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Generation and Quantitative Imaging of Enteroid Monolayers

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Intestinal Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2171))


The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease. Recently, we developed an enteroid monolayer (2D) culture system that recapitulates important features of 3D organoids and the in vivo intestinal epithelium such as tissue renewal, representation of diverse epithelial cell types, self-organization, and apical–basolateral polarization. Enteroid monolayers are cultured in microtiter plates, enabling high-throughput experiments. Furthermore, their 2D nature makes it easier to distinguish individual cells by fluorescent microscopy, enabling quantitative analysis of single cell behaviors within the epithelial tissue.

Here we describe experimental methods for generating enteroid monolayers and computational methods for analyzing immunofluorescence images of enteroid monolayers. We outline experimental methods for generating enteroid monolayers from freshly isolated intestinal crypts, frozen intestinal crypts, and 3D organoids. Fresh crypts are easily obtained from murine or human intestinal samples, and the ability to derive enteroid monolayers from both frozen crypts and 3D organoids enables genetic modification and/or biobanking of patient samples for future studies. We outline computational methods for identifying distinct epithelial cell types (goblet, stem, EdU+) in immunofluorescence images of enteroid monolayers and, importantly, individual nuclei, enabling truly single cell measurements of epithelial cell behaviors to be made. Taken together, these methods will enable detailed studies of epithelial homeostasis and intestinal disease.

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  1. Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265

    Article  CAS  Google Scholar 

  2. van de Wetering M, Francies HE, Francis JM et al (2015) Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161:933–945

    Article  Google Scholar 

  3. Farin HF, Jordens I, Mosa MH et al (2016) Visualization of a short-range Wnt gradient in the intestinal stem-cell niche. Nature 530:340–343

    Article  CAS  Google Scholar 

  4. Thorne CA, Chen IW, Sanman LE et al (2018) Enteroid monolayers reveal an autonomous WNT and BMP circuit controlling intestinal epithelial growth and organization. Dev Cell 44:624–633.e4

    Article  CAS  Google Scholar 

  5. Tian H, Biehs B, Warming S et al (2012) A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature 482:120

    Article  CAS  Google Scholar 

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This work was supported by NIH GM112690 (S.J.A.), NCINIH R01 CA184984 (L.F.W.), the UCSF Program for Breakthrough Biomedical Research which is partly funded by the Sandler Foundation (L.F.W.), NIH NRSA fellowship F32DK120102 (L.E.S.), NSF GRFP fellowship 1650113 (I.W.C.), and NIH R00 DK10312 (C.A.T.).

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Correspondence to Curtis A. Thorne , Lani F. Wu or Steven J. Altschuler .

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Sanman, L.E., Chen, I.W., Bieber, J.M., Thorne, C.A., Wu, L.F., Altschuler, S.J. (2020). Generation and Quantitative Imaging of Enteroid Monolayers. In: Ordóñez-Morán, P. (eds) Intestinal Stem Cells. Methods in Molecular Biology, vol 2171. Humana, New York, NY.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0746-6

  • Online ISBN: 978-1-0716-0747-3

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