P0022 Development of primary human fibroblast cell cultures for fibrosis modelling in Inflammatory Bowel Diseases

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Abstract Background Inflammatory Bowel Diseases (IBD) are chronic immune-mediated diseases of the gastrointestinal tract. Fibrosis is frequent in IBD driven by inflammatory cytokines. During fibrosis, extracellular matrix proteins accumulate in the tissue, causing loss of epithelial function and stenosis, leading to malabsorption and intestinal motility problems. This can ultimately lead to serious complications, strictures, fistulas, or ileus. Anti-inflammatory drugs cannot target fibrosis, only surgical intervention can be used. No relevant primary in vitro cell culture model is available for intestinal fibrosis modelling. Therefore, we aimed to establish and optimize human primary fibroblast cell culture from intestinal biopsies of control and IBD patients. We characterized and compared the fibrosis phenotype and gene expression as well. Methods Fibroblast cell cultures were generated from colonic biopsy samples by enzymatic digestion. In a suitable medium, the cells attach to the bottom of the vessel and proliferate, at 90% confluence the cultures were passaged. The phenotype was confirmed by immunofluorescent staining. Target proteins: COL1A1 (Collagen type 1 alpha 1), TGF-ß1 (Transforming growth factor beta), PAI-1 (Plasminogen activator inhibitor type 1), α-SMA (Alpha smooth muscle actin), and PHH3 (Phosphohistone H3). For gene expression analysis RNA was isolated and qRT-PCR technique was used for the following target genes: COL1A1, ACTA2 (Actin alpha 2), TGF-ß1, FN1 (Fibronectin 1), PAI-1, H3C4 (H3 clustered histone 4). We evaluated the collected data and performed statistical analysis. Results We can efficiently create and maintain human colon primary fibroblast cell cultures from colonic biopsies of healthy and CD origins. Cell morphology differences were observed from control and diseased patient samples. At the staining, the fluorescent intensity of fibrotic markers (COL1A1, TGF-ß1, PAI1) and the mitosis marker PHH3 were significantly higher in the CD samples, indicating that the cells maintained fibrotic phenotype, and the diseased fibroblasts had a higher proliferation rate. The myofibroblast marker, α-SMA didn’t show a significant difference. Interestingly, significant differences were not noted between the two groups in the qRT-PCR measurements. Conclusion In conclusion, we can generate primary human intestinal fibroblast cell cultures and maintain them to passage number 3. Fibrotic protein markers and morphology distinguish the diseased samples from the healthy subjects, therefore it could be useable for in vitro fibrosis modelling. However, the gene expression analysis doesn’t discriminate between the two groups at passage number 3. In the future, we would like to observe the fibrotic properties in earlier passage numbers.