Determination of the Electroporation Threshold for Pulses from 95 ns to 20 μs G. Saulis *1 , S. Balevicius ** , R. Saule*, V. Stankevic ** , and N. Zurauskiene ** * Department of Biology, Vytautas Magnus University 58 K. Donelaicio str., Kaunas 44248, Lithuania, 1 sg@kaunas.omnitel.net ** Laboratory of High Power Pulses, Institute of Semiconductor Physics 11 A. Gostauto str., Vilnius 01108, Lithuania ABSTRACT The dependences of the fraction of electroporated mouse hepatoma MH-22A cells on the pulse intensity were obtained for the cells exposed to a single square-wave electric pulse with the duration from 95 ns to 20 μs. The amplitude of an electric pulse was varied from 0.4 to 12 kV/cm. Increasing the intensity of the electric field pulse increased the fraction of electroporated cells. The electric field, which lead to a given percentage of electroporation, decreased with increasing the pulse length. The dependence of the amplitude of the electric pulse required to electroporate 50 % of mouse hepatoma MH-22A cells on the pulse duration was also determined. Keywords: potassium ions, potassium selective electrode, electropermeabilization, mouse hepatoma, nanosecond pulses 1 INTRODUCTION The permeability of the cell membrane can be modified by exposing of cells to high-voltage electric pulses leading to the formation of nanometer-sized pores in the cell membrane (electroporation or nanoporation) [1]. This phenomenon is widely used in cell biology, biotechnology, and medicine [1-3]. For optimization of practical applications and comparison with theoretical modeling, it is important to know whether and how many of cells have become electroporated as a result of a particular electric treatment. There are theoretical models allowing to obtain theoretical relationships between the parameters of the electric treatment resulting in cell electroporation for any type of an electric treatment [4,5]. However it is still difficult to predict individual responses of different cells to electric treatment [6,7]. This is because there are very few studies, in which the dependence of the electroporation threshold on the pulse duration would be determined [8,9]. Either the electric field parameters needed for the increase of the cell membrane permeability to a particular substance (electropermeabilization) [6] or cell viability [7] have been determined for several cell lines. As a result, the fraction of electroporated cells still needs to be determined empirically for each cell line [7]. Up to now, almost there are no studies in which the dependence of the amplitude of the electric pulse required to electroporate 50 % would be determined for the electric pulses with the durations shorter than 1 μs [8] and there is no study in which this would be done for pulses shorter than 320 ns. The main reason of this is, that it is difficult to detect the threshold of electroporation, because the pores created in the plasma membrane can be small [10-13]. Only recently, the electroporation threshold was compared for different cell lines [9]. However, this was done for the electric pulses in the range from 20 μs to 2 ms. Here, the dependences of the fraction of electroporated mouse hepatoma MH-22A cells on the pulse intensity were obtained for the cells exposed to a single square-wave electric pulses with the durations from 95 ns to 20 μs. 2 MATERIALS AND METHODS The culture medium consisted of Dulbecco’s modified Eagle’s medium (cat. no. D5546, Sigma-Aldrich Chemie, Steinheim, Germany) supplemented with 10 % fetal bovine serum (cat. no. F7524, Sigma-Aldrich Chemie), 1 % L- glutamine (cat. no. G7513, Sigma-Aldrich Chemie), 100 U/ml penicillin, and 100 μg/ml streptomycin (cat. no. P0781, Sigma-Aldrich Chemie). As an electroporation medium, the culture medium was used. Calibration solutions containing 0.2-100 mM KCl were prepared by diluting a stock solution of 100 mM KCl and adding 150 mM sodium chloride and 8 mM sodium benzoate [14]. The NaCl is added to keep sodium ion concentration close to that in electroporation medium. The mouse hepatoma MH-22A cells were grown in monolayer cultures in 75-cm 2 flasks at 37 o C and 5 % CO 2 in a water-jacketed incubator. When cells reached confluence they were trypsinazed, suspended in the culture medium at approximately 2-5×10 7 cells/ml, and kept for 60–70 min at room temperature (20–21 o C) [15]. During this time, the cells restored the normal level of the intracellular concentration of potassium ions. Then the cells were electroporated within 15-20 min. For electroporation, single square-wave pulses with the duration of 100 μs and 2 ms and the amplitude ranging from 0.2 to 2.4 kV/cm were used. A 50-μl droplet of cell suspension was placed between a pair of flat stainless-steel electrodes and was subjected to a single square-wave electric pulse. NSTI-Nanotech 2010, www.nsti.org, ISBN 978-1-4398-3415-2 Vol. 3, 2010 506