Comparative 3D models show context-dependent responses in lung fibroblast-monocyte co-cultures

Lung fibroblast-monocyte interactions are implicated in aberrant repair and fibrosis. We developed complementary 3D in vitro co-culture models to study how direct versus indirect monocyte-fibroblast interactions shape inflammatory and extracellular matrix (ECM) responses under injurious conditions relevant to fibrotic lung disease. Human fetal lung fibroblasts (MRC-5) and monocyte-like THP-1 cells were incorporated into 6% or 10% gelatin methacrylate (GelMA) hydrogels that mimic ECM environments, either in a direct co-culture format or in an indirect Transwell-based format. Cultures were exposed to 20% cigarette smoke extract (CSE) or left unstimulated. Readouts included LDH release as a measure of cellular stress, collagen I release, and cytokine secretion by ELISA, CD86-expression through immunoblotting, and broad secretome profiling using a 48-plex Luminex assay with multivariate analyses. In direct co-cultures, THP-1 monocytes were closely associated with MRC-5 fibroblasts. After CSE exposure, LDH release increased in both co- and monocultures, with no significant effect of GelMA concentrationRStudio. CSE-induced TGF-β release observed in monocultures was absent in co-cultures, whereas CD86 expression increased with CSE exposure in direct co-culture conditions. Multiplex profiling identified IL-8, MCP-1, GROα, IL-6, and FGF-2 as the most abundant analytes. Dimensionality reduction revealed modest separation by CSE exposure and GelMA concentration, but clearer separation by culture condition, with monoculture versus co-culture emerging as a strong contributor to cytokine profiles. Together, these complementary co-culture platforms show that fibroblast monoculture and co-culture conditions can produce different responses across GelMA concentrations and CSE exposure conditions. This model may support future mechanistic studies of monocyte-fibroblast signalling relevant to fibrotic lung disease.