Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Definitions

• This invention relates to a method and apparatus for transfecting cells with exogenous materials by electroporation.
• Electroporation is a technique in which cells are electrically stimulated to take up materials such as DNA and drugs from their surrounding medium. This is achieved by placing the medium containing the material and cells between two electrodes and subjecting the cells to an electrical impulse (Chang, D.C., Guide to Electroporation and Electrofusion, Academic Press, San Diego, CA, 1992).
• a suggested mechanism for the electroporation phenomena involves the formation of transient holes or pores in the cell membrane through which the surrounding medium containing the material may enter the cell (Neuman e al., (1982) E BO J., VoLl, 841-845, Wong etal(1982) Biochem and Biophys. Research Commun. 107, 584 - 587).
• cells are electroporated in suspension by placing cells between two uniformly spaced parallel electrodes.
• electroporation devices for the electroporation of cells in suspension are commercially available cuvettes that are fitted with parallel electrodes and electrode assemblies such as the one disclosed in the US patent 5,422,272 (Papp etal, 1995).
• attachment-dependent cells are normally grown on the inner surface of a non-conductive container such as a petri dish or a tissue culture bottle.
• In situ electroporation of such cells can be carried out by, for example, positioning an electrode assembly close to the plated cells consisting of a pair of electrodes which serve as the "ground” ( - ) and "high voltage” (+) electrodes.
• the electrodes can be in the form of parallel plates or concentric rings. In addition to parallel plates and concentric rings, other configurations such as multiple plates and multiple concentric rings can be used. Electrode assemblies with these characteristics have been disclosed by Chang (US patent 4,822,470, 1989). Other types of electrode assembly comprising electrode array patterns that may be formed by depositing gold or platinum on a non-conductive support have also been disclosed by Meyer (PCT application, WO 98 / 12310, March 1998). This method allows the fabrication of much smaller electrodes on the bottom of an electrode carrier for in situ electroporation of cells with smaller electrode separations in the range of 50 -100 ⁇ m.
• Electroporation has been used in vivo to introduce foreign material such as DNA and drugs into living cells in body.
• electroporation has been used in vivo in a process termed electrochemotherapy for introducing anti-cancer agents into tumour cells ( Mir etal. , European Journal of Cancer (1991), Nol.27 , 68-72).
• Electrochemotherapy is carried out by infusing an anticancer drug directly into the tumour and applying an electric field to the tumour between a pair of electrodes.
• Electrodes are provided in various configurations such as, for example, a calliper that grips the epidermis overlying a region of cells to be treated or needle-shaped electrodes that may be inserted into the patient, to access more deeply located cells. Plate-type electrodes aligned in parallel have been proposed to provide a uniform electric field for electrochemotherapy delivered transcutaneously ( U.S. Pat.No. 5,468,223 to Mir) and U.S.Pat. No.
• Electroporation is particularly promising for chemotherapy because some of the most effective anti-cancer drugs, such as Bleomycin, cannot successfully penetrate the membranes of certain cancer cells under normal conditions. With the application of an electric field near tumours, however, it is possible to insert the Bleomycin effectively into the cells. Electroporation in this application is especially beneficial because it can help minimize the amount of implant agent used and thus provides a means for preventing the harmful effects associated with administration of anticancer or cytotoxic agents.
• a high electric field is applied between electrodes to induce membrane breakdown in the targeted cells.
• the electric field is normally applied in a pulsed form to prevent irreversible cell damage.
• Waveforms which are often used, include rectangular pulse ("square pulse") normally generated by a function generator, and the exponential decay pulse which is generated by discharging a capacitor that has been precharged at a high voltage.
• the pulse width is characterized by the decay constant ⁇ , which depends on the capacitor selected and the ionic conductivity of the electroporation medium.
• Successful electroporation is critically dependent on both biological variables and the characteristics of the applied pulse.
• the major electrical parameters affecting the success or efficiency of electroporation include the electric field strength, pulse wave shape, duration of the pulse (pulse length), and number of pulses applied.
