Modular millifluidic platform for controlled generation of triple emulsions with tunable architecture

Triple emulsions are powerful model systems in synthetic biology and biomedicine since their nested architecture and multicompartmental structure resembles the organization of living cells and human organs. The preparation of triple emulsions by microfluidics is associated with the delicate control of fluid flow rates and low droplet productivity. Therefore, novel millifluidic techniques with improved simplicity, flexibility, and productivity are needed to generate triple emulsions with tailored properties in meaningful quantities. In this study, millimetre-sized, monodisperse O/W/O/W and W/O/W/O triple emulsions with tuneable droplet morpholog were prepared in a modular millifluidic platform by combining coaxial glass capillaries with CNC-milled, LEGO-inspired interlocking blocks. The method allows independent regulation of the number and morphology of droplets at each hierarchical level. The encapsulation of up to several hundred innermost droplets in each double emulsion droplet and up to 8 double emulsion droplets in each triple emulsion droplet was achieved with 100 % efficiency. The number of innermost droplets (N 1 = f 2 /f 1 ) and the number of double emulsion droplets (N 2 = f 3 /f 2 ) in each compound droplet was precisely controlled by adjusting fluid flow rates. A self-synchronisation of droplet generation in consecutive steps was observed due to the tendency for surface energy minimisation, which helped to achieve a consistent morphology of compound droplets without any secondary droplet break-up. The maximum throughput of triple emulsion droplets exceeded 50 mL/h, which is one order of magnitude higher than in microfluidic systems. Prepared triple emulsion droplets can be used to create encapsulated droplet interface bilayers (eDIBs) and multicompartmental scaffolds for stem cell growth and self-organization into complex organoid structures.