In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.
Biosciences Institute, University Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Goblet Cells, Respiratory Mucosa, Epithelial Cells, Humans, Cystic Fibrosis, Aminopyridines, H(+)-K(+)-Exchanging ATPase, Cystic Fibrosis Transmembrane Conductance Regulator, Culture Media, Diffusion Chambers, Culture, Sequence Analysis, RNA, Cell Differentiation, Gene Expression Regulation, Cell Lineage, Hydrogen-Ion Concentration, Benzodioxoles, Primary Cell Culture, Transcriptome