Karel Flisar

Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses: Part I. Increased efficiency of permeabilization

The paper presents a comparative study of electropermeabilization of cells in suspension by unipolar and symmetrical bipolar rectangular electric pulses. While the parameters of electropermeabilization by unipolar pulses have been investigated extensively both in cell suspensions and in tissues, studies using bipolar pulses have been rare, partly due to the lack of commercially available bipolar pulse generators with pulse parameters suitable for electropermeabilization. We have developed a high-frequency amplifier and coupled it to a function generator to deliver high-voltage pulses of programmable shapes. With symmetrical bipolar pulses, the pulse amplitude required for the permeabilization of 50% of the cells was found to be approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing the death of 50% of the cells. Bipolar pulses also led to more than 20% increase in the uptake of lucifer yellow. We show that these results have a theoretical background, because bipolar pulses (i) counterbalance the asymmetry of the permeabilized areas at the poles of the cell which is introduced by the resting transmembrane voltage, and (ii) increase the odds of permeabilization of cells having a nonspherical shape or a nonhomogeneous membrane. If similar results are also obtained in tissues, bipolar pulse generators could in due course gain a wide, or even a predominant use in cell membrane electropermeabilization.

The Role of Electrophoresis in Gene Electrotransfer

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LVxa0+xa0HV and HVxa0+xa0LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HVxa0+xa0LV, while for the reversed sequence, LVxa0+xa0HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.

Testing a prototype pulse generator for a continuous flow system and its use for E. coli inactivation and microalgae lipid extraction.

Among other applications, electroporation is used for the inactivation of pathogens and extraction of substances from microorganisms in liquids where large scale flow systems are used. The aim of our work was therefore to test a pulse generator that enables continuous pulsed electric field (PEF) treatment for Escherichia coli inactivation and microalgae lipid extraction. In the continuous flow PEF system, the flow rate was adjusted so that each bacterial cell received a defined number of pulses. The results of PEF flow treatment showed that the number of pulses influences E. coli inactivation to the same extent as in the previously described cuvette system, i.e., batch system. The continuous flow PEF system was also tested and evaluated for lipid extraction from microalgae Chlorella vulgaris. In control experiments, lipids were extracted via concentration of biomass, drying and cell rupture using pressure or an organic solvent. In contrast, electroporation bypasses all stages, since cells were directly ruptured in the broth and the oil that floated on the broth was skimmed off. The initial experiments showed a 50% oil yield using the electroporation flow system in comparison to extraction with organic solvent.

Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA–membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long‐duration, short‐duration and combinations of electropermeabilizing short (high‐voltage; HV) and electrophoretic long (low‐voltage; LV) pulses were performed. The effect of Mg2+ ion concentrations on electrotransfer and their effect on DNase activity were explored.

Metastatic potential of melanoma cells is not affected by electrochemotherapy.

Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin and cisplatin has shown its effectiveness in controlling local tumour growth in the treatment of malignant melanoma. However, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Prevention of metastasis is an important aspect of successful treatment; however, it is known that metastasis can be induced by different treatment modalities. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with cisplatin on the metastatic potential of human malignant melanoma cells. Cells treated by electrochemotherapy with cisplatin were tested for their ability to migrate and invade through Matrigel-coated porous membrane. In addition, RNA was isolated from cells after treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with cisplatin on gene expression. There were no significant changes observed in cell migration and invasion of melanoma cells after electrochemotherapy. In addition, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis showed that a very low number of genes were differentially expressed after electrochemotherapy with cisplatin. Two genes, LAMB3 and CD63 involved in cell migration, were both downregulated after electrochemotherapy with cisplatin and the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy does not increase the metastatic behaviour of human melanoma cells.

Enhanced cytotoxicity of bleomycin and cisplatin after electroporation in murine colorectal carcinoma cells

Enhanced cytotoxicity of bleomycin and cisplatin after electroporation in murine colorectal carcinoma cells Background. Electrochemotherapy is a local treatment combining application of electric pulses and chemotherapy. Two chemotherapeutic drugs, bleomycin and cisplatin, have proved to be effective in electrochemotherapy. The effectiveness of electrochemotherapy was demonstrated in the treatment of various cutaneous and subcutaneous tumours in cancer patients. Only a few preclinical studies were performed in colorectal carcinoma, mostly using bleomycin. Our aim was to evaluate the sensitivity of the murine colorectal carcinoma cell line CMT-93 to electrochemotherapy with bleomycin and cisplatin for potential use in preclinical and clinical studies. Methods. CMT-93 cells were exposed to either the chemotherapeutic drug alone or electrochemotherapy. A clonogenic assay was used to determine cell survival after treatment. Apoptosis was measured by caspase-3/7 activity, necrosis by changes in cell morphology and cell viability by the MTS assay 16 hours after electrochemotherapy. Results. Cells treated with electrochemotherapy were 500-fold more sensitive to bleomycin and 2.8-fold more sensitive to cisplatin compared to cells treated with the drugs alone. At the highest concentrations, a significant reduction in cell viability, increase in caspase-3/7 activity and necrotic cells were observed after electrochemotherapy. Conclusions. Exposure of cells to electric pulses enhanced cytotoxicity of both bleomycin and cisplatin. Reduced cell viability was due to apoptotic and necrotic cell death. Furthermore, electrochemotherapy with bleomycin was more cytotoxic than electrochemotherapy with cisplatin in this colorectal carcinoma cell line.

Cell membrane electropermeabilization with arbitrary pulse waveforms

We present a detailed design of a system for in vitro electropermeabilization with arbitrary waveforms. A low-voltage signal is generated by a programmable function generator and amplified by a bipolar amplifier circuit built from commercial components. We describe the general outline of the setup, give the scheme of the amplifier circuit, and present the frequency characteristics of the system. Unlike the commercially available devices used for electropermeabilization, the system presented in this article provides a custom choice of the pulse waveform, with the amplitude from 0 up to 260 V (520 V peak-to-peak) with a shape distortion below 5% for the band from 500 Hz up to 35 kHz, and below 15% up to 55 kHz. The circuit can deliver currents up to 5.2 A, which, at the maximum output voltage, is obtained on a resistive load of 50 W. For larger loads (lower resistivity), the performance of the circuit is reduced, with a possibility of malfunction. The total cost of the amplifier circuit components is less than US

Electrofusion of B16-F1 and CHO cells: The comparison of the pulse first and contact first protocols

400, and with programmable function generators starting at approximately US

Extraction of Non-polar Molecules from Green Alga Chlorella vulgaris by Electroporation

1,000, this makes the presented design attainable to any laboratory with interest in electropermeabilization.

Extraction of Sugar Solution from Sugar Beet Cossettes by Electroporation and Compressive Load

High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.