Xenogeneic-Free Culture of Human Intestinal Stem Cells on the Vapor-Phase Synthesized Polymers for Regeneration of Intestinal Diseases

Intestinal stem cells (ISCs) which reside at crypt base in the intestinal epithelium, served as a promising cell source for regenerative medicine in the context of gastrointestinal diseases. Recently, human Intestinal organoid (hIO) culture was a prominent technology for investigating the molecular and cellular nature of ISCs <i>in vitro</i>. However, it was challenging to efficiently and rapidly expand ISCs in vitro because it constituted a small portion of the cells within the large population of cells in the hIOs. Previously, we developed a novel 2D culture method of ISC^3D-hIO which supported fast expansion, long-term maintenance, and cryopreservation of ISC^3D-hIO by enriched culture of ISCs population and improved luminal side accessibility through in vitro differentiation into 2.5 dimensional (2.5D) intestinal epithelium.<br/>However, due to the undefined nature of basement membrane extract (BME), such as Matrigel, the use of BME for ISC culture posed a significant barrier that had to be overcome for the development of clinical-grade cell therapies. To address limitations posed by traditional culture system, we developed Xenogeneic-Free Dish for ISC (XF-DISC) using initiated chemical vapor deposition (iCVD) process on cell culture substrates. The XF-DISC not only facilitated the rapid expansion with a 24-fold propagation in 30 days, but also enabled long-term expansion of 30 sequential passages. Additionally, XF-DISC supported stable stock banking system of ISCs^3D-hIO stored for over 3 years and enabled efficient differentiation into intestinal epithelium, with no discernible distinctions compared to Matrigel-coated surfaces. Furthermore, ISCs^3D-hIO cultured on XF-DISC were capable of engrafting and regenerating of intestinal epithelium of the EDTA-induced injury and DSS-induced colitis models by direct transplantation into colon of mouse. This innovative culture method for ISCs^3D-hIO presented here underscored its potential for developing intestinal stem cell therapies applicable to regenerative medicine in human intestinal diseases.