Studies of persistent poration dynamics of cell membranes induced by electric pulses

Summary form only given. Experimental studies have been conducted to investigate the plasma membrane response of HL-60 human promyelocytic leukemia cells to pulsed electric fields (PEFs) with pulse widths ranging from 125 nanoseconds to 1 millisecond. Both single and trains of monopolar PEFs were delivered to HL-60 cells suspended in micro-fabricated micro-cuvettes. The cells were monitored in real-time via optical microscopy. Four different types of experiments were performed by monitoring the uptake of trypan blue (TB) into the cell cytoplasm: 1) single pulses with pulse widths from 125 ns to 1 ms; 2) a pulse train of ten 1 mus pulses with different pulse intervals from 250 ns to 1 s at 1 Hz, and a single 10 mus pulse; 3) pulse trains with different numbers of pulses (from 1 to 10) for different pulse widths (from 1 mus to 100 mus); 4) one or ten pulses for different ion (Ca ²⁺ and Mg²⁺) concentrations of the suspension media. Experimental results qualitatively agree with previous experimental observations as well as theoretical predictions on pulse accumulation effects due to persistence of large pores, and rapid relaxation times of transient small pores. For single pulses, estimates of the required pulse ohmic dissipation energy delivered to cell populations were analyzed, resulting in a pulse energy curve exhibiting a local minimum. This local minimum can be related to both the pore relaxation time and the pore diffusion constant, a fundamental parameter associated with the rapidity with which pore size can change in response to a driving force. Also, based on the transient aqueous pore theory for electric field induced cell membrane dynamics, a quantitative estimate for the product of the pore diffusion constant and the line-tension pore energy parameter can be inferred from our data. Meanwhile, variable interpulse spacing was employed to examine different attributes of membrane dynamics (pore generation, evolution, size distribution, an- resealing time). These measurements indicated a transient pore relaxation or resealing time of approximately 250 ns for HL-60 cells, in close agreement with the value inferred from the pulse-energy-versus-pulse-width data. For pulse trains, the required electric field decreased monotonically with the number of pulses. This can be explained in terms of a pulse accumulation effect. Finally, the effect of increasing the cell suspension medium´s Ca²⁺ and Mg ²⁺ ionic concentration was observed to impede electroporation