简介：
Overview
This article presents a modified protocol for culturing human enteroid or colonoid monolayers that maintain intact barrier function. This model allows for the study of host epithelial-microbiota interactions at the cellular and biochemical level.
Key Study Components
Area of Science
- Neuroscience
- Microbiology
- Cell Biology
Background
- 3D intestinal organoids limit the study of host-pathogen interactions.
- Access to the lumen for bacteria or virulence factors is restricted.
- Sampling of secreted materials from 3D cultures is challenging.
- Colonoid monolayers can provide a new model for studying mucus biology.
Purpose of Study
- To develop a tractable model for studying intestinal host-pathogen interactions.
- To facilitate ex vivo studies of pathogen-mucus interactions.
- To explore mucus biology relevant to inflammatory bowel disease and microbiome studies.
Methods Used
- Conversion of 3D human enteroids or colonoids to monolayers.
- Use of a mini cell scraper and orbital shaker for cell dislodging.
- Immunostaining protocols for monitoring monolayer formation.
- Bright field microscopy and confocal imaging for analysis.
Main Results
- Colonoid monolayers secrete a thick apical mucus layer.
- Confluent monolayers exhibit continuous apical surfaces and F-actin perijunctional rings.
- Progressive loss of proliferation during jejunal monolayer differentiation was observed.
- Fragmentation of colonoids is critical for successful attachment to filters.
Conclusions
- The modified protocol allows for effective study of mucus biology.
- Colonoid monolayers provide insights into host-pathogen interactions.
- This method can be applied to other mucus-secreting organs.
What are colonoid monolayers?
Colonoid monolayers are cultured cells derived from human enteroids or colonoids that maintain barrier function and secrete mucus.
Why is studying mucus biology important?
Mucus biology is crucial for understanding host-pathogen interactions and the role of mucus in diseases like inflammatory bowel disease.
How does the modified protocol differ from traditional methods?
The modified protocol allows for the conversion of 3D cultures to monolayers, enabling better access for experimental analysis.
What techniques are used to monitor monolayer formation?
Bright field microscopy and immunofluorescence staining are used to monitor the progress of monolayer formation.
Can this method be applied to other organs?
Yes, the method can be adapted for use with other mucus-secreting organs such as the upper GI tract and respiratory system.
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