Volume 7 (2012) Issue 4 Pages 589-602
Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.