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. 2021 Mar 9;11:5479. doi: 10.1038/s41598-021-84955-7

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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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Characterization of the maturation process of the 3T3-L1 3D organoid with (DIF+) or without adipogenesis (DIF−). Representative phase contrast microscopy (PC) images at several time points (0, 1, 3, or 6 h, or Day1, 3, 5 or 7) of 3T3-L1 3D organoids without adipogenesis (DIF−) are shown in (A) (Scale bar: 100 µm). Electron microscope (EM) images at several time points (Day1, 3, 5 or 7) of the 3T3-L1 organoids without (DIF−) or with adipogenesis (DIF+) (B, Scale bar: 100 µm). Since adipogenic differentiation was induced from Day 1, the EM image of DIF+ at Day 1 is omitted. A single 3D organoid was compressed by a micro-squeezer, and the compression process was continuously monitored by a microscopic camera (C left, S: sensor, P: plate, O: organoid). As the physical stiffness at several time points (Day 3, 5 or 7) of 3D organoids without (DIF−) or with adipogenesis (DIF+), the force (μN) required to induce deformation until their half diameter (μm) was reached were measured, and force/displacement (μN/μm) was potted (C right). ****P < 0.001, and other pairs were not statistically significant (ANOVA followed by a Tukey’s multiple comparison test).