FN-silk membrane enables alveologenesis processes and self-organization of the H441 epithelial cell line into native-like alveolar morphology

In vitro alveolar-capillary models based on co-culturing the alveolar epithelial cell line H441 and primary pulmonary microvascular endothelial cells (HPMEC) are a widely used platform for evaluating the function of the alveolar barrier. However, the relatively thick synthetic membranes that are used as substrates in most approaches fail to mimic the properties of the natural basement membrane, thereby decreasing the physiological relevance of those models. We investigated the potential of the FN-silk membrane to support an in vitro alveolar-capillary model. The FN-silk membrane is micrometer-thin, fibrillar, constructed from a functionalized recombinant spider silk protein, and has been previously shown to be a potent basement membrane mimic supporting physiologically relevant in vitro models of various barrier tissues (i.e., blood vessel, skin, BBB, and kidney). Herein, it supported alveolar epithelial and endothelial barrier formation, surfactant protein B and C (SPB, SPC) production, and epithelial cell polarization, detected with immunofluorescence. Notably, we also demonstrated for the first time, to our knowledge, that key events related to alveologenesis (i.e., cell hollowing, lumen formation, septation, and α-SMA expression) can take place in an in vitro model. This further highlights the ability of the FN-silk membrane to recapitulate the complicated alveolar milieu and expanding the known potential of the H441 cell line, which to our knowledge, has not been previously reported to enable modeling the alveolar tissue 3D morphology. We propose the FN-silk-based alveolar-capillary model as an advanced in vitro model that can be used as a potent tool in respiratory, developmental biology, and regenerative medicine research.