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. 2017 Oct 26;7:14085. doi: 10.1038/s41598-017-14542-2

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The histological analysis of ex vivo-fabricated liver-like tissue. (a,b,c) The histological analysis of the structures on the needles (3days after 3D printing) by hematoxylin-eosin staining, Masson’s trichrome staining and immunostaining using anti-human CD31 antibodies. (b) During tissue construction, the cells self-produced ECM. (c) HUVECs (indicated by the arrowheads) covered the surface of the tissue. (d,e) Fluorescence images of the liver-like tissue on the 7^th day in culture. hHeps were stained with anti-human albumin antibodies (green) and HUVECs were stained with anti-human CD31 antibodies (red). The production of albumin by hHeps and the formation of reticular endothelial networks inside the liver-like tissue were verified. Scale bars, 200 μm. (f) An HE-stained cross-section on the 7^th day in culture. The hepatocytes were viable, even in the center of the tissue, the thickness of which was approximately 600 μm. Scale bars, 100 μm. (g) Immunostaining of the liver-like tissue on the 24^th day in culture. CK19-positive cells and a duct-like morphology were observed. The arrowheads indicate the CK19-positive cells with a duct-like morphology. Scale bars, 200 μm. (h) Fluorescence images of the bile duct-like structure in the liver-like tissue.