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. 2021 Oct 22;4:1213. doi: 10.1038/s42003-021-02719-5

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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 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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Fig. 1 — a Schematic diagram of conditions used to induce brain organoid by orbital mixing (upper schema) and by vertical mixing (lower schema). b, c Immunostaining for neural progenitor (SOX2, magenta) and neuron (MAP2, green) in brain organoid generated by orbital mixing (b) or vertical mixing (c) on Day 56. d Quantification of SOX2-positive area in the peripheral region of brain organoid on Day 56. The peripheral region was defined as 100 µm inside from the edge of brain organoid. Brain organoid from vertical mixing showed higher percentage of SOX2-positive area in peripheral region in comparison with brain organoid from orbital mixing. Data represent mean ± SD (n = 6 for brain organoids by orbital mixing, n = 7 for brain organoids by vertical mixing). Difference between the two conditions was analyzed by Student’s two-tailed t-test (*p < 0.0001). e, f Immunostaining for markers of neural progenitors (SOX2, blue), intermediate neural progenitors (TBR2, gray), and cortical neurons (CTIP2, red; MAP2, green) in brain organoids by orbital mixing (e) or vertical mixing (f) on Day 56. g, h Immunostaining for markers of ventricular neuroepithelial cells (N-CADHERIN: N-CAD, gray), neural progenitors (SOX2, magenta), and cortical neurons (MAP2, green) in brain organoids by orbital mixing (g) or vertical mixing (h) on Day 56. Note the apical side of organoids generated by orbital mixing is located inside organoids, while that of organoids generated by vertical mixing is located at the surface of organoids. i Schematic diagram shows the difference of brain organoids by orbital mixing and by vertical mixing. Scale bars = 500 µm (b, c), 100 µm (e, f), 50 µm (g, h).