GENERATION OF HUMAN INTESTINAL ORGANOIDS FROM INTESTINAL INFLAMED TISSUES

MARIA BELÉN POLO; EMANUEL BARBIERA ROMERO; JULIÁN VACCARO; MANUELA ILLID; LORENA MENENDEZ; ANABELLA ZOSI; LUCIANA GUZMAN; VIVIANA BERNEDO; EUGENIA M. ALTAMIRANO; GUSTAVO J. CORREA; MARTÍN YANTORNO; ALICIA SAMBUELLI; ANDRÉS ROCCA; MARÍA BELÉN SARACHO; KARINA COLLIA; MARÍA VIRGINIA GENTILINI; GABRIEL GONDOLESSI; RENATA CURCIARELLO; GUILLERMO H. DOCENA; CECILIA I. MUGLIA

San Luis

Congreso; Reunión Anual de la Sociedad Argentina de Inmunología (SAI); 2023

Intestinal organoids are self-organized three-dimensional structures that partiallyrecapitulate the identity, cellular heterogeneity, and behavior of the originalintestinal tissue in vitro. Intestinal stem cells are located in crypts and are capableof self-renewal and differentiate into major epithelial lineages. As such, organoidshave emerged in recent years as valuable models for studying intestinaldevelopment, homeostasis, diseases, and regeneration. In this study, our aimwas to obtain and characterize human intestinal organoids using stem cellsderived from samples under various pathological inflammatory conditions.We firstly quantified stem cells in intestinal biopsies (n=23) and surgicalspecimens (n=6) from inflamed regions and adjacent tissue of adult patients withcolorectal cancer (CRC), inflammatory bowel diseases (IBD) (n=19), and healthycontrols (n=8). We also analyzed samples from colorectal polyps of pediatricpatients sensitized to cow’s milk protein (n=7) and biopsies of the surroundingareas (n=2). Epithelial cells were obtained by incubation of samples in HBSSsupplemented 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 theaforementioned samples and embedding them in Corning® Matrigel® Matrix withIntestiCult™ Organoid Growth Medium. Organoids were passaged every 7 to 10days and subjected to analysis via fluorescence microscopy.Our results revealed a higher frequency of LGR5+ cells in inflamed IBD tissuecompared to non-inflamed IBD tissue and healthy controls (p=0.007). Conversely,a smaller proportion of LGR5+ stem cells was observed in polyp samplescompared to the control biopsies surrounding the polyps (p=0.04). We obtainedorganoids from IBD biopsies, surgical samples of the small intestine, colorectalpolyp tissues, and biopsies surrounding the polyps. We verified the expression ofthe epithelial marker Epcam and the stem cell marker Lgr5 through fluorescencemicroscopy. Furthermore, we detected proliferating cells using the Ki-67 marker.In conclusion, we effectively generated human intestinal organoids from healthyand inflammatory samples of adult and pediatric patients. These organoids werecharacterized 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 indifferent inflammatory settings.