Three-dimensional mesenchymal stromal cell spheroids potently suppress neutrophil extracellular trap formation

Background/Aims Neutrophil extracellular traps (NETs) contribute to tissue injury and organ dysfunction in infectious and inflammatory diseases. Although targeting NET formation is an attractive therapeutic strategy, the clinical use of direct NET inhibitors remains uncertain. Mesenchymal stromal cells (MSCs) possess potent immunomodulatory effects, but their functional activity is strongly influenced by culture conditions. We hypothesized that MSCs cultured as three-dimensional (3D) spheroids would suppress endotoxin-induced NET formation more effectively than MSCs expanded in conventional two-dimensional (2D) culture. Methods Bone marrow–derived MSCs (BM-MSCs) and neutrophils were isolated from C57BL/6 mice. Neutrophils were studied under four conditions: unstimulated (NE), lipopolysaccharide-stimulated (NE+LPS), and LPS-stimulated cocultured with either 2D cultured MSCs (NE+LPS+MSC2D) or 3D MSC spheroids (NE+LPS+MSC3D). NET formation was quantified by extracellular DNA release and confirmed by immunofluorescence microscopy. Comparative proteomic analysis was performed to identify candidate mediators underlying the differential immunomodulatory activity of MSC spheroids. Results MSCs cultured as 3D spheroids markedly reduced endotoxin-induced NET formation compared with MSCs grown in 2D culture. This effect was evident by lower extracellular DNA concentrations and reduced NET structures on immunofluorescence microscopy. Proteomic analysis revealed a distinct secretory profile in MSC spheroids enriched for proteins implicated in neutrophil regulation, including SerpinB1, an inhibitor of neutrophil elastase, and developmental endothelial locus-1 (EDIL-3), a mediator of anti-inflammatory signaling. Conclusions A 3D MSC spheroid culture significantly enhances the ability of MSCs to suppress NET formation. This effect is associated with a distinct proteomic signature enriched in neutrophil-modulating proteins. These findings highlight 3D MSC culture as a promising strategy to optimize MSC-based therapies targeting diseases characterized by excessive NET formation.