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. 2022 Apr 28;23(9):4888.
doi: 10.3390/ijms23094888.

Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells

Esther Marhuenda  1   2 , Alvaro Villarino  1 , Maria Leonor Narciso  1   3 , Marta Camprubí-Rimblas  2   4 , Ramon Farré  1   2   5 , Núria Gavara  1   2   3 , Antonio Artigas  2   4 , Isaac Almendros  1   2   5 , Jorge Otero  1   2   3
Affiliations

Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells

Esther Marhuenda et al. Int J Mol Sci. .

Abstract

One of the main limitations of in vitro studies on lung diseases is the difficulty of maintaining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype maintenance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a longer time as compared with those conventionally cultured. Interestingly, cells successfully grew when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which the hydrogel promotes type II phenotype maintenance. These results regarding protein expression strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research on lung diseases.

Keywords: YAP; alveolar cells; extracellular matrix; hydrogels; type II phenotype.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rat primary alveolar cells were cultured either on porcine lung-derived hydrogel (A) or on a plate (B). Bright field images were taken every 24 h from day 1.
Figure 2
Figure 2
Three-dimensional representation of primary alveolar type II cells cultured for three days on hydrogel (A) and on a plate (B). Nuclei are stained in blue and actin cytoskeleton in red. In the case of the hydrogel-cultured cell, it can be distinguished by a cuboidal morphology and the presence of microvilli, indicated by arrows. In the case of the cell cultured on a plate, a more spread out and wider cytoplasm can be observed showing increased size.
Figure 3
Figure 3
ATII cells were cultured either on hydrogel (HG) or a tissue culture plate (TCP). ATII typical marker surfactant proteins C (sftpc) and B (sftpb) and ATI typical marker aquaporin 5 (aqp5), podoplanin (pdpn) were studied at different time points (from day 2 to day 5) by qPCR (A). Relative gene expression is shown. Expression of surfactant protein C (SPC) was studied using immunofluorescence on ATII cells cultured on hydrogel at day 2 (B) and day 4 (D) and on a plate at day 2 (C) and day 4 (E). * p < 0.05, *** p < 0.001.
Figure 4
Figure 4
Alveolar type II cells were cultured on hydrogel (A) or on a plate (B) and stained for YAP protein (green). YAP nuclear and cytoplasmic expressions were quantified and expressed as the nuclei/cytoplasm fluorescence ratio in both conditions hydrogel (HG) and tissue culture plate (TCP) (C), *** p < 0.001.
Figure 5
Figure 5
Representative images of focal adhesions (FAs) in cells cultured on hydrogels (A) and plate (B). Red: phalloidin, green: paxillin, blue: nuclei. Quantification of the FA length (C) and the intensity of surfactant protein C (SPC) of alveolar type II cultured on hydrogels for 3 days with (Y27632) and without (CTRL) the addition of the ROCK inhibitor (D), * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Alveolar type II cells were cultured on hydrogels for three days. After that time, they were subcultured on plates and stained for surfactant protein C (red) and EpCAM (green).
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