Fig. 3
3D colon organoids and organoid-derived colonic epithelial monolayers resemble the typical appearance of colonic epithelium and empower co-cultivation with commensal bacteria. (A-B) Representative pictures of 3D colonic epithelial organoids (colonoids) and cellular composition of organoid-derived monolayers of control individual (A) and patient with active UC (B). Hoechst 33,342 (blue) was used in all cases as a counterstain for cell nuclei. Areas of proliferation are identified by Ki67 (green) expressing proliferating cells. Epithelial barrier integrity is defined by detection of tight junction protein ZO-1 (orange). Absorptive colonocytes are defined by positive Cytokeratin 20 (green) staining. Mucin-producing Goblet cells are identified by positive Mucin 2 (red) staining. Hormone-producing enteroendocrine cells are defined by positive Chromogranin A (green) staining. (C-D) Representative pictures of colonic epithelial monolayers co-cultured with Escherichia coli(C) and Phocaeicola vulgatus(D). (E) Expression analysis of marker genes (x-axis), representing host response to pathogen recognition (TLR4), tight junction regulation (ZO1) and stress stimuli indication (HSPA1A and HSPB1). Expression estimates (Ct) were normalized to ATCB (delta-Ct) and were inverted (as log2(2^−deltaCT)) to recapitulate direction of the effect