High Pulsed Electric Fields Research Articles

Using non-thermal irreversible electroporation to create an in vivo niche for exogenous cell engraftment.

The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. We treated the livers of mice with NTIRE and subsequently implanted exogenous congenic hepatocytes within the zone of cell ablation. Donor hepatocytes engrafted and integrated with host liver parenchyma pre-treated with NTIRE. This new approach should have value for studying the effects of a native matrix scaffold on in vivo cell growth and may pioneer a new type of minimally-invasive regenerative surgery.

Effect of low and high pulsed electric field on the quality and nutritional minerals in cold boned beef M. longissimus et lumborum

The present research investigated the effects of low PEF (LPEF, 2.5 kV, 200 Hz and 20 μs) and high PEF (HPEF, 10 kV, 200 Hz and 20 μs) on the quality of cold-boned beef loins at 1 and 14 days of post-treatment. HPEF increased (P < 0.001) the temperature of the beef M. longissimus et lumborum (LL) samples. HPEF samples had higher (P = 0.007) shear force than LPEF samples and control samples were not different from either. HPEF beef LL samples had higher L* values and lower a* values (P < 0.001) compared to LPEF and control samples. Higher lipid oxidation (P = 0.013) was found in HPEF samples compared to LPEF samples. Lower (P < 0.01) P, K and Fe concentrations were found in HPEF samples compared to LPEF samples. The results suggest that high intensity PEF treatment can negatively affect the quality of beef.

A Full-Bridge Submodule-Based Modular Unipolar/Bipolar High-Voltage Pulse Generator With Sequential Charging of Capacitors

Repetitive high pulsed electric field (PEF) is an effective method to kill microorganisms and bacteria in water treatment applications. The PEF can be generated by applying high-power electromagnetic pulse across the sample to be treated. There are two main types of high-voltage pulse generators, namely, unipolar and bipolar. In this paper, a full-bridge submodule-based modular high-voltage pulse generator, having the ability to generate unipolar and bipolar high-voltage pulses with different shapes from a relatively low-voltage input dc supply, is proposed. In the proposed configuration, relatively low-voltage insulated gate bipolar transistors (IGBTs) are required to generate the high-voltage bipolar pulses. A thyristor rated at the level of the pulsed output voltage is required in the proposed configuration to bypass the load during the charging process of capacitors. In the proposed approach, a thyristor is used instead of the self-commutated high-voltage switch (e.g., series-connected IGBTs), as thyristors are available with high-voltage ratings and possess inherent reverse voltage blocking capability. A detailed illustration of the proposed configuration and its operational concept are introduced in this paper. Simulation and experimental results are presented to validate the proposed approach.

Thermal characterization of Bacillus subtilis endospores and a comparative study of their resistance to high temperature pulsed electric fields (HTPEF) and thermal-only treatments

Heat characterization of Bacillus subtilis DSM 618 endospores at temperatures ranging from 90 to 120 °C was performed in water and reconstituted skimmed milk. DT values showed that B. subtilis endospores were more heat resistant in skimmed milk than in water and the calculated z-values indicated the same thermo-dependency regardless of the treatment medium used. Moreover, the effect of High-Temperature Pulsed Electric Fields (HTPEF) treatments on the inactivation of B. subtilis endospores in water and its comparison to thermal-only treatments by means of equal FT values at different temperatures was studied. HTPEF treatment consisted in bipolar square-wave pulses (10 μs, 38 kV/cm, 466 Hz) at maximum processing temperatures up to c. 123 °C. Inactivation values greater than c. 4.5 log10 cycles were obtained for both HTPEF and heat treatments with no significant differences between them. However, further studies at higher electric field strengths and temperatures could be performed to establish if greater inactivation levels can be achieved and to observe whether PEF has an additional effect over heat.

Electrotransfection of Polyamine Folded DNA Origami Structures.

