Extended Data Fig. 1: HepOrg grown in optimized medium expand long-term.
From: Mouse liver assembloids model periportal architecture and biliary fibrosis
a. Experimental design. b. Immunofluorescence staining for bile canaliculi (CD13, green) and F-actin (Phall, grey) in 2D-hepatocytes cultured as sandwich culture, HepOrg cultures grown in Hu et al. media, Peng et al. media, and in liver tissue sections. Note the difference in organoid size and length of the bile canaliculi when compared to the optimized medium in Fig. 1d. DAPI stained nuclei (blue). Representative images from n = 3 independent experiments. Scale bar, 50 µm. c. Schematic illustration of the 3-dimensional (3D) nature of bile canaliculi network within HepOrg. d. Schematic representation of the different measurements used to describe the bile canaliculi network. e. Graph represents the length of the largest network in HepOrg cultured as indicated. Dot, measure per organoid. Line, mean. Kruskal-Wallis-test followed by Dunn’s multiple comparison post-hoc test. f. Graph represents the number of quadruple junctions for the largest network in tissue and HepOrg cultured as indicated. Line, mean. Dot, measure per organoid. Kruskal-Wallis-test followed by Dunn’s multiple comparison post-hoc test. g-h. Bright-field pictures of HepOrg at passage 0 (P0, 9 days after seeding) (g), or at passage 2 (P2, 29 days after seeding) (h), seeded as sparse culture (1000 cells/well) and cultured under the indicated media conditions. Representative images from n = 3 independent experiments. Scale bar, 500 µm; zoom-in 100 µm. i. Graph shows HepOrg expansion over time. Each line represents an independent biological replicate. Cultures were split at a 1:2 split ratio. Dot, time of passage. j. Growth curves of HepOrg grown in the indicated media. Values represent total number of cells at the indicated passage expressed as mean ± SEM from n = 4 independent biological replicates with n = 2 technical replicates. Statistics are provided between HM-Wnt and the other conditions and presented colour-coded for the condition they compare to; multiple unpaired t-tests, two-sided.
