3D cell spheroid inoculated with bacteria: An in vitro model for assessing antimicrobial efficacy

Abstract

Bacterial infections persist as a significant global health challenge, intensifying the demand for novel antimicrobial agents capable of overcoming persistent infections and mitigating the spread of drug-resistant strains. Traditional 2D cell culture assays, prone to bacterial contamination, fail to recapitulate the complex 3D architecture of in vivo tissues, rendering them inadequate as in vitro models for evaluating antimicrobial efficacy. This study investigates the effectiveness of 3D spheroids inoculated with bacteria, hypothesizing that 3D spheroids allow for assessment of antibacterial agents. Human prostate cancer cells (DU145) were cultured into 7-day-old spheroids. The morphology and migration ability of the 3D spheroids before and after inoculation with the model bacterium E. coli were analyzed. Through fluorescence microscopy and single-cell multimode analysis, it was found that E. coli can penetrate into the central region of the 3D spheroid while maintaining its viability. The complex multicellular structure of the 3D spheroids is retained under bacterial growth pressure, and the migration function of the 3D spheroids is preserved. The antimicrobial efficacy of the commercial antibiotic gentamicin and the functional material ZIF-8@Ag was evaluated by counting viable E. coli within the 3D spheroids. The results showed that both gentamicin and ZIF-8@Ag can penetrate the 3D spheroids, eradicating the embedded E. coli while preserving the spheroid’s structure. These findings highlight the importance of 3D spheroid-bacteria co-culture models as valuable tools for evaluating the permeability and efficacy of antimicrobial agents.