A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10-30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.