Background

Airway disease modeling and drug discovery have benefited greatly from the development and use of primary airway epithelial cultures grown on permeable transwell filters at air-liquid interface (ALI). This model has several advantages over immortalized cell lines in that the primary epithelial cells can differentiate into an airway mucosa that features multiple epithelial cell types including ciliated, serosal, and basal cells and their arrangement is quite reflective of in vivo cellular organization. Primary ALI models exhibit functional micro-physiological processes including beating cilia and the ability to secrete mucus, features which are notably absent in the cell line-derived epithelial monolayer. Further, primary cells do not rely on artificial immortalization or transformation, as cell line-derived epithelial cells do, and therefore are unencumbered by the potential deranged signaling seen in cell lines, which can misrepresent processes occurring in airway epithelium. Despite these notable limitations, immortalized cell lines are widely employed to model and investigate the airway epithelium because primary airway epithelial cells present their own set of challenges. Primary epithelial cells fail to replicate after a few passages and must be continuously harvested and isolated to complete each set of studies. In addition, molecular biology techniques to alter or delete the expression of genes of interest are difficult to achieve and sustain with primary epithelial cells. These disadvantages, creating both cost and technical hurdles, have hindered the widespread usage of primary ALI cultures despite their obvious advantages for investigating the airway mucosa.

Recently, transforming growth factor-β superfamily signaling (BMP/TGFβ/SMAD signaling) activity was found to be suppressed in p63⁺ basal cell compartments, but highly active in differentiated apically positioned cells (Mou et al., 2016). These observations facilitated the establishment of a novel culture platform with the use of dual SMAD inhibition to overcome the growth arrest and irreversible differentiation encountered in standard culturing of primary cells. Importantly, epithelial basal cell culture no longer relies upon co-culture of primary epithelial cells with mitotically inactive fibroblast feeder layers, a technique established in the 1970’s to enhance epithelial cell proliferation by improving the capacity of cultured cells to escape senescence (Rheinwald and Green, 1975). With this newly discovered ability to expand airway basal stem cells, patient-specific cells can be isolated and preserved from small biopsies of the airway, generating a virtually limitless supply of patient-specific airway epithelium in vitro (Mou et al., 2016). When differentiated on air-liquid interface, airway basal stem cell-derived epithelium forms a polarized mucociliary layer that exhibits proper epithelial architecture, physiology and response to clinically relevant pharmacologic agents (Mou et al., 2016; Yonker et al., 2017a and 2017b). Further, airway disease can be studied by generating basal stem cell-derived epithelium from individuals with airway disease, such as cystic fibrosis (CF), a genetic disease caused by a mutation in the CFTR gene, resulting in progressive respiratory failure. Primary ALI mucosa can be used to explore and understand cellular and molecular mechanisms that contribute to CF disease pathology.

Chronic infection and aberrant inflammation are hallmarks of CF (Yonker et al., 2015). Persistent infection with Pseudomonas aeruginosa, a typical pathogen in the CF airway, contributes to overzealous inflammation marked by pathological neutrophilic breach of the airway mucosa. A better understanding of cellular and molecular mechanisms mediating neutrophil trans-epithelial migration has the potential to inform and improve the efficacy of anti-inflammatory therapeutic strategies critically needed to treat inflammation mediated lung damage associated with CF. Recently, we have applied our advanced culture method in a co-culture system with neutrophils to investigate neutrophil-airway epithelium interaction and P. aeruginosa-induced neutrophil trans-epithelial migration (Yonker et al., 2017a and 2017b), Our findings from cultured human airway basal stem cells differentiated on ALI are consistent with prior molecular mechanistic studies implicating epithelial-derived neutrophil chemoattractant hepoxilin A3, an arachidonic acid metabolite synthesized by 12-lipoxygenase, as key in driving P. aeruginosa-induced trans-epithelial migration (Yonker et al., 2017a and 2017b). Additionally, these studies revealed previously unknown cellular mechanisms associated with neutrophilic breach of the airway mucosal barrier. Neutrophils applied to the basolateral aspect of the infected airway mucosal ALI organize in clusters before migrating through the mucosal barrier in response to epithelial infection. These organized clusters of neutrophils persist along the apical surface following trans-epithelial migration before individual neutrophils begin detaching from clusters into the airspace compartment (Yonker et al., 2017a). These results demonstrate that our expandable airway basal stem cell culturing and ALI differentiation system has tremendous potential to be exploited in a variety of ways to better understand micro-anatomy, physiology, interaction with pathogens, and innate immunity at the airway mucosa.

Below, we describe, in detail, a protocol for the generation of unlimited airway basal cells for the development of functional airway mucosal models that exhibit relevant physiological functioning. This protocol overcomes several barriers associated with investigations involving primary cells and represents a robust cellular platform for human disease modeling and drug development.