (NCN): UMO-2020/37/B/NZ4/03920, and DEC-2021/41/B/NZ7/03786, and EMBO Scientific Exchange Grant (no. 10698), and Polish National Agency for Academic Exchange (NAWA): PPI/APM/2019/1/00089/U/00001, and Jane & Aatos Erkko Foundation, project “Matrix Matters”, and Academy of Finland “Phenotypic Screening for Cancer Drug Discovery/Consortium: PESCADoR (309372). This work was also supported by Institut Pierre-Gilles de Gennes ANR-10-EQPX-34, EU Horizon 2020 Grant ERC PoC (101100823) and PSL QLife initiative.