Elsevier

Bioelectrochemistry

Volume 74, Issue 1, November 2008, Pages 52-57
Bioelectrochemistry

The temperature effect during pulse application on cell membrane fluidity and permeabilization

https://doi.org/10.1016/j.bioelechem.2008.04.012Get rights and content

Abstract

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1.

While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 μs and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 μl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 °C) to 4 °C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.

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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