Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Abstract

AbstractGiant unilamellar vesicles (GUVs) are widely used as three-dimensionalin vitromodel membranes in biophysics and as cell-sized containers in bottom-up synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Over the years, numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity in recent years. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To gain a deeper understanding of GUV formation in cDICE, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. Based on our observations and theoretical modelling of the fluid dynamics within the system, we find that the transfer of these droplets through the oil-water interface exhibits a size-selectiveness toward GUV-sized droplets. Finally, we demonstrate that protein in the inner solution affects GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE and, by extension, in emulsion-based methods in general, but ultimately also aid the development of more reliable and efficient methods for GUV production.Graphical abstractWe developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.