The number of molecules taken up by electroporated cells: quantitative determination

Fluorescent and fluorescent-labeled molecules were used with calibrated flow cytometric fluorescence measurements of electrically pulsed cells (intact yeast: Saccharomyces cerevisiae) to demonstrate a method for determining the net number of molecules transported into electroporated cells. For the conditions used, a single pulse of width 50 microseconds and magnitude 8.0 +/- 0.5 kV/cm resulted in an average net molecular uptake which is large, n = 1.4 x 10(5) molecules of 70 kDa FITC-dextran (supplied extracellular concentration of 500 microM), and n = 1.0 x 10(8) molecules of 660 Da propidium iodide (PI; 80 microM). Both molecules were present in pulsed cells at less than equilibrium values, consistent with a transient uptake mechanism. Intracellular FITC-dextran is present in soluble form, while PI is predominantly bound to nucleic acids. A broad, statistically significant distribution of molecular uptake was also observed. Such quantitative determinations should be important for guiding applications of electroporation, and for testing models of electroporation mechanisms. Further, the use of PI, which is well established as a membrane exclusion dye, provides additional support for the interpretation that both PI and FITC-dextran were internalized as a result of an electrical pulse.     

The loading of human erythrocytes with small molecules by electroporation

The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight below 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius below 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4 degrees C, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37 degrees C, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments.

Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells.     

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.     

Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells

Several non-viral techniques involving the use of liposomes, particle bombardment and electroporation have been used for efficient transfection of plasmids and other molecules into cells. Current approaches target whole or bulk regions of tissue, lacking the desired spatial control over the transfection process. In this study, we present a novel approach using microsystems to achieve spatial and temporal control over the transfection process in adherent cells. A 6x6 MEA (microelectrode array) with 100 microm microelectrode dimension was developed on a silicon substrate using standard microfabrication procedures and passivated with a biocompatible layer. Using finite element models, electric field intensities were simulated and locations of optimal electroporation zones in the cell culture on the microelectrode surface were predicted. The MEA was subsequently tested using 3T3 fibroblasts cultured on the MEA surface for 96 h and stimulation voltages in the range of 2-5 V in the presence of propidium iodide (PI), a cell impermeant dye. Maximum electric field intensities in the z-direction were estimated to be in the range of 320-820 V/cm for applied differential voltages in the range of 2-5 V. Cells directly on the top and on the edges of the stimulating microelectrodes in the MEA were preferentially transfected with PI as predicted by the simulations. The results of these experiments demonstrate that spatial and temporal control of desired regions of transfection in vitro can be achieved using MEAs and electroporation.     

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.     

Self-electroporation as a model for fusion pore formation

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237-1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than approximately 3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells

Radiolabeled or biologically functional DNA molecules were introduced into cells by electroporation in a variety of forms: double stranded circles, linearized double stranded fragments and single stranded circular molecules. Molecules rapidly entered cells after exposure to a high field-strength electric pulse and then redistributed between the cytoplasm and the nucleus. Maximal intranuclear levels approximated 10(4) molecules per cell. Introduced DNA persisted in a biologically active form with a half-life of 15-24 h. There was no evidence for biologically significant alteration of two double stranded gene sequences. Single stranded DNA molecules also retained biological activity.     

Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

Previous studies have shown greater fluorophore uptake during electroporation on the anode-facing side of the cell than on the cathode-facing side. Based on these observations, we hypothesized that hyperpolarizing a cell before electroporation would decrease the requisite pulsed electric field intensity for electroporation outcomes, thereby yielding a higher probability of reversible electroporation at lower electric field strengths and a higher probability of irreversible electroporation (IRE) at higher electric field strengths. In this study, we tested this hypothesis by hyperpolarizing HL-60 cells using ionomycin before electroporation. These cells were then electroporated in a solution containing propidium iodide, a membrane integrity indicator. After 20 min, we added trypan blue to identify IRE cells. Our results showed that hyperpolarizing cells before electroporation alters the pulsed electric field intensity thresholds for reversible electroporation and IRE, allowing for greater control and selectivity of electroporation outcomes.     

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.     

Modeling the electromobility of ions in a target tissue

Electroporation is a clinical and laboratory technique for the delivery of molecules to cells. This method imposes electric fields onto cells or tissues through the use of electrodes and a set of electrical parameters to ultimately incorporate molecules into the cells. Clinical applications may include using directional fields to bring therapeutics to the target tissues before triggering an electroporation event. The choice of applicator may also have a significant influence on this molecular flow. Modeling ionic flow in tissues will yield insight into selecting the appropriate parameters or electroporation signature for a desired target application. In this paper, the motion of tissue injected ions was modeled for two common electroporation applicator configurations-the parallel plate, and the four needle electrodes. This electric field induced fluid flow model predicts that the parallel plate applicator ultimately directs the movement of an ionic therapeutic in a forward manner with side motion due only to obstruction, while the four-needle applicator directs anisotropic flow within the field ultimately forcing the therapeutic into a mound at the fringes of the induced electric field.     

Quantitative study of electroporation-mediated molecular uptake and cell viability

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.     

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.     

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm⁻¹, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G₁ cells is less affected by electroporation than that of cells in late S and G₂/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.     

Three dimensional electrode array for cell lysis via electroporation

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.     

The cell-cycle promoter cdc2aAt from Arabidopsis thaliana is induced in the lateral roots of the actinorhizal tree Allocasuarina verticillata during the early stages of the symbiotic interaction with Frankia

The symbiosis between the actinorhizal tree Allocasuarina verticillata and the actinomycete Frankia leads to the formation of root nodules inside which bacteria fix atmospheric nitrogen. Actinorhizal nodule organogenesis starts with the induction of cell divisions in the root cortex and in the pericycle cells opposite protoxylem poles near Frankia -infected root hairs. To study the ability of Frankia to induce progression through the cell cycle, we monitored the expression of the β-glucuronidase ( gus ) gene driven by the promoter from cdc2aAt , an Arabidopsis cyclin-dependent kinase gene that displays competence for cell division, during plant growth and nodule ontogenesis. In non-symbiotic tissues, the gus gene was mainly expressed in primary and secondary meristems of roots and shoots. Auxins and cytokinins were found to induce reporter gene activity in the root system of whole plants, showing that the promoter cdc2aAt displayed the same regulation by hormones in Allocasuarina as that reported in Arabidopsis . In transgenic nodules, gus expression was found to be restricted to the phellogen. During the early stages of the interaction between Frankia and the plant root system, cdc2aAt was strongly induced in the lateral roots surrounded by hyphae of the actinomycete. Histochemical analysis of β-glucuronidase activity revealed that cells from the pericycle opposite protoxylem poles were very deeply stained. These data indicate that upon Frankia infection, cells from the lateral roots, and notably pericycle cells that can give rise to a nodule or a root primordium, prepare to re-enter the cell cycle.     

Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.     

Membrane electroporation--fast molecular exchange by electroosmosis.

Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.