期刊
BIO-PROTOCOL
卷 12, 期 2, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.4295
关键词
Intestinal organoids; Intestinal epithelium; Organoid-derived monolayers; TEER measurement; Cell culture inserts; Intestinal pathogens; Intestinal infection modeling
类别
资金
- Deutsche Forschungsgemeinschaft (DFG) [GRK 2046]
- Sonnenfeld foundation
- Robert Koch-Institute
- DFG
- European Union [773830]
- [TRR 241]
This study describes a protocol aimed at harmonizing the seeding conditions for creating electrophysiologically tight epithelial barriers using three-dimensional intestinal organoids. The protocol provides detailed descriptions of media compositions and culture conditions, allowing even inexperienced researchers to obtain reproducible results.
In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two- dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological processes and tissue barrier functions, where easy experimental access of pathogens to the luminal and/or basolateral cell surface is required. This has resulted in an increasing number of publications reporting different protocols and media compositions for organoid manipulation, precluding direct comparisons of research outcomes in some cases. With this in mind, here we describe a protocol aimed at the harmonization of seeding conditions for three-dimensional intestinal organoids of four commonly used research species onto cell culture inserts, to create organoid-derived monolayers that form electrophysiologically tight epithelial barriers. We give an in-depth description of media compositions and culture conditions for creating these monolayers, enabling also the less experienced researchers to obtain reproducible results within a short period of time, and which should simplify the comparison of future studies between labs, but also encourage others to consider these systems as alternative cell culture models in their research. Graphic abstract: [GRAPHICS] Schematic workflow of organoid-derived monolayer generation from intestinal spheroid cultures. ECM, extracellular matrix; ODM, organoid-derived monolayer.
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