Figure 1.

Liver organoids generated by multiple cells. To generate self-assembled liver organoids, Huh-7, liver sinusoidal endothelial (LSECs), LX-2, and macrophage cells were seeded in a Matrigel-coated 96-well plate and cultured for three days. Fibrotic liver organoids were produced by thioacetamide (TAA) treatment for an additional three days. (A) Schematic diagram of the experimental protocol. (B) Representative images of 2D-cultured cells at the indicated time points (days 0–3). The medium was replaced with fresh medium every two days. (C) Representative images at indicated time points (days 0 to 6). The self-assembled liver organoid was cultured for six days without subculturing while exchanging with fresh medium every two days. (D) Viability of 2D-cultured cells and organoids up to six days. * p < 0.05. (E) Cells required for self-assembly of liver organoids. Liver organoids were prepared by excluding one cell type from the Huh-7, LSECs, LX-2, and macrophage cells or by replacing LSECs with human umbilical vein endothelial cells (HUVECs). (F) Images of representative liver organoids generated using immortalized primary hepatocytes and LSECs instead of Huh-7 and LSECs. Scale bar for (B,C,E,F): 1 mm. (G) Immunofluorescence images of liver organoids. To evaluate the cell distribution, antibodies against CK8, CD31, EMR1, and PDGFRα/β were applied to liver organoid sections obtained from day 6 without TAA treatment to detect Huh-7, LSECs, macrophages, and LX-2 cells, respectively. Red arrowheads indicate PDGFRα/β-positive cells. Green arrowheads indicate CK8-, CD31-, or EMR1-positive cells. Scale bars: 500 µm. Data shown for (B–G) are representative of three independent experiments.