Giant unilamellar vesicles (GUVs) are membrane models used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol, dry lipid film is formed. The drying of the lipid film induces the cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals which do not participate in the formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose a novel electroformation protocol that includes rapid solvent exchange (RSE), plasma cleaning, and spin-coating methods to produce GUVs. We tested the protocol, focusing on vesicles with a high Chol content using different spin-coating durations and vesicle type deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles created from the phosphatidylcholine/Chol mixture was 30 s. Multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by the extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores, were deposited on an electrode for 1.5/1 Chol/phosphatidylcholine (POPC) lipid mixture, and the results were compared. Electroformation using all three deposited vesicle types resulted in a high GUV yield, but the deposition of LUVs obtained by the extrusion of MLVs through 100 nm membrane pores provided the most reproducible results. Using the deposition of these LUVs, we produced high yield GUVs for six different Chol concentrations (from 0% to 71.4%). Using a protocol that included dry lipid film GUVs resulted in lower yields and induced the Chol demixing artifact, proving that the lipid film should never be subjected to drying when the Chol content is high.
Keywords: GUV; cholesterol; cholesterol demixing artifact; damp lipid film; electroformation; lipid film deposition.