WO1999043782A1 - Procede et dispositif d'electropermeabilisation - Google Patents

Procede et dispositif d'electropermeabilisation Download PDF

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Publication number
WO1999043782A1
WO1999043782A1 PCT/GB1999/000613 GB9900613W WO9943782A1 WO 1999043782 A1 WO1999043782 A1 WO 1999043782A1 GB 9900613 W GB9900613 W GB 9900613W WO 9943782 A1 WO9943782 A1 WO 9943782A1
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electric field
electrodes
electropermeabilisation
membrane
cell
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PCT/GB1999/000613
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English (en)
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John Edward Osborne
Martin David Bootman
Peter Lipp
Fedja Bobanovic
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The Babraham Institute
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Publication of WO1999043782A1 publication Critical patent/WO1999043782A1/fr

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    • 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

  • the invention relates to an electropermeabilisation method and apparatus, with particular application to the electropermeabilisation of the membranes of biological cells.
  • the plasma membrane of a biological cell is a continuous macromolecular structure composed of a lipid bilayer with immersed proteins. It defines the outer dimensions of the cell, and possesses anchor points for the cellular cytoskeleton, thus providing structural integrity. As it is essentially composed of lipid, the plasma membrane is virtually impervious to water-soluble molecules and ions . They can usually only traverse the plasma membrane via specific channels formed by proteins located in the bilayer. The study and control of cellular processes often requires manipulation of the intracellular environment. Methods currently in use to allow the introduction of non membrane-permeant molecules include micro injection and patch-clamping of cells. These techniques are limited in that they are time-consuming and allow only a relatively few cells to be manipulated. Alternative techniques, which permit many cells to be manipulated simultaneously, involve permeabilisation of the membrane using weak amphipathic detergents, liposome- mediated delivery, scrape-loading and electric fields (known as electropermeabilisation or electroporation) .
  • Exposure of cells to high-intensity electric fields causes transient localised disruptions, or rupture, of the lipid bilayer (the outer cellular membrane) , which give rise to short-lived pores through which molecules can pass relatively unhindered.
  • This is a widely-used technique in laboratory and clinical environments, to enable rapid and selective introduction of non-membrane-permeant materials.
  • the efficiency of electrical permeabilisation is essentially related to two parameters: (i) the area of the cell membrane which has been permeabilised and (ii) the properties of the created pores, in particular pore diameter and life-time.
  • a cell In conventional electropermeabilisation a cell is placed in a uniform electric field generated, for example, by applying a voltage between two electrodes, a cathode and an anode.
  • the field interacts with the cell membrane and causes changes in the membrane potential. If these changes exceed several hundred millivolts, membrane breakdown occurs and non-selective pores start to open (electropermeabilisation) .
  • the pore life-time and the pore dimensions are directly affected by the strength of the field. The electric field strength must be increased to increase the permeabilisation effect.
  • an electrically-stimulated cell may be considered as a thin-walled sphere made of pure dielectric.
  • a uniform electric field applied to the cell then induces a change of the membrane potential ( ⁇ V) which varies with position on the cell surface according to the following simplified equation (1) :
  • Equation (2) E is the electric field intensity
  • r is the radius of the cell
  • the total membrane potential V consists of the resting potential V r ⁇ t (which exists when no external field is applied) and the superimposed field-induced ⁇ V, as expressed by equation (2) :
  • V V xeat - ⁇ V (2)
  • the external field E causes a hyperpolarisation ( ⁇ ) at the cell hemisphere nearest the anode and a depolarisation (V ⁇ ) at the opposite cell hemisphere, nearest the cathode.
  • ⁇ hyperpolarisation
  • V ⁇ depolarisation
  • FIGs 1 and 2 attached hereto show the membrane potential V( ⁇ ) as a function of the angle ⁇ in polar coordinates in unexposed (Fig. 1) and exposed (Fig 2) spherical cells.
  • Each figure illustrates the voltages in the cell membrane around a section through an ideal, spherical cell.
  • the cell is shown schematically at the centre of each drawing. The origin of the co-ordinates is at the centre of the cell. Since the resting voltage is negative for most cells, hyperpolarising voltages are shown in the radially outward direction and depolarising voltages in the radially inward direction.
