Ulcerative colitis (UC) is characterized by chronic relapsing inflammation starting from the rectum and distal colon, which in severe disease cases may affect the entire colon. Intestinal stem cells (ISCs) directly isolated from inflamed UC colonic tissue specimens have been found to present an inflammatory gene expression profile. However, a critical issue is whether these cells retain memory of exposure to inflammation and/or therapeutics. Here, we aimed to investigate whether human intestinal epithelial cells retain the inflammatory state observed in vivo when expanded in vitro as 3D cultured organoids to assess their suitability for therapeutic transplantation. ISCs were isolated from noninflammatory bowel disease controls (noninflamed; n = 18), as well as from colonoscopy-obtained biopsies of the sigmoid colon from individuals diagnosed with UC (inflamed), who were glucocorticoid naive (n = 19). Moreover, ISCs were collected from all patients with inflammatory bowel disease following prednisolone treatment. Epithelial cells were cultured as 3D intestinal organoids in media to support stem cell maintenance and differentiation. Subsequently, the 3D intestinal organoids were harvested at the end of passage 2 for bulk RNA sequencing. The data revealed that the cellular phenotype of in vitro-cultured epithelial cells isolated from inflamed tissue did not maintain the hallmarks of inflammation observed in the ulcerated environment from which the cells were initially obtained. Our findings indicate that the autologous reinsertion of in vitro-expanded ISCs in active stages of UC may aid in intestinal healing, which calls for future clinical studies. Additionally, a link between organoid morphology and the inflammatory state of the tissue of origin was identified, as organoids derived from inflamed colon exhibited a lower degree of circularity.