Giant unilamellar vesicles (GUVs) show great potential as biosensors and artificial cells and have been utilized extensively as cellular models for studies on dynamic membrane properties, mechanical measurements, phospholipid domain formation, and membrane protein integration. Macroscale methods to make GUVs are widespread, but suffer from polydispersity, multilamellarity, and low encapsulation efficiency. While the control of droplet production provided by microfluidic technologies have been able to overcome many of these limitations, widespread usage of GUVs outside of research settings has been hindered by their fragility and short shelf life, rendering them difficult to ship and commercialize. A similar structure, known collectively as multisomes, can be suitable for the same applications as GUVs, but their potential has been undervalued and they also suffer similar storage limitations as GUVs. In order for GUVs and multisomes to realize their vast potential, it is paramount that they can be produced in bulk, and stored for a reasonable amount of time without structural degradation.

Numerous microfluidic methods to produce GUVs employ water-in-oil-in-water (W/O/W) double emulsion templates that quickly convert into lipid vesicles via dewetting and/or solvent extraction. These methods generally use harsh, volatile solvents in the sheath (oil) phase that can potentially harm both cells and encapsulated biological materials. Furthermore, the immediate removal of the solvent to form GUVs limits their useful lifetime. Nearly all the work to improve GUV shelf life has been focused on modifying their storage conditions after production, which can be damaging.

In this work, we present a method to circumvent the storage limitations and fragility of GUVs and multisomes. Double emulsions templates are produced utilizing a single junction co-focusing microfluidic device to ensure monodispersity, and are composed of only biocompatible surfactants and fatty acids. The design allows for double emulsions to be produced with diameters ranging from 9-50µm and generation rates up to 3 kHz by simply varying the fluid flow rates. The templates can be stored for up to a year and converted into multisomes or GUVs on demand within minutes by a simple solution change that alters the spreading coefficients of the system. We have also uncovered the interfacial parameters that control the stability of double emulsions for storage, and their subsequent dewetting to form multisomes or GUVs. Furthermore, multisomes were surface functionalized to demonstrate their applicability as artificial cells. In particular, they were surface modified to promote insulin secretion in pancreatic B cells through surface interactions with neuroligin-2. Ultimately, we envision that these multisomes and GUVs can be utilized as blank templates for a variety of applications, including drug delivery vehicles, artificial antigen presenting cells, and biosensors.