  • the membrane potential levels at which permeabilisation first occurs i.e. the threshold levels
  • the threshold levels are shown as outer and inner circles 4, 6 respectively. Since V I ⁇ st is negative for most cells, the total membrane potential during electric field exposure has the highest absolute values at the hyperpolarising side of the cell. This results in practice in a highly uneven permeabilisation effect between the two sides of the spherical cell; membrane disruption will always be higher at the end of the cell facing towards the anode.
  • the field intensity has to be increased by at least 200%.
  • a significant limitation of conventional electropermeabilisation arises from these figures.
  • An increase in electropermeabilisation effect may be achieved by increasing the intensity of the electric field applied to the cells. On average, this increases the area of the cell membrane permeabilised, the diameter of the pores and the pore life-time.
  • an increase in field strength does not necessarily lead to an increased efficiency of electropermeabilisation, since high- mtensity electric fields can lead to a loss of cell viability by creating irreversible pores that do not re- seal. Therefore, although the chances of permeabilismg cells are higher with more intense electric fields, the actual efficiency of the process may be lower because the cells die.
  • a further significant drawback of conventional electropermeabilisation is that unlike, for example, chemical permeabilisation, where the entire cell membrane s rather evenly affected, the electric field causes permeabilisation primarily restricted to the areas of the cell facing the electrodes (particularly the hyperpola ⁇ sing side of the cell which faces the anode) .
  • Only perfectly spherical cells undergoing spontaneous movement allow a homogeneous electropermeabilisation of populations of cells, and unfortunately not all cells can be grown under conditions in which they retain a spherical shape. Instead, many cells need to adhere to a basal surface to maintain their characteristic morphology and physiology. Such adherent cells usually flatten considerably and occupy unpredictable irregular shapes.
  • the invention provides an electropermeabilisation method and apparatus as defined in the appendent independent claims. Preferred or advantageous features of the invention are defined in dependent subclaims.
  • the invention preferably uses low-intensity, high- frequency, multipolar electric fields to achieve more effective electropermeabilisation at lower field strengths than in the prior art.
  • the invention may apply a moving or rotating electrical field to cells.
  • the electrical field may advantageously move or rotate stepwise. This may allow more extensive, or more uniform, electropermeabilisation of cells at lower field strengths than if a uniform or unidirectional alternating field is used as in the prior art.
  • the preferred rate or frequency of movement of the electric field may vary depending on the type of cell being electroporated, on the electric field strength applied and on the duration, or length, of the electric field pulse applied in each direction.
  • Large cells such as mammalian cells, are easier to porate than small cells, such as bacteria, and so may require lower frequencies, lower field strengths and/or shorter pulse durations .
  • Smaller cells may require higher-energy poration. Higher energy poration may also advantageously be used to uptake larger molecules into porated cells.
  • the invention may advantageously use frequencies of about 1kHz or less, such as from about 100Hz up to radio frequencies. For higher frequency applications, frequencies of 100 kHz or more may be used. (Frequency is defined with relation to the time period taken to complete a cycle of operation of the invention, when the field has been applied in all available directions, equivalent to a 360° rotation if a rotating field is used) .
  • the invention may also advantagsouly use electric field strengths of about 100 V/cm up to about 1000 V/cm or more. Particularly preferably field strengths of 500 V/cm to 1000 V/cm may be used.
  • pulse lengths of less than microsecond to about 1 ms may be used, although shorter pulses are preferred, such as about 1 to 10 microseconds.
  • the electric field may be applied in each of a number of different directions in the form of a square pulse.
  • opposite voltages may be applied to electrodes, or pairs or groups of electrodes, on opposite sides of a cell or cells to be electropermeabilised. If such voltages are applied across single pairs of electrodes, equal and opposite voltages may advantageously be applied to respective electrodes.
  • the invention may allow an advantageously larger area of a cellular membrane to undergo permeabilisation, for example as the applied field changes direction and affects different portions of the membrane .
  • the invention may also advantageously improve control of the size of pores formed during membrane breakdown. This is a consequence of three factors. One is the use of a multi-polar device to produce a more uniform distribution of pore sizes than in the prior art. The second is the ability to use low electric-field strengths while achieving an adequate degree of electroporation. The third is the use of square pulses of applied electric field, which may allow close control of the electroporation field applied to any portion of a cell wall while reducing collateral damage to the cell. These factors, taken individually or preferably in combination, may advantageously enable discrimination between molecules of differing sizes, to differentially control their entry across the cellular membrane, and/or to control the longevity of pores.
