Figure 1.

Generation of Long-Term Canine Hepatic Organoid Cultures Derived from Adult Liver Tissue by Duct Isolation

(A) Scheme of organoid culture. Canine liver tissue obtained from Tru-Cut biopsies, fine-needle aspiration biopsies, or wedge biopsy was enzymatically digested to achieve biliary duct fragments. Isolated ducts were cultured as described. See also Figures S1 and S2.

(B) Representative images of K19, HepPar1, α-SMA, MAC384, and Factor VIII immunostaining in isolated fraction at day (d) 0, showing the presence of biliary duct fragments, hepatocytes, stellate cells, macrophages, and endothelial cells, respectively, in the initial culture fraction, and K19 staining of organoids after 1 week in culture. HepPar1, α-SMA, MAC384, and Factor VIII stainings were negative in organoids 1 week after isolation.

(C) Representative phase contrast images of biliary ducts fragments and growing hepatic organoids in culture at indicated time points and passage numbers. The lower right image is the 3D overlay of Ki67 and DAPI immunofluorescence staining, indicating strong proliferation in organoids.

(D) Representative karyotype of a cell with normal chromosome number n = 78. The graph shows the percentage of cells with chromosome counts in organoids after 1, 3, and 5–8 months in culture (n = 3 independent organoid isolations per time point, 100 cells counted per culture); 1–3 loss (n = 75, 76, and 77); and 1 gain (n = 79).

See also Table S1.