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Introduction Intestinal organoids are three-dimensional in vitro structures derived from stem cells that have emerged as a new modality to study cellular processes and intestinal function. They mimic cellular heterogeneity, morphology and mucosal functions. Here, we have brought together a multidisciplinary team to study inflamed and non-inflamed organoids derived from the Birmingham IBD Inception Cohort. We aim to develop a chronic inflammatory mucosal organoid model with mucus production in ulcerative colitis (UC) by sampling inflamed and non-inflamed mucosal tissue. These can be used to study the host-microbiota interface. More specifically, we aim to culture pathobionts identified from the STOP-colitis clinical trial with host mucus, epithelium and autologous macrophages to determine the mechanisms underpinning inflammatory pathology. (Quraishi MN, 2024)Methods So far, 14 patients were sampled (7 UC, 2 Crohn’s disease and 5 Non-IBD patients). A 2D air liquid interface (ALI) epithelial monolayer was generated using various media to assess mucus production and thickness.Mucosoid cultures were co-infected with Anaerobutyricum hallii at a multiplicity of infection of 10:1 for 2 h under 2% oxygen. Fixed monolayers were stained using combined immunohistochemistry and fluorescent in situ hybridisation, with antibodies against E-cadherin and Muc2 on specific RNAscope probes targeting the bacterial 16S rRNA gene, respectively. Transwell filters were mounted onto glass microscope slides in DAPI-containing mounting medium, before viewing with confocal microscopy. Organoids were also co-cultured with monocyte-derived macrophages (MDMs) from IBD patients.Results Organoids were consistently generated from Inception IBD patients (from non-inflamed sites). Time to monolayer confluency formation was optimal at 1x10 6 cells/mL (3-5 days) and cultures were stable for over 6 weeks. Mucus formation was achieved 21 days after ALI cultures in differentiation media, as confirmed by visualisation and removal of mucus from the top chamber of the ALI. Co-culture of epithelial monolayer with MDMs was confirmed by visualisation of cellular morphology under light microscopy. Infection of epithelial monolayers with A. hallii is ongoing.Conclusion We have successfully established ALI cultures from the Inception cohort, with 100% efficacy. We are currently interrogating the host mucus, epithelial and macrophage response to pathobionts and commensals shown to be clinically relevant to UC pathogenesis from a range of FMT-IBD clinical trials.