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<div><p>Patient-derived organoids provide a unique model system to explore disease-causing mutations <i>ex vivo</i>. By using organoids from duodenal or colonic biopsies of pediatric patients with intestinal epithelial disorders, we can directly assay the patient cells to tailor treatment to their unique disease state. The advent of organoid technology from patients with severe intestinal disorders such as Congenital Diarrhea Enteropathies (CoDE) and Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) has allowed for rapid advances in the understanding of and the treatment of these monogenic disorders. Still, the expansion of these lines for scalable studies is not trivial, and success rates of expansion are variable between groups, and even lab members within the same group. These protocols have been validated on patients with CoDE or VEO-IBD and age-matched control patients. Here, we present our recommended protocols for the cultivation of organoids from pediatric patients with CoDE and VEO-IBD. These protocols have been validated on organoids generated from the duodenum (duodenoids), ileum (ileoids), colon (colonoids) and iPSC-derived intestinal colonoids from pediatric healthy donors or donors with CoDE or VEO-IBD (Gwilt et al., 2023). Using our modified culture media, extended culture times from biopsy preparation and thawing frozen lines, gentle passaging techniques with the incomplete removal of the organoids from the matrigel, and modified monolayer protocols (Maeda et al., 2023; Maeda et al., 2022), we have been able to successfully culture and expand several lines for more than 5 years. The conditions and protocols used here provide a basis for reproducible phenotypes, scaling for larger functional studies on patient lines, and for reproducibility of results between several investigators. We provide a useful starting point and troubleshooting guidelines for the optimization of culturing organoids from any patient with novel disease pathology.</p></div>