A Versatile Microfluidic Device for High-throughput Combinatorial Drug Screening

Abstract

Combination therapies can improve anticancer efficacy, but identifying effective drug pairs and dose combinations requires systematic exploration of multidimensional concentration spaces. Here, we extend a diffusion-driven, flowless microfluidic concentration gradient generator (CGG) to enable quantitative combinatorial drug screening without external pumps or continuous flow. The platform comprises a 5 x 5 array of interconnected culture nodes coupled to four peripheral reservoirs, in which overlapping diffusion fields generate spatially defined single- and multidrug exposures. Computational modelling was used to assign local drug concentrations to individual nodes, enabling direct correlation of the predicted exposure landscape with cellular response. Using MCF-7 breast cancer cells and 5-fluorouracil (5-FU) and doxorubicin (DOX) as model therapeutics, the platform resolved concentration-dependent single-agent responses, yielding IC50 values of 3.46 M for 5-FU and 1.22 M for DOX. Combinatorial loading generated 25 spatially defined 5-FU-DOX concentration pairs within a single device, which were resolved into two-dimensional concentration-response landscapes. Bliss independence analysis revealed concentration-specific drug interactions, with synergy predominating at low-to-intermediate concentrations and a transition towards additive and antagonistic responses at higher exposures. These findings establish a pump-free microfluidic strategy that integrates computational concentration mapping with spatially resolved pharmacological analysis to identify effective drug-combination windows within a single platform.