Oil-mediated high-throughput generation and sorting of water-in-water droplets

Aqueous two-phase system (ATPS) droplets have demonstrated superior compatibility over conventional water-in-oil droplets for various biological assays. However, the ultralow interfacial tension hampers efficient and stable droplet generation, limiting further development and more extensive use of such approaches. Here, we present a simple strategy to employ oil as a transient medium for ATPS droplet generation. Two methods based on passive flow focusing and active pico-injection are demonstrated to generate water-water-oil double emulsions, achieving a high generation frequency of ~2.4 kHz. Through evaporation of the oil to break the double emulsions, the aqueous core can be released to form uniform-sized water-in-water droplets. Moreover, this technique can be used to fabricate aqueous microgels, and the introduction of the oil medium enables integration of droplet sorting to produce single-cell-laden hydrogels with a harvest rate of over 90%. We believe that the demonstrated high-throughput generation and sorting of ATPS droplets represent an important tool to advance droplet-based tissue engineering and single-cell analyses.