The temperature effect during pulse application on cell membrane fluidity and permeabilization
Introduction
Cell membrane permeabilization of an intact cell (exposed to external electric field) is caused by the application of high intensity electric pulses of short duration. The parameters of electric pulses need be chosen properly in order to obtain desired permeabilization of the cell membrane and at the same time not to significantly affect the cell survival [1], [2]. The process of electropermeabilization consists of at least two separate phases: pore formation that takes place during, and resealing which happens after the pulse application [3].
The interest on the temperature effect on electropermeabilization was mainly focused on the Joule heating that takes place during the electric pulse application [4], [5], [6], [7], [8]. A significant localized Joule heating was observed, which increased with the voltage and the duration of electropermeabilization pulses in human stratum corneum [5]. For exponential pulses with pulse amplitude 70 V and duration of 1 ms, the temperature rise above physiological temperature for 19 °C has no significant role in stratum corneum permeability increase [7]. In in vitro experiments it was found that the spatial temperature gradient that changes with time could be an important factor for post-pulse recovery processes but not for the electropermeabilization. For pulse durations used for electropermeabilization and pulses of rather large amplitude (4.5 kV/cm), no considerable heating was observed when applied to cell suspension at 25 °C [4].
On the other hand, in some studies researchers focused on the chilling effect on electropermeabilization of erythrocytes, alga and porcine skin [9], [10], [11], [12]. Experiments with erythrocyte showed that the temperature had no significant effect on electropermeabilization, as the breakdown voltage was 0.8 V at 25 and 37 °C and 0.87 V at 4 °C [9]. However, in alga Valonia, electropermeabilization at 5 °C required higher voltage, because its breakdown potential was 1 V, while at 35 °C it was 0.65 V [10]. Similar results were obtained with porcine stratum corneum. The chilling of the stratum corneum also had a significant effect on electropermeabilization. Decreasing the temperature from 25 °C to 4 °C required higher voltages for electropermeabilization, suggesting that elevated temperature facilitated the formation of electro pores. Authors further proposed that lipid fluidity, which is controlled by the temperature, is the major factor affecting electropermeabilization of the stratum corneum [12].
As the temperature has an effect on cell membrane electropermeabilization and on cell membrane fluidity, the aim of our study was to correlate both effects. We determined the effect of temperature decrease from physiological temperature 37 °C to 4 °C on average cell membrane fluidity and on the membrane domain structure of two different cell lines.
Section snippets
Cell culture and media
Cell lines V-79 Chinese hamster lung fibroblasts and B16-F1 Murine melanoma were grown in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) in the incubator (Kambič, Slovenia) at 37 °C in a humidified 5% CO2 atmosphere. From confluent cultures a cell suspension was prepared by 0.25% trypsin/EDTA solution (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). To obtain cell pellet, cell suspension was centrifuged at 1000 rpm
Results
Two cell lines, V79 and B16F1 were used to determine the effect of the temperature on cell membrane fluidity and electropermeabilization at different temperatures. By comparing the experimental EPR spectra measured at 4 °C and 37 °C of both cell lines it is evident that the average cell membrane fluidity significantly decreases (by chilling the cells) (Fig. 1). The spectra at 4 °C are typical for highly restricted motion of spin probe, while the spectra at 37 °C are typical for less restricted
Discussion
The main goal of our study was to determine the effect of cell membrane fluidity and membrane domain structure on electropermeabilization by changing the temperature of the cell suspension during the application of electric pulses. Our results show that cell membrane permeabilization depends on the temperature, which has a significant effect on cell membrane fluidity and domain structure.
In discussing the membrane domain structure as measured by EPR, we would like to stress that EPR labelling
Acknowledgments
This work was financially supported by Slovenian Research Agency (ARRS).
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