A Macrophage-Integrated 3D Spheroid Model Inoculated with Bacteria for Simultaneous Evaluation of Antibacterial...

Conventional antimicrobial-material testing typically relies on separate bacterial assays and two-dimensional (2D) mammalian cell cultures. Although essential as first-line screening tools, these approaches do not capture the three-dimensional (3D) tissue architecture, diffusion constraints, extracellular-matrix remodeling, and innate immune interactions encountered by host-contacting antimicrobial materials. Herein, this study introduces a macrophage-containing 3D spheroid model designed for the simultaneous evaluation of antimicrobial efficacy, host-cell cytotoxicity, and immune responses. The tripartite spheroids comprised DU 145 epithelial cells, THP-1-derived M0 macrophages, and E. coli. The influence of macrophage integration on spheroid architecture was characterized using scanning electron microscopy and scanning ion conductance microscopy, revealing significant biophysical remodeling marked by enhanced matrix compaction and surface densification. These structural alterations were further supported by changes in electrostatic properties and reduced FITC-dextran penetration, indicating enhanced barrier function. Following E. coli challenge, resident macrophages exhibited functional plasticity, polarizing toward an M1-like phenotype to suppress bacterial colonization. The platform was then used to evaluate PA-PQ-37, a cationic antimicrobial coating. In contrast to 2D monolayers, macrophage-containing spheroids retained structural integrity and live-cell esterase activity on the coating while exhibiting reduced recoverable bacterial burden. The model further differentiated the macrophage-associated responses elicited by a cationic polymer, ampicillin, and azithromycin, including inflammatory and pro-resolution-like marker profiles. This animal-free platform complements conventional antibacterial and cytotoxicity assays by supporting integrated evaluation of host-contacting antimicrobial biomaterials in a tissue-like, immune-responsive microenvironment. STATEMENT OF SIGNIFICANCE: Antimicrobial biomaterials are typically evaluated using simplified bacterial assays and 2D mammalian cell cultures, which do not reproduce the tissue architecture, diffusion barriers, extracellular matrix remodeling, or immune surveillance encountered in vivo. This limitation can lead to inaccurate assessment of both antimicrobial efficacy and host-cell compatibility. In this study, we developed a macrophage-integrated 3D infected spheroid model that incorporates resident immune cells into a tissue-like microenvironment for biomaterial evaluation. The model captures macrophage-driven spheroid compaction, matrix remodeling, bacterial restriction, and infection-responsive immune polarization. Using this platform, we demonstrate that cationic PA-PQ-37 coatings appear highly cytotoxic in 2D assays but are well tolerated by macrophage-competent 3D spheroids while retaining antibacterial activity. The model also distinguishes antimicrobial agents with different immunomodulatory profiles, including inflammatory, neutral, and pro-resolution responses. This work provides a physiologically relevant strategy for assessing antimicrobial biomaterials beyond conventional bacterial killing and cytotoxicity endpoints.