Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. 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), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, 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 >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

Creating an artificial cell from the bottom up is a long-standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar vesicles (GUVs) have emerged as a suitable container with many methods available for their production. Well-studied swelling-based methods offer a wide range of lipid compositions but at the expense of limited encapsulation efficiency. Emulsion-based methods, on the other hand, excel at encapsulation but are only effective with a limited set of membrane compositions and may entrap residual additives in the lipid bilayer. Since the ultimate artificial cell will need to comply with both specific membrane and encapsulation requirements, there is still no one-method-fits-all solution for GUV formation available today. This review discusses the state of the art in different GUV production methods and their compatibility with GUV requirements and operational requirements such as reproducibility and ease of use. It concludes by identifying the most pressing issues and proposes potential avenues for future research to bring us one step closer to turning artificial cells into a reality.

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.