Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

Abstract

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion

“…Compared to vesicles grown using the gentle hydration method, the electroformation method reduces the compositional heterogeneity of the vesicles [ 6 ] and increases the proportion of unilamellar vesicles [ 3 ]. The method has evolved significantly over the years, with many potential pitfalls identified and protocol modifications tested [ 7 , 8 , 9 ]. The most important issues are related to the use of organic solvents during lipid film deposition, reproducibility of the conventional film deposition technique, and the step in which the lipid film is completely dried.…”

Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures

“…The dried film is then hydrated, and an alternating current applied to detach the lipid film from the surface and form GUVs. Although quite simple at first glance, the method is influenced by many parameters, requiring optimization depending on the experimental setup [ 2 ]. We focus on the effect of lipid composition, lipid film thickness, and electrical parameters (frequency and voltage) using a ternary mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/Chol) ( Figure 1 ).…”

DisGUVery: A Versatile Open-Source Software for High-Throughput Image Analysis of Giant Unilamellar Vesicles

“…First, we investigated if our methods could be used to track GUV size and number during swelling-based GUV formation. GUV swelling methods include electroformation and gel-assisted swelling and are by far the most popular formation methods, as they are fast and easy and yield large numbers of GUVs. ,,, In these experiments, lipids are first dried on a surface, which can be a hydrogel, an electrode, glass, Teflon, or a porous material. Subsequent addition of a swelling solution leads to swelling of the lipid film and formation of large numbers of GUVs that are closely packed above the swelling surface (Figure A, top).…”

“…Despite the experimental ease of producing and imaging GUVs, their quantitative image analysis has received relatively little attention . Typically, GUVs are either manually detected in the image and afterward (manually) processed to extract data, ,− or custom-made scripts are used to process specific data sets and generate a predefined set of output parameters. ,,− While general image analysis software (e.g., ImageJ) can be used with GUV microscopy data, there are no dedicated modules or plugins for GUV detection and analysis, requiring for the user to adapt the built-in methods and, in case of automated analysis, programming knowledge is often needed.…”