DNA origami structures are artificial molecular nanostructures in which DNA double helices are forced into a closely packed configuration by a multitude of DNA strand crossovers. We show that three different types of origami structures (a flat sheet, a hollow tube, and a compact origami block) can be formed in magnesium-free buffer solutions containing low (<1 mM) concentrations of the condensing agent spermidine. Much like in DNA condensation, the amount of spermidine required for origami folding is proportional to the DNA concentration. At excessive amounts, the structures aggregate and precipitate. In contrast to origami structures formed in conventional buffers, the resulting structures are stable in the presence of high electric field pulses, such as those commonly used for electrotransfection experiments. We demonstrate that spermidine-stabilized structures are stable in cell lysate and can be delivered into mammalian cells via electroporation.

Effect of guard cells electroporation on drying kinetics and aroma compounds of Genovese basil ( Ocimum basilicum L.) leaves

Abstract In this study the effect of the application of pulsed electric fields as pre-treatment before air drying of basil leaves at 50 °C was investigated. The parameters of the electric treatment were designed in such a way that they either (i) electroporated the tissue reversibly (tissue remains viable) and without electroporating the guard cells in the stomata complex (65 pulses of 600 V/cm, 120 μs pulse width, 500 μs between pulses), (ii) electroporated the tissue reversibly causing the guard cells to irreversibly open the stomata (65 pulses of 600 V/cm, 150 μs pulse width, 760 μs between pulses), or (iii) irreversibly electroporated the tissue leading to cell death (65 pulses of 1500 V/cm, 150 μs pulse width, 760 μs between pulses). Results demonstrate that dehydration of basil leaves at 50 °C can be aided by the application of pulsed electric fields (PEF). If the applied PEF conditions are such that stomata are irreversibly opened while the rest of the tissue remains viable, the drying process is faster and the product keeps a better colour, is richer in aroma compounds and has better rehydration capacity than the untreated control. Irreversible tissue damage provoked by high intensity PEF drastically decreased the drying time but the measured quality characteristics of the dried leaves were inferior compared to those of the dried product when the stomatal function of the fresh leaf was selectively targeted. Industrial relevance This paper addresses the ever-increasing global demand from consumers for high-quality foods, reporting a novel methodology which improves the quality of dried aromatic herbs using pulsed electric fields.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

BackgroundActivated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma.AimsTo determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity.MethodsPRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin.ResultsPRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone.ConclusionsPEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Comparison of the application of high pressure and pulsed electric fields technologies on the selective inactivation of endogenous enzymes in tomato products

Application of novel technologies such as high pressure (HP) or pulsed electric fields (PEF) on the remaining activity of endogenous tomato pectinolytic enzymes such as Pectinmethylesterase (PME) and Polygalacturonase (PG), responsible for tomato products texture was studied. HP combined with temperature (200–800 MPa @ 55–75 °C), PEF (5.5–12.5 kV/cm, 0–12 ms treatment time) and thermally treated (55–75 °C) samples were studied. After thermal treatment, PG appeared to be more resistant than PME. Opposite behavior was observed for HP treated samples. For PME inactivation more intense P-T process conditions were necessary compared to PG. For PEF treatment, 98% inactivation was observed at 12.5 kV/cm and 6 ms for PME, and at 5.5 kV/cm and 11 ms for PG. PME appeared to be more HP and PEF resistant compared to PG. The results support the potential application of HP and PEF to selectively inactivate PG while partially retaining PME in tomato juices, aiming in improved tomato products' textural characteristics. The aim of the tomato industry is to produce tomato products of desired textural and sensorial characteristics while increasing the yield by decreasing the evaporated water. This can be achieved by applying novel technologies such as high pressure (HP) processing or pulsed electric fields (PEF) that affect the remaining activity of the endogenous pectinolytic enzymes such as Pectinmethylesterase (PME) and Polygalacturonase (PG), responsible for the final texture leading to products with improved quality characteristics such as viscosity, color and consistency. However, HP treatment is a batch process and makes it difficult for the treatment of large quantities (production of small quantities of superior products could be the target of the application of HP technology), while PEF technology could be applied in line with the typical production flow of that kind of products before the cold break step.

