Massively multiplexed microfluidics maps combinatorial andsequential antibiotic responses in 3D
Current microbial culture and antibiotic susceptibility testing platforms lack dynamic chemical control and do not scale to high-throughput 3D culture. We present an automated microfluidic system that creates 512 independently programmable 3D hydrogel-culture chambers and delivers each chamber a distinct combinatorial and time-varying drug protocol. Automated workflows execute thousands of micro-pipetting operations and track 10,000 bacterial colonies via live-cell microscopy, and generate half a million single colony images to quantify growth, drug response and morphology. Using this system, we study how hydrogel stiffness and nutrient timing alter colony architecture and modulate antibiotic susceptibility and resistance. We then profile the efficacy of antibiotic pairs using simultaneous and sequential (temporally ordered) dosing across 2,700 drug-dose combinations, revealing synergy, antagonism and order dependent shifts in drug efficacy. This platform integrates serial and parallel multiplexing, automation, and dynamic 3D microenvironments to map the chemical and mechanical determinants of antibiotic response and drug interactions.