Effects of Submicrosecond, High Intensity Pulsed Electric Fields on Living Cells -Intracellular Electromanipulation

摘要

Development of technology to produce nanosecond duration pulsed electric fields has allowed examination of the effects of ultrashort duration, high intensity electric fields on living cells. Theoretically, high intensity (MV/m) electric field applications with durations of less than one microsecond, when shortened toward nanoseconds, should increasingly affect intracellular rather than surface membranes of living cells. Experimentally, square-wave, 60 ns duration, high energy (36-53 kV/cm) pulses applied in trains of 1-10 pulses result in progressive increases in the numbers of permeabilized intracellular granules in a human eosinophil cell model - without large surface membrane effects. Electron micro-graphic examination of cells treated in this way demonstrates alteration of intracellular granule morphology consistent with permeabilization of granule membrane, i.e., intracellular electromanipulation. Continuous microscopic examination of individual living cells exposed to long or short duration pulsed electric field applications shows that permeabilization of surface membrane (median 5 minutes) with anodic preference (electroporation) and prompt cellular swelling follow a single, long duration (100 microsecond) pulse. In contrast, after a single short duration (60 ns) pulse, onset of surface membrane permeability is delayed (median 17 minutes), the increased permeability shows no anodic preference, and cellular swelling is absent suggesting that these effects are due to intracellular electromanipulation rather than direct effects on the surface membrane. Submicrosecond, intense pulsed electric fields applied to living cells achieve preferential effects on intracellular rather than surface membranes, potentially providing new approaches for selective/generalized cell or tissue ablation, growth stimulation and tissue remodeling.