Book of abstract 2008

The influence of hypoosmolar medium and electric field on cell volume
Marko Ušaj, Katja Trontelj, Maša Kandušer, Damijan Miklavčič
Faculty of electrical engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia
Cell electrofusion is a process where two or more cells exposed to electric field, which are
in close contact, fuse. The consequence of exposure to pulses is transient and nonselective
permeabilization of cell membranes, which is necessary for fusion of cell membranes.
Besides optimal electrical treatment, good physical contact between cells is necessary
for achieving cell fusion. In this context, it is understandable that fusion of adherent
cells is common event, while it is difficult to obtain sufficient contact between cells in
suspension.
Cell swelling facilitates the processes of electrofusion. For achieving contact between cells
in the process of electrofusion, different methods are used, which use either electrical (in
dielectrophoresis) or mechanical forces (in centrifuge, on filters…). Cells swell, when they
are suspended in hypoosmolar medium. Knowledge of swelling kinetics could contribute
to optimization of the methods for contact achievement.
We determined the kinetics of cell swelling for three different cell lines: Chinese hamster
ovary cells CHO, Mouse melanoma cells B16F1 and Chinese hamster lung fibroblasts
V79. All cells under investigation start swelling immediately after exchanging the
isoosmolar medium (10 mM phosphate buffer, 1 mM MgCl
p46
2
, 250 mM sucrose, pH 7,2
and low conductivity 0,12 S/m) with hypoosmolar (75 mM sucrose). However, they reach
their maximum size value at different time after the change (between 2 to 5 minutes) and
differ in the speed of shrinking as well. We observed diameters of cells for 30 minutes after
the medium exchange. In this period, cells shrunk to their original size due to regulatory
volume decrease. When we kept cells in isoosmolar medium, they did not change their
size.
Cell swelling is also observed after electropermeabilization. We determined the influence
of combination of electropermeabilization and hypoosmolar medium on cell size. We
electroporated cells 1 minute after exchanging the medium, that is before cells reach
their maximum size. We used electrical parameters, which are optimal for transient
permeabilization of cells in isoosmolar medium. Electropermeabilization abolished the
effect of regulatory volume decrease during the 30 minutes of our observation.
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