[Show abstract][Hide abstract] ABSTRACT: Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
[Show abstract][Hide abstract] ABSTRACT: Nanosecond (ns) electric pulses of sufficient amplitude can provoke electroporation of intracellular organelles. This paper investigates whether such pulses could provide a method for controlled intracellular release of a content of small internalized artificial lipid vesicles (liposomes). To estimate the pulse parameters needed to selectively electroporate liposomes while keeping the plasma and nuclear membranes intact, we constructed a numerical model of a biological cell containing a nucleus and liposomes of different sizes (with radii from 50 nm to 500 nm), which were placed in various sites in the cytoplasm. Our results show that under physiological conditions selective electroporation is only possible for the largest liposomes and when using very short pulses (few ns). By increasing the liposome interior conductivity and/or decreasing the cytoplasmic conductivity, selective electroporation of even smaller liposomes could be achieved. The location of the liposomes inside the cell does not play a significant role, meaning that liposomes of similar size could all be electroporated simultaneously. Our results indicate the possibility of using ns pulse treatment for liposomal drug release.
[Show abstract][Hide abstract] ABSTRACT: Electrical breakdown of cell membranes (electroporation) can be either reversible of irreversible, each
having many applications in medicine and biotechnology. Here, we describe electroporation on the molecular and cellular level.
[Show abstract][Hide abstract] ABSTRACT: Simulations probing the conductivity changes of three-dimensional models of biological tissues consisting of random ternary core-shell sphere packings with different spatial scales are described. We investigate the temporal evolution of the electric conductivity of these packings during application of an electric field with magnitude either below or above the value leading to cell membrane electroporation. The fraction of electroporated cells can be described by a hyperbolic tangent function of the electric field. The collective physical processes causing the transient permeability of the cell membranes can be understood by analogy with the physics of a two-state system with an external field.
[Show abstract][Hide abstract] ABSTRACT: Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca(2+). With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.
[Show abstract][Hide abstract] ABSTRACT: Gene electrotransfer is an established method for transfer of genes into cells, however, the mechanism of transfer of DNA across the cell membrane is still not known. Some studies suggest that DNA is translocated through membrane pores while others propose that DNA enters the cell via electro-endocytosis, but no direct observation was performed. In this paper we investigated the second hypothesis. Cells were stained with membrane dye FM 1-43FX, which is used for observation of endocytosis, and then exposed to electric pulses. We analyzed if endocytosis was stimulated by applying electric pulses with intensities below and above the threshold value for gene electrotransfer. No increase in endocytosis from 20 min or even up to 2h after the pulse delivery was observed, regardless of the electric field strength. These observations do not correlate with electrotransfer efficiency, which increases with field strength and is observed only above the threshold value. Our results suggest that electro-endocytosis is not a crucial mechanism for gene electrotransfer and that the hypothesis of DNA entry by translocation through permeabilized membrane is more plausible. The presented results are important for better understanding of the mechanisms of gene electrotransfer and for its optimization for clinical applications.
[Show abstract][Hide abstract] ABSTRACT: An exposure of a cell to an external electric field results in the induced transmembrane voltage (ΔΨm) that superimposes to the resting voltage. This can have a range of effects, from modification of the activity of voltage-gated channels to membrane electroporation, and accurate knowledge of spatial distribution and time course of ΔΨm is important for the understanding of these effects. In this chapter, we present the analytical, numerical, and experimental methods of determination of ΔΨm, and combine them with the monitoring of electroporation-induced transmembrane molecular transport (TMT) in Chinese Hamster
Ovary (CHO) cells. Potentiometric measurements are performed using di-8-ANEPPS, and TMT is monitored using propidium iodide. In isolated cells, we combine analytical derivation (for spherical cells) and numerical computation of ΔΨm (for irregularly shaped cells) with potentiometric measurements to show that the latter are accurate and reliable. Monitoring of TMT in these same cells shows that it is confined to the regions with the highest |ΔΨm|. We then review other parameters influencing electroporation of isolated cells, and proceed, through the intermediate case of dense suspensions, to cells in direct contact with each other. We use the scrape-loading test to show that the CHO cells
in a monolayer are interconnected, and then study ΔΨm and TMT in a cluster of four such cells. With low pulse amplitudes, the cluster behaves as one big cell, with ΔΨm continuous along its outer boundary, reflecting the interconnections. With interconnections inhibited, the cells start to behave as individual entities, with ΔΨm continuous along the plasma membrane of each cell. With the cluster exposed to porating (higher amplitude) pulses, TMT occurs in the membrane regions for which computations predict the highest |ΔΨm| if the cells are modeled as insulated, suggesting that the interconnections are blocked by supraphysiological ΔΨm, either directly by voltage gating or indirectly through changes in ionic concentrations caused by electroporation.
