Figure 2.

Viable frozen tissue processing and generation of primary airway lung organoids

(A) Schematic experimental design for primary human lung organoid generation: viable frozen tissue is thawed and digested to obtain a single cell lung suspension containing airway cell progenitors. The cell lung suspension is resuspended in Cultrex, dispensed as a dome, and cultured in a medium containing factors allowing airway organoid expansion and lung epithelium enrichment. After each passage dissociated organoids are cryopreserved.

(B) Representative photomicrographs of primary lung organoids at different passages were captured using a bright-field microscope. Images were generated using ImageJ. Scale bars 500 μm, in black on the left corner.

(C) Representative immunofluorescent (IF) images of whole mounted lung organoids showing markers for epithelial cells (PAN-CK, green), extracellular matrix (Fibronectin, red), and nuclei (DAPI, blue). Pan-CK is present exclusively in epithelial cells and fibronectin allows visualization of the presence of the remaining extracellular matrix from tissue dissociation. The fibronectin disappears after four passages and characterizes the enrichment of lung epithelial cells. Scale bar 80 μm, in white in the left corner. Note: Figure 2C reprinted with permission from Diana Cadena Castaneda et al. 2023¹ (Cell Press, Open Access).