High-throughput bioprinted 3D cultures for probing host–pathogen interactions in bioinspired microenvironments

The microenvironment of immune cells is an important regulator of their function and fate. Three-dimensional (3D) culture systems provide opportunities for probing immune cell responses to invading pathogens in microenvironments with biophysical and biochemical properties inspired by human tissues. Yet, the low throughput and manual preparation of many 3D culture models present challenges for translation of assays and their broad and accessible use for studying host-pathogen interactions. To address this, we established a high-throughput macrophage-bacteria co-culture model that mimics lung tissue stiffness across healthy and diseased conditions. Using bioprinting, human THP-1 monocytes were encapsulated and differentiated into macrophages within synthetic extracellular matrices (ECMs) fabricated with well-defined polymer and peptide bioinks in a 96-well plate format. Macrophages retained viability and displayed immunocompetence, including phenotype, phagocytosis, and response to stimuli. Macrophages in fibrosis-inspired 'stiffer' (storage modulus (<i>G</i>') ∼4.8 kPa) microenvironments exhibited higher basal expression of both inflammation and traditional fibrosis associated genes compared to more compliant (<i>G</i>' ∼1.1 kPa) synthetic ECMs inspired by healthy lung microenvironments. We applied our model 3D cultures to study immune response to invasion of a bacterial pathogen implicated in hospital born lung infections and mortality, <i>Pseudomonas aeruginosa</i>. Macrophages exhibited differential responses to <i>P. aeruginosa</i> in stiff microenvironments, with decreased cytokine secretion of IL-6 and IL-1β and elevated IL-10 and TNF-α compared to healthy compliant microenvironments, suggesting that microenvironment properties may shape initial immune responses. This high-throughput, accessible controlled platform provides opportunities for understanding human host-pathogen interactions and a foundation for identifying therapeutic strategies for bacterial infections in well-defined physiologically relevant microenvironments.