Toward a Philosophy and Theory of Volumetric Nonthermal Processing.

Nonthermal processes for food preservation have been under intensive investigation for about the past quarter century, with varying degrees of success. We focus this discussion on two volumetrically acting nonthermal processes, high pressure processing (HPP) and pulsed electric fields (PEF), with emphasis on scientific understanding of each, and the research questions that need to be addressed for each to be more successful in the future. We discuss the character or "philosophy" of food preservation, with a question about the nature of the kill step(s), and the sensing challenges that need to be addressed. For HPP, key questions and needs center around whether its nonthermal effectiveness can be increased by increased pressures or pulsing, the theoretical treatment of rates of reaction as influenced by pressure, the assumption of uniform pressure distribution, and the need for (and difficulties involved in) in-situ measurement. For PEF, the questions include the rationale for pulsing, difficulties involved in continuous flow treatment chambers, the difference between electroporation theory and experimental observations, and the difficulties involved in in-situ measurement and monitoring of electric field distribution.

Difference of Cell Death of Budding Yeast for Frequency Components of Pulsed Electric Field

Pulsed power technology has possibility to be able to provide a new method for tissue ablation. Applying a pulsed high electric field with nanosecond duration time onto a cancer cell can induce apoptosis into that. In this method, a frequency component of the electric field applied to the cell is an important factor. This paper focuses on the investigation of frequency characteristics of a difference of a cell death caused by applying the pulsed high electric field. The budding yeast that is a eukaryotic cell has been used as a model organism of a human cell. Difference of the cell death of the yeast for frequencies of the pulsed electric field by using molecular probes, propidium iodide, Annexin-V, and FLICA has been investigated. The frequency of the pulsed electric field was changed from 200 kHz to 70 MHz. The low frequencies, 200 kHz and 3.5 MHz, under 45 MHz yielded both necrosis and apoptosis in the cells. The electric field of frequency of 200 kHz with an intensity of 30 kV/cm only induced necrosis by breakdown in most cells. At the frequencies of 200 kHz with 16 kV/cm and 3.5 MHz, both apoptosis and necrosis occurred. The apoptotic pathway at 3.5 MHz went via caspase. At the frequency of 70 MHz, only apoptosis, not necrosis, was induced in the cells. However, the apoptosis via caspase was not dominant for the cell death. A signal for the cell death passed through other pathway of apoptosis, which was different from that of 3.5 MHz. At 45 MHz, the cell death did not occur. 45 MHz is the boundary where necrosis occurs, and the apoptotic pathway is different.

Developments in osmotic dehydration technique for the preservation of fruits and vegetables

Abstract In recent years much attention has been focused on maintaining the freshness of fruits and vegetables by immersion of cellular materials containing water in an osmotic solution. It results in the development of intermediate moisture products having lower water activity, which is imparted by solute gain and water loss. During the process, chemical, physical and biological activities, which deteriorate the foods, are lowered considerably; hence extends the shelf life of food products. In this process moisture is withdrawn from the product at ambient temperature by diffusion, so phase change has been avoided. Besides, it helps to improve the nutritional and sensory attributes of food products and is less energy intensive process as compared to other drying techniques. Osmotic dehydration is influenced by various factors such as osmotic agent, time and temperature, solute concentration, solution to sample ratio, agitation and geometry of the materials. Recently, osmotic dehydration has been combined with several other methods namely, pulsed high electric field, high hydrostatic pressure, ultrasound, centrifugal force, vacuum and gamma irradiation. These techniques have been employed during or after osmotic treatment to enhance osmotic dehydration performance by increasing the cell membrane permeability and mass transfer rate. These combined operations reduce the drying time, minimizing further energy costs. In this study, various segments of osmotic dehydration techniques and its application in food processing as well as recent advances in osmotic dehydration have been reviewed. Industrial relevance The osmotic dehydration technique is gaining popularity as a mean of obtaining minimally processed food. This review paper deals with the kinetics and mechanisms of osmotic dehydration technique for the preservation of fruits and vegetables. The various factors effecting osmotic mass transfer rate in food have been reviewed. In this paper, the combined effect of osmotic dehydration and several other innovative techniques (pulsed high electric field, high hydrostatic pressure, ultrasound, centrifugal force, vacuum and gamma irradiation) on the quality and shelf life of fruits and vegetables have been reviewed. These techniques have been employed during or after osmotic treatment to enhance osmotic dehydration performance by increasing the cell membrane permeability. These combined operations reduce the drying time, minimizing further energy costs as well as improving the quality of fruits and vegetables during storage.

