Fibroblast-like culture from bronchoalveolar lavage of patient with idiopathic pulmonary fibrosis

Abstract

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease (ILD) in which fibroblast dysfunction is responsible for dysregulated extracellular matrix deposition and fibrotic processes.

Aims and objectives: The aim of the study was the development and characterization of human lung fibroblast cultures isolated by bronchoalveolar lavage (BAL), derived from patients with a diagnosis of IPF.

Methods: Starting from BAL alveolar macrophages, we isolated these cells after dyspase digestion, and seeded with complete Fibroblast medium and fibroblast growth factor type 2 (FGF-2) for 28 days. Flow cytometry analysis was performed to phenotype the cells with the use of fibronectin, collagen type I, Endoglin for fibroblast-like cells, Vimentin and Human alpha-Smooth Muscle (α-SMA) antibodies identifying myofibroblasts. The viability dye assay, the oxidative and peroxidative state of the cells were performed with novel developed flow cytometric probes. Cell sorting with anti-fibroblast micro-beads were performed.

Results: After staining with antibodies, more than 70% of cells have a fibroblast-like phenotype and co-expressed fibronectin, collagen type I and Endoglin. About 45% of the cells showed a myofibroblast phenotype. Viability tests showed that after 28 days of culture, 80% of cells resulted live. The oxidative state of our cell culture resulted higher than negative control (NAC), but also from positive control with TBHP oxidative agents.

Conclusion: The protocol developed from cell line of BAL, resulted useful working matrix to study IPF model and will provide new knowledge of pathogenetic mechanisms.