Hierarchical biofilm models using sodium alginate beads containing bacteria embedded in a cellulose-chitosan hydrogel matrix

In biofilm studies, a stable model is crucial for exploring infection mechanisms, antibiotic resistance, and evaluating materials' antibiofilm performance. Cultured biofilms often face challenges, such as slow maturation or rapid bacteria dispersion. Therefore, developing a stable, mature-stage biofilm model is critical for effective biofilm research. In this study, we report a beads-in-hydrogel biofilm model, in which sodium alginate (SA) hydrogel beads that contain bacteria are embedded within a chitosan-cellulose hydrogel film to simulate natural bacterial biofilms. This model can retain bacteria for a relatively long period of time, preventing their dispersion to the surrounding areas while keeping them viable. The reliability of the model was validated by measuring functional molecules, including extracellular DNA and biofilm-forming related proteins. Overall, this study presents a stable 3D beads-in-hydrogel biofilm model that effectively replicates natural biofilms, providing a reliable platform for exploring infection mechanisms, antibiotic resistance, and evaluating antibiofilm strategies.