Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells

O’Hare, Michael J.; Ormerod, M.G.; Imrie, P.R.; Peacock, J.H.; Asche, W.

doi:10.1007/978-1-4899-2528-2_20

Cited by 22 publications

(14 citation statements)

References 21 publications

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“…As we and others have shown that different intrinsic sensitivities to electroporation exist between different cell types in vitro, this might also be the case in solid tumors in vivo. 37,38 It has already been shown that electroporation, in addition to the direct effect on cell membranes, also affects the cytoskeleton and chromosomal DNA, and therefore might trigger different cellular processes leading to apoptosis or necrosis. 39,40 Another factor, which should be taken into account for gene delivery in vivo, is the amount of hypoxia (low oxygen ) present in the solid tumors.…”

Section: Discussionmentioning

confidence: 99%

Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes, and integrin-targeted vector

et al. 2002

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In this study, we measured transfection efficiency in vitro and in vivo using the following nonviral approaches of gene delivery: injection of plasmid DNA, electroporation -assisted, liposome -enhanced, and integrin -targeted gene delivery, as well as the combination of these methods. Four histologically different tumor models were transfected with a plasmid encoding the green fluorescent protein ( GFP ) ( B16 mouse melanoma, P22 rat carcinosarcoma, SaF mouse sarcoma, and T24 human bladder carcinoma ) using adherent cells, dense cell suspensions, and solid tumors. Emphasis was placed on different electroporation conditions to optimise the duration and amplitude of the electric pulses, as well as on different DNA concentrations for effective gene delivery. In addition, transfection efficiency was correlated with cell density of the tumors. The major in vivo findings were: ( a ) electroporationassisted gene delivery with plasmid DNA, employing long electric pulses with low amplitude, yielded significantly better GFP expression than short electric pulses with high amplitude; ( b ) electroporation combined with liposome -DNA complexes yielded the highest percentage of transfected tumor area in B16F1 tumor ( 6% ); ( c ) transfection efficiency of electroporation -assisted plasmid DNA delivery was dependent on tumor type; ( d ) integrin -targeted vector, alone or combined with electroporation, was largely ineffective. In conclusion, our results demonstrate that some nonviral methods of gene delivery are feasible and efficient in transfecting solid tumors. Therefore, this makes nonviral methods attractive for further development.

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Section: Discussionmentioning

confidence: 99%

Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes, and integrin-targeted vector

et al. 2002

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“…While part of the differences can be attributed to the differences in cell size and shape, already mentioned before, some differences are related to biological characteristics of the treated cell (O'Hare et al 1989;Rols and Teissié 1992a;Rouan et al 1991;Čegovnik and Novaković 2004;Kandušer et al 2006). Among such biological factors that affect cell membrane electroporation are membrane fluidity, cell cytoskeleton, and cell wall in bacteria, yeast, and plant cells.…”

Section: Understanding Electroporation Of Different Cell Typesmentioning

confidence: 96%

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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“…Our technique differs in principle from the usual use of fluorescein diacetates (administered after stressing the membrane to see whether large molecules-and specifically esterases-have leaked out of the cell) (24). We used CF diacetate to load the cells before electromanipulation as a means of measuring the pulse-induced loss of CF.…”

Section: Fluorescent Staining Of Cellsmentioning

confidence: 99%

“…Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible uptake, ghosts with high uptake of dextran, and intact nonpermeabilized cells (9).…”

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Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmolkell) before electropulsation (0.5-3.0 kV/cm, 40 ps) in medium containing 25-50 pg/ml PI at 21-23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form >95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (73 of about 2 min. In 150 mOsm medium, resealing was faster (typically T~ -0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius de-10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C,), was taken into account. Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism. Other permeabilization methods (e.g., the use of bacterial toxins, detergents, hypertonic treatment) can markedly affect cell viability and cause undesirable leakage of the cytoplasm or extensive cell lysis ( 2 5 3 0 ) .Plasma membrane electropermeabilization within statistically representative cell samples can be assessed by measuring membrane crossing of tracer molecules in a flow cytometer. Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible upta...

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Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells

Cited by 22 publications

References 21 publications

Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes, and integrin-targeted vector

Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes, and integrin-targeted vector

Electroporation in Biological Cell and Tissue: An Overview

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

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