高電界パルスによる大腸菌殺菌に及ぼす水溶液中のアミノ酸の影響

高電界パルスによる大腸菌殺菌に及ぼす水溶液中のアミノ酸の影響

Associations between nutritional composition and farming type of organically and conventionally grown cereals

technologies offer unprecedented opportunities and challenges for the food industry to market safe, high quality health-promoting foods. Those innovative food processing is often perceived as an alternative to thermal food processing, yet there are many nonthermal preparatory unit operations as well as food processing and preservation opportunities and challenges that require further investigation by the food industry. Current researches devote attention to improving food functionality with high hydrostatic pressure (HHP) and pulsed electric fields (PEF).&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; HHP and PEF processed foods meet consumer expectations for a minimally process, safety, fresh-like quality and convenience.

Introduction to Innovative Food Processing and Technology

Consumers, the food industry and the regulatory agencies demand the innovative technologies to provide safe and stable foods. Nonthermal processing technologies offer unprecedented opportunities and challenges for the food industry to market safe, high quality health-promoting foods. Those innovative food processing is often perceived as an alternative to thermal food processing, yet there are many nonthermal preparatory unit operations as well as food processing and preservation opportunities and challenges that require further investigation by the food industry. Current researches devote attention to improving food functionality with high hydrostatic pressure (HHP) and pulsed electric fields (PEF). HHP and PEF processed foods meet consumer expectations for a minimally process, safety, fresh-like quality and convenience.

Simultaneous Impact of High Pressures and Pulsed Electric field on Saccharomyces cerevisiae Model System

Results of simultaneous high pressure (HPP) and pulsed electric field (PEF) preservation treatments on Saccharomyces cerevisiae model suspension are presented. They are based on the novel implementation of PEF method, associated with the sequence of sine- wave pulses of the strong electric field. The simultaneous HPP + PEF enabled reaching the pasteurization threshold for pressure P = 200 MPa and the intensity of the electric field E < 10 kV/cm well below typical values for separate HPP and PEF treatments. Notable is the negligible parasitic heating for the new PEF implementation. The supplementary broad band dielectric spectroscopy (BDS) frequency scan for HPP, PEF and HPP + PEF revealed qualitative differences, particularly notable for the complex conductivity representation.

Potential of high isostatic pressure and pulsed electric fields to improve mass transport in pea tissue

The aim of this study was to investigate and evaluate the potential of high isostatic pressure (HP) and pulsed electric fields (PEF) for implementation in innovative legume processing concepts. Whole peas were subjected to HP (400MPa, 40°C, 10min) and PEF (5kVcm−1, 125kJkg−1) treatments. The impact on the pea tissue was assessed by means of microscopic changes, release of protein and oligosaccharides during treatment and rate of subsequent drying and rehydration steps. HP and PEF resulted in altered cell wall structures and improved mass transport in comparison to the control samples (20°C, 10min). Effects on the extent of diffusion depended on the molecular size of the cell compounds. More than 10% of raffinose equivalent sugars were removed during treatments while preserving more than 99.9% of nutritionally valuable protein. Hence, both emerging technologies may be implemented in pea processing for a targeted reduction of flatulence causing oligosaccharides. The drying and rehydration rates of whole peas were increased to a higher extent by HP and PEF than by conventional thermal treatments (80°C, 10min). These innovative pre-treatments may therefore be applied to enhance the efficiency of industrial mass transfer processes or to decrease the preparation time of meals with whole dried peas and thus increase the flexibility for the consumer.

