Abstract: Intestinal stem cells (ISCs), located at the crypt base of the intestinal epithelium, hold significant promise as a cell source for regenerative medicine targeting gastrointestinal diseases. While human Intestinal organoid (hIO) cultures have emerged as a powerful tool for studying ISCs in vitro, efficient and scalable expansion of ISCs remains challenging due to their limited portion within the diverse cellular population of hIOs. To overcome these limitations, we previously developed a novel 2-dimensional (2D) culture method for ISCs derived from hIOs (ISCs3D-hIO), enabling rapid expansion, long-term maintenance, and cryopreservation through enriched ISC populations and enhanced luminal accessibility via in vitro differentiation into 2.5 dimensional (2.5D) intestinal epithelium. However, the reliance on undefined basement membrane extract (BME) like Matrigel posed a critical barrier to the development of clinical-grade cell therapies. Here, we introduce the xenogeneic-free dish for ISC (XF-DISC), a scalable and clinical-grade culture platform deposited by using initiated chemical vapor deposition (iCVD) process on cell culture substrates. The XF-DISC supported the rapid expansion of ISCs3D-hIO, achieving a 24-fold increase in 30 days, while maintaining long-term expansion over 30 sequential passages. Additionally, the XF-DISC enabled robust stock banking, with ISCs3D-hIO cryopreserved for over 3 years, and facilitated efficient differentiation into intestinal epithelium comparable to Matrigel-coated surfaces. Furthermore, ISCs3D-hIO cultured on XF-DISC demonstrated their clinical potential through successful engraftment and regeneration of the intestinal epithelium in EDTA-induced injury and DSS-induced colitis mouse models via direct transplantation. This innovative xenogeneic-free culture method offers a scalable and clinical-grade solution, underscoring its potential as a therapeutic tool for regenerative medicine in human intestinal diseases.
