Understanding the dynamics of macromolecular assemblies in solution, such as Liquid-Liquid Phase Separation (LLPS), represents technologic and fundamental challenges in many fields. In cell biology, such dynamics are of great interest, because of their involvement in subcellular processes. In our study, we aimed to control the assembly of macromolecules in aqueous semi-permeable vesicles, that we named osmosomes, using microfluidics. We developed a microfluidic chip that allows for producting and trapping Giant Unilamellar Vesicles (GUVs) encapsulating macromolecules. This device also allows for modification of the composition of the inner phase and of the membranes of the trapped GUVs. The vesicles are produced from water-in-oil-in-water (w/o/w) double emulsions in less than 20 min after discarding the oil phase. They are highly monodisperse and their diameter can be modulated between 20 and 110 mu m by tuning the flow rates of fluid phases. Their unilamellarity is proofed by two techniques: (1) fluorescence quenching experiments and (2) the insertion of the alpha-hemolysin membrane protein pore. We demonstrate that the internal pH of osmosomes can be tuned in less than 1 min by controlling solvent exchanges through the alpha-hemolysin pores. The detailed analysis of the exchange kinetics suggests that the microfluidic chip provides an efficient pore formation due to the physical trapping of vesicles and the constant flow rate. Finally, we show a proof of concept for macromolecular assembly within osmosomes by pH-triggered LLPS of wheat proteins within a few minutes. (C) 2020 Elsevier Inc. All rights reserved.