Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 130, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/56960
Keywords
Developmental Biology; Issue 130; human intestinal organoids; microinjection; epithelial barrier permeability; enteroids; 3D tissue culture; in vitro imaging
Categories
Funding
- Intestinal Stem Cell Consortium [U01DK103141]
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
- National Institute of Allergy and Infectious Diseases (NIAID)
- NIAID Novel, Alternative Model Systems for Enteric Diseases (NAMSED) consortium [U19AI116482]
- National Institute of Allergy and Infectious Disease (NIAID) [T32AI007528]
- Clinical and Translational Science award [UL1TR000433]
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Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.
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