• the most critical electrical factor for efficient electroporation is known to be the peak electric field strength generated by the electrical voltage and it is generally accepted that a threshold field intensity is required to observe the occurrence of permeabilization. It has been observed that when an electrical pulse of very short duration is used, the field strength required for the effective poration is usually so high that the cell viability becomes unacceptably low. On the other hand, when the pulse length is large, the required field strength for successful electroporation of cells is low (Chu et.
• the conductivity of the electroporation medium also plays an important role in the successful electroporation of cells.
• alteration in the ionic strength of the electroporation medium can significantly affect the characteristics of the electric pulse delivered and may also change the optimal voltage required for efficient electroporation of cells. It is therefore important to monitor the shape, duration and voltage of each impulse by a storage oscilloscope to ensure that the characteristics of the delivered pulse remain unchanged from experiment to experiment.
• a solid substratum e.g. a glass plate or a micro- porous membrane
• attachment-dependent cells are normally grown on the inner surface of a non-conductive container such as a petri dish or a tissue culture bottle.
• In situ electroporation of such cells can be carried out by, for example, positioning an electrode assembly very close to the plated cells consisting of a pair of electrodes which serve as the "ground” ( - ) and "high voltage” (+) electrodes.
• the electrodes can be in the form of parallel plates or concentric rings.
• the distance between electrodes and the cell monolayer is normally about 1 mm or less and in some cases electrodes are placed directly on the cell monolayer( Liang etal., BioTechniques, Vol.6, No.6, (1988), 550-558). In such cases, the gas bubbles generated can severely affect the viability of cells in close contact with the electrodes. In particular, in electroporation devices where cells are cultured on the electrode surface, such as those disclosed by Baer (US patent 5,128,257, 1992) and Casnig (US patent 5,134,070 , 1992), gas bubbles can seriously affect the attachment of the cells to the electrodes, and this may in turn adversely affect normal cellular functions and cell viability.
• Gas bubbles therefore, form at the nucleation sites on the electrodes in a random fashion and grow in size. Gas bubbles may interact and finally touch each other and may cover the whole or part of the electrode surface, forming an unstable gas film ("bubble curtain") which rapidly collapses and reforms. Gas bubbles attached to the electrodes' surfaces reduce the active surface area of the electrodes and result in a non-uniform distribution of current and the establishment of a non-uniform electric field during the course of the electroporation process. The coverage of the electrodes' surfaces with gas bubbles can be especially serious in cases where the dimensions of the electrodes and the spacing between them are comparable with the size of gas bubbles (i.e., when the dimensions of electrodes are in the range of 20 - 100 ⁇ m).
• Examples of small electrodes of this type are electrodes disclosed by Baer (US patent 5,128,257 , 1992) .
• Gas evolution can be particularly critical in electroporation devices such as those described by Klenchin etal ( Biophysical Journal, (1991), Vol.60, 804 - 811) and Yang etal.( Nucleic Acids Research (1995), Vol.23, No.15, 2803 - 2810) where cells are cultured on a microporous membrane and placed between two flat electrodes during the electroporation process. In such devices gas bubbles may get entrapped between the microporous membrane and the electrodes and this may result in substantial changes in the interfacial electrical resistance during the electroporation process.
• This can be specially critical in electroporation devices where the microporous membrane is in close contact with one or both electrodes as in the electroporation device described by Klenchin etal (Biophysical Journal, (1991), Vol.60, 804 - 811).
• the problem associated with gas bubbles can also be critical in devices which are designed for the in situ electroporation of cells and which are equipped with horizontal electrodes facing downward.
• An example of electroporation devices of this kind is the device described by Meyer (PCT application, WO 98 / 12310, March 1998) that is equipped with an interlinked set of electrodes in the range of 120 to 340 ⁇ m formed on the bottom of an electrode carrier.
• Electrical pulses of long duration are applied to such electrodes, gas bubbles may accumulate under the electrodes as a result of the buoyancy forces acting on bubbles and thus, electrodes may become partially isolated from the electroporation medium.
• gas bubbles may depart from the electrodes when they have reached a sufficient size and may remain dispersed in the electroporation medium in the interelectrode spaces and this may provide an additional local and non-steady state condition within the electroporation device.
• the electric conductivity of gas bubbles is practically equal to zero, the current conducting sectional area in such cases would be restricted to the liquid phase.
