Culturing primary alveolar epithelial cells on lung hydrogels preserves the type II phenotype

Abstract

<b>Introduction:</b> One of the main limitations of in vitro studies on alveolar diseases is the difficulty of maintaining the type II phenotype of primeary alveolar epithelial cells. This fact has been previously related to the translocation of mechanosensing Yes-associated protein (YAP) to the nuclei and the Rho signaling pathway. In this work we cultured primary type II alveolar epithelial cells on lung-derived hydrogels. <b>Methods:</b> Primary alveolar type II cells were isolated from rat lungs and seeded over extracellular matrix hydrogels obtained from the decellularization of porcine lungs until they formed a monolayer. Types I and II populations were identified by qPCR and the expression of relevant proteins (surfactant protein C and paxillin) was studied by immunofluorescence. Samples were stained for YAP expression and a set of experiments using a Rho kinase inhibitor (Y-27632) were conducted. <b>Results:</b> Cells cultured on hydrogels formed monolayers and maintained type II phenotype for longer times. Moreover, cells cultured on plate showed the active form of YAP protein and the formation of stress fibers and focal adhesions, unlike hydrogel cultured ATII. The use of Rho inhibitor partially reversed the effect caused by the plate substrate, strongly suggesting that one of the mechanisms by which the hydrogel promotes type II phenotype maintenance could be a partial inhibition of the Rho signaling pathway. <b>Conclusion:</b> The use of lung-derived hydrogels allows type II phenotype maintenance of primary alveolar epithelial cells.