Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

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.

Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.