Lungosphere Assay: 3D Culture of Lung Epithelial Stem/Progenitor Cells.

Lung epithelium contains distinctive subpopulations of lung stem/progenitor cells (LSPCs) that are essential for lung epithelial maintenance and repair in vivo. Hence, LSPCs are in the center of interest of lung biology due to their promising therapeutic applications. To reach this goal, proper characterization of LSPCs, understanding of their proliferation and differentiation potentials and elucidation of mechanisms that control them are necessary. Therefore, development of reliable in vitro clonogenic assays has been needed. We established lungosphere assay, an in vitro sphere-forming 3D culture assay that enables to evaluate stem/progenitor cell activity, self-renewal and differentiation capacity of LSPCs and to conveniently test the effect of various treatments on LSPCs. Here we provide a detailed description of procedures for isolation of adult mouse lung epithelial cells, their culture in non-adherent conditions to form LSPC-derived spheroids (lungospheres) and for embedding of lungospheres into 3D extracellular matrix to model processes of lung tissue maintenance in a physiologically relevant microenvironment.