Fig. 2. Hepatocytes in the organoids are strongly polarized. MhAT3F cells were grown either in a monolayer or included in Matrigel or a collagen sandwich. The cells had an epithelial phenotype, visualized by the cortical actin distribution (phalloidin-TRITC, red), both in monolayer (2D) and in the organoids (3D). (A) Radixin (fluorescein-isothiocyanate labelled, green), a marker of the apical membrane domains in hepatocytes, showed a strong apical labelling of a subset of monolayer cells, easily visible in 3D-reconstructed (3D reconst.) image and a z-axis sections (below). It decorated the lumen-exposed membranes of all cells in 3D organoids. Nuclei are stained with Hoechst 33258 (blue). 3D-reconstructed images are shown, except for overlay panels, which show single confocal stacks. (B) ZO-1 (fluorescein-isothiocyanate labelled, green), a tight-junction marker, had a heterogeneous distribution in monolayer cells but was strongly localized around the lumen in the organoids. (C) Transmission electron microscopy of monolayer (a,b) and organoids (c,d) allows visualization of microvilli (mv) and tight junctions (tj). Multiple small (a) or larger (b) lumens (IL) lined with microvilli can be seen in the organoids. n, nucleus. (D) Phase-contrast microscopy of cells in the collagen-sandwich culture. Two types of structures can be seen: spheroid organoids (arrows) and extended sheets of cells. (E) Immunofluorescence analysis of collagen-sandwich cultures. Radixin labelling (green) is punctate in extended cells (top) and strongly localized to the interior of the organoids (bottom). Bars, 20 µm (A,B,E), 1 µm (C), 40 µm (D).