Fibroproliferative Remodeling of Immune‐Vascular Co‐Culture Spheroid Models in Response to Atherogenic Stimulation

Cell-based models are highly valuable platforms for preclinical studies of atherosclerosis, offering controllable environments to investigate disease mechanisms. Three-dimensional (3D) spheroids, a class of cell-based models, enable the study of atherosclerotic disease phenomena under physiologically relevant yet controlled conditions. In this study, we examined the lipid-induced changes in a co-culture spheroid model comprising THP-1-derived macrophages and primary human coronary artery smooth muscle cells (HCASMCs) following treatment with 20 or 40 µg/mL oxidized LDL (ox-LDL), a known atherogenic agent. Over a 10-day period, we assessed changes in spheroid morphology (size and compactness), cell viability, metabolic activity, intracellular composition, gene expression, pro-inflammatory cytokine release and extracellular matrix deposition, relative to the untreated control. Lipid exposure increased the abundance of macrophages in ox-LDL-treated spheroids (LS20-3 h: 35.5 ± 21.9%; LS40-3 h: 38.3 ± 8.7%) compared with controls (21.8 ± 0.7%) as early as day 3. Upregulation of fibronectin (LS20-3 h: ~1.8-fold; LS40-3 h: ~2.1-fold), CD36 (~1.8-fold), and ABCA-1 (~1.9-fold) was also observed. Moreover, spheroids exposed to 40 µg/mL ox-LDL exhibited reduced viability and compactness compared with those treated with 20 µg/mL by day 10. While lipid-treated spheroids showed non-statistically significantly higher levels of pro-inflammatory IL-1β cytokine expression at day 0 which declined (> 85% decrease) by day 3, all spheroid groups showed comparable global fibronectin ECM secretion indices. Collectively, these findings reveal lipid-induced proliferative changes to spheroid composition & functionality and show their potential as biomimetic models for the study of atherosclerotic disease phenomena.