Using the Droplet Transfer Method to Reliably Prepare Giant Unilamellar Vesicles.

Over the past two decades, the droplet transfer method, also known as inverted emulsion, double emulsion, phase transfer, or emulsion transfer, has proven advantageous for the preparation of Giant Unilamellar Vesicles (GUVs) and particularly, for loading them with different cargoes, thus playing a crucial role in synthetic biology. Because of the efficiency of encapsulation and the simplicity of execution, it has been broadly used for the development of artificial cells. A large variability in several parameters is observed in the literature, which leads to extremely variable outcomes. This is partially due to the adjustments required for different needs and applications of this versatile method, yet it may prove disorienting for researchers approaching this technique for the first time. To provide beginners with a basic understanding of the method and the role of critical parameters, a protocol is presented alongside hints on the fundamental physicochemical principles underlying GUV formation through droplet transfer. This step-by-step guide for the preparation of GUVs thus includes considerations and practical suggestions based on literature and direct experience. Considering possible sources of variability, a few aspects are identified as critical: the sensitivity of phospholipids to light, oxidation, and hydrolysis; the choice of the oil to dissolve phospholipids (the lipid solution or LS); and the recovery of GUVs after centrifugation. Cost-effective measures are proposed to minimize the interference of atmospheric oxygen and humidity. To help identify suitable combinations of oil and phospholipids, the general protocol is complemented with a discussion on the relevant physicochemical properties of the LS. Finally, to avoid impractical procedures, a straightforward and safe method is proposed to completely remove the oil phase at once and recover a clean GUV dispersion. These measures speed up the implementation of adjustments to new GUV compositions, potentially widening their adoption in synthetic biology and neighboring fields, including microrobotics.