Clinical Aspects of Electroporation, Edited by S.T. Kee, J. Gehl, E.W. Lee, 01/2011: chapter 3: pages 19-29; Springer., ISBN: 978-1-4419-8362-6
[Show abstract][Hide abstract] ABSTRACT: Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, DWm) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of DWm and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between DWm and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of DWm are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.
[Show abstract][Hide abstract] ABSTRACT: Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, DeltaPsi (m)) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of DeltaPsi (m) and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between DeltaPsi (m) and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of DeltaPsi (m) are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.
[Show abstract][Hide abstract] ABSTRACT: In this paper we examine the influence of gap junction inhibition on the amplitude and distribution of the induced transmembrane
voltage (ITV) and electroporation of cells in clusters. Cell clusters were used as they represent simple models of cells in
tissues and thus enable the study of the effects of the external electric field on the level of individual cells. We demonstrated
that cells in clusters respond differently to the electric field exposure, depending on the field parameters. Namely, when
exposed to long, low voltage pulses (such as during the measurements of the ITV) cells in clusters behave as one giant, single
cell. At short, high voltage pulses (such as during electroporation) they behave as individual cells. Different response of
cells in clusters was attributed to the changes in the properties of gap junctions, specifically, their opening and closing.
This was demonstrated by pre-treating the cells with gap junction inhibitor, which caused the cells in clusters to respond
as individual cells, regardless of the pulse parameters.
KeywordsGap junctions–Transmembrane Potential–Electropermeabilization–Lucifer Yellow–di-8-Anepps
[Show abstract][Hide abstract] ABSTRACT: 3.1 The Cell and the Induced Transmembrane Voltage
3.2 Analytical Derivation
Laplace’s Equation • Spherical Cells • Spheroidal, Ellipsoidal,
and Cylindrical Cells • High Frequencies and Very Short Pulses
3.3 Numerical Computation
Computational Methods • Irregularly Shaped Cells •
Cells in Dense Suspensions and Tissues
3.4 Experimental Determination
Potentiometric Dyes • Image Acquisition and Data Processing
Advanced Electroporation Techniques in Biology and Medicine, Edited by A.G. Pakhomov, D. Miklavčič, M.S. Markov, 01/2010: chapter 3: pages 51-70; CRC Press., ISBN: 978-1-4398-1906-7
[Show abstract][Hide abstract] ABSTRACT: Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by DeltaPhi, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of DeltaPhi on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of DeltaPhi. This video shows the protocol for measuring DeltaPhi using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of DeltaPhi.
Journal of Visualized Experiments 11/2009; DOI:10.3791/1659 · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe a finite-element model of a realistic irregularly shaped biological cell in an external electric field that allows the calculation of time-dependent changes of the induced transmembrane voltage (ΔΨ) and simulation of cell membrane electroporation. The model was first tested by comparing its results to the time-dependent analytical solution for ΔΨ on a nonporated spherical cell, and a good agreement was obtained. To simulate electroporation, the model was extended by introducing a variable membrane conductivity. In the regions exposed to a sufficiently high ΔΨ, the membrane conductivity rapidly increased with time, leading to a modified spatial distribution of ΔΨ. We show that steady-state models are insufficient for accurate description of ΔΨ, as well as determination of electroporated regions of the membrane, and time-dependent models should be used instead. Our modeling approach also allows direct comparison of calculations and experiments. As an example, we show that calculated regions of electroporation correspond to the regions of molecular transport observed experimentally on the same cell from which the model was constructed. Both the time-dependent model of ΔΨ and the model of electroporation can be exploited further to study the behavior of more complicated cell systems, including those with cell-to-cell interactions.
[Show abstract][Hide abstract] ABSTRACT: For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.
[Show abstract][Hide abstract] ABSTRACT: The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 µs to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 µs, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of milliseconds, hundreds of milliseconds, and tens of seconds, respectively.
[Show abstract][Hide abstract] ABSTRACT: Electrochemotherapy is a local drug delivery approach aimed at treatment with palliative intent of cutaneous and subcutaneous tumour nodules of different histologies. Electrochemotherapy, via cell membrane permeabilising electric pulses, potentiates the cytotoxicity of non-permeant or poorly permeant anticancer drugs with high intrinsic cytotoxicity, such as bleomycin or cisplatin, at the site of electric pulse application.
An overview of preclinical and clinical studies is presented, and the treatment procedure is further critically evaluated.
In clinical studies electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumour nodules. The treatment response for various tumours (predominantly melanoma) was approximately 75% complete and 10% partial response of the treated nodules.
Electrochemotherapy is a new, clinically acknowledged method for the treatment of cutaneous and subcutaneous tumours. Its advantages are high effectiveness on tumours with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment.
European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 03/2008; 34(2):232-40. DOI:10.1016/j.ejso.2007.05.016 · 2.89 Impact Factor