  • the ability to generate smaller pores than in the prior art may also advantageously shorten the membrane re- sealing time, reduce collateral damage and substantially improve the prospects for cell viability.
  • the pulses may advantageously be spaced in time so that, between the pulses, no electric field is applied. This may further improve control of electroporation conditions because pores created by a pulse may persist for some time after the electric field has been removed, and if this means that no electric field need be applied during parts of the electropermeabilisation process, cell damage may advantageously be decreased by switching off the electric field at such times .
  • Adherent cells which are usually non-spherical, provide the worst-case scenario for electropermeabilisation.
  • the chances of permeabilising irregularly- shaped cells may advantageously be greatly improved.
  • aspects of the invention may find particular advantage in medical applications and in vivo applications. For example there may be advantage in administering drugs to cells in vivo in a selective manner such that the size and number of ions or molecules admitted to cells is controlled by controlling the electropermeabilisation conditions .
  • One aspect of the invention relates to such a medical use, providing an apparatus comprising three or more electrodes for contacting or penetrating body tissue surrounding cells for electropermeabilisation, and administering desired ions, chemicals, drugs or the like to those cells.
  • An example of such a use may be to treat melanoma.
  • Figure 1 is a diagram of potential vs. angle, in polar coordinates, in a theoretical model of a spherical cell, with no applied electrical field;
  • Figure 2 is a diagram of potential vs. angle, in polar coordinates, in a theoretical model of a spherical cell, with applied uniform electrical field E, as in the prior art;
  • Figures 3 to 8 are diagrams of potential vs. angle, in polar coordinates, in a theoretical model of a spherical cell, with applied fields in successively rotated orientations, according to a first embodiment of the invention
  • Figure 9 illustrates the electropermeabilised region of an ideal spherical cell in an applied unipolar field, as in the prior art
  • Figure 10 illustrates the electropermeabilised regions of an ideal spherical cell in a rotating or moving field according to the embodiment of Figures 3 to 8.
  • FIG 11 is a block diagram of an electropermeabilisation apparatus according to the embodiment of Figures 3 to 8;
  • Figure 12 is a schematic plan view of the chamber of the apparatus of Figure 11;
  • Figure 13 is a schematic cross-section on A-A of the chamber of Figure 12;
  • FIGS 14 to 18 illustrate various sequences for applying voltage pulses to the electrodes of the embodiments of figures 11, 12, 13, 19, 20 and 21;
  • Figure 19 is a perspective view of an apparatus embodying the invention for electroporation of skin cells
  • Figure 20 is a cross-section of the embodiment of figure 19 in use
  • Figure 21 is a schematic view of a second apparatus embodying the invention for electroporation of skin cells.
  • Figure 22 is a plot of voltage vs. time showing two bipolar square voltage pulses output by the apparatus of figure 11.
  • Figures 1 and 2 have been described above and will not be discussed in detail again here.
  • the direction of the electric field is switched through a succession of six directions, which are coplanar and separated by 60°.
  • Figures 3 to 8 are similar to Figures 1 and 2 but illustrate the voltage distribution 10 in the cell wall as each of the six successive field directions is applied.
  • a sufficient field strength is applied to cause permeabilisation in the cell surface nearest the anode.
  • a new area of permeabilisation is correspondingly induced.
  • electropermeabilisation generally causes a highly uneven permeabilisation effect between the two sides of a spherical cell; membrane disruption will always tend to be higher at the side of the cell facing towards the anode. This condition is illustrated in
  • Figure 9 for a cell in a uniform field (as in the prior art), such as the cell illustrated in Figure 2.
  • the electroporated region 12 of the cell 14 is shown in black and covers approximately 60° of the circumference of the cell at the side nearest the anode. This corresponds to an applied field strength exceeding the threshold for electropermeabilisation by about 15%, as described above.
  • Figure 10 shows the corresponding degree of electroporation when the cell is exposed to the rotating electric field illustrated in Figures 3 to 8. In each of the 6 field directions, a portion 16 of the cell wall is permeabilised and therefore, as the field rotates, a region around substantially the entire circumference of the cell in the plane of exposure is permeabilised.
