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.

水性两相系统 (ATPS) 液滴在各种生物测定中表现出优于传统油包水液滴的兼容性。然而，超低的界面张力阻碍了高效稳定的液滴生成，限制了此类方法的进一步发展和更广泛的使用。在这里，我们提出了一种简单的策略，使用油作为 ATPS 液滴生成的瞬态介质。两种基于被动流聚焦和主动微微注射的方法被证明可以产生水-水-油双乳液，实现约 2.4 kHz 的高产生频率。通过油的蒸发打破双乳液，可以释放水核以形成大小均匀的水包水滴。此外，该技术可用于制备水性微凝胶，并且油介质的引入使得液滴分选能够集成，以产生单细胞负载的水凝胶，收获率超过90%。我们相信，ATPS 液滴的高通量生成和分选代表了推进基于液滴的组织工程和单细胞分析的重要工具。