Engineering dynamically tunable 3D co-culture models of human pulmonary fibrosis

To advance in vitro modeling of pulmonary fibrosis, we engineered a biomaterial microenvironment that supports induced pluripotent stem cell derived alveolar epithelial type II (iATII) cells and fibroblasts in 3D and assessed the impact of exogenous stimulation and dynamic stiffening on fibrotic activation. Magnetically aggregated iATIIs were embedded in soft or stiff cell-degradable hydrogels with lung fibroblasts and cell-adhesive peptides. Soft constructs were dynamically stiffened through a secondary di-tyrosine crosslinking reaction. Samples were cultured in CKDCI+1% FBS, with either a fibrosis cocktail (FC) or cocktail control (CC), that was replenished every 48 h (A. Jacob, et al. Nat Protoc, 2019; V. Ptasinski, et al. Life Sci Alliance, 2023) (Fig 1A). Column isolations split epithelial cells from fibroblasts. Viability was evaluated and RT-PCR of each cell type measured iATII (SFTPC, LAMP3) and fibroblast (COL1A1, CTGF, FN1, LTBP2) gene expression. Elastic moduli values matched healthy and fibrotic lung tissue (Fig 1B). iATII clusters formed distal lung geometries in 3D (Fig 1C). Viability was over 70% for 7 days. Relative to CC, iATII markers were downregulated at days 4 and 7 in stiff FC, while fibroblast genes were upregulated. Ongoing experiments will identify mechanisms and drugs that reduce fibrotic markers in vitro. Engineered models of pulmonary fibrosis could enhance effectiveness of early-stage drug discovery.