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Abstract The need for basement membrane extract (BME) with undefined constituents, such as Matrigel, for intestinal stem cell (ISC) culture in traditional systems poses a significant barrier that must be overcome for the development of clinical-grade, scalable, ready-to-use ISCs. Here, we propose a functional polymer-based xenogeneic-free dish for the culture of intestinal stem cells (XF-DISC), ensuring substantially prolonged maintenance of ISCs derived from 3-dimensional human intestinal organoids (ISCs3D-hIO). XF-DISC enables remarkable expandability, exhibiting a 24-fold increase in cell numbers within 30 days, with long-term maintenance of ISCs3D-hIO for more than 30 consecutive passages (>210 days). In addition, XF-DISC is fully compatible with a cell banking system. Notably, human pluripotent stem cell-derived ISCs3D-hIO cultured on XF-DISC are successfully transplanted into intestinal injury and inflammation mouse models, leading to engraftment and regeneration of damaged mouse intestinal epithelium. As a reliable and scalable xenogeneic-free ISC3D-hIO culture method, XF-DISC is highly promising for the development of regenerative ISC therapy for human intestinal diseases.