  • the method of the first embodiment of the invention allows permeabilisation of a very much larger area of the cell membrane than in the prior art with substantially no increase in the strength of the applied field.
  • to increase the area of the porated region in a unipolar field, as in the prior art would require a very significant increase in field strength, which would be very likely to prejudice cell viability.
  • the electric field in the first embodiment can be advantageously switched at high frequencies, for example at 100kHz or 200kHz, to achieve a quasi-continuous electropermeabilisation around the entire cell circumference, although frequencies as low as 1 kHz or 10 kHz may be used to achieve quasi-continuous poration if the resealing time is long.
  • This rapidly repeated application of low-energy short pulses to each area of the cell surface may generate pores more gradually giving better cell viability. Pores created under low energy conditions may reseal better than pores created under high energy conditions.
  • the exposure time required to cause electroporation varies with a number of parameters, including the applied field strength, the conductivity of the medium surrounding the cell, and the response time of the cell membrane itself.
  • the field must be applied for a few microseconds (for example l ⁇ s to lO ⁇ s) .
  • the electropermeabilised area is made up of many, smaller pores and the size and other properties of each pore may vary depending on the applied field strength perpendicular to the cell membrane at the location of each pore.
  • the pores at the centre of the electropermeabilised area, where the effective field strength is greatest, will be larger than those at the edge. Therefore, as the applied field strength of a unipolar field is increased in order to increase the electropermeabilised are, the field strength and the pore size at the centre of the area will become very large. Such pores are much more likely to prejudice cell viability than smaller pores created by smaller applied fields .
  • the resealing time of the pores generated in conventional electropermeabilisation is usually much longer, of the order of milliseconds, and can be even longer, of the order of seconds or even minutes. This will vary with the type of the cell membrane as well the pore size, which is related to the applied field strength as described above.
  • the invention aims to use these factors to increase the area of electropermeabilisation by moving the applied field so as to permeabilise different areas of a cell membrane in a rapid succession. Because the resealing time of a pore is greater than the time taken to create a pore, then the invention may allow a large area of a cell membrane to be permeabilised continuously or quasi- continuously . For example, if the invention is embodied in a method applying electric fields successively in several different directions to a cell, then pores in the portions of the cell membrane permeabilised by all the field directions will remain open continuously as long as the interval between the repeated application of the field in each direction is less than the pore resealing time.
  • the field must be applied in each direction more often than once in every 10ms, that is at greater than 100Hz. If the field is applied at even time intervals in each direction, it must therefore be switched from one direction to the next at more than 600Hz.
  • the field In each direction, the field must be applied for long enough to electropermeabilise the membrane which, as discussed above, may be l ⁇ s to lO ⁇ s. This can easily be achieved as, at 600Hz, 1.67ms is available before the field must be switched to the next direction.
  • a switching frequency of 6kHz is required, but this still allows an interval of 167 ⁇ s in each field direction to apply an electric field pulse of l ⁇ s to lO ⁇ s duration.
  • the example discussed uses six field directions. More or less directions may be used, which would raise or lower the required minimum switching frequency accordingly if quasi-continuous poration is to be achieved.
  • six coplanar field directions is a practical embodiment because it is possible to generate an electropermeabilised area covering about 60° of a cell circumference by exceeding the electropermeabilisation threshold only by about 15% as described above, and with six field directions this advantageously achieves permeabilisation around an entire circumference of a cell.
  • Non-coplanar field directions may be used to electropermeabilise areas of a cell membrane not permeabilised by a set of coplanar field directions. This may entail the use of more field directions and correspondingly higher switching frequencies. In principle, this may even enable quasi-continuous poration over the entire surface of a cell, even if the cell is non-spherical.
  • the model of the principle behind the invention described above assumes that the areas permeabilised by fields applied in different directions do not overlap. It also does not consider the option of applying a continuously- moving field. However, the necessary modifications to the model required to accommodate these factors can easily be made by the skilled person without inventive effort.
  • an electric field pulse of the order of microseconds duration may be applied, for example as a square wave pulse or any other convenient pulse shape.
  • a square wave pulse is expected to optimise permeabilisation while reducing the likehood of damaging a cell.
