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–fibroblast co‐culture platform for head and neck squamous cell carcinoma (HNSCC) that overcomes these limitations via a dual strategy: APTES‐mediated surface silanization anchors the ECM to the chip, combined with Genipin‐based crosslinking, which modestly increases hydrogel stiffness and progressively reinforces the network without compromising cell viability, as confirmed by time‐ and frequency‐resolved rheology. Fourier‐transform infrared spectroscopy (FTIR) verified successful collagen crosslinking while preserving reactive ─OH and ─NH 2 groups, enabling covalent bonding to the APTES‐functionalized chip. The platform further integrates semi‐automated segmentation and high‐content imaging to quantify dynamic phenotypic drug responses at both single‐cell and multicellular/tissue organization levels. Drug chemosensitivity assays, including co‐culture with patient‐derived CAFs, enabled quantitative assessment of clinically relevant chemoprotective effects. By combining biomaterial engineering with functional microfluidic design, this system enables reproducible, physiologically relevant modeling of tumor–fibroblast interactions, offering a scalable tool for preclinical drug chemosensitivity screening and clinical translation.