Influence of Culture Substrates on Morphology and Function of Pulmonary Alveolar Cells In Vitro

Abstract

Cell's microenvironment has been shown to exert influence on cell behavior. In particular, matrix-cell interactions strongly impact cell morphology and function. The purpose of this study was to analyze the influence of different culture substrate materials on phenotype and functional properties of lung epithelial adenocarcinoma (A549) cells. A549 cells were seeded onto two different biocompatible, commercially available substrates: a polyester coverslip (Thermanox™ Coverslips), that was used as cell culture plate control, and a polydimethylsiloxane membrane (PDMS, Elastosil^® Film) investigated in this study as alternative material for A549 cells culture. The two substrates influenced cell morphology and the actin cytoskeleton organization. Further, the Yes-associated protein (YAP) and its transcriptional coactivator PDZ-binding motif (TAZ) were translocated to the nucleus in A549 cells cultured on polyester substrate, yet it remained mostly cytosolic in cells on PDMS substrate. By SEM analysis, we observed that cells grown on Elastosil^® Film maintained an alveolar Type II cell morphology. Immunofluorescence staining for surfactant-C revealing a high expression of surfactant-C in cells cultured on Elastosil^® Film, but not in cells cultured on Thermanox™ Coverslips. A549 cells grown onto Elastosil^® Film exhibited morphology and functionality that suggest retainment of alveolar epithelial Type II phenotype, while A549 cells grown onto conventional plastic substrates acquired an alveolar Type I phenotype.

Keywords: cell function; cell morphology; lung epithelial cells; mechanobiology; substrate properties.

Figure 1

(A,B) Stress-strain curves obtained through a tensile testing for Thermanox™ Coverslips and Elastosil^® Film, respectively, and (C) the corresponding elastic modulus values calculated as the slope of the linear elastic region (*** p < 0.001, n = 5). (D–G) Immunostaining of fibronectin: Thermanox™ Coverslips with FN coating and without, D and E respectively; Elastosil^® Films with FN coating and without, F and G respectively (n = 2 for each condition).

Figure 2

(A) Cell metabolic activity (resazurin assay, RFU: relative fluorescence units) of A549 cultured on Thermanox™ Coverslips and on Elastosil^® Film (there were no statistical differences between samples, TCPS was used as standard control, n = 12). (B–E) Phase-contrast images of A549 cells cultured on Thermanox™ Coverslips (B), magnification 5×; (C), magnification 20× or Elastosil^® Film (D), magnification 5×; (E), magnification 20×.

Figure 3

Scanning electron microscopy images of A549 cells cultured on Thermanox™ Coverslips (A,C) and Elastosil^® Film (B,D) after 72 h of culture. Arrows indicate surfactant on the apical surface of cells.

Figure 4

Immunofluorescence images of A549 cells stained for F-Actin (red) and caspase-3 (green) cultured on Thermanox™ Coverslips (A–C) and on Elastosil^® Film (D–F) after 72 h of culture. Magnification 40× (n = 20, representative images are here reported).

Figure 5

(A–D) Immunofluorescence images of A549 cells stained for F-actin (red) and focal adhesions (green) cultured on Thermanox™ Coverslips (A,B) and Elastosil^® Film (C,D) after 72 h of culture (magnification 40×, inset scale bars represent 20 μm). The sections on z-axis are reported in images B and D for Thermanox™ Coverslips and Elastosil^® Film respectively. (E,F) Quantification of focal adhesion observed on Thermanox™ Coverslips and Elastosil^® Film (** p < 0.01, n = 10).

Figure 6

(A) Immunofluorescence images of A549 cells stained for YAP-1 (red) cultured on Thermanox™ Coverslips (first row) and on Elastosil^® Film (second row). Nuclei are stained in blue. Merged images are shown on the right. Magnification 63× (B) Percentage of nuclear YAP-1 in A549 cells cultured on Thermanox™ Coverslips or Elastosil^® Film (*** p < 0.001, n = 16).

Figure 7

Immunofluorescence images of A549 cells stained for Prosurfactant Protein C (red) and nuclei (blue) cultured on Thermanox™ Coverslips (A) and on Elastosil^® Film (B) after 72 h of culture. Magnification 40×.