• a bubble-liquid mixture can be considered as a random dispersion of spherical bubbles.
• the Bruggman equation can be used to calculate its ohmic resistance (De La Rue_R.E and Tobias, C.W, J. Electrochem. Soc. Nol 106 (1959)).
• the Bruggman equation is :
• R is the ohmic resistance of the bubble-containing solution of a void fraction
• ⁇ volume fraction of gas in liquid
• Rp is the ohmic resistance of the bubble-free solution
• the resistance of the medium may be significantly altered as the volume fraction of gas in the liquid reaches a high level.
• the volume fraction of gas in the electroporation medium can especially reach to a critical level when one uses a small volume of electroporation medium.
• An example of a device which is especially designed for electroporation of cells in a small volume of medium is the device described by Teruel e al. (Biophysical Journal, Vol.73 (1997), 1785-1796 ).
• the small volume of the medium allows minimizing the high cost associated with the exogenous materials such as drugs, DNA etc.
• the volume fraction of gas in such a small volume of medium can be considerably high when one uses electrical pulses of long duration.
• R is the resistance of the medium and C is the capacitance.
• the fast removal of gas bubbles can be similarly restricted in devices which are normally used for the in situ electroporation of cells and which are equipped with horizontal electrodes facing downward such as the devices described by Casnig (US patent 5,134,070 , 1992) , Meyer ( PCT application, WO 98 / 12310 , March 1998), and Klenchin etal ( Biophysical Journal, (1991 ), Vol.60, 804 - 811).
• devices which are normally used for the in situ electroporation of cells and which are equipped with horizontal electrodes facing downward
• Casnig US patent 5,134,070 , 1992
• Meyer PCT application, WO 98 / 12310 , March 1998)
• Klenchin etal Biophysical Journal, (1991 ), Vol.60, 804 - 811).
• gas bubbles may get entrapped under the electrodes and the removal of gas bubbles by conventional methods in a short time period would be extremely difficult if not impossible.
• Accumulation of adherent hydrogen and / or oxygen bubbles at the electrode- membrane interface can result in the blockage of a significant number of pores during the electroporation process. This is because gas bubbles normally have a relatively large diameter in the range of 20 to 100 ⁇ m and can cover a significant number of pores. This in turn results in significant local heterogeneities in the electric field during the electroporation of cells. Thus some cells may not receive the optimum field strength that is required for electropermeabilization .
• the problems associated with gas evolution may also be encountered during in vivo electroporation of cells particularly when a train of low intensity pulses with long durations are applied for the effective electroporation of cells. Under these conditions a significant amount of electrical energy would be converted into chemical energy through electrochemical reactions at the electrode-tissue interface. Substances dissolved in the tissue are consumed in the electrode reactions and new species that may have toxic effects on healthy tissue and cells are produced. These include formation of oxygen gas bubbles and toxic radicals at the anode and the evolution of hydrogen gas at the cathode. Gas evolution at the electrode-tissue interface can promote the transport and spreading of toxic species to the surrounding healthy tissues by convection.
• a method for the electroporation of cells comprising the steps of placing cells and a medium containing exogenous materials between two electrodes, said electrodes comprising a cathode made of palladium (Pd) metal and an anode made of palladium metal charged with hydrogen ( Pd- H) , and applying an electrical pulse across said electrodes for electroporating cells.
• Another aspect of the present invention is a device for the in vitro electroporation of cells comprising means for holding cells and a medium containing exogenous materials between two electrodes, and means for delivering an electrical pulse across said electrodes, said electrodes comprising a cathode made of palladium (Pd) metal and an anode made of palladium metal charged with hydrogen (Pd-H).
• Pd palladium
• Pd-H palladium metal charged with hydrogen
• Yet another aspect of the present invention is a device for the in vivo electroporation of cells for use in electrochemotherapy and gene therapy, comprising a plurality of anodes and cathodes made of palladium (Pd) and palladium charged with hydrogen (Pd-H) respectively.
• Electrodes in prior art electroporation devices have been selected from a range of conductive materials such as aluminium, stainless steel, platinum, gold, silver, and transparent semiconductors such as indium tin oxide.
