Primary airway epithelial cell culture from lung transplant recipients

Abstract

Long-term survival in lung transplantation is limited by the development of obliterative bronchiolitis, a condition characterised by inflammation, epithelial injury, fibroproliferation and obliteration of bronchioles leading to airflow obstruction. To investigate the role of the bronchial epithelium in the pathogenesis of obliterative bronchiolitis the current study aimed to establish primary bronchial epithelial cell cultures (PBEC) from lung allografts.Four to six bronchial brushings were obtained from sub-segmental bronchi of lung allografts. Cells were seeded onto collagen-coated plates and grown to confluence in bronchial epithelial growth medium.Bronchial brushings (n533) were obtained from 27 patients. PBECs were grown to confluence from 12 out of 33 (39%) brushings. Failure to reach confluence was due to early innate infection. Bacteria were usually isolated from both bronchoalveolar lavage and culture media, but a separate population was identified in culture media only.Primary culture of bronchial epithelial cells from lung transplant recipients is feasible, despite a high rate of early, patient-derived infection. Latent infection of the allograft, identified only by bronchial brushings, may itself be a persistent stimulus for epithelial injury. This technique facilitates future mechanistic studies of airway epithelial responses in the pathogenesis of obliterative bronchiolitis.

Azithromycin Attenuates Effects of Lipopolysaccharide on Lung Allograft Bronchial Epithelial Cells

“…We have previously reported the difficulty of establishing PBECs in lung transplant patients with both occult and sub-clinical infection and the possibility that the bronchial brushing technique may disturb biofilms. 48 Although the culture medium used in our series of experiments contained additional anti-microbials (1% penicillin, streptomycin, gentamycin and amphotericin), the methodology used compared the effects of azithromycin with control cells, which were also cultured with these anti-microbials. Nevertheless, there remains the possibilty that azithromycin interacted with one of these agents to exert the observed effects.…”

“…48 Briefly, after bronchoscopy, suspended brushing samples were centrifuged and the ensuing cell pellet dissolved in supplemented basal epithelial growth medium (BEBM [Clonetics] supplemented with BEGM single qouts [Clonetics] and 1% penicillin/streptomycin) and transferred to a 25-cm 2 Petri dish pre-coated with collagen (Vitrogen 100; Cohesion). This was placed in a CO 2 -enriched incubator (37°C/5% CO 2 ) and PBECs grown to approximately 70% to 80% confluence.…”

Inflammation and Epithelial to Mesenchymal Transition in Lung Transplant Recipients: Role in Dysregulated Epithelial Wound Repair

“…PBECs from stable lung transplant recipients were isolated by airway brushing at bronchoscopy as previously described (23 Figure 7ii) and TNF-a (Figure 7iii) secretion. …”

Pseudomonas aeruginosa accentuates epithelial-to-mesenchymal transition in the airway

“…Primary bronchial epithelial cells (PBECs) from stable lung transplant recipients were isolated by airway brushing at bronchoscopy, as described previously [25]. Cells were cultured in Small Airway Growth Medium (Lonza, Slough, UK) on collagen coated flasks.…”