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Development and characterization of mouse small intestinal enteroids KCI 등재
- 언어KOR
- URLhttps://db.koreascholar.com/Article/Detail/395390
pp.62-68
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.
서 론
재료 및 방법
실험동물
마우스 소장 유래 크립트(crypt) 분리 및 오가노이드(organoid) 형성
오가노이드(organoid) 계대 배양
정량적 중합효소연쇄반응(quantitative real-time RT-PCR,qRT-PCR)을 이용한 유전자 분석
통계학적 분석
결 과
마우스 소장 유래 크립트 분리 및 U자형 형상
마우스 소장 크립트 유래 오가노이드 형성
마우스 소장 오가노이드의 장 특이 마커 발현
고 찰
REFERENCES