• conductive materials such as aluminium, stainless steel, platinum, gold, silver, and transparent semiconductors such as indium tin oxide.
• factors such as electrical conductivity, chemical stability, mechanical stability, biological inertness and cost have been considered.
• electrodes are constructed from materials that are non-toxic to cells, resistant to electrochemical attack and mechanically stable.
• anode material for electroporation devices is especially important because it determines the type of anodic reactions and the nature of the toxic species generated during the electroporation process. If the anode material is electrochemically soluble (e.g. aluminium and stainless steel), both metal dissolution and oxygen evolution can take place at the anode- electrolyte interface during the pulsing process. However, a large part of the anodic current in this case will consist of the current associated with the metal dissolution. For example, when aluminium anodes are used in an electroporation device, the following anodic reactions can occur:
• the extent of each of the above reactions would primarily depend upon factors such as pulse intensity, pulse duration, number of pulses applied, and the conductivity of the electroporation medium.
• the metal dissolution reaction can adversely affect the electroporation process in a number of ways. For example, it has been reported that electric discharge through solution of biological macromolecules such as DNA, RNA and proteins, using aluminium anode plates, can cause precipitation of significant portions of these macromolecules. The precipitation of macromolecules is a consequence of the interaction of macromolecules with the metal ions solubilized from the anode by the electric pulse (Stapulions, Bioelectrochemistry and Bioenergetics (1999), Vol.48, 249 - 254).
• Toxic metal ions can further adversely affect both the properties of cell membranes ( Gimmier etal., J. Plant Physiol. (1991 ), Vol.138, 708 - 715) and the yield of viable permeabilized cells ( Friedrich etal., Biochemistry and Bioenergetics(l 998), Vol. 47, 103 - 111 ).
• toxic metal ions initially form locally at the anode- electrolyte interface, they can be transported to the entire electroporation medium by convection in a short time. This is because gas bubbles can enhance the mass transfer of toxic metal ions by agitating the solution.
• the Pd cathode and Pd-H anode of the device in the present invention have the capability of effectively eliminating hydrogen and oxygen gas bubbles respectively during the electroporation process, and thus can prevent the problems associated with gas bubbles discussed above.
• the device of the present invention offers an exceptional and hitherto non-existing means of subjecting cells to an electric field using electric pulses of long duration without having the disadvantages and limitations of prior art electroporation devices.
• Palladium is known to have an exceptionally high capacity for absorbing hydrogen.
• the palladium electrode effectively prevents the interfacial concentration of hydrogen reaching the saturation level that is thermodynamically required for the formation of gaseous hydrogen in the form of gas bubbles.
• the rapid diffusion of hydrogen atoms to the surface of the electrode ensures a rapid conversion of hydrogen atoms to hydrogen ions at the electrode surface and results in fast consumption of the electrical charge. Because of its rapid rate, the above reaction effectively becomes the predominant reaction at the electrode surface and consumes a substantial part of the electrical charge that is passed through the electroporation device. Thus, it effectively eliminates the oxygen evolution reaction that would otherwise take place at the electrode surface in the absence of the stored hydrogen in palladium. In addition, other anodic reactions such as metal dissolution reaction, anodic formation of toxic radicals, and generation of chlorine at the anode will be substantially suppressed. Description of the Preferred Embodiment
• the negative electrode of the present invention can be made substantially of palladium.
• properties such as hydrogen permeability and hydrogen storage capacity of the electrode are of paramount importance
• conventional methods known to those skilled in the art such as grain refinement, surface modifications and the addition of alloying elements can be considered as a means of improving the properties of palladium.
• a palladium alloy composed of 77% palladium and 23% silver is known to have better properties than pure palladium in terms of the hydrogen absorption and can be used for this purpose.
• palladium with nano-meter size grains is known to have improved properties concerning hydrogen permeability and, thus, can be used for this application.
• a palladium electrode can be coated with palladium black to increase its surface area by electro-deposition method at 50 mA cm "2 in a solution of 1 g of PdCl 2 in 100 ml of 0.1 mol dilute aqueous HC1 at room temperature.
• the negative electrode of the present invention may be made in the form of plates, flat screens, cylinders, wires and needles in an appropriate size and dimension.
