Negative Pressure Actuated Microfluidic Droplet Generation Enables High-Throughput and Robust Synthesis of Cell-Laden Alginate Microgels

Hydrogel microparticles (microgels) have significant potential for use as building blocks in tissue engineering, as bioinks for 3D bioprinting, and as drug and cell carriers for cell-based therapies targeting damaged and diseased tissues. Various fabrication techniques have been developed for producing microgels with predefined shapes and sizes. However, for practical applications in biological laboratories and clinics, it is necessary to reduce time costs and simplify instrumentation and synthesis protocols, improving their reproducibility and reliability. Here we demonstrate a three-step experimental approach to develop microfluidic flow-focusing droplet generators that enable the introduction of all liquids by creating negative pressure in the outlet reservoir for the generation of spherical, core-shell, and Janus alginate microgels with living cells. This approach allows the use of a simple experimental setup that is easy to operate and robust and provides highly reproducible results, achieving a synthesis performance of up to 200 μL of microgels per hour. The size and the structure of the microgels were determined by the chip design and remained stable under pressure variations within the operating range of -7 to -15 kPa. This enabled the reliable and reproducible encapsulation of CT26 and HepG2 cells into core-shell and Janus alginate microgels with diameters ranging from 80 to 120 μm, maintaining over 80% cell viability during long-term incubation. Our findings offer a new perspective for the automation and scaling of multicomponent alginate microgel fabrication, paving the way for their implementation in tissue engineering and 3D bioprinting.