Generation of double emulsion droplets with controllable intra- and extra-droplet trehalose concentration in a six-way microfluidic chip

Drying preservation of mammalian cells presents a promising alternative to cryopreservation, with intracellular vitrification being the central challenge. Trehalose, a critical desiccation protectant, is essential for achieving this vitrification, but effective intracellular delivery and concentration control remain major hurdles. Current microfluidic methods for generating Giant Unilamellar Vesicles (GUVs, cell models) rely heavily on glycerol. This glycerol dependence fundamentally impedes the study and application of trehalose-mediated vitrification, as glycerol’s low glass transition temperature and difficulty of removal interfere with trehalose’s protective function. This study develops a glycerol-free microfluidic strategy that directly utilizes trehalose as the core component to enable stable generation of monodisperse water-in-octanol-in-water (W/O/W) double emulsions (GUV precursors) with precisely programmable intra- and extra-vesicular trehalose concentrations. Using a PDMS microfluidic chip with a six-way junction, the effects of replacing glycerol with varying trehalose concentrations (0–1.2 M) on double emulsion generation were investigated. Results demonstrate that trehalose effectively compensates for the destabilization caused by glycerol removal by increasing inner aqueous phase viscosity and reducing inner interfacial tension, significantly broadening the stable dripping regime. Droplet size exhibited non-monotonic variation with inner trehalose concentration, while increasing outer aqueous trehalose consistently reduced droplet size. This work is the first to demonstrate that trehalose can fully replace glycerol for generating double emulsions with spatially controlled trehalose loading. This glycerol-free approach overcomes the limitations of conventional methods, providing a vital experimental platform for investigating trehalose-mediated intracellular vitrification mechanisms and advancing trehalose-based anhydrous cell preservation technologies.