Volume 39, Issue 5, Pages 716-723 (November 2003) Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells  Keisuke Harada, Toshihiro Mitaka, Shigeki Miyamoto, Shinichi Sugimoto, Shinichiro Ikeda, Hiroshi Takeda, Yohichi Mochizuki, Koichi Hirata  Journal of Hepatology  Volume 39, Issue 5, Pages 716-723 (November 2003) DOI: 10.1016/S0168-8278(03)00412-4

Fig. 1 Experimental protocol: SH colonies were isolated from the culture dishes at 12–14 days after plating and replated on collagen sponges (CS) or collagen-coated dishes (C). Black arrowheads indicate the days for collecting the medium, whereas white arrowheads indicate the days of 2 mM NH4Cl treatment. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 2 Phase-contrast photographs of SH colonies replated on a new collagen-coated dish. The cells within the same field marked by a needle are shown. (A) Day 3. SH colonies attached on the dishes. NPCs are shown by arrows. (B) Day 6. The proliferation of SHs can be clearly observed and NPCs also proliferate around the colonies. (C) Day 9. (D) Day 13. The proliferation of NPCs is suppressed, whereas SHs continue growing. (E) Day 16. (F) Day 28. All photographs show the same magnification. Scale bar, 50 μm. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 3 Photographs of replated SH colonies on a collagen sponge. (A) Day 7 after replating. Hematoxylin-eosin staining was carried out. SH colonies attached on the collagen fibers. (B) Day 56. The dark violet shows the cell aggregates (arrowheads). They seem to be hepatic organoids. Scale bar, 150 μm. (C) DNA synthesis of the cells replated on a collagen sponge is shown by the immunocytochemistry for BrdU. The cells were treated with 40 μM BrdU at day 7 and, 24 h later, fixed with cold absolute ethanol. The cells with brown nuclei incorporated BrdU into their DNA. Scale bar, 50 μm. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 4 Perpendicular sections of the collagen sponge are shown. Hematoxylin-eosin staining (A–C) and immunocytochemistry for albumin (D), CK8 (E), CK19 (F), desmin (G), ED1/2 (H), and SE-1 (I) were carried out. (A) Day 7. (B) Day 21. (C) Day 56. Scale bars, 30 μm. Albumin and CK8 were used as markers for hepatocytes, CK19 for bile duct cells, desmin for stellate cells, ED1/2 for Kupffer cells, and SE1 for sinusoidal endothelial cells. The cells positive for the antibody are stained green. E-cadherin or β-catenin (red) was used for the identification of epithelial cells and the cells positive for the antibody are stained red. DAPI (blue) was used for the identification of nuclei. The images of these triple stainings were taken by fluorescent microscopy and analyzed with the CELLScan system. (D–I) Day 28. Scale bar, 20 μm. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 5 Transmission electron micrographs of a perpendicular section of cells in a collagen sponge at day 21. The cells have many mitochondria, rER, peroxisomes, and glycogen granules in their cytoplasm. (A) Arrows show bile canaliculi between cells. Scale bar, 2 μm. (B) Large peroxisomes with a crystalline nucleoid (arrows) are observed in the cytoplasm. Scale bar, 500 nm. (C) Arrows show gap junctions and arrowheads show desmosomes between hepatocytes. Scale bar, 200 nm. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 6 (A) Albumin secretion into the culture medium of the cells replated on collagen sponges; 1.25×103 (circle), 2.5×103 (square), 5×103 (diamond), and 10×103 (triangle) colonies were replated on collagen sponges. The details are described in Materials and methods. Three independent experiments were performed. The points show the average of three dishes and the bars S.D. (B) Albumin secretion of the cells cultured in collagen-coated dishes (white) and in collagen sponges (black); 2.5×103 (squares) and 1.25×103 (circles) colonies are shown. Three independent experiments were performed. The points show the average of three dishes and the bars S.D. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 7 Western blot analysis for transferrin, haptoglobin, and fibrinogen of the medium (A) and CPS and CYP 1A1 of the cells cultured on the collagen sponge (B); 2.5×103 colonies were replated on collagen sponges (CS) and on collagen-coated dishes (C). Samples (medium, 1 μl/lane; cells, 10 μg/lane) were separated by 10% SDS-PAGE. NS, normal rat serum or plasma (×1000 diluted with PBS); P, primary MHs at 3 h after plating. (C) Northern blot analysis for albumin and transferrin mRNAs of the cells cultured on the collagen sponge. For each lane, 20 μg of total RNA was applied and hybridized with each cDNA probe after blotting. Details are described in Materials and methods. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 8 Urea synthesis of the cells cultured in collagen sponges (squares) and collagen coated dishes (circles). The medium was replaced with that supplemented with 2 mM NH4Cl at days 7 and 14 after replating. Twenty-four hours later, the medium was collected. The methods are described in Materials and methods. Three independent experiments were performed. The points show the average of three dishes and the bars S.D. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)

Fig. 9 Corresponding phase-contrast (A and C) and fluorescent images (B and D). Uptake and metabolization of FD by cells in the sponges at day 56. Secretion of fluorescence into bile canaliculi is clearly shown. BC are organized between the cells cultured in collagen sponges (arrows). All photographs show the same magnification. Scale bar, 30 μm. Journal of Hepatology 2003 39, 716-723DOI: (10.1016/S0168-8278(03)00412-4)