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. 2021 Jan 12;11:515. doi: 10.1038/s41598-020-80019-4

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Optimized 3D collagen culture conditions promote spheroid formation and the maintenance of hepatic functions over time. (A) TPEF images of Hepoid after 8 days of culture. 3D reconstruction (xyz) of (left) 250 μm and (middle left) 100 μm depth images stack (TPEF stack) at 10× and 60× magnification, respectively; (middle right) and (right) are TPEF images taken at 20× and 60× magnification, respectively. (B) Quantification of the gene expression patterns by RT-qPCR of E-cadherin and N-cadherin in FIH (black), 2D cultures (light grey) and 3D cultured PHH (dark grey), 4 (d4), 15 (d15) and 28 days (d28) after seeding. The results are from at least three independant experiments and are expressed according to the 2D culture level (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test, GraphPad Prism v.7.0). (C) Immunostaining of E-cadherin or N-cadherin (red), Albumin (green) and nuclei (blue) in PHH spheroids at d4, d15 and d28. (D) Immunostaining of MKL1 (pink) and nuclei (blue) in 2D and 3D cultures. Scale bar = 25 μm.