Experimental Studies of Persistent Poration Dynamics of Cell Membranes Induced by Electric Pulses

Experiments have been conducted that investigate the plasma membrane response of HL-60 human promyelocytic leukemia cells to pulsed electric fields (PEFs) with pulsewidths ranging from 125 ns to 1 ms. Both single and trains of monopolar PEFs were delivered to HL-60 cells while suspended in microcuvettes where they were monitored in real-time via optical microscopy. By monitoring the uptake of trypan blue (TB) into the cell cytoplasm, the formation of pores that persist long after the electric stimulus was recorded as a function of pulsewidth, pulse amplitude, and interpulse spacing. 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. The resealing or relaxation time was estimated to be approximately 250 ns for the transient pores and 1 s or more for the persistent pores. The presence of calcium and magnesium in the cell suspension fluid impeded persistent pore formation, particularly at shorter pulsewidths. Finally, the authors report the first experimental measurement of two fundamental membrane poration parameters: the pore diffusivity and the pore-perimeter line-tension-energy coefficient