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. 2018 Jan 10;8:230. doi: 10.1038/s41598-017-18431-6

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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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In vivo CAM assay. The angiogenic potential of spheroids in both conditions was tested using chick embryonic membrane. (A) Brightfield images of spheroids inside the CAM O-ring; (B) photomicrographs acquired for counting newly formed vessels; (C) Angiogenic effect of spheroids grown in the standard versus XF conditions, expressed by number of newly formed vessels (n ≥ 12, two independent experiments, ***p < 0.0004, Mann-Whitney test was used for statistical comparison). (D) HE staining of CAM interface and spheroids after 72 h incubation time (Scale bar 100 μm). (E) Immunofluorescence of equivalent sections represented in D (Scale bar 50 μm). vWF (green) was used to access endothelial organization, HuNu (red) to discriminate human nuclei and DAPI (blue) for total nuclei. In images (D) and (E), insets (and respective 4× magnifications, depicted on the right) highlight the direct contact between spheroids and the CAM. Yellow arrows are pointing to organized OEC inside the compacted spheroids.