171 | Development of a 3D ex vivo model of human intestinal organoids for the study of inflammatory bowel diseases

Giulia Maria Giorgia Lo Piano¹, Massimo Biondo¹, Adelaide Carista¹, Domiziana Picone¹, Stefano Burgio^2|3, Alberto Fucarino⁴, Francesca Rappa¹, Alessandro Pitruzzella¹, Fabio Bucchieri¹ | ¹Department of Biomedicine, Neuroscience and Advanced Diagnostics BIND, University of Palermo, Italy; ²Euro-Mediterranean Institute of Science and Technology IEMEST, Palermo, Italy; ³Department of Medicine and Surgery, Kore University of Enna, Enna, Italy; ⁴Department of Theoretical and Applied Sciences, eCampus University, Novedrate, Como, Italy.

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, encompassing ulcerative colitis (UC) and Crohn's disease (CD), arising from complex interactions between genetic predisposition, environmental factors, and immune dysregulation [1]. Despite significant advances in understanding the intricate pathophysiology of IBD, research in this field is currently limited by the lack of experimental intestinal models that accurately recapitulate the complex three-dimensional (3D) architecture and cellular heterogeneity of the human intestinal mucosa [2]. To address this gap, we established an ex vivo cellular model using patient-derived human intestinal organoids (HIOs) starting from duodenal biopsies. Lgr5+ stem cells were isolated from intestinal crypts and cultured using two distinct culture models: traditional Matrigel-embedded droplets and a Transwell-based system. Organoid morphology was regularly monitored by phase-contrast microscopy, while histological and immunohistochemical analyses evaluated structural integrity, mucus production, and the presence of specialized epithelial lineages. Both culture systems supported organoid growth and maturation, with organoids transitioning from spherical cystic structures to complex budding phenotypes. These morphological changes reflected the native crypt-villus architecture and demonstrated differentiation into multiple intestinal epithelial cell types (enterocytes, Goblet cells, Paneth cells, and enteroendocrine cells), alongside maintenance of a localized stem cell compartment [3]. However, the Transwell system facilitated downstream analyses, including paraffin embedding, microtome sectioning, and immunostaining. Preliminary immunohistochemical analysis of HIOs cultured on Transwell PET membranes highlighted the presence of a proliferative compartment (Ki67+) in crypt-like regions, as well as the detection of enteroendocrine cells (Chromogranin A+). In addition, Alcian Blue-PAS staining revealed areas of mucin production, consistent with the presence of differentiated mucus-producing Goblet cells. Taken together, these preliminary results validate the Transwell-based HIO model as a reliable platform for intestinal disease modeling. Current efforts focus on comprehensive molecular characterization of organoid cellular composition and functional maturation, as well as implementation of inflammatory stimulation protocols (cytokine treatment, bacterial component exposure) to model IBD-specific conditions. This platform will enable investigation of intestinal epithelial-immune cell interactions and support development of personalized therapeutic strategies for IBD patients.