In vivo electroporation of newborn mouse retina

In vivo electroporation is the process referred to a transient increase in the permeability of cell membranes upon high electric field pulses and delivery of engineered constructs into cells. Compared with the viral-mediated gene transfer system, the in vivo electroporation technique has several advantages, such as safe, high efficiency, rapid, stable and wide application. Thus, this technique has been widely used in the studies of many tissues or organs, including the eye. Here, we report the application of in vivo electroporation in the newborn mouse retina. DNA plasmid of GFP expression vector with high concentration was directly injected into the subretinal space of neonatal mouse pups. The DNA was then transfected into the retinal cells after several pulses of high voltage. Transfected GFP allowed clear visualization of cell morphologies in cryo-sections and the GFP was highly expressed in retinal outer nuclear layer. The results showed that this technique can effectively transfer the genes into retinal cells. In vivo electroporation will be a useful tool for the study of retinal development and function.

Use of surface enhanced blocking (SEB) electrodes for microbial cell lysis in flow-through devices.

By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells.

A new sterilization technique to Escherichia coli combined with high pulse electric field and thermal sterilization technology

High Pulse Electric Field (PEF) , which was highly developed in recent years, is a potential non-thermal sterilization technology. However, because of its complicated parameters, such as field strength, pulse width, frequency, time, pulse number and other parameters, the best sterilization parameters are still unknown. In this study a combination sterilization technique was designed, which took the advantages of both PEF sterilization technique and the traditional thermal sterilization technology, to achieve a simple, energy-saving, fast, safe and efficient sterilization technology. Through the experiment, heating Escherichia coli (E. coli) at 45℃ for 5min before the high PEF sterilization could decrease the survival rate to 13.69%. In conclusion, this new combination sterilization technique could not only save energy, but also apply to the food sterilization which could not be heated up to 45℃.

The Effect of High Voltage, High Frequency Pulsed Electric Field on Slain Ovine Cortical Bone

High power, high frequency pulsed electric fields known as pulsed power (PP) has been applied recently in biology and medicine. However, little attention has been paid to investigate the application of pulse power in musculoskeletal system and its possible effect on functional behavior and biomechanical properties of bone tissue. This paper presents the first research investigating whether or not PP can be applied safely on bone tissue as a stimuli and what will be the possible effect of these signals on the characteristics of cortical bone by comparing the mechanical properties of this type of bone pre and post expose to PP and in comparison with the control samples. A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses (up to 500 V and 10 kHz). The functional behavior of bone in response to pulse power excitation was elucidated by applying compressive loading until failure. The stiffness, failure stress (strength) and the total fracture energy (bone toughness) were determined as a measure of the main bone characteristics. Furthermore, an ultrasonic technique was applied to determine and comprise bone elasticity before and after pulse power stimulation. The elastic property of cortical bone samples appeared to remain unchanged following exposure to pulse power excitation for all three orthogonal directions obtained from ultrasonic technique and similarly from the compression test. Nevertheless, the compressive strength and toughness of bone samples were increased when they were exposed to 66 h of high power pulsed electromagnetic field compared to the control samples. As the toughness and the strength of the cortical bone tissue are directly associated with the quality and integrity of the collagen matrix whereas its stiffness is primarily related to bone mineral content these overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength and toughness augmentation, it apparently did not affect the mineral phase of the cortical bone material. The results also confirmed that the indirect application of high power pulsed electric field at 500 V and 10 kHz through capacitive coupling method was safe and did not destroy the bone tissue construction.