title: Isolation of fibroblasts and epithelial cells in bronchoalveolar lavage (BAL).
abstract: The long-term outcome of lung transplants is poor with 60%-70% of patients developing chronic rejection. Chronic rejection is manifested histologically by obliterative bronchiolitis with bronchiolitis obliterans syndrome (BOS), the clinical surrogate. Recent studies suggest that fibroblasts and epithelial cells present in bronchoalveolar lavage (BAL) may be a clinically relevant biomarker for BOS. The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types. Fibroblasts and epithelial cells were isolated from BAL using attachment methods and the phenotype of the cells confirmed using immunostaining for vimentin (fibroblasts) and epithelial cell adhesion molecule (EpCAM, epithelial cells). Both fibroblasts and epithelial cells were isolated in every sample of BAL processed with the frequency of fibroblasts ranging from 0.03% to 0.48% and epithelial cells ranging from 0.05% to 1.5% of the total sample. Additional studies were performed using cytospins of cells after macrophages were depleted; cells exhibiting characteristics of both fibroblasts and epithelial cells were observed. The frequency of the cells of interest suggests that conventional methods of immunomagnetic isolation will not be effective in isolating these subpopulations. Finally, some of the low-frequency cells isolated via cytospin exhibit characteristics of epithelial to mesenchymal transition (which was not observed in plating incubations), indicating that the epithelial to mesenchymal cell transition fibroblasts may be nonadherent. In future studies, this technique and dataset may be of use to statistically correlate low-frequency cell type abundance to the onset and development of BOS.
authors.affiliation: Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Pollock
authors.forename: Luke
authors.initials: L
authors.lastname: Albares
authors.forename: Chris
authors.initials: C
authors.lastname: Wendt
authors.forename: Allison
authors.initials: A
authors.lastname: Hubel
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D001989:Bronchiolitis Obliterans / Q000175:diagnosis / Q000209:etiology / Q000473:pathology; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D000071858:Epithelial Cell Adhesion Molecule; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D006084:Graft Rejection / Q000473:pathology; D006801:Humans; D016130:Immunophenotyping; D016040:Lung Transplantation / Q000009:adverse effects / Q000473:pathology; D014746:Vimentin / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D014746:Vimentin