Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses.

BACKGROUND: Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. METHODS: The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. RESULTS: The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. DISCUSSION: The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers.

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms.

Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses.

High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100µs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1µs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.

Mathematical Models Describing Chinese Hamster Ovary Cell Death Due to Electroporation In Vitro.

Electroporation is a phenomenon used in the treatment of tumors by electrochemotherapy, non-thermal ablation with irreversible electroporation, and gene therapy. When treating patients, either predefined or variable electrode geometry is used. Optimal pulse parameters are predetermined for predefined electrode geometry, while they must be calculated for each specific case for variable electrode geometry. The position and number of electrodes are also determined for each patient. It is currently assumed that above a certain experimentally determined value of electric field, all cells are permeabilized/destroyed and under it they are unaffected. In this paper, mathematical models of survival in which the probability of cell death is continuously distributed from 0 to 100 % are proposed and evaluated. Experiments were performed on cell suspensions using electrical parameters similar to standard electrochemotherapy and irreversible electroporation parameters. The proportion of surviving cells was determined using clonogenic assay for assessing the ability of a cell to grow into a colony. Various mathematical models (first-order kinetics, Hülsheger, Peleg-Fermi, Weibull, logistic, adapted Gompertz, Geeraerd) were fitted to experimental data using a non-linear least-squares method. The fit was evaluated by calculating goodness of fit and by observing the trend of values of models' parameters. The most appropriate models of cell survival as a function of treatment time were the adapted Gompertz and the Geeraerd models and, as a function of the electric field, the logistic, adapted Gompertz and Peleg-Fermi models. The next steps to be performed are validation of the most appropriate models on tissues and determination of the models' predictive power.

Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination.

High voltage electric pulses cause electroporation of the cell membrane. Consequently, flow of the molecules across the membrane increases. In our study we investigated possibility to predict the percentage of the electroporated cells in an inhomogeneous electric field on the basis of the experimental results obtained when cells were exposed to a homogeneous electric field. We compared and evaluated different mathematical models previously suggested by other authors for interpolation of the results (symmetric sigmoid, asymmetric sigmoid, hyperbolic tangent and Gompertz curve). We investigated the density of the cells and observed that it has the most significant effect on the electroporation of the cells while all four of the mathematical models yielded similar results. We were able to predict electroporation of cells exposed to an inhomogeneous electric field based on mathematical modeling and using mathematical formulations of electroporation probability obtained experimentally using exposure to the homogeneous field of the same density of cells. Models describing cell electroporation probability can be useful for development and presentation of treatment planning for electrochemotherapy and non-thermal irreversible electroporation.