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 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.