  • the square wave pulse shape is preferred because it reduces collateral damage to the cell.
  • the need is to create transient pores so that substances such as a chemical, DNA or other molecule can be taken up into the cell and the cell envelope, or wall, repairs so that the cell remains viable.
  • the greater the voltage and the greater the time (pulse length) the larger the pores and the longer they typically last.
  • Having wave-forms with significant tailing, such as a sinusoidal or decaying pulse in which the voltage reduces relatively slowly from its maximum, can cause additional damage to the cell without aiding poration.
  • the embodiment allows finely controlled field strengths to achieve effective electroporation based on transient pores, minimal cell death and hence maximum cell viability post electroporation. This has been exemplified in experiments by the applicant with 99% viability in respect of electroporation with Ca 2+ and >90% viability when molecules of 1,000 daltons were electroporated into cells.
  • the ability to control the pulse shape and intensity enables control of the longevity of pore formation; hence transient or longer-lasting pores may advantageously be selectively generated.
  • Varying the pulse parameters enables the creation of pores of different sizes. This can be used to define pore sizes and hence control the size of molecules entering a cell. In this way size-exclusion principles can be employed for selective uptake. This has been exemplified in experiments by the applicants in which molecules of different sizes were successfully selectively electroporated into a mammalian cell line. Differential field strengths were used to enable Ca 2+ (small pore sizes) and Fura-2 or ruthenium red (1,000 daltons; medium pore sizes) to gain entry into cells. It was observed that lower field strengths were required to get Ca 2+ into the cells as compared to Fura-2 or ruthenium red.
  • Figure 11 shows a schematic plan view of an electropermeabilisation chamber 20 containing 6 electrodes 22, 24, 26, 28 30 and 32 and associated circuitry.
  • the electrodes are positioned at 60° intervals around a central portion of the chamber in which cells may be placed.
  • the electrodes are thus arranged as three pairs of electrodes facing each other.
  • Each electrode can be coupled via a respective high voltage switch 34, 36, 38, 40, 42 and 44 either to a high positive voltage, or to earth, or to a high negative voltage.
  • This preferred apparatus applies square voltage pulses to the electrodes; other embodiments may apply other pulse types if required) .
  • the high positive and negative voltages are generated by respective voltage sources 46, 48 coupled to respective capacitor banks 50, 52.
  • the outputs of each capacitor bank are connected in parallel to inputs of each high voltage switch.
  • a PC 54 controls the high voltage sources and, via a microprocessor 56, the high voltage switches.
  • Figure 22 illustrates the production of two successive bipolar square voltage pulses by a practical implementation of this apparatus tested by the applicant.
  • the voltage supply to each opposite pair of electrodes is switched using an H-bridge configuration to connect each electrode either to a positive voltage or a negative voltage, enabling high voltages to be rapidly switched to the electrodes in a bipolar, biphase (push- pull) mode.
  • a field may be generated by applying a high voltage to electrode 22 and an opposite voltage to the opposite electrode 28.
  • Figures 14 and 15 illustrate possible sequences of voltage pulses to be delivered to each electrode to implement this embodiment. The figures differ in that shorter pulses are used in figure 14, leaving quiescent periods between pulses in order to minimise cell damage.
  • a field may be generated by applying voltages to four opposite electrodes at a time.
  • FIG. 16 illustrates a possible sequence of voltage pulses to be delivered to each electrode to implement this embodiment.
  • the apparatus may therefore be designed with electronic elements rated at half of the voltage and power capacity that can actually be generated by the apparatus .
  • FIG. 17 illustrates a possible sequence of voltage pulses to be delivered to each electrode to implement this embodiment. It should be noted that figure 17 illustrates an electric field rotation of only about half a revolution. Further alternatives in which voltages are applied to groups of more than two adjacent electrodes may also be useful. For example, in a further preferred embodiment, the voltages may be applied to adjacent groups of three electrodes opposite each other.
  • the PC can be programmed to control various electric field exposure sequences by controlling the high voltage sources and switches appropriately. It should be noted that although figures 14 to 17 illustrate rotating fields, any desired sequence of different field directions may be used, and controlled by suitable programming of the PC. There may be advantage in not using a rotating field in some cases if it is preferred not to apply the electric field successively to adjacent cell-wall regions. This may increase cell viability.
