期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 146, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/59357
关键词
Immunology and Infection; Issue 146; Human enteroid or colonoid monolayers; host-pathogen interaction; apical infection; apical and basolateral secretion; colonic mucus; intestinal organoid monolayer
资金
- NIH [P01 AI125181, K01 DK106323, K01 DK113043]
- Integrated Physiology and Imaging Cores of the Hopkins Conte Digestive Disease Basic and Translational Research Core Center [P30 DK089502]
Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.
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