Book of abstract 2008

High-voltage nanosecond electrical pulses affect plasma membrane of
cultured cells
Tina Batista Napotnik1, Matej Reberšek1, Danilo Vrtačnik2, Slavko Amon2, Damijan
Miklavčič1
1Laboratory of Biocybernetics, Faculty of Electrical Engineering, Tržaška 25, 1000 Ljubljana, Slovenia;
2Laboratory of Microsensor Structures and Electronics, Faculty of Electrical Engineering, Tržaška 25,
Ljubljana, Slovenia
The exposure of cells to an electric field results in poration of the plasma membrane
(i.e. electroporation). The electroporation is already in use in clinical treatments in
electrochemotherapy and gene electrotransfection. Typical pulses that are used for the
electroporation are in micro- to millisecond time range and with electrical field strength
of 100 to a few 1000 V/cm.
High-voltage nanosecond electrical pulses (with duration from 1 to 100 ns and the electric
field strength up to a few 100 kV/cm) can be achieved by high-voltage generators, usually
derived from a Blumlein pulse-forming network. These pulses are short enough and high
enough to induce sufficiently high induced voltage across the intracellular membranes. The
nanosecond pulses have greater effect on cell organelles than on the plasma membrane:
they can induce pore formation in membranes of cell organelles. This can lead to elevating
cell calcium, DNA damage and apoptosis. When pulse parameters (duration, voltage, and
number of pulses) are augmented, also the effects on plasma membrane increase.
We have designed a modified Blumlein generator for generating high-voltage nanosecond
electrical pulses of various durations (10 to 300 ns) and pulse frequency in a pulse train
(up to very high frequencies of a few 100 kHz). The electrodes made of gold are deposited
onto a microscope cover glass and placed under a fluorescence microscope in order to
observe immediate effects on cultured cells. The gap between the two electrodes is 100 μm
so very high field strength
p1
can be achieved (up to 100 kV/cm).
The B16 F1 mouse melanoma cells were exposed to high-voltage nanosecond pulses
(60 ns, 44 kV/cm) of various repetitition frequency (up to 100 Hz) and number of pulses
(up to 60) in a pulse train. We observed cell uptake of propidium iodide (PI) after the
pulsing. We confirmed that higher number of pulses and higher frequency of pulses in a
pulse train cause rise of plasma membrane permeability, therefore cells become positive for
PI staining. These results show that high-voltage nanosecond pulses in higher frequencies
and number of pulses in a pulse train affect plasma membrane of cultured cells.
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