Audrius Grainys

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2–3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100–250 μs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

High-frequency submicrosecond electroporator

ABSTRACT In this work, we present a novel electroporator which is capable of generating single and bursts of high power (3 kV, 60 A) square wave pulses of variable duration (100 ns to 1 ms) with predefined repetition frequency (1 Hz to 3.5 MHz). The proposed synchronized crowbar implementation ensures a constant pulse rise and fall times, which are independent from the load, thus highly relevant in electroporation. The electroporator was successfully tested for the inactivation of the human pathogen Candida albicans. The device is compatible with standard commercial electroporation cuvettes.

Irreversible magnetoporation of micro-organisms in high pulsed magnetic fields.

Electroporation is an appealing way of stimulating living cells, which causes permanent or temporary nanoporosities in the structure of the biological objects. However, the technique has a disadvantage such as a requirement of contact between the electrodes and the cell medium. In this review, a methodology of contactless treatment of the biological objects using pulsed magnetic fields is proposed. The eukaryotic micro-organisms Achlya americana and Saprolegnia diclina have been used in the study and magnetic fields up to 7 T were applied, which caused effects similar to irreversible electroporation resulting in the death of the species. The proposed technique is applicable for different types of the biological cells or micro-organisms and possibly can be used in the area of cancer, antifungal treatment and other biotechnological fields.

Programmable Pulsed Magnetic Field System for Biological Applications

In this paper, a pulsed magnetic field system developed for biological applications is presented. The proposed pulsed power setup generates repetitive controlled frequency and width square wave 0-4 kV electrical pulses and supports currents up to 1000 A. As a load, a solenoid-type inductor is used with integrated plastic cuvette for biological cells. Homogeneous repetitive microsecond range pulsed magnetic field up to 15 T inside the effective volume of the cuvette can be generated. The development process, the structure of the pulsed magnetic field setup, and the solutions implemented to achieve biomedical applicability are overviewed in this paper. The presented experimental results showing contactless permeabilization of biological cells in pulsed magnetic field have an excellent potential for the development of a new biomedical treatment technique and applicability of the setup.

Microsecond pulsed magnetic field improves efficacy of antifungal agents on pathogenic microorganisms

Control and treatment of the emerging filamentous and yeast fungal diseases are of high priority in the biomedical field. This study investigated the influence of the pulsed magnetic field combined with common antifungal agents on the viability of various pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Trychophyton rubrum. Repetitive microsecond pulsed magnetic fields up to 6.1 T were applied in the study. The synergistic effect of co-applying drugs and magnetic treatment to different fungi species causing various human mycoses showed the potential for more effective and less toxic therapy.

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.

Compact Electro-Permeabilization System for Controlled Treatment of Biological Cells and Cell Medium Conductivity Change Measurement

Abstract Subjection of biological cells to high intensity pulsed electric field results in the permeabilization of the cell membrane. Measurement of the electrical conductivity change allows an analysis of the dynamics of the process, determination of the permeabilization thresholds, and ion efflux influence. In this work a compact electro-permeabilization system for controlled treatment of biological cells is presented. The system is capable of delivering 5 μs - 5 ms repetitive square wave electric field pulses with amplitude up to 1 kV. Evaluation of the cell medium conductivity change is implemented in the setup, allowing indirect measurement of the ion concentration changes occurring due to the cell membrane permeabilization. The simulation model using SPICE and the experimental data of the proposed system are presented in this work. Experimental data with biological cells is also overviewed

High power facilities for electroporation of biological cells in pulsed magnetic fields

High power pulsed generator for non-contact cell electroporation application in pulsed magnetic fields is presented. Finite element method analysis of the magnetic field and the analysis of the Jurkat T lymphoblasts electroporation experimental data are overviewed. The dependence of cell electroporation efficiency on the structure of the microcoil was acquired experimentally. Possible problems of the technique, further developments and result overview are discussed.

High-power bipolar multilevel pulsed electroporator

ABSTRACT A novel, compact, high-voltage, bipolar electroporator is reported that produces single or multiple, symmetrical or asymmetrical, high-power square wave pulses up to ±1 kV and 100 A. A wide 1 μs to 10 ms pulse duration is provided with high resolution of 1 V and 0.5 μs. The device provides increased flexibility due to enhanced control of the pulse shape and delivered energy.

Controlled inactivation of Trichophyton rubrum using shaped electrical pulse bursts: Parametric analysis.

The dermatophytes infect the skin by adherence to the epidermis followed by germination, growth, and penetration of the fungal hyphae within the cells. The aim of this study was to investigate the efficacy of the pulsed electric fields (PEF) of controlled inactivation of Trichophyton rubrum (ATCC 28188). In this work, we have used bursts of the square wave PEF pulses of different intensity (10–30 kV/cm) to induce the irreversible inactivation in vitro. The electric field pulses of 50 µs and 100 µs have been generated in bursts of 5, 10, and 20 pulses with repetition frequency of 1 Hz. The dynamics of the inactivation using different treatment parameters were studied and the inactivation map for the T. rubrum has been defined. Further, the combined effect of PEF with the antifungal agents itraconazole, terbinafine, and naftifine HCl was investigated. It has been demonstrated that the combined effect results in the full inactivation of T. rubrum colony.