Fine-tuning cell-mimicking polyacrylamide microgels: Sensitivity to microscale reaction conditions in droplet microfluidics

• Developed a droplet microfluidics approach to produce polyacrylamide (PAAm) microgels with tightly controlled size and tunable elasticity. • Demonstrated that flow conditions strongly influence microgel elasticity independent of droplet size and pre-gel composition by modulating catalyst transport at the oil–water interface. • Identified two distinct production regimes where varying the PAAm-to-oil flow rate ratio alters polymerization efficiency, bead swelling, and Young’s modulus. • Provided a clear framework for future design of microgel beads with tailored physical properties through precise flow control. • Addressed reproducibility challenges in PAAm microgel production by revealing overlooked microscale interfacial transport effects that critically affect polymerization and microgel properties. • Moved beyond trial-and-error approaches to establish a predictive, robust protocol for standardized, scalable fabrication of microgels with well-defined size and elasticity. • Demonstrated that precise control of flow conditions is essential to regulate polymerization efficiency, bead swelling, and elasticity in droplet microfluidics, unlike bulk polymerization. Shaping polyacrylamide (PAAm) hydrogels via droplet microfluidics enables production of microgels that mimic cellular physical properties, advancing mechanobiology research. Controlling microgel size and elasticity is essential but challenging, as multiple factors influence polymerization and network formation. Although chemical reactions in microdroplets are generally faster and more uniform than in bulk, these microreactors are highly sensitive: small changes in chemical or physical conditions can cause significant variations in microgel properties. Our study identifies flow conditions as a crucial factor affecting both microgel elasticity and size by modulating interfacial transport during gelation. Using a flow-focusing microfluidic chip, we generated pre-gel droplets with the same composition in an oil phase, systematically varying the PAAm-to-oil flow rate ratio while maintaining a constant total flow rate. This method produced droplets with minimal size variation (