By using electrostatic
interactions as driving force to assemble
vesicles, the droplet-stabilized method was recently applied to reconstitute
and encapsulate proteins, or compartments, inside giant unilamellar
vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized
approach exhibits low production efficiency associated with the troublesome
release of the GUVs from the stabilized droplets, corresponding to
a major hurdle for the droplet-stabilized approach. Herein, we report
the use of pH as a potential trigger to self-assemble droplet-stabilized
GUVs (dsGUVs) by either bulk or droplet-based microfluidics. Moreover,
pH enables the generation of compartmentalized GUVs with flexibility
and robustness. By co-encapsulating pH-sensitive small unilamellar
vesicles (SUVs), negatively charged SUVs, and/or proteins, we show
that acidification of the droplets efficiently produces dsGUVs while
sequestrating the co-encapsulated material. Most importantly, the
pH-mediated assembly of dsGUVs significantly improves the production
efficiency of free-standing GUVs (i.e., released from the stabilizing-droplets)
compared to its previous implementation.