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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.44 No.2 pp.62-68
DOI : https://doi.org/10.13041/jpvm.2020.44.2.62

Development and characterization of mouse small intestinal enteroids

Siu Lee#,Na-Yeon Gu#,Se-A Lee,Jienny Lee,Yoon-Hee Lee,Bang-Hun Hyun
Viral Disease Research Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660 Republic of Korea
# These authors equally contributed.
Corresponding Author. Jienny Lee, Tel: +82-54-912-0809, Fax: +82-54-912-0812, E-mail: roska@korea.kr

Abstract

The culture of the intestinal epithelium into three dimensional (3D) structures typically termed organoid culture. Organoid culture is based on the ability of intestinal stem cells (ISCs), at the base of the crypt, perpetually to divide and produce a fully differentiated, polarized epithelium. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) positive ISCs isolated from the intestine can form organoids in long-term culture. Thus, when cultured under the appropriate 3D conditions, single Lgr5+ ISCs undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal epithelial cells (IECs). In this study, we performed isolation, characterization and consecutive subculture of small intestinal crypts from BALB/c-nude mouse. Briefly, isolated mouse crypts were embedded in matrigel, cast into 40 μL droplets at the bottom of well in a 48-well plate. Following polymerization, the gels were overlaid with ISCs expansion medium containing B27, N2, N-acetylcysteine, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse crypt-derived ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs-related specific genes (Lgr5, sox9) and IECs-related specific genes (chromogranin A, defensin-5, mucin-1, mucin-2, and villin) was maintained at eight passages or more. Thus, we observed that expression of specific markers and consecutive self-renewing in the mouse small intestinal crypt-derived organoids.

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