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. 2017 Jul 21;7:6163. doi: 10.1038/s41598-017-06369-8

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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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3D lacrimal gland (LG) cell spheroids formation and culture. (A–D) Phase contrast and F-actin staining (Phalloidin) images of LG cell spheroids in Matrigel at different stages. (A,C) day 2; (B,D) day 5. (E,F) IHC showing the expression of LG specific markers (red; counterstained by DAPI). (E) Pan-cytokeratin; (F) lysozyme. Scale bar: 400 μm (A,B); 200 μm (C); 100 μm (D); 50 μm (E,F). (G) Schematics of LG cultured as either 2D monolayer or 3D spheroids embedded in Matrigel. (H) Gene expression (RT-qPCR; fold change is expressed as 2^−ΔΔCT) and (I) β-hexosaminidase assay comparing LG cells culture under 2D and 3D conditions. Both supernatant and cell lysate were collected for hexosaminidase assay and the value (units) was normalized to total protein content (mg). *p < 0.05; **p < 0.001.