The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy

Pucihar, Gorazd; Mir, Lluis M.; Miklavčič, Damijan

doi:10.1016/s1567-5394(02)00116-0

Cited by 113 publications

(77 citation statements)

References 17 publications

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“…One can take advantage of this slow resealing to deliver the successive pulses at a 1kHz frequency to reduce the duration of the treatment in ECT. 32 These observations are in agreement with a model where the target of the field is under the control of the pulse duration. Due to their internal flexibility (mainly at the level of the polar heads), phospholipids are sensitive to short pulses.…”

Section: Sieving Of Electropermeabilizationsupporting

confidence: 85%

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

Teissié¹,

Escoffre²,

Rols³

et al. 2008

Radiology and Oncology

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Background. Electropulsation is one of the non-viral methods successfully used to transfer drugs and genes into living cells in vitro as in vivo. This approach shows promise in field of gene and cellular therapies. This presentation first describes the temporal factors controlling electropermeabilization to small molecules (< 4kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings our attention on the processes occurring before (s), during (ms) and after electropulsation (ms to hours) ofhas the main advantages of being easy to use, fast, reproducible and safe.While during 30 years due to technological limits, pulse duration was always larger than 1 microsecond, the recent availability of high voltage (tens of kV) nanosecond long pulse generators opens the way to a new approach. Very fast perturbations under strong fields are induced in the membrane organization. 16,17 A new field of development is now present for electropermeabilization and promising results for clinical applications were reported.One of the limiting problems remains that very few is known on the physicochemical mechanisms supporting the reorganisation of the cell membrane. The molecular target of the field effect remains unclear.The present review focuses on the critical role played by the pulse duration in the electropermeabilization to small molecules (< 4kDa) and on its support to the processes associated to DNA transfer in vitro. Pulse durations are easy to adjust for an optimization of the clinical target: electrochemotherapy, irreversible electropermeabilization or gene therapy as suggested as a final conclusion. Electropermeabilization Theory of membrane potential difference modulation.An external electric field modulates the membrane potential difference. 18 From the physical point of view, a cell can be described as a spherical capacitor which is charged by the external electrical field. The transmembrane potential difference induced by the electric field, ΔΨi is a complex function g(λ) of the specific conductivities of the membrane (λ m ), the pulsing buffer (λ out ) and the cytoplasm (λ cyt ), the membrane thickness and the cell size. Thus, 1 ΔΨi = f. g (λ). r. E.cosθ [1] in which θ designates the angle between the direction of the normal to the membrane at the considered point on the cell surface and the field direction, E the field intensity, r the radius of the cell and f, which is a shape factor (a cell being a spheroid). Therefore, ΔΨi is not uniform on the cell surface. It is maximum at the positions of the cell facing the electrodes. These physical predictions were checked experimentally by videomicroscopy by using the potential difference sensitive fluorescent probes. [19][20][21] The pulse duration plays a critical role when shorter than the capacitive loading time of the membrane. In the previous part of the paper, it was considered that the pulse was long enough to bring the potential steady state value. The loading time τ load brings a limit in this description. 1 ΔΨi = f. ...

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Section: Sieving Of Electropermeabilizationsupporting

confidence: 85%

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

Teissié¹,

Escoffre²,

Rols³

et al. 2008

Radiology and Oncology

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“…A 50-ml droplet of MSC suspension was electrotransfected with 8 mg of plasmid DNA using established techniques. 32 After electroporation, the cells were collected, suspended in 50 ml electropulsation buffer (to prevent drying) and allowed to recover at 22 1C (room temperature) for 10 min. Subsequently, culture medium was added and cells were incubated for 48 h under standard cell culture conditions (37 1C, 95% CO 2 , 5% O 2 ).…”

Section: Optimization Of the Rat Msc Electroporation Parametersmentioning

confidence: 99%

“…31 To achieve efficient DNA electrotransfer the critical electric pulse parameters are frequency, intensity, duration and number. 32 Technical solutions for studies aiming at the optimization of these parameters were addressed in another study. 33 Later, a pulse generator was developed (Cliniporator, IGEA, Carpi, Italy) that allows independent assessment of the permeabilization and electrophoresis achieved by means of combinations of pulses and by adjusting the critical parameters of these pulses.…”

Section: Introductionmentioning

confidence: 99%

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

et al. 2008

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Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm À1 with pre-incubation in Spinner's minimum essential medium buffer at 22 1C. Under these conditions b-galactosidase expression was achieved in 29 ± 3% of adherent cells 48 h post transfection. The kinetics of b-galactosidase activity revealed maintenance of b-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

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“…When pulses are applied with high repetition frequency, above 1 kHz, the pause between two consecutive pulses is too short and does not allow cell membrane to return to pre-pulse state. From the experimental results it can be concluded that cell viability and cell membrane electroporation is optimal in the frequency range from 0.5 to 10 Hz and decreases at higher frequencies (Vernhes et al 1999;Pucihar et al 2002;Pavlin et al 2005). For reversibility of electroporation, the membrane potential has to be kept below the critical value.…”

Section: Introduction Of Small Moleculesmentioning

confidence: 99%

Electroporation in Biological Cell and Tissue: An Overview

Kandušer

Miklavčič²

2008

Electrotechnologies for Extraction From Food Plants and Biomaterials

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In this chapter, basics and mechanisms of electroporation are presented. Most important electric pulse parameters for electroporation efficiency for different applications that involve introduction of small molecules and macromolecules into the cell or cell membrane electrofusion are described. In all these applications, cell viability has to be preserved. However, in some biotechnological applications, such as liquid food sterilization or water treatment, electroporation is used as a method for efficient cell killing. For all the applications mentioned above, besides electric pulse parameters, other factors, such as electroporation medium composition and osmotic pressure, play significant roles in electroporation effectiveness. For controlled use of the method in all applications, the basic mechanisms of electroporation need to be known. The phenomenon was studied from the single-cell level and dense cell suspension that represents a simplified homogenous tissue model, to complex biological tissues. In the latter, different cell types and electric conductivity that change during the course of electric pulse application can significantly affect the effectiveness of the treatment. For such a complex situation, the design and use of suitable electrodes and theoretical modeling of electric field distribution within the tissue are essential. Electroporation as a universal method applicable to different cell types is used for different purposes. In medicine it is used for electrochemotherapy and genetherapy. In biotechnology it is used for water and liquid food sterilization and for transfection of bacteria, yeast, plant protoplast, and intact plant tissue. Understanding the phenomenon of electroporation, its mechanisms and optimization of all the parameters that affect electroporation is a prerequisite for successful treatment. In addition to the parameters mentioned above, different biological characteristics of treated cell affect the outcome of the treatment. Electroporation, gene electrotransfer and electrofusion are affected by cell membrane fluidity, cytoskeleton, and the presence of the cell wall in bacteria yeast and plant cells. Thus, electroporation parameters need to be specifically optimized for different cell types.

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The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy

Cited by 113 publications

References 17 publications

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Electroporation in Biological Cell and Tissue: An Overview

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