Bronchoalveolar Lavage Derived Fibroblasts From Interstitial Lung Disease Patients: A Chance to Exploit 2D/3D Model of Pulmonary Fibrosis In Vitro.

Abstract

Background: Bronchoalveolar lavage (BAL) constitutes a valuable diagnostic approach for the differential diagnosis of various pulmonary fibrotic diseases. BAL fluids from patients with interstitial lung diseases (ILDs) can also be utilized for research purposes, offering cell populations suitable for functional and phenotypical studies. In this study, we demonstrate the feasibility of isolating a discrete number of fibroblasts/myofibroblasts in vitro from the BAL fluid from ILD patients, a procedure typically performed during the early stages of disease when high-resolution computed tomography does not yield a definitive diagnosis.

Methods: We obtained BAL samples from a total of 43 patients. Fibroblasts were successfully derived in vitro from 20 patients, with larger quantities of cells from 11 patients. Whenever possible, the cells were cultured and expanded until passage 12-15. Fibroblasts could be expanded to passage 36 in only one case. The expression of typical fibrotic markers, such as type I collagen, α-smooth muscle actin, and fibronectin-extra domain A or B (FN-EDA/-EDB), was therefore compared in fibroblasts obtained from ILD-patients with fibroblasts derived from non-diseased controls by quantitative RT-PCR, immunofluorescence, and cytofluorographic analysis. The rate of proliferation, migration, and response to the anti-fibrotic drug pirfenidone was further determined in 2D and in 3D models of in vitro cultures.

Results: A specific morphological heterogeneity among fibroblasts/myofibroblasts derived from patients with fibrotic or non-fibrotic ILD was observed, such as enlarged and flattened shaped cells vs spindle-shaped cells. Moreover, a higher expression of α-smooth muscle actin (α-SMA), type I collagen (collagen I), and fibronectin was demonstrated in ILD fibroblasts than in control fibroblasts. The anti-fibrotic drug pirfenidone was effective in inhibiting the growth and migration of ILD-fibroblasts both in 2D and 3D in vitro models.

Conclusions: Collectively, the present study suggests that BAL-derived fibroblasts from ILD patients may serve as a useful in vitro model for studying and assaying pulmonary fibrosis. This approach has the potential to improve our understanding of ILD pathogenesis and overcome ethical and availability concerns associated with biopsy-derived tissues.