Hepatocyte-derived cultures form bilayers in two-dimensional cell cultures. (A) Organization of tubule-forming epithelia and hepatocytes. Lumina are in red. (B) MDCK, WIF-B9 and HepG2 cells grown in 2D cultures were fixed and analyzed for the distribution of the apical protein Ezrin and the TJ marker ZO-1. (C) x-y-t brightfield time-lapse sequence of MDCK, WIF-B9, and HepG2 cells plated at equivalent density. Cells were imaged for 25 h, then fixed and stained as indicated. Confocal sections corresponding to the brightfield area (bottom). (B and C) Note the presence of cells not contacting the substratum indicated by asterisks in the nuclei. Arrowheads show hepatocytic lateral lumina.