Establishing a wound healing model in well differentiated cultures of primary human nasal epithelial cells

Abstract

The nasal epithelium is the initial contact between the external environment and the respiratory tract. Its response to noxious stimuli and how it repairs after damage is important in many respiratory diseases, including Asthma. Growing airway epithelial cells in culture at the air-liquid interface allows for a physiologically relevant model of the human upper airways. The aim of this thesis was to characterize human primary nasal epithelial cells grown at the air-liquid interface and establish a model for use in wound healing assays. This study determined the time required for full differentiation of nasal epithelial cells in an air-liquid interface culture to be at least 7 weeks using the standardised ALI differentiation techniques in two media types B-ALI and PC-ALI. Using this model, nasal epithelial cultures from healthy, atopic, non-atopic asthmatic and atopic asthmatic subjects were differentiated at air-liquid interface and manually wounded. Wounds were monitored over time until complete closure using a time lapse imaging microscope and with cultures identified to have a rate of wound healing (%/hour) independent of initial wound size. No significant difference in the wound healing rate in cells differentiated in B-ALI (3.7 ± 1.8 %/hour) and PC-ALI (5.3 ± 1.9 %/hour) when compared. Testing robustness of the model by EGFR inhibition caused the rate of wound healing to drop a significant 3.6%/hour with there being no closure of the wound after 48 hours. There was significant difference in the rate of repair for atopic subjects (2.9 ± 1.8 %/hour) when compared to healthy controls (4.3 ± 1.9 %/hour), and for non-atopic asthmatic (4.1 ± 1.1 %/hour) and with a significant difference in barrier polarization between non-atopic asthmatic and atopic asthmatic subject cultures.Restoration of the faulty repair mechanisms were monitored by the addition of EGF to the ALI cultures over time of wound healing. EGF was added 7 days prior to wounding and caused no change in the rate of wound closure. Remodelling of the epithelium was reduced by the addition of IL-13 but inhibition of the cytokine had no effect on wound repair. Lastly, respiratory infection that occurs at the airway epithelium was tested by infecting the ALI cultures with green fluorescent-tagged RSV-A2 at MOI 1 and 0.1 and monitored for the effect on wound healing. A significant decrease in rate of wound healing was seen for all phenotypes at a MOI 1 after a 6 day infection with no significant difference in viral titre in subject groups over the infection period, leading to further analysis into a steroid responsive, Th-2 driven defect in wound healing. The robust wound healing model established in this study will be essential for studying factors influencing wound healing, including host disease status and environmental exposures in the future.