Comparative study of the effects of PM2.5 organic extracts in 2D and 3D models of human bronchial epithelial cells.

The respiratory system is a primary target of exposure to fine particulate matter (PM2.5), and the solvent-extractable organic fraction contains bioavailable constituents that may contribute substantially to PM-associated toxicity. In this study, we systematically compared the responses of human bronchial epithelial cells (16HBE) cultured as two-dimensional (2D) monolayers or three-dimensional (3D) spheroids following exposure to organic extracts of well-characterized atmospheric PM2.5 collected during three sampling periods. The level of agreement between culture formats depended strongly on the biological endpoint. Changes in WST-1 metabolic activity, reactive oxygen species-associated fluorescence, and extracellular metabolite exchange were more pronounced in 2D cultures, whereas changes in pro-inflammatory cytokine secretion were more evident in spheroids under selected conditions. Cell death responses were dominated by necrosis rather than apoptosis in both formats. These results show that 2D and 3D cultures provide complementary, endpoint-dependent information, with monolayers favouring detection of direct cytotoxic, oxidative and metabolic effects, and spheroids highlighting responses influenced by spatial organization, exposure gradients and intercellular communication.