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. 2023 Jul 26;13:12130. doi: 10.1038/s41598-023-39425-7

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© The Author(s) 2023

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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Development and characterization of a cyst enriched-organoid. (a) Scheme of the development of a cyst enriched-organoid and the bright-field images of organoids. Scale bar = 100 µm. (b) The bright-field images of organoids derived from single cells prepared by day 4 conventional organoids and cyst-enriched organoids. Growth of the organoids was analyzed with CellTiter-Glo^® 3D Cell Viability Assay and the bright-field image analysis by cellSens. Data represents mean ± s.d. (n = 3, biological replicates). Scale bar = 1 mm (c) The bright-field images of organoids in the growth factor reduced conditions and the results of the growth factor dependency analysis using CellTiter-Glo^® 3D Cell Viability Assay. Scale bar = 100 µm. Data represents mean ± s.d. (n = 4, biological replicates). Δ means omitting a specific growth factor from the complete medium. (d) Whole mount immunostaining of cyst-enriched organoid for Sox9 (green) and F-actin (white), Ki-67 (red) and F-actin (white), merge with DAPI (blue), CK20 (green) and P-gp (red) with DAPI (blue), phospho-Ezrin (p-Ezrin, red) and DAPI (blue). Scale bar = 100 µm. **p < 0.01, ****p < 0.0001. Student t test for organoid formation assay, and Dunnett’s test compared with control for growth factor dependency analysis. W_20% 20% Afamin/Wnt3a CM, E epidermal growth factor, N Noggin, R R-spondin 1, A A83-01, I IGF-1, F FGF-2, Y Y-27632, g Gastrin.