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 alveolar 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.

Figure 1.

Emulsion polymerization produced hydrogel microspheres for templating alveolar micro-architecture. A) An overview of the microsphere fabrication process detailing chemical components and emulsion parameters. Created with BioRender.com. B) Representative fluorescent image of polymerized microspheres showed spherical geometry. Scale bar, 100 μm. C) Image analysis of microsphere diameter from fluorescent microscope images demonstrated that collecting 85-100 μm filtrate yielded physiologically relevant microspheres sizes (median diameter = 171 ± 42 μm; N = 4). D) Parallel plate rheology confirmed that microsphere stiffness was within the range of healthy lung tissue (E = 4.62 ± 1.01 kPa; N = 8).

Figure 2.

Formation of 3D acinar structures. A) Overview of the 3D acinar structure fabrication process. Created with BioRender.com. B) Confocal image showing ATII cells (green) dispersed across the surface of aggregated microspheres (red) in three dimensions. Scale bar, 50 μm. C) Schematic depicting the embedding hydrogel formulation and polymerization process. These materials were crosslinked with an MMP2-degradable dithiol crosslinker to enable fibroblast-mediated matrix remodeling. C) Parallel plate rheology confirmed that embedding hydrogel formulations recapitulated the elastic modulus of healthy (E = 2.7 ± 0.31 kPa) or fibrotic lung tissue (E = 18.1 ± 1.34 kPa). N = 8.

Figure 3.

3D lung models supported cell viability of both A) epithelial cells (imaged in the central area containing microsphere templates) and B) fibroblasts (imaged in the peripheral embedding hydrogel). Data are reported as percent live cells out to at least 3 weeks post-embedding in both hydrogel conditions. Representative confocal images (50 μm z-stacks displayed as a maximum intensity projection) showed even distribution of live (green) C) epithelial cells at the center of 3D lung models with preserved pulmonary architecture and D) fibroblasts evenly distributed through the embedding hydrogel with relatively low numbers of dead (red) cells (n=5-7). Scale bar, 100 μm.

Figure 4.

Epithelial cell identity shifted over time. A) Representative fluorescent images of immunostaining of ATII (SFTPC), ATI (PDPN), and transitional (KRT8) markers in 3D lung models containing primary murine fibroblasts. B) Image analysis of the epithelial cell population showed the majority of the ATII cell population retained that identity, with some ATII cells transitioning to an ATI or ATII-ATI transitional phenotype at 3 weeks post-embedding (n = 3). Scale bar, 50 μm.

Figure 5.

Fibroblasts activated in response to stiffness and epithelial cell presence. A) Fibroblast activation assessed up 3 weeks post-embedding revealed that fibroblast-epithelial cell co-culture and a stiff extracellular environment synergistically promoted fibroblast activation as measured by the percentage of αSMA and/or Col1a1 expressing reporter fibroblasts in culture (n = 6). B) Within the activated cell population, the proportion of αSMA+ myofibroblasts increased over time in all conditions, but increased more quickly in co-culture conditions, suggesting that the presence of epithelial cells provokes a more rapid fibrotic response than mechanical cues alone (n = 6). C) Representative confocal images (maximum intensity projection of 100 μm z-stacks) showed dual reporter fibroblasts within 3D lung models increased expression of activation reporters αSMA and Col1a1 in co-culture within a stiff extracellular environment, particularly at three weeks. Scale bar, 50 μm.

Figure 6.

Fibrotic gene network analysis in 3D lung models. Expression of 84 fibrosis-associated genes was assessed using a Qiagen RT² Profiler PCR array. A subset of genes with a fold change greater than two in at least one condition relative to the soft, fibroblast-only condition is displayed. Within each gene, expression relative to the housekeeper (2^(−ΔCT)) was normalized across the four groups as a percent of maximum expression. This analysis revealed coordinated regulation of functionally related genes based on cellular co-culture and microenvironmental stiffness. Specifically, stiffness tended to promote the expression of genes related to cell-matrix interactions, while presence of epithelial cells resulted in enhanced expression of growth factors, pro-proliferative transcription factors, and TGF-β signaling pathway members (n = 3).