Air liquid interface (ALI)-grown immortalized human cell lines: a promising tractable model for human airway

Abstract

The airway epithelium consists of multiple specialised cell types that generate an efficient innate defensive shield. Models of this epithelium are most generated using primary epithelial cells grown at the air liquid interface (ALI). Such cultures have a finite lifespan and are not easily genetically modified. To overcome these limitations, we have validated a tractable cell line-based model based on a human immortalized epithelial cell line. HBEC3-KT cells (an human immortalized bronchial epithelial cell line) were differentiated at the ALI up to three weeks. RT-PCR and qPCR undertaken with a number of cell-type specific markers (eg BPIFA1, BPIFB1, SCGB1A1, TEKT1) confirmed that differentiation was initiated within 7 days of ALI culture. IF microscopy and western blotting was used to show that these gene expression changes were translated into protein production. Similar changes were seen in differentiating primary HBECs cultured in a similar manner. To investigate the tractability of the cells they were used to gene edit the master regulator of ciliogenesis, FOXJ1 using CRISPR-cas9. HBEC3-KT cells were transiently transfected using a double guide RNA approach, GFP expressing cells were selected by FACS and clonal cell lines derived by limiting dilution. Genomic PCR and sequencing identified mutant lines including one with an in-frame deletion of the FOX DNA binding domain. Loss of FOXJ1 reduced TEKT1 in differentiated cells but did not reduce secretory cell markers. Our data shows that HBEC3-KT can serve as a tractable model of the human airway and can be used for studies of cellular differentiation.