Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface

Abstract

Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model.

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica

“…Submerged monolayer cell cultures fail to recapitulate these fundamental processes 25,36,37 , and therefore are poorly suited to the study of colonisation by pathogenic species. We recently developed well-differentiated primary OTECs for use in modelling interactions of relevant pathogens with the sheep respiratory epithelium in vitro 24,25 . The model forms a heavily ciliated, mucus-producing cell layer with similar physiology to that of the native respiratory tissue.…”

“…The complex cellular architecture of this tissue plays a role in such defences by physically occluding passage of bacteria and viruses 33 , entrapping particles in goblet cell-secreted mucus 34 and clearing mucus globules via the coordinated beating of ciliated epithelial cells 35 . Submerged monolayer cell cultures fail to recapitulate these fundamental processes 25 , 36 , 37 , and therefore are poorly suited to the study of colonisation by pathogenic species. We recently developed well-differentiated primary OTECs for use in modelling interactions of relevant pathogens with the sheep respiratory epithelium in vitro 24 , 25 .…”

“…Cultures of differentiated OTECs were prepared as previously described 24,25 . Briefly, OTECs were extracted from tracheal epithelia using protease XIV from Streptomyces griseus, at 4 °C overnight.…”

“…Primary antibodies were labelled using an anti-mouse-HRP polymer and detected using REAL EnVision Peroxidase/DAB + Detection System (Dako, Immunofluorescence microscopy. Immunofluorescence microscopy was carried out as described previously 24,25 with some alterations. Infected OTEC cultures were fixed by incubating in 4% (w/v) paraformaldehyde for 15 min.…”

“…With this in mind, our group have developed highly optimised in vitro tissue engineered models of both cattle and sheep respiratory epithelia [24][25][26][27] . By optimising medium, growth factor concentrations, substrate pore density, atmospheric gas composition and period of differentiation, these models best reflect the thickness, ciliation level, mucus production and cellular composition of their source of isolation.…”