  • each pair or group of opposed electrodes is used first to apply an electric field with one polarity and then, later on, with reversed polarity. This is achieved by applying opposite voltages to the electrodes in each case.
  • Each electrode may therefore be termed biphase, such that the electrodes operate in a push-pull mode.
  • bipolar, biphase system of applying voltage pulses to the electrodes described above is the preferred option for generating electric fields for electroporation in the embodiment
  • alternatives are possible.
  • a unipolar voltage pulse could be applied to each electrode in turn around the ring of electrodes in figure 12, while each opposed electrode is grounded, or earthed.
  • Figure 21 illustrates a possible sequence of voltage pulses to be delivered to each electrode to implement this embodiment .
  • This method would have the disadvantages that the voltage generator (s) for generating the pulses and the associated cables would need to be rated at twice the voltage of the voltage generators in the biphase system, making them more dangerous and more expensive, and that the electrode opposite the electrode at high voltage would need to be connected to OV.
  • This might be done using a 2000 Ohm resistor but this would be in series with the electroporated sample and would limit the current flow through the sample. For example with a 2000V voltage and 2000 Ohm resistor, only a maximum of 1 amp could pass through the sample.
  • each pair of opposite electrodes could be permanently connected to 0V while biphase (alternately positive and negative) voltages are applied to the other of each pair of electrodes in sequence.
  • This method would again suffer the disadvantage of needing voltage generator (s) and cables rated at twice the voltage of those in the bipolar system, and would lack flexibility in rotating the electrical field if half of the electrodes are permanently grounded.
  • this problem could be reduced by only connecting to 0V the electrode opposite the electrode at high or low voltage at any time.
  • the electropermeabilisation chamber 20 is illustrated in more detail in Figures 12 and 13.
  • the body 60 of the chamber is made of a non-conducting, transparent material.
  • the six electrodes are arranged around a compartment 62 in which cells 64 may be placed for exposure.
  • a respective channel 66, 68, 70, 72, 74 and 76 leads radially outwards from the exposure compartment 62 past each electrode and then leads to the upper surface 78 of the chamber.
  • a central channel 80 leads from the exposure compartment 60 to the upper surface 78 of the chamber.
  • the channels thus provide perfusion pathways leading past each electrode and through the exposure compartment for passing fluids across cells held therein.
  • a respective electrical connection 82 couples each electrode via the upper surface 78 of the chamber to a respective high voltage switch.
  • Perfusion media may or may not be electrically conducting. If the medium is conducting, the efficiency of permeabilisation may be increased as a greater electric field and current flow may be experienced by the cell membrane .
  • fluids passed through the chamber may be electrolysed by the electrodes to produce hazardous or toxic products.
  • the channels containing the electrodes may be filled with agar bridges to provide protection from such possible toxic products, although perfusion would then only be possible along the central channel 80.
  • two central channels may be provided to enhance perfusion flow.
  • Further optional measures to avoid electrolysis products include providing a constant flow of fluid perfused though the compartment, as well as the application of charge-balanced exposure (the application of opposite high and low voltages to opposite electrodes) and the use of inert platinum electrodes.
  • the cells in the chamber are cultured on glass coverslips 24mm in diameter. Such cover slips are commonly used by researchers and are readily available from commercial suppliers.
  • the bottom of the chamber is arranged to mount such a cover slip with attached cells.
  • the design of the electropermeabilisation chamber is based on the typical fluid volumes and cell culture materials currently used by researchers. However, it could be modified into other arrangements as required.
  • the chamber of Figures 12 and 13 is designed for use with adherent cells attached to a substrate. Clearly the design would need to be modified, in particular regarding the perfusion of fluid, for use with free-floating cells. Such modifications are, however, within the ability of the skilled man.
  • the electrodes in the chamber of figures 12 and 13 are arranged on a circle of radius approximately 20mm. Total voltages of lkV to 2kV are typically applied between opposed electrodes and currents of between 1A and 10A may flow between opposed electrodes. These parameters may vary significantly with the application of the apparatus.
  • a number of variations from the embodiments described above may be incorporated.
  • a greater or smaller number of electrodes may be used to generate the changing electric field.
