Tailoring Polymer Surface for Long-term Reliable Xenogeneic-free Culture of Intestinal Stem Cells via Regulated Dynamic Migratory Behavior

Regenerative therapy employing intestinal stem cells (ISCs) holds a great promise for reliable epithelial restoration. However, achieving this goal requires scalable and robust, but fully defined culture platforms that eliminate the use of xenogeneic components while preserving stem cell function. Most existing systems usually rely on the use of Matrigel, complicating clinical translation and limiting mechanistic understanding of stem cell-material interactions. In this study, a poly(ethyleneglycoldimethacrylate) (pEGDMA)-based synthetic culture surface is precisely refined through N2 plasma treatment thereon, enabling tailored modulation of surface properties. This modification enhances wettability of the polymer surface and introduces N-containing functional groups, resulting in the PoLymer-coated Ultra-stable Surface (PLUS) that significantly improves ISC attachment under xenogeneic-free conditions. Remarkably, PLUS maintains its ISC-supportive function even after three years of ambient storage. Gene expression analysis and comparative proteomic profiling clearly reveal the upregulation of factors involved in actin dynamics and cytoskeletal reorganization, indicating a structural basis for enhanced colony expansion. Consistently, colonies on PLUS exhibit a broader migratory range and migrate 1.8-fold faster during random movement than those on pristine pEGDMA. Functional validation using small-molecule perturbation assays confirms the involvement of cytoskeletal remodeling by exquisitely mediating ISC-substrate interaction. Furthermore, PLUS enables progressive wound closure via dynamic migration by up to 46.7% within 144 h through actin-dependent mechanisms, supporting the facilitated epithelium regeneration. By providing a long-term reliable bioactive surface that sustains stemness and regenerative capacity of ISCs, 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 intestinal stem cell-based regenerative medicine.

采用肠道干细胞（intestinal stem cells, ISCs）的再生疗法，在实现可靠的上皮组织修复方面展现出巨大潜力。然而，达成这一目标需要构建可规模化、稳定性优异且成分完全明确的培养平台，在保留干细胞功能的同时，彻底消除异种成分的使用。目前多数现有培养体系均依赖基质胶（Matrigel），这一做法不仅给临床转化带来了复杂阻碍，也限制了对干细胞-材料相互作用机制的深入解析。本研究针对聚乙二醇二甲基丙烯酸酯（poly(ethyleneglycoldimethacrylate), pEGDMA）基合成培养表面，通过氮气等离子体处理实现了表面性质的精准调控。该改性工艺可提升聚合物表面的润湿性，并引入含氮官能团，由此制备得到的聚合物包覆超稳定表面（PLUS），可在无异种成分的培养条件下显著促进肠道干细胞的黏附。值得注意的是，PLUS即使在室温环境储存三年后，仍可维持其支持肠道干细胞生长的核心功能。基因表达分析与对比蛋白质组学分析结果清晰表明，肌动蛋白动力学与细胞骨架重排相关因子的表达显著上调，这为菌落扩增能力的提升提供了结构层面的分子基础。实验结果一致显示，PLUS表面的干细胞菌落展现出更广泛的迁移范围，其随机运动时的迁移速度较原始pEGDMA表面高出1.8倍。通过小分子扰动实验开展功能验证，证实该体系可通过精准介导肠道干细胞-底物相互作用，依赖细胞骨架重排实现调控效果。此外，PLUS可通过肌动蛋白依赖的机制，在144小时内将动态迁移介导的伤口闭合率提升至多46.7%，进而有效促进上皮组织再生。综上，PLUS作为一种长期可靠的生物活性表面，可维持肠道干细胞的干性与再生能力，其通过以细胞骨架系统作为干细胞-底物相互作用的核心介导因子，部分通过促进肌动蛋白依赖的细胞骨架重排，使其成为可规模化、具备临床转化潜力的基于肠道干细胞的再生医学培养平台。