• the negative electrode can also be made by, for example, depositing a thin film of palladium on a solid substrate made of metals, transparent glass and plastics in various forms. For example, by controlling the thickness of the palladium film, it would be possible to form an optically transparent and electrically conductive film of palladium on glass and plastics.
• the electrode can also be prepared by ion implantation of palladium atoms onto the surface of an electrically conductive substrate made of, for example, titanium, stainless steel or a glass substrate coated with gold or silver.
• palladium atoms and nano-meter size palladium particles can be incorporated into thin films of oxides and ceramics that can be deposited onto glass and plastics.
• palladium atoms and small particles of palladium can be incorporated into sol-gel derived thin film oxides such as titanium oxide (Tour, J.M et. al., Chem.Mater, Vol.2 (1990) 647-649) and silicone dioxide.
• the sol-gel derived oxide films containing palladium can be deposited onto conductive glass such as a glass substrate coated with gold and silver to form transparent and conductive electrodes for in situ electroporation of cells.
• the negative electrode of the electroporation device is fabricated by depositing a thin film of palladium on a flat transparent substrate made of glass or plastics.
• Optically transparent Pd films with sufficient electrical conductivity can be deposited on glass and plastics by a range of conventional techniques.
• Cells cultured on the palladium film can be subjected to in situ electroporation while they are attached to the palladium film.
• In situ electroporation of cells can be carried out by, for example, positioning a Pd-H electrode or a conventional positive electrode such as a platinum electrode close to the plated cells and applying a voltage between the two electrodes.
• the palladium electrode can rapidly absorb hydrogen atoms generated during electroporation, cells can be subjected to electrical pulses of long duration without being disturbed by hydrogen bubbles.
• the palladium film can be further covered by a microporous film of an inorganic material such as titanium oxide or indium tin oxide.
• the positive electrode of the present invention can also be made substantially of palladium in various forms similar to the negative electrode as discussed above except that it is charged with hydrogen to form a Pd-H alloy such as an ⁇ - phase ( [H / Pd] ⁇ 0.33 )or ⁇ - phase ( [H Pd ] ⁇ 0.66 ).
• Charging of the palladium electrode with hydrogen can be carried out by various conventional methods such as exposing palladium to a gaseous hydrogen environment or by the electrolytic method.
• the electrolytic method is preferred since palladium and palladium alloys can be conveniently charged with hydrogen in electrolytes under normal temperatures in a very short time.
• a significant amount of hydrogen (up to a [H / Pd ] ⁇ 1 ) can be stored in palladium by this method (Krueger, F and Gehm ,G , Ann. Phys., Vol.78, P.72 ,1925).
• the palladium electrode is preferably charged with hydrogen shortly before it is used as a positive electrode in the electroporation process, although under normal conditions, hydrogen can be stored in palladium for a long time.
• the electrodes of the electroporation device in the present invention can be used independently of each other or together in various combinations and configurations for the electroporation of cells.
• the Pd electrode of this invention can be used as a negative electrode (cathode) in an electroporation device together with a conventional anode to absorb hydrogen atoms and to eliminate hydrogen gas bubbles during the electroporation process.
• the Pd-H electrode of the present invention can be used as a positive electrode (anode) in an electroporation device together with a conventional cathode to suppress oxygen evolution reaction.
• the Pd and Pd-H electrodes of this invention can be used together as a pair of negative and positive electrodes in an electroporation device to eliminate both hydrogen and oxygen gas bubbles.
• the Pd and Pd- H electrodes of this invention can be used together as a pair of parallel plate electrodes for the electroporation of cells in suspension similar to the parallel electrodes described by Andreason etal. (BioTechniques (1988), Vol.6, No.7, 650-659) and as described by Papp et al (US patent 5,422.272, 1995).
• both electrodes can be made of solid palladium or palladium alloys.
• one of the electrodes can be charged with hydrogen to serve as the positive electrode for the electroporation of cells.
• Charging of the palladium electrode with hydrogen can be performed by, for example, dipping the pair of electrodes in an electrolyte or by filling the gap between the two electrodes with an electrolyte and applying a voltage to the electrodes for a short time using, for example, a D. C power supply.