  • the electrodes need not be arranged in a plane.
  • effective electropermeabilisation using six electrodes in opposed pairs may be achieved if the pairs are arranged on three axes at right angles to each other.
  • a ring of six electrodes in a plane may be supplemented by one or more electrodes above and/or below the plane.
  • Varying or moving electric fields may also be generated by varying the voltages applied to electrodes, either continuously or in steps, rather than the simple switching on and off of electrodes described above. For example, if four electrodes are spaced at 90° intervals around a circle, and sinusoidal alternative voltages in antiphase are applied to one opposed pair of electrodes and cosinusoidal alternating voltages in antiphase are applied to the other opposed pair of electrodes, cells between the electrodes will experience a smoothly rotating electric field.
  • the electrodes or the cells being electroporated may be physically moved in order to produce a moving field.
  • the embodiments of the invention may thus give a superior efficiency of electropermeabilisation.
  • they may provide better control over pore position, size and resealing time, as well as causing minimal disturbance to the cells.
  • This increase in efficiency of electropermeabilisation may be particularly beneficial under conditions where cell viability is paramount, and may significantly reduce the pain associated with the use of electropermeabilistation in the clinical environment.
  • the technique can be used for introduction into cells of a variety of chemical agents including proteins, antibodies, drugs, enzymes, DNA, RNA and antisense oligonucleotides .
  • the invention may find application in a variety of medical treatments.
  • One example is in the treatment of skin conditions such as melanoma, in which electroporation may broaden the range of substances which can be administered into the cells of the melanoma.
  • Two suitable apparatuses for enabling this are illustrated in figures 19, 20 and 21.
  • Figure 21 illustrates the use of a hexagonal ring of needles 122, 124, 126, 128, 130, 132 which are held in a support (not shown) from which the sharp ends of the needles extend in a hexagonal arrangement.
  • the opposite ends of the needles are connected to circuitry similar to that of figure 11 or the other embodiments discussed above.
  • the needles pierce the skin 134 of a patient, surrounding a melanoma 136, the substance to be introduced to the cells is placed over the melanoma and a moving or rotating electric field is applied as described herein.
  • Figure 19 and 20 illustrate an alternative apparatus which does not require the patient's skin to be punctured. It consists of a bowl-shaped vessel 220 of non-conducting material, such as a transparent plastic. Electrodes 222, 224, 226, 228, 230, 232 are set into the inner surface of the bowl. The electrodes are arranged at 60° intervals around the inner rim of the bowl, set back slightly from the rim. The electrodes are connected to circuitry similar to that of figure 11 or other embodiments discussed above (not shown) . Two channels 234, 236 extend from the outside to the inside of the bowl and are connected in use to a source of a perfusion medium.
  • Figure 20 is a cross-section of the apparatus of figure 14 in use.
  • the inverted bowl-shaped vessel 220 is placed on the skin 238 of a patient over a melanoma 240.
  • Perfusion medium 242 is passed through the interior of the vessel via the channels 234, 236 and the pressure of the medium is controlled to be below atmospheric pressure such that the skin of the patient is drawn into the bowl and into contact with the electrodes .
  • Electroporation can then be performed as described herein.
  • the method and apparatus of the embodiments permit significant advantages over conventional electropermeabilisation, they also retain the existing advantages of electropermeabilisation over other techniques.
  • control of the electric field permits permeabilisation for only a short controllable period of time after exposure.
  • Ability to manipulate the recovery time of the plasma membrane will allow introduction of a desired quantity of non-permeable molecules. For larger quantities, long-lasting pores can be created while, for introduction of smaller quantities, the electrical field can be controlled to allow formation of pores for only a short period.
  • the cells being exposed may be monitored directly by a microscope or microscope-based video acquisition software.
  • Pore size and duration may be controlled by the frequency, intensity, duration time and signal shape of an applied electric field.
  • the technique described herein may be suitable for broad- based use in cell physiology. In particular, it may be applicable in the plant science field where ester loading of cells is not practical and where all such work conventionally has to take place with micro-injection. It may be useful for loading non-permeant species which currently require expensive and time-consuming micro- injection. It may be useful for rapid loading of large populations of cells for biochemistry and molecular biology applications or for in situ loading of cells in tissue slices which is not possible at present.

