Fig 1: Characterization of long-term cultured colonic organoids embedded in an ECM matrix. (A) Morphological changes of colonic organoids during long-term culture. (B) Detection of Mucin proteins (MUC) within the organoids at week 6 using MUC2, MUC3, and MUC4 antibodies. (C) Representative FACS plot for EpCAM-expressing cells of colonic organoids. (D) Immunostaining confirmed the formation of an epithelial layer expressing EpCAM, CD133, or Villin on the outer layer of the organoids. (E) Detection of marker proteins representing various cell types within the epithelial layer, including goblet cells (MUC3, MUC4), enterocytes (Villin), Paneth cells (DEFA5), enteroendocrine cells (CHGA), and colonic stem cells (LGR5). (F) Swelling assay confirming the presence of functional epithelial cells in the organoids treated after forskolin. All scale bars=50 μm. CO: colonic organoid.