Micro-hydrogel molding-assisted fabrication of a PDMS-based microfluidic concentration-gradient generator for dynamic anticancer drug testing

Traditional drug testing <i>via</i> polystyrene or glass-based cell culture platforms exposes cells to static drug doses and mechanically rigid environments [stiffness in gigapascals (GPa)], which do not accurately replicate physiological conditions. To address these limitations, we developed a polydimethylsiloxane (PDMS)-based microfluidic concentration gradient generator (μCGG) with six integrated cell culture chambers, using a cost-effective and frugal micro-hydrogel molding-assisted technique that eliminates the need for cleanroom infrastructure, specialized equipment, or advanced expertise. This platform facilitates dynamic drug exposure to cells cultured in chambers with flexible PDMS bases [stiffness in kilopascal (kPa) range], providing a scalable and accessible approach for drug dose-response analysis under physiologically relevant conditions, thereby improving accuracy. μCGG utilized a pressure-driven flow design that repeatedly split, mixed, and recombined fluid streams owing to the presence of the mesh-like geometry of the microchannels. This generated a stable and predictable drug concentration gradient across six outlet chambers, as validated through COMSOL simulations, fluorescence microscopy, and UV-Vis spectroscopy using 5-fluorouracil (5-Fu) as a model drug. MDA-MB-231 breast cancer cells were then cultured in the outlet chambers and exposed to six distinct dynamically generated concentrations of 5-Fu. Cellular viability assessed <i>via</i> live/dead assays yielded an IC₅₀ value of 41 ± 4 μM, closely matching the results from conventional multiwell plates using manually pipetted gradients under static conditions (IC₅₀: 36 ± 3 μM). Additional validation was carried out using immunocytochemistry and flow cytometry to assess apoptotic markers and treatment responses. Overall, our study presents a simple, frugal, and scalable microfluidic platform that addresses the major limitations of traditional drug testing platforms by incorporating dynamic chemical gradients, physiologically relevant mechanical environments, and low-barrier fabrication methods, paving its way for broader adoption in preclinical drug evaluation and dose-response assays.