Transitional alveolar epithelial cells and microenvironmental stiffness synergistically drive fibroblast activation in three-dimensional hydrogel lung models

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that progressively and irreversibly alters the lung parenchyma, eventually leading to respiratory failure. The study of this disease has been historically challenging due to the myriad of complex processes that contribute to fibrogenesis and the inherent difficulty in accurately recreating the human pulmonary environment in vitro. Here, we describe a poly(ethylene glycol) PEG hydrogel-based three-dimensional model for the co-culture of primary murine pulmonary fibroblasts and alveolar epithelial cells that reproduces the micro-architecture, cell placement, and mechanical properties of healthy and fibrotic lung tissue. Co-cultured cells retained normal levels of viability up to at least three weeks and displayed differentiation patterns observed in vivo during IPF progression. Interrogation of protein and gene expression within this model showed that myofibroblast activation required both extracellular mechanical cues and the presence of transitional epithelial cells. Differences in gene expression indicated that cellular co-culture induced TGF-β signaling and proliferative gene expression, while microenvironmental stiffness upregulated the expression of genes related to cell-ECM interactions. This biomaterial-based cell culture system serves as a significant step forward in the accurate recapitulation of human lung tissue in vitro, and highlights the need to incorporate multiple factors that work together synergistically in vivo into models of lung biology of health and disease.

Hydrogel-embedded precision-cut lung slices supportex vivoculture ofin vivo-induced premalignant lung lesions

“…Norbornene-conjugated eight-arm 10 kg/mol PEG was generated as described previously. 22, 233 The purified product was characterized by 1H NMR (Bruker DPX-400 FT NMR spectrometer, 300 MHz) and only product that was at least 90% functionalized was used in experiments.…”

Tissue-engineered models of lung cancer premalignancy

“…Eight-arm 10 kg/mol PEG-hydroxyl (PEG-OH; JenKem Technology) was conjugated with norbornene as described previously 44,45 . Lyophilized PEG-OH (5 g) and 4-Dimethylaminopyridine (DMAP, 0.002 mol; Sigma-Aldrich) were dissolved in anhydrous dichloromethane (DCM; Sigma-Aldrich) and then Pyridine (0.02 mol; Fisher Scientific) was injected to the mixture.…”