Regenerative therapy employing intestinal stem cells (ISCs) holds promise for epithelial restoration. However, it requires scalable and robust, but fully defined culture platforms free of xenogeneic components. Most existing systems, however, typically rely on Matrigel, which complicates clinical translation and limits understanding of stem cell-material interactions. Here, a poly(ethyleneglycoldimethacrylate) (pEGDMA)-based synthetic culture surface is exposed to N₂ plasma treatment for tailored modulation of surface properties. This modification enhances surface wettability and introduces N-containing functional groups, resulting in a PoLymer-coated Ultra-stable Surface (PLUS) that improves ISC attachment under xenogeneic-free culture conditions. Remarkably, PLUS maintains its ISC-supportive function even after 3 years of ambient storage. Gene expression and proteomic analyses reveal upregulation of factors involved in actin dynamics and cytoskeletal reorganization. Consistently, colonies on PLUS exhibit a broader migratory range, with a 1.8-fold increase in migration speed compared to pristine pEGDMA. Small-molecule perturbation assays confirm the involvement of cytoskeletal remodelling by mediating ISC-substrate interaction. Furthermore, PLUS supports dynamic, actin-dependent migration, enabling up to 46.7% wound closure within 144 h. These findings demonstrate that PLUS engages cytoskeletal machinery as a central mediator of ISC-substrate interaction, in part by promoting actin-dependent cytoskeletal reorganization, positioning it as a scalable, translational platform for ISC-based regenerative medicine.