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Concentration gradients of soluble molecules are ubiquitous within the living body and known to govern a number of key biological processes. This has motivated the development of numerous  in vitro  gradient-generators allowing researchers to study cellular response in a precise, controlled environment. Despite this, there remains a current paucity of simplistic, convenient devices capable of generating biologically relevant concentration gradients for cell culture assays. Here, we present the design and fabrication of a compartmentalized polydimethylsiloxane diffusion-based gradient generator capable of sustaining concentration gradients of soluble molecules within thick (5 mm) and thin (2 mm) agarose and agarose-collagen co-gel matrices. The presence of collagen within the agarose-collagen co-gel increased the mechanical properties of the gel. Our model molecules sodium fluorescein (376 Da) and FITC-Dextran (10 kDa) quickly established a concentration gradient that was maintained out to 96 h, with 24 hourly replenishment of the source and sink reservoirs. FITC-Dextran (40 kDa) took longer to establish in all hydrogel setups. The steepness of gradients generated are within appropriate range to elicit response in certain cell types. The compatibility of our platform with cell culture was demonstrated using a LIVE/DEAD® assay on terminally differentiated SH-SY5Y neurons. We believe this device presents as a convenient and useful tool that can be easily adopted for study of cellular response in gradient-based assays.

A Macroscopic Diffusion-Based Gradient Generator to Establish Concentration Gradients of Soluble Molecules Within Hydrogel Scaffolds for Cell Culture