We present two novel microfluidic flow cells developed to provide reliable control of flow distributions and chemical gradients in biofilm studies. We developed a single-inlet microfluidic flow cell to support biofilm growth under a uniform velocity field, and a double-inlet flow cell to provide a very smooth transverse concentration gradient. Both flow cells consist of a layer of polydimethylsiloxane (PDMS) bonded to glass cover slips and were fabricated using the replica molding technique. We demonstrate the capabilities of the flow cells by quantifying flow patterns before and after growth of Pseudomonas aeruginosa biofilms through particle imaging velocimetry, and by evaluating concentration gradients within the double-inlet microfluidic flow cell. Biofilm growth substantially increased flow complexity by diverting flow around biomass, creating high- and low-velocity regions and surface friction. Under a glucose gradient in the double-inlet flow cell, P. aeruginosa biofilms grew in proportion to the local glucose concentration, producing distinct spatial patterns in biofilm biomass relative to the imposed glucose gradient. When biofilms were subjected to a ciprofloxacin gradient, spatial patterns of fractions of dead cells were also in proportion to the local antibiotic concentration. These results demonstrate that the microfluidic flow cells are suitable for quantifying flow complexities resulting from flow–biofilm interactions and investigating spatial patterns of biofilm growth under chemical gradients. These novel microfluidic flow cells will facilitate biofilm research that requires flow control and in situ imaging, particularly investigations of biofilm–environment interactions. Biotechnol. Bioeng. 2014;111: 597–607. © 2013 Wiley Periodicals, Inc.
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