Generation of Human Intestinal Organoids from Intestinal Inflamed Tissues

POLO BELEN; BARBIERA ROMERO, EMANUEL; VACCARO, JULIÁN; ILID, MANUELA; MENENDEZ LORENA; BOROBIA PAULA; ZUBIRI CECILIA; ZOSI ANABELLA; GUZMAN, LUCIANA; ALTAMIRANO EUGENIA; CORREA GUSTAVO; YANTORNO MARTÍN; SAMBUELLI ALICIA; ROCCA ANDRES; SARACHO MARIA; COLLIA KARINA; GENTILINI MARIA; GONDOLESI GABRIEL; CURCIARELLO, RENATA; DOCENA GUILLERMO; MUGLIA, CECILIA ISABEL

Congreso; Sociedad Argentina de Inmunología; 2023

Intestinal organoids are self-organized three-dimensional structures that partially recapitulate the identity, cellular heterogeneity, and behavior of the original intestinal tissue in vitro. Intestinal stem cells are located in crypts, and are capable of self-renewal and differentiate into major epithelial lineages. As such, organoids have emerged in recent years as valuable models for studying intestinal development, homeostasis, diseases, and regeneration. In this study, our aim was to obtain and characterize human intestinal organoids using stem cells derived from samples under various pathological inflammatory conditions.We firstly quantified stem cells in intestinal biopsies (n=23) and surgical specimens (n=6) from inflamed regions and adjacent tissue of adult patients with colorectal cancer (CRC), inflammatory bowel diseases (IBD) (n=19), and healthy controls (n=8). We also analyzed samples from colorectal polyps of pediatric patients sensitized to cow’s milk protein (n=7) and biopsies of the surrounding areas (n=2). Epithelial cells were obtained by incubation of samples in HBSS supplemented with EDTA 0,5 mM. Crypts were detached by vigorous shaking. Stem cells were identified and quantified as Lgr5+ cells using flow cytometry. Subsequently, we initiated organoid cultures by isolating intestinal crypts from the aforementioned samples and embedding them in Corning® Matrigel® Matrix with IntestiCult™ Organoid Growth Medium. Organoids were passaged every 7 to 10 days and subjected to analysis via fluorescence microscopy.Our results revealed a higher frequency of LGR5+ cells in inflamed IBD tissue compared to non-inflamed IBD tissue and healthy controls (p=0.007). Conversely, a smaller proportion of LGR5+ stem cells was observed in polyp samples compared to the control biopsies surrounding the polyps (p=0.04). We obtained organoids from IBD biopsies, surgical samples of the small intestine, colorectal polyp tissues, and biopsies surrounding the polyps. We verified the expression of the epithelial marker Epcam and the stem cell marker Lgr5 through fluorescence microscopy. Furthermore, we detected proliferating cells using the Ki-67 marker.In conclusion, we effectively generated human intestinal organoids from healthy and inflammatory samples of adult and pediatric patients. These organoids were characterized based on the expression of distinct markers. Further analyses, encompassing functional studies, RNA expression profiling, and co-culture experiments are planned to gain deeper insights into the role of epithelial cells in different inflammatory settings.