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Available from: Matej Rebersek, Sep 03, 2014
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    • "Based on this research, electroporation's advantages compared to other extraction techniques are considered to be shorter process time (in a microsecond to millisecond range), no need for additional procedures to obtain targeted molecule and/or adding undesired chemicals into product. Since electrical pulse parameters are affecting cell membrane permeability (Rols and Teissie 1990; Pucihar et al. 2011) and undesired membrane contaminants, such as endotoxins could be released from the outer membrane of bacteria cells, pulse treatment conditions have to be adjusted in order to extract a maximum quantity of intracellular product by means of electroporation, with high cell viability. Namely, membrane contaminants, such as endotoxins could be released from damaged membrane, and additional purification steps are needed, which at large scales would represent up to 80 % of the production costs. "
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    ABSTRACT: Extracting proteins by means of electroporation from different microorganisms is gaining on its importance, as electroporation is a quick, chemical-free, and cost-effective method. Since complete cell destruction (to obtain proteins) necessitates additional work, and cost of purifying the end-product is high, pulses have to be adjusted in order to prevent total disintegration. Namely, total disintegration of the cell releases bacterial membrane contaminants in the final sample. Therefore, our goal was to study different electric pulse parameters in order to extract as much proteins as possible from E. coli bacteria, while preserving bacterial viability. Our results show that by increasing electric field strength the concentration of extracted proteins increases and viability reduces. The correlation is reasonable, since high electric field destroys bacterial envelope, releasing all intracellular components into surrounding media. The strong correlation was also found with pulse duration. However, at longer pulses we obtained more proteins, while bacterial viability was not as much affected. Pulse number and/or pulse repetition frequency at our conditions have no or little effect on concentration of extracted proteins and/or bacterial viability. We can conclude that the most promising pulse protocol for protein extraction by means of electroporation based on our experience would be longer pulses with lower pulse amplitude assuring high protein yield and low effect on bacterial viability.
    Full-text · Article · Jul 2015 · Journal of Membrane Biology
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    • "6(b)]. It was shown, that out of several theoretical equations proposed for the description of such a relationship, the best fit to the experimental data was given by (1)–(4) [25]. In Fig. 6(c), the dependences of the amplitude of a squarewave electric pulse required to electroporate 50% of cells, E 0.5 , on the pulse duration are presented for human erythrocytes [18], [26], CHO [26], and mouse hepatoma MH-22A cells. "
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    ABSTRACT: Here, theoretical relationships between the parameters of the electric pulse, which is necessary to porate the cell by electric pulse of various shapes, have been obtained. The theoretical curves were compared with the experimental relationships. Experiments were carried out with human erythrocytes and mouse hepatoma MH-22A cells. The fraction of electroporated MH-22A cells was determined from the extent of the release of intracellular potassium ions and erythrocytes – from the extent of their hemolysis after long (20–24 h) incubation in 0.63% NaCl solution at 4 oC. The dependence of the fraction of electroporated cells on the amplitude of the electric field pulse was determined for pulses with the duration from 95 ns to 2 ms. The shapes of theoretical dependencies are in agreement with experimental ones. The cell poration time depended on the intensity of the pulse: the shorter the pulse duration, the higher the electric field strength has to be. This dependence is much more pronounced for pulses shorter than 1 μs. For example, if the pulse amplitude required to electroporate 50% of human erythrocytes increased from 1.0 to 1.76 kV/cm, when the duration of a square-wave pulse was reduced from 2 ms to 20 μs, it increased from 3 to 12 kV/cm, when the pulse duration was reduced from 950 to 95 ns. The relationships between the electric field strength required for electroporation and the frequency of the applied ac field were calculated for different pulse lengths. It has been obtained that although the electric field strength is constant for frequencies less than 10 kHz but its value depends on the pulse length decreasing with increasing pulse duration. At higher frequencies electric field strength is dependent on the frequency of the ac field.
    Full-text · Article · Oct 2013 · IEEE Transactions on Plasma Science
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    • "electroporation pulses) [1-3]. The efficiency of electroporation strongly depends on pulse parameters such as pulse amplitude, pulse duration, number of pulses and repetition frequency [4]. In order to successfully electropermeabilize a cell membrane the induced transmembrane voltage needs to exceed a given value that depends on cell or tissue type (e.g. "
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    ABSTRACT: Background: Electroporation based therapies and treatments (e.g. electrochemotherapy, gene electrotransfer for gene therapy and DNA vaccination, tissue ablation with irreversible electroporation and transdermal drug delivery) require a precise prediction of the therapy or treatment outcome by a personalized treatment planning procedure. Numerical modeling of local electric field distribution within electroporated tissues has become an important tool in treatment planning procedure in both clinical and experimental settings. Recent studies have reported that the uncertainties in electrical properties (i.e. electric conductivity of the treated tissues and the rate of increase in electric conductivity due to electroporation) predefined in numerical models have large effect on electroporation based therapy and treatment effectiveness. The aim of our study was to investigate whether the increase in electric conductivity of tissues needs to be taken into account when modeling tissue response to the electroporation pulses and how it affects the local electric distribution within electroporated tissues. Methods: We built 3D numerical models for single tissue (one type of tissue, e.g. liver) and composite tissue (several types of tissues, e.g. subcutaneous tumor). Our computer simulations were performed by using three different modeling approaches that are based on finite element method: inverse analysis, nonlinear parametric and sequential analysis. We compared linear (i.e. tissue conductivity is constant) model and non-linear (i.e. tissue conductivity is electric field dependent) model. By calculating goodness of fit measure we compared the results of our numerical simulations to the results of in vivo measurements. Results: The results of our study show that the nonlinear models (i.e. tissue conductivity is electric field dependent: σ(E)) fit experimental data better than linear models (i.e. tissue conductivity is constant). This was found for both single tissue and composite tissue. Our results of electric field distribution modeling in linear model of composite tissue (i.e. in the subcutaneous tumor model that do not take into account the relationship σ(E)) showed that a very high electric field (above irreversible threshold value) was concentrated only in the stratum corneum while the target tumor tissue was not successfully treated. Furthermore, the calculated volume of the target tumor tissue exposed to the electric field above reversible threshold in the subcutaneous model was zero assuming constant conductivities of each tissue.Our results also show that the inverse analysis allows for identification of both baseline tissue conductivity (i.e. conductivity of non-electroporated tissue) and tissue conductivity vs. electric field (σ(E)) of electroporated tissue. Conclusion: Our results of modeling of electric field distribution in tissues during electroporation show that the changes in electrical conductivity due to electroporation need to be taken into account when an electroporation based treatment is planned or investigated. We concluded that the model of electric field distribution that takes into account the increase in electric conductivity due to electroporation yields more precise prediction of successfully electroporated target tissue volume. The findings of our study can significantly contribute to the current development of individualized patient-specific electroporation based treatment planning.
    Full-text · Article · Feb 2013 · BioMedical Engineering OnLine
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