Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Abstract

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to approximately 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigmae is smaller than that of the cytosol sigmai. The stretching force vanishes when sigmae is equal to or larger than sigmai. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).

Ultrashort electric pulse induced changes in cellular dielectric properties

“…Using a lower conductivity buffer also reduces the electroporation threshold for nsPEFs [27]. Applying a single 11 ns, 160 kV/cm pulse to a cell suspension with conductivity of approximately 0.1 S/m resulted in slightly less than 10% of the cells taking in propidium iodine [27]. Here, we applied five pulses with similar parameters, likely increasing the fraction of cells electropermeabilized.…”

“…While 10 ns pulses are generally too short to induce electroporation [4], applying multiple pulses charges the cell membrane more, increasing the likelihood of exceeding r crit [26]. Using a lower conductivity buffer also reduces the electroporation threshold for nsPEFs [27]. Applying a single 11 ns, 160 kV/cm pulse to a cell suspension with conductivity of approximately 0.1 S/m resulted in slightly less than 10% of the cells taking in propidium iodine [27].…”

Effects of extracellular calcium on cell membrane resealing in sonoporation

“…The process may be the same as that involved in the cell repair of the plasma membrane disruption occurring during normal physiological functions (e.g., cardiac contraction) and injury. The pores generated in electroporation reseal themselves in the absence of extracellular Ca 2+ probably only because of the extremely small size of the disruptions observed, which usually are below 1 μm [37][38][39] and mostly in the range of 20-120 nm [40]. In such cases, resealing of the small holes, besides being driven by the energetically unfavourable situation, may also involve a process similar to the fast process observed in this study, which may bring complete resealing at very low extracellular [Ca 2+ ] when the disruption is extremely small.…”

Electro-mechanical manipulation of mammalian cells in suspension

“…This work has been continued recently by some of the same authors [48]. ED has also been reported, on several occasions, by U. Zimmermann and co-workers, among their very extensive electro-manipulation literature, which includes such topics as: DEP and ER [89], the effects of ED on EP [38], and the effects of ultra-short pulses [90]. They have also reported ED of non-RBC mammalian (murine Sp2 myeloma) cells [46].…”