Cryobanking of human distal lung epithelial cells for preservation of their phenotypic and functional characteristics

The epithelium lining airspaces of the human lung is maintained by regional stem cells including basal cells of pseudostratified airways and alveolar type 2 pneumocytes (AT2) of the gas-exchange region. Despite effective techniques for long-term preservation of airway basal cells, procedures for efficient preservation of functional epithelial cell types of the distal gas-exchange region are lacking. Here we detail a method for cryobanking of epithelial cells from either mouse or human lung tissue for preservation of their phenotypic and functional characteristics. Flow cytometric profiling, epithelial organoid-forming efficiency, and single cell transcriptomic analysis, were used to compare cells recovered from cryobanked tissue with those of freshly dissociated tissue. Alveolar type 2 cells within single cell suspensions of enzymatically digested cryobanked distal lung tissue retained expression of the pan-epithelial marker CD326 and the AT2 cell surface antigen recognized by monoclonal antibody HTII-280, allowing antibody-mediated enrichment and downstream analysis. Isolated AT2 cells from cryobanked tissue were comparable with those of freshly dissociated tissue both in their single cell transcriptome and their capacity for in vitro organoid formation in 3D cultures. We conclude that the cryobanking method described herein allows long-term preservation of distal human lung tissue for downstream analysis of lung cell function and molecular phenotype, and is ideally suited for creation of an easily accessible tissue resource for the research community. Single cell RNA-seq using 10x chromium system on freshly isolated and cryo-preserved lung explant tissue. The barcoded sequencing libraries were quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing libraries were loaded on a NovaSeq 6000 (Illumina).