Abstract
Colonic organoids, three-dimensional colonic crypts grown in vitro that show realistic microanatomy, have many potential applications for studying physiology, developmental biology, and pathophysiology of intestinal diseases including inflammatory bowel disease and colorectal cancer. Here, we describe detailed protocols for mouse colonic crypt isolation, organoid culture, and downstream applications. Specific culture strategies including growth factor enriched Matrigel and Wnt and R-spondin conditioned media serve as key factors for enhancing the growth and cost efficiency of colonic organoid cultures.
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References
Sato T, Clevers H (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340:1190–1194
Zachos NC, Kovbasnjuk O, Foulke-Abel J, In J, Blutt SE, de Jonge HR et al (2016) Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J Biol Chem 291:3759–3766
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265
Sato T, Clevers H (2013) Primary mouse small intestinal epithelial cell cultures. Methods Mol Biol 945:319–328
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007
Zhao J, Li S, Trilok S, Tanaka M, Jokubaitis-Jameson V, Wang B et al (2014) Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells. Development 141:3848–3858
Ohata H, Ishiguro T, Aihara Y, Sato A, Sakai H, Sekine S et al (2012) Induction of the stem-like cell regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating cells. Cancer Res 72:5101–5110
Ahmad AA, Wang Y, Gracz AD, Sims CE, Magness ST, Allbritton NL (2014) Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications. J Biol Eng 8:9
Fan YY, Davidson LA, Callaway ES, Goldsby JS, Chapkin RS (2014) Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells. Carcinogenesis 35:606–612
Fan YY, Davidson LA, Callaway ES, Wright GA, Safe S, Chapkin RS (2015) A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells. Am J Physiol Gastrointest Liver Physiol 309:G1–G9
DeClercq V, McMurray DN, Chapkin RS (2015) Obesity promotes colonic stem cell expansion during cancer initiation. Cancer Lett 369:336–343
Acknowledgement
This work was supported by funding from NIH R35CA197707 and P30ES023512. We would like to thank Drs. Hans Clevers, Nick Barker, and Toshiro Sato for initial guidance in organoid culture technique.
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© 2016 Springer Science+Business Media New York
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Fan, YY., Davidson, L.A., Chapkin, R.S. (2016). Murine Colonic Organoid Culture System and Downstream Assay Applications. In: Turksen, K. (eds) Organoids. Methods in Molecular Biology, vol 1576. Humana, New York, NY. https://doi.org/10.1007/7651_2016_8
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DOI: https://doi.org/10.1007/7651_2016_8
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-7616-4
Online ISBN: 978-1-4939-7617-1
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