Analysis of Inner Content and Phospholipid Membrane Fusion during Two Step Freezing Induced Liposome Fusion

Giant unilamellar vesicles (GUVs), which are comparable in size to living cells, can serve as artificial cell models by encapsulating biochemical reactions such as transcription and translation within their phospholipid bilayers. However, because these lipid membranes are impermeable to macromolecules, the encapsulated reactions are limited by substrate depletion, halting over time. To address this limitation, repeated freeze-thaw (F/T) cycles have been shown to promote vesicle fusion, thereby replenishing internal contents and enabling sustained reactions inside GUVs. Fusion of GUVs by the F/T method is typically attributed to membrane rupture caused by internal volume expansion during freezing and subsequent membrane reassembly during thawing. In this study, we discovered that subjecting GUVs to further cooling after initial freezing of both internal and external solutions significantly enhances the fusion efficiency between vesicles. This result suggests that not only the freezing of aqueous compartments, but also the phase state of lipid molecules constituting the bilayer, plays a crucial role in membrane fusion. Notably, for GUVs composed of POPC, freezing at -20 °C followed by further cooling to -196 °C increased fusion efficiency while minimizing internal leakage, indicating that this approach can be used to optimize artificial cell culturing.