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A1972 - Primary Bronchial Epithelial Cell Culture from Bronchial Brushings of Bronchiectasis Patients: An International Collaboration
Author Block: M. J. McDonnell1, I. Haq2, B. Verdon2, A. Aldhahrani2, J. P. Pearson2, J. Lordan3, A. De Soyza4, M. Brodlie2, R. M. Rutherford1, J. G. Laffey5, C. Ward2; 1Respiratory Medicine, Galway University Hospitals, Galway, Ireland, 2Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, United Kingdom, 3Respiratory and Transplant Medicine, Freeman Hospital, Newcastle, United Kingdom, 4Respiratory and Transplant Medicine, Newcastle University, Newcastle, United Kingdom, 5Lung Biology Group, NUI Galway, Galway, Ireland.
Rationale: The pathogenesis of bronchiectasis lung disease remains poorly understood. Experimental models are therefore vital for use in research to elucidate mechanistic studies of airway epithelial responses in the pathogenesis of this condition and for the discovery and evaluation of potential therapeutic compounds. Although immortalised cell lines are valuable in the early stages of high throughput screening, they have inherent limitations. Primary bronchial epithelial cells (PBECs) derived from bronchoscopic bronchial brushings of individual bronchiectasis patients are a valuable but technically and logistically challenging source of cells that more accurately recapitulates the situation in vivo. Cells are traditionally processed within 2-4 hours of sample collection. The current study aimed to establish PBECs from bronchial brushings of bronchiectasis patients to investigate the role of the bronchial epithelium in its pathogenesis, and to assess the feasibility of international transfer of brushings for culture after a 48-72 hour window. Methods: Brushings were obtained from sub-segmental bronchi of patients with bronchiectasis undergoing bronchoscopy. The brush was detached on completion of the procedure and dispersed into 5mL Roswell Park Memorial Institute medium and 500µL 1% penicillin/streptomycin for international transport from Ireland to the UK. On arrival after 48-72 hours, cells were seeded onto collagen-coated flasks and grown to confluence in bronchial epithelial growth medium for submerged culture, or onto semi-permeable membranes for air-liquid interface (ALI) culture models for electrophysiological studies. Results: PBECs were successfully cultured from 14/22 (64%) bronchiectasis patients (mean (SD) age 61.1 (14.7), 59% female). 12/22 (55%) patients were found to have positive microbiology on bronchoalveolar lavage (BAL). Cultured cells were characterized by their morphology under light microscopy, with cytokeratin and hematoxylin-eosin staining in a representative proportion. Failure to reach confluence was due to early infection in eight unsuccessful cultures, 6 (75%) of whom had positive microbiology on BAL. Cells remained viable after storage in liquid nitrogen, facilitating further cell culture experiments at passage 2 and 3. Cilliogenesis, mucus secretion and demonstration of normal airway electrophysiology was observed at ALI. Conclusion: Primary culture of PBECs from bronchiectasis patients is feasible, even after a 48-72 hour window allowing international transfer of samples, despite the potential for early, patient-derived infection. To the best of our knowledge, this is the first description of a program to culture PBECs in bronchiectasis patients on an international platform. This provides an important model to facilitate future mechanistic studies to elucidate pathogenic mechanisms in bronchiectasis and to investigate potential therapeutic targets.