Impaired Wound Healing of Alveolar Lung Epithelial Cells in a Breathing Lung-On-A-Chip

Abstract

The lung alveolar region experiences remodeling during several acute and chronic lung diseases, as for instance idiopathic pulmonary fibrosis (IPF), a fatal disease, whose onset is correlated with repetitive microinjuries to the lung alveolar epithelium and abnormal alveolar wound repair. Although a high degree of mechanical stress (>20% linear strain) is thought to potentially induce IPF, the effect of lower, physiological levels of strain (5-12% linear strain) on IPF pathophysiology remains unknown. In this study, we examined the influence of mechanical strain on alveolar epithelial wound healing. For this purpose, we adopted the "organ-on-a-chip" approach, which provides the possibility of reproducing unique aspects of the in vivo cellular microenvironment, in particular its dynamic nature. Our results provide the first demonstration that a wound healing assay can be performed on a breathing lung-on-a-chip equipped with an ultra-thin elastic membrane. We cultured lung alveolar epithelial cells to confluence, the cells were starved for 24 h, and then wounded by scratching with a standard micropipette tip. Wound healing was assessed after 24 h under different concentrations of recombinant human hepatic growth factor (rhHGF) and the application of cyclic mechanical stretch. Physiological cyclic mechanical stretch (10% linear strain, 0.2 Hz) significantly impaired the alveolar epithelial wound healing process relative to culture in static conditions. This impairment could be partially ameliorated by administration of rhHGF. This proof-of-concept study provides a way to study of more complex interactions, such as a co-culture with fibroblasts, endothelial cells, or immune cells, as well as the study of wound healing at an air-liquid interface.

Keywords: air–blood barrier; cyclic stretch; idiopathic pulmonary fibrosis; organ-on-a-chip; wound healing.

Figure 1

(A) Breathing concept in vivo. When the diaphragm contracts, negative thoracic pressure is generated and the lungs and lung alveoli expand. (B) Breathing concept in vitro. A microdiaphragm deflects in a defined microcavity. Due to the negative pressure generated, the alveolar membrane, where the cells are cultured, deflects downward. Schematic adapted from (Guenat and Berthiaume, 2018). (C) Image of the lung-on-a-chip with three culture chambers. Scale bar: 3 mm. (D) Immunofluorescence image of A549 cells cultured on the membrane. Cell nuclei are stained in blue, and ZO-1 (a marker of tight junctions) is stained in red. Scale bar: 20 μm. (E,F) Scanning electron micrograph of A549 on non-porous (top) and porous (bottom) membranes. Scale bars: 50 μm (top), 20 μm (bottom).

Figure 2

(A) Scheme of the scratch wound assay. The wound is created by scratching the cell-covered surface with a standard pipette tip. (B) Micrograph of wounded A549 cells on a porous membrane. (C,D) Mosaic image of an entire wound immediately after wounding (C) and 24 h later (D). (E) Wound closure on non-porous membranes, with or without cyclic stretch (CS, 10% linear, 0.2 Hz), recombinant human hepatic growth factor (rhHGF, in ng/mL), or 10% fetal bovine serum (FBS). Wound closure is promoted in a dose-dependent fashion by rhHGF, and impaired when CS is applied. (F) Wound closure is impaired when cells are cultured under CS or on a porous membrane. Scale bars: 100 μm.