A 3D-Printed Customizable Platform for Multiplex Dynamic Biofilm Studies

Abstract

Biofilms are communities of microbes that colonize surfaces. While several biofilm experimental models exist, they often have limited replications of spatiotemporal dynamics surrounding biofilms. For a better understanding dynamic and complex biofilm development, this manuscript presents a customizable platform compatible with off-the-shelf well plates that can monitor microbial adhesion, growth, and associated parameters under various relevant scenarios by taking advantage of 3D printing. The system i) holds any substrate in a stable, vertical position, ii) subjects samples to flow at different angles, iii) switches between static and dynamic modes during an experiment, and iv) allows multiplexing and real-time monitoring of biofilm parameters. Simulated fluid dynamics is employed to estimate flow patterns around discs and shear stresses at disc surfaces. A 3D printed peristaltic pump and a customized pH measurement system for real-time tracking of spent biofilm culture media are equipped with a graphical user interface that grants control over all experimental parameters. The system is tested under static and dynamic conditions with Streptococcus mutans using different carbon sources. By monitoring the effluent pH and characterizing biochemical, microbiological, and morphological properties of cultured biofilms, distinct properties are demonstrated. This novel platform liberates designing experimental strategies for investigations of biofilms under various conditions.

Keywords: 3D printing; biofilms; hydrodynamics; multiplex system; real-time pH measurement.

Figure 1.

Platform for performing dynamic biofilm investigations. Custom 3D printed parts as used in the experimental setup with a 24-well plate (clear), 4-hole trays (blue), 6-hole trays (purple), tubing (gray), fluidic channel caps (yellow), disc clamps (brown), and discs (white). A) Integrated assembly for the dynamic experiments. B) Assembly of clamping system with 4-hole tray and tubing. C) Assembled clamping system with inlet and outlet channels. D) 4-hole tray with banana plug-shaped holders for a sturdy fit with interwell voids in the plates. E) Compressing grip of the clamps to hold the discs vertically. F) Custom pH electrode reservoir with an elevated outlet for stable, continuous measurement of effluent pH. G) Assembled plate ready for experimental use. H) 3D printed fluidic channel cap. I) 3D printed clamp. J) 3D printed clamp with 12 mm disc. K) 3D printed clamp with 7 mm disc. L) 3D printed pH electrode reservoir parts.

Figure 2.

General schematic of dynamic biofilm experiments and components for assembly. A) Schematic depicting dynamic experiments with multiple inlet sources pumping nutrient media into multiple wells that are then subjected to real-time effluent pH monitoring and collection for analysis or waste. B) Simultaneous runs in 8 different conditions (4 static and 4 dynamic modes). C) Schematic depicting the universal cap for testing multiple replicates under different conditions. D) Estimated uniform flow in each channel by computational fluid dynamics. E) 3D printed universal ca F) Simultaneous run with 4 replicates for 4 different conditions. G) 3D printed downstream flow distributor. H) pH probe with tubing in the customized reservoir for downstream in-line measurements.

Figure 3.

Simulations for flow around discs within the experimental setups. Computational Fluid Dynamics simulations for 100 μL min⁻¹ flow of media at 45° to disc. A) Streamline volume plots depicting fluid dynamics around the disc as XYZ, XY, YZ, and ZX views B) ZX surface velocity across the disc surface. C) YZ surface velocity plot around the disc. D) Shear stress plot across the disc surface.

Figure 4.

Schematic of GUI and communication with pump and pH meter.

Figure 5.

Validation of platform for static and dynamic biofilm experiments. Biofilm parameters after 18 h experiments with various sugar contents (basic medium with no added sugar (No sugar), basic medium + 0.5% glucose + 0.5% fructose (Glc+Frc), basic medium + 1.0% sucrose (Suc), and basic medium + 1% sucrose + 0.5% glucose + 0.5% fructose (Suc+Glc+Frc)) depicting A) End-point pH values B) Dry-weight C) Biofilm CFU, and D) Planktonic CFU for static experiments. E) Representative XY confocal images for static biofilms grown in E) No sugar, F) Glc+Frc, G) Suc, and H) Suc+Glc+Frc. Scale bars: 50 μm. Corresponding panels on the right side of the figure represent dynamic biofilm experiments I–P). Statistics: t-tests with * representing p < 0.05 for comparisons against no sugar control (n = 3).

Figure 6.

Continuous measurement of pH with dynamic biofilm experiments. Real-time pH of the effluent from dynamic biofilm experiments was continuously monitored for 18 h in 4 different conditions – no sugar (black), a combination of glucose + fructose (blue), sucrose (red), and a combination of sucrose + glucose + fructose (green) (n = 3).