Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.