Isolation, Culture, and Phenotypic Analysis of Murine Lung Organoids

Three-dimensional (3D) organoid cultures retain self-renewing stem cells that differentiate into multiple cell types that display spatial organization and functional key features, providing a highly physiological relevant system. Here we describe a strategy for the generation of 3D murine lung organ...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 2805; p. 3
Main Authors Evans, Kelly, Dabrowska, Catherine, Ng, Minn E, Brainson, Christine F, Lee, Joo-Hyeon
Format Journal Article
LanguageEnglish
Published United States 2024
Subjects
Animals
Cell Culture Techniques - methods
Cell Differentiation
Cell Separation - methods
Coculture Techniques - methods
Epithelial Cells - cytology
Lung - cytology
Mice
Organoids - cytology
Phenotype
Stem Cells - cytology
Stem Cells - metabolism
Trachea - cytology
Lung organoids
Lung stem cells
Wholemount staining
Organoid co-culture
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Summary:Three-dimensional (3D) organoid cultures retain self-renewing stem cells that differentiate into multiple cell types that display spatial organization and functional key features, providing a highly physiological relevant system. Here we describe a strategy for the generation of 3D murine lung organoids derived from freshly isolated primary tracheal and distal lung epithelial stem cells. Isolated tracheas are subjected to enzymatic digestion to release the epithelial layer that is then dissociated into a single cell suspension for organoid culture. Lung epithelial cells are obtained from dissected lobes, which are applied to mechanical and enzymatic dissociation. After flow sorting, organoids are established from tracheal basal, secretory club, and alveolar type 2 cells in the defined conditioned medium that is required to sustain organoid growth and generate the differentiated cells. Multi-cell-type organoid co-culture replicates niches for distal epithelial stem cells to differentiate into bronchiolar and alveolar cell types. Established organoids can be fixed for wholemount staining and paraffin embedding, or passaged for further culture. Taken together, this protocol provides an efficient and validated approach to generate murine lung organoids, as well as a platform for further analysis.
ISSN:1940-6029
DOI:10.1007/978-1-0716-3854-5_1