Comparison of different clearing and acquisition methods for 3D imaging of murine intestinal organoids
An organoid is a three-dimensional multicellular structure that shows realistic micro-anatomy of an organ. This in vitro model mimics the in vivo environment, architecture and multi-lineage differentiation of the original organs and allows to answer many interesting biological questions. For these reasons, they are widely used in stem cell, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. In order to study organoids, microscopy is very useful: It is possible to make three-dimensional reconstruction of serial sections but it is time consuming and error-prone. Here we propose an alternative solution: Tissue clearing reduces the dispersion of light because it homogenizes the refractive index of the tissue, allowing sample observation throughout its thickness. We have compared different clearing techniques on mouse intestinal organoids using different acquisition methods.
1. Sato T, Clevers H. Primary mouse small intestinal epithelial cell cultures. Methods Mol Biol. 2013;
2. Sato T, Vries RG, Snippert HJ, Van De Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;
3. Ehman EC, Johnson GB, Villanueva-meyer JE, Cha S, Leynes AP, Eric P, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2011;470(7332):105–9.
4. Kretzschmar K, Clevers H. Organoids: Modeling Development and the Stem Cell Niche in a Dish. Dev Cell [Internet]. 2016;38(6):590–600.
5. Zhang RR, Koido M, Tadokoro T, Ouchi R, Matsuno T, Ueno Y, et al. Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids. Stem Cell Reports [Internet]. 2018;10(3):780–93.
6. Nigro G, Hanson M, Fevre C, Lecuit M, Sansonetti PJ. Intestinal Organoids as a Novel Tool to Study Microbes–Epithelium Interactions. In 2016.
7. Fasciano AC, Mecsas J, Isberg RR. New Age Strategies To Reconstruct Mucosal Tissue Colonization and Growth in Cell Culture Systems. Microbiol Spectr. 2019;7(2).
8. Dedhia PH, Bertaux-Skeirik N, Zavros Y, Spence JR. Organoid Models of Human Gastrointestinal Development and Disease. Gastroenterology. 2016.
9. Sato T, Clevers H. SnapShot: Growing Organoids from Stem Cells. Cell [Internet]. 2015;161(7):1700-1700.e1.
10. Nagaishi K, Arimura Y, Fujimiya M. Stem cell therapy for inflammatory bowel disease. Journal of Gastroenterology. 2015.
11. Vigouroux RJ, Belle M, Chédotal A. Neuroscience in the third dimension: Shedding new light on the brain with tissue clearing. Mol Brain. 2017;10(1):1–10.
12. Tainaka K, Kuno A, Kubota SI, Murakami T, Ueda HR. Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling. Vol. 32, Annual Review of Cell and Developmental Biology. 2016. 713–741 p.
13. Silvestri L, Costantini I, Sacconi L, Pavone FS. Clearing of fixed tissue: a review from a microscopist’s perspective. J Biomed Opt. 2016;21(8):081205.
14. Richardson DS, Lichtman JW. Clarifying Tissue Clearing. Cell. 2015;162(2):246–57.
15. Aoyagi Y, Kawakami R, Osanai H, Hibi T, Nemoto T. A rapid optical clearing protocol using 2,2′-thiodiethanol for microscopic observation of fixed mouse brain. PLoS One. 2015;10(1):1–13.
16. Lai HM, Liu AKL, Ng HHM, Goldfinger MH, Chau TW, Defelice J, et al. Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues. Nat Commun. 2018;9(1).
17. Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc [Internet]. 2015;10(11):1709–27.
18. SunJin Lab, 2015. Available from : https://www.sunjinlab.com/wp-content/uploads/One_Step_Tissue_Clearing_Reagents.pdf
19. SunJin Lab, 2017. Available from : https://www.sunjinlab.com/wp-content/uploads/datasheet.pdf
20. Sato T, Stange DE, Ferrante M, Vries RGJ, Van Es JH, Van Den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology [Internet]. 2011;141(5):1762–72.