Senescent Alveolospheres: A Preliminary 3D Model for Exploring Epithelial Senescence and Pro-Fibrotic Signaling

Idiopathic pulmonary fibrosis (IPF) is one of the most severe forms of idiopathic interstitial pneumonia. Increasing evidence indicates that the gradual accumulation of senescent fibroblasts and alveolar epithelial cells contributes significantly to IPF pathogenesis, suggesting senescence as a potentially targetable process. Recurrent injury to the alveolar epithelium promotes senescence in epithelial cells, impairing their regenerative capacity and thereby predisposing the tissue to fibrotic degeneration. Although epithelial senescence is strongly implicated in the initiation and progression of lung fibrosis, the mechanisms through which it drives IPF remain challenging, partially due to the lack of physiologically relevant in vitro models capable of recapitulating lung architecture under both normal and pathological conditions. The objective of the present study was to develop a reproducible alveolosphere model in healthy and senescent conditions as a preliminary approach to investigate epithelial features that may be relevant to aspects of the IPF microenvironment. An alveolosphere system was generated by culturing alveolar epithelial cells with or without basement membrane components in combination with alveolar/epithelial optimized medium. Cultures were maintained for 3, 6, and 8 days, and cell viability together with morphological assessment confirmed the absence of cytotoxicity. The expression of keratin 8/18 and AQP5 was consistent with the maintenance of epithelial and alveolar-associated features. Cellular senescence was induced by exposing alveolospheres to doxorubicin for 24 h. Subsequent analyses of viability, along with the expression of senescent and pro-fibrotic markers, inflammatory mediators, and tissue remodeling factors, such as MMPs, were carried out in senescent 3D structures. The results demonstrated robust cell viability at all time points, supported by morphological observations. Marker expression suggested preservation of key epithelial characteristics, while senescence-inducing conditions were associated with an increase in senescence-associated, pro-fibrotic, inflammatory, and matrix-modulating markers. Collectively, these findings describe the preliminary establishment of a cost-effective and reproducible alveolosphere platform that may represent a useful starting point for studying epithelial senescence and its potential association with pro-fibrotic signaling relevant to aspects of IPF pathogenesis. Furthermore, this model may provide a basis for the preliminary evaluation of senotherapeutic compounds aimed at delaying or preventing the onset of cellular senescence.