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Abstract

L'invention concerne un dispositif d'électroperméabilisation comportant une chambre centrale qui reçoit des cellules biologiques. Cette chambre est entourée de six électrodes (20, 22, 24, 26, 28, 30). On peut immerger les cellules dans un fluide de perfusion. Chaque électrode peut être couplée à une tension positive élevée ou à une tension négative élevée, ou bien reliée à la terre via un commutateur correspondant dans un groupe de commutateurs (34, 36, 38, 40, 42, 44), sous le contrôle d'un ordinateur personnel (54) et d'un microprocesseur (56). On applique ensuite des tensions positives et négatives aux différentes paires opposées d'électrodes en séquence, de manière à exposer les cellules à un champ électrique d'électroperméabilisation, dont la direction se déplace en cours d'exposition.
PCT/GB1999/000613 1998-02-27 1999-03-01 Procede et dispositif d'electropermeabilisation WO1999043782A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9804246.8 1998-02-27
GBGB9804246.8A GB9804246D0 (en) 1998-02-27 1998-02-27 Electropermeabilisation method and apparatus

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WO1999043782A1 true WO1999043782A1 (fr) 1999-09-02

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004025046A1 (de) * 2004-05-18 2005-12-15 Forschungszentrum Karlsruhe Gmbh Einrichtung zum Elektroporieren von biologisch pflanzlichem Prozeßgut
WO2021151124A1 (fr) 2020-01-21 2021-07-29 Luong Thi Hong Lien Dispositif à plasma froid portatif

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0126389A2 (fr) * 1983-05-13 1984-11-28 Bayerische Julius-Maximilians-Universität Würzburg Enceinte pour le traitement de cellules dans un champ électrique
JPS60251874A (ja) * 1984-05-30 1985-12-12 Hitachi Ltd 微粒子取扱い装置
JPS60251877A (ja) * 1984-05-30 1985-12-12 Hitachi Ltd 細胞融合装置
JPS61111680A (ja) * 1984-11-07 1986-05-29 Hitachi Ltd 細胞取扱い装置
JPS6349071A (ja) * 1986-08-19 1988-03-01 Shimadzu Corp 多極電極型細胞融合チヤンバ
US5304486A (en) * 1987-10-09 1994-04-19 Baylor College Of Medicine Method of and apparatus for cell portion and cell fusion using radiofrequency electrical pulse
WO1998056893A1 (fr) * 1997-06-10 1998-12-17 Walters Richard E Procede et appareil pour traiter des matieres avec des champs electriques a orientations differentes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0126389A2 (fr) * 1983-05-13 1984-11-28 Bayerische Julius-Maximilians-Universität Würzburg Enceinte pour le traitement de cellules dans un champ électrique
JPS60251874A (ja) * 1984-05-30 1985-12-12 Hitachi Ltd 微粒子取扱い装置
JPS60251877A (ja) * 1984-05-30 1985-12-12 Hitachi Ltd 細胞融合装置
JPS61111680A (ja) * 1984-11-07 1986-05-29 Hitachi Ltd 細胞取扱い装置
JPS6349071A (ja) * 1986-08-19 1988-03-01 Shimadzu Corp 多極電極型細胞融合チヤンバ
US5304486A (en) * 1987-10-09 1994-04-19 Baylor College Of Medicine Method of and apparatus for cell portion and cell fusion using radiofrequency electrical pulse
WO1998056893A1 (fr) * 1997-06-10 1998-12-17 Walters Richard E Procede et appareil pour traiter des matieres avec des champs electriques a orientations differentes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 010, no. 128 (C - 345) 13 May 1986 (1986-05-13) *
PATENT ABSTRACTS OF JAPAN vol. 010, no. 296 (C - 377) 8 October 1986 (1986-10-08) *
PATENT ABSTRACTS OF JAPAN vol. 012, no. 270 (C - 515) 27 July 1988 (1988-07-27) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004025046A1 (de) * 2004-05-18 2005-12-15 Forschungszentrum Karlsruhe Gmbh Einrichtung zum Elektroporieren von biologisch pflanzlichem Prozeßgut
WO2021151124A1 (fr) 2020-01-21 2021-07-29 Luong Thi Hong Lien Dispositif à plasma froid portatif

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