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. 2019 Mar 19;9:4863. doi: 10.1038/s41598-019-41273-3

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© The Author(s) 2019

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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(a) Hematoxylin and eosin stained thin sections of early-stage spheroids at day 3 with loosely connected cell clusters (black arrows), mid-stage spheroids at day 6 with smooth spheroid surfaces and late-stage HepG2 spheroids starting at day 12 post-seeding with disintegrated spheroid surfaces (arrow). (b) Transmission electron microscopy micrographs of early-stage spheroids at day 3 with extracellular space between plasma membranes (black arrows), mid-stage spheroids at day 6 with tight junctions (black arrows) and late-stage HepG2 spheroids starting at day 12 post-seeding with blebbing of the cell surface (arrowhead), condensed chromatin (arrow) and apoptotic bodies (AB). Scale bar, 100 µm (top panel) and 1 µm (bottom panel).