• the electrolyte for example, can be a physiologically acceptable solution such as the solutions that are normally used for cell culture and for the electroporation of cells.
• the electrolyte contains strongly reducing agents such as sodium hypophosphite.
• the Pd electrode that is connected to the negative terminal of the DC power supply would act as a cathode and would be charged with hydrogen whereas the other Pd electrode would serve as a counter electrode.
• the time required to charge the Pd electrode with hydrogen would primarily depend on the pH of the electrolyte and the voltage applied during the charging process. If a high voltage is used, the charging time can be as low as a few seconds or less. In a preferred embodiment, the charging process is carried out at a relatively low voltage (eg 20 - 30 volts) for less than 300 seconds and more preferably about 60 seconds.
• the power supply can be, for example, a D.C power source or a function generator capable of generating an electric pulse with a predetermined amplitude and pulse length.
• a power generator similar to those commonly used for the electroporation of cells may be employed for the charging process.
• charging can be carried out using, for example, one or more exponential-decay pulses.
• charging of the Pd electrode with hydrogen is carried out using a low voltage square pulse with a pulse length of about 60 seconds.
• a train of square pulses having short pulse lengths can be employed for charging the Pd electrode with hydrogen.
• the electrical set-up for charging the palladium electrode may be equipped with means for controlling the charging process such that the palladium electrode can be electrolytically charged with hydrogen to a pre-determined level (e.g. charged to a preselected H/Pd atomic ratio).
• the electrodes of the present invention can also be advantageously incorporated into a wide variety of conventional devices suitable for the in situ electroporation of attachment-dependent cells.
• An example of electroporation device for in situ electroporation of cells is the device described by Klenchin etal (Biophysical Journal, (1991), Vol.60, 804 - 811) in which cells are plated on a microporous membrane that is positioned between two closely spaced and parallel electrodes.
• both electrodes can be made of palladium or palladium alloys.
• one of the electrodes Prior to electroporation of cells, one of the electrodes can be charged with hydrogen to serve as the positive (anode) electrode during the electroporation of cells.
• the charging step can be carried out conveniently and in a very short time prior to the electroporation step by placing an electrolyte between the two closely spaced Pd electrodes and applying a voltage between the two electrodes as described above.
• the use of the Pd and Pd-H electrodes of the present invention in this application is particularly beneficial since it results in effective elimination of hydrogen and oxygen gas bubbles that are normally generated at the interface of the microporous membranes and electrodes.
• the electrodes of this invention can also be used in other types of conventional devices suitable for in situ electroporation of attachment- dependent cells such as devices described by Chang (US patent 4,822,470, 1989). For example, cells can be cultured on a solid, non-conductive substrate such as a glass slide.
• In situ electroporation of cells can then be carried out by positioning an electrode assembly close to the plated cells consisting of a pair of electrodes made of Pd and Pd-H that serve as the cathode and the anode respectively.
• the electrodes can be in the form of parallel plates or concentric rings. In addition to parallel plates and concentric rings, other configurations such as multiple cathodes and anodes can be used.
• Pd cathodes and Pd-H anodes can be arranged in alternating fashion into two groups. One group would be connected to the negative terminal, while the other group would be connected to the positive terminal of a high voltage power source (pulse generator) during the electroporation process.
• Electrode assembly comprising electrode array patterns that may be formed by depositing palladium on a non-conductive support can also be used. This method allows fabrication of much smaller electrodes on the bottom of an electrode carrier for the in situ electroporation of cells with smaller electrode separations.
• the Pd and Pd-H electrodes of the present invention can be used in devices suitable for in vivo electroporation of cells for application in electrochemotherapy and gene therapy such as electrode assemblies described by Mir( U.S. Pat.No. 5,468,223 ) and Hofinann (U.S Pat. No. 5,439,440).
• the electrodes can be made of palladium or palladium alloys.
• one or a group of the Pd electrodes Prior to the electroporation of cells, one or a group of the Pd electrodes can be charged with hydrogen to serve as the positive electrode(s) for the electroporation of cells.
• Charging of the palladium electrode(s) with hydrogen can be carried out as described previously by, for example, dipping the electrode assembly in an electrolyte and applying a voltage between the electrodes for a very short time.

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