Swelling‐Programmed Topographical Guidance for Dynamic Spheroid Self‐Assembly via a Mechanochemical Hydrogel Niche

ABSTRACT Precise and reproducible spheroid formation remains challenging for cell types with weak intrinsic cohesion, and post‐formation size heterogeneity often compromises downstream analyses. Here, we present a micropatterned interpenetrating polymer network hydrogel substrate designed as a mechanochemical niche that guides robust spheroid self‐assembly. The line‐patterned hydrogel undergoes swelling‐mediated topographical evolution, resulting in progressive groove narrowing. This process increases local cell‐cell contact while preserving space for cell migration. Using this platform, uniform and viable spheroids were generated from both L929 fibroblasts, which exhibit limited spontaneous aggregation, and HCT116 colorectal cancer cells. Mechanistic analyses revealed cell/substrate interactions, collective compaction, and enhanced extracellular matrix (ECM) deposition, accompanied by upregulation of ECM‐ and adhesion‐related genes. Transcriptomic profiling demonstrated cell‐type‐specific remodeling, with L929 spheroids enriching extracellular matrix and cytoskeletal programs, whereas HCT116 spheroids displayed pronounced immune‐ and stress‐response signatures, including interferon‐stimulated and immune‐evasion genes. These molecular features were consistent with increased resistance to doxorubicin and paclitaxel. To reduce variability in downstream analyses, we developed a hand‐powered, cord‐driven centrifugal microfluidic platform that selectively enriches spheroids ranging from approximately 150 to 250 µm without motors or electronics. We believe this integrated system offers a scalable, physiologically relevant, and size‐controlled pipeline for advanced 3D culture and tumor modeling studies.