A Technique to Harvest Viable Tracheobronchial Epithelial Cells from Living Human Donors

Abstract

The ability to obtain airway epithelial cells from the lower respiratory tract in living human donors will facilitate study of the biologic properties of these cells. We report our experience harvesting tracheobronchial epithelial cells from living human donors by brushing the mucosal surface of the trachea and mainstem bronchi. Cells were obtained on 21 occasions from 18 healthy adult subjects under direct vision with a brush-tipped catheter during fiberoptic bronchoscopy. The average number of cells harvested per subject was 14 +/- 2 x 10(6), and cell viability determined by trypan blue exclusion averaged 36 +/- 4%. Of note, cell viability was significantly enhanced when lidocaine was confined to the nares. Lidocaine was also observed to diminish cell viability in vitro in a dose-dependent fashion. Morphologic and staining properties were used to classify harvested cells into the three major cell types present in the mucosa (i.e., ciliated, secretory, and basal cells). All three subtypes were obtained. The percentage of ciliated, secretory, and basal-like cells was 24 +/- 2%, 11 +/- 1%, 29 +/- 1%, respectively, while the remaining 36% were difficult to type. In one subject in whom brushing was performed on three occasions over a 7-wk period, the percentage of each of the three subtypes was similar across procedures. Harvested cells could be successfully placed in primary culture with a plating efficiency of 50 to 60% and could be subcultured for up to seven passages. Acutely dissociated cells could be used to study the beta-adrenergic receptor adenylyl cyclase system since they produced cAMP in response to isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystic Fibrosis F508del Patients Have Apically Localized CFTR in a Reduced Number of Airway Cells

“…This is mainly because of the invasiveness and risk of most techniques aimed at collecting human cells (eg, biopsies), the small number of cells thus collected, or the limited number, poor quality, and nonrepresentative nature of samples resulting from surgery (such as nasal polypectomies or lung transplants). Brushing of the respiratory tract, however, a noninvasive method originally proposed for ciliary studies (Kelsen et al, 1992;Rutland and Cole, 1980;Rutland et al, 1982), allows the easy sampling of numerous, representative, wellpreserved, and dissociated cells from the superficial mucosa (Bridges et al, 1991;Chapelin et al, 1996;Danel et al, 1996). The present study was thus performed on cell samples obtained by nasal brushings of F508del homozygous patients with CF, F508del carriers, and non-CF control subjects.…”

Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation

“…The percentage of apoptotic airway epithelial cells was significantly higher for patients with COPD than controls for each of the techniques used ( Disruption of epithelial cells from the basement membrane has been reported to induce apoptosis [22]. In addition, the local anaesthetic, lignocaine, has been reported to diminish epithelial cell viability, in vitro [14]. Staining with Annexin V and 7-AAD relies on changes to the cell membrane, but it was considered that any confounding factor due to technical issues would similarly affect the COPD and control groups.…”

“…Brushings and BAL were obtained from the right-sided airways, unless there was other pathology in this area, when the left-sided airways were sampled. Particular attention was given to use a minimal amount of lignocaine (100 mg) in the airways in view of the adverse effect it has on cell viability [14].…”

Myeloid‑derived suppressor cells in bronchoalveolar lavage fluid in patients with chronic obstructive pulmonary disease

“…A special effort was made to use as low dose of lignocaine as possible. 23,24 The bronchoscope was wedged in the segmental or subsegmental bronchus of the middle lobe. The bronchus was lavaged with 50 -ml aliquots of sterile saline solution at a temperature of 37°C, and then the fluid was aspirated.…”