A 3D printed microfluidic flow-cell for microscopy analysis of in situ-grown biofilms.

Biofilm phenomena ranging from metabolic processes to attachment, detachment and quorum sensing are influenced by the fluid flow across the biofilm. A number of commercially available flow-cells allow for microscopy analysis of laboratory biofilms under flow, but there is a lack of shear controlled microfluidic devices that accommodate biofilms grown in situ on carriers or tissue samples. Therefore, we developed a flow-cell with adjustable geometry for microscopy analysis of in situ-grown biofilm samples under shear-controlled flow. The flow-cells were designed as one-piece disposable models, 3D-printed in resin and sealed with a coverslip after insertion of the biofilm sample. As a proof of concept, we studied the impact of stimulated saliva flow on pH developments in in situ-grown dental biofilms exposed to sucrose. Under static conditions, pH dropped in the biofilms, with pronounced differences between individual biofilms, but also between different microscopic fields of view within one biofilm. pH in the top layer of the biofilms tended to be lower than pH in the bottom layer. Under conditions of stimulated saliva flow (5 mm/min), pH rose to neutral or slightly alkaline values in all biofilms, and the vertical gradients were reversed, with the biofilm bottom becoming more acidic than the top. Hence, the present work demonstrates the importance of flow for the study of pH in dental biofilms.