EP0266418A1 - Dispositif et procede de formation de pores dans des vesicules, ainsi que de chargement et de fusion desdites vesicules - Google Patents

Dispositif et procede de formation de pores dans des vesicules, ainsi que de chargement et de fusion desdites vesicules

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Publication number
EP0266418A1
EP0266418A1 EP87903578A EP87903578A EP0266418A1 EP 0266418 A1 EP0266418 A1 EP 0266418A1 EP 87903578 A EP87903578 A EP 87903578A EP 87903578 A EP87903578 A EP 87903578A EP 0266418 A1 EP0266418 A1 EP 0266418A1
Authority
EP
European Patent Office
Prior art keywords
chamber
vesicles
electric field
liquid suspension
charge
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP87903578A
Other languages
German (de)
English (en)
Other versions
EP0266418A4 (fr
Inventor
John Marshall, Iii
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Electropore Inc
Original Assignee
Electropore Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Electropore Inc filed Critical Electropore Inc
Publication of EP0266418A1 publication Critical patent/EP0266418A1/fr
Publication of EP0266418A4 publication Critical patent/EP0266418A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • 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

  • Field of the Invention - This invention relates to apparatuses and methods for use in the treatment of biological and non-biological vesicles which utilize electric field pulses to prealign vesicles, to introduce pores in and through the membranes of vesicles for the purpose of loading or unloading materials into the vesicles or for the purpose of fusing of two or more vesicular structures together and, in particular, to apparatuses and methods which utilize homogeneous and uniform electric fields to treat vesicles.
  • cells are suspended in any liquid media, electrolyte, non- electrolyte, or mixtures of electrolytes and non- electrolytes and then subjected to an electric field pulse.
  • Pulse lengths that is the time that the electric field has been applied to such cell suspensions, have varied in length, for example anywhere from about 10 nanoseconds to about 100 milliseconds.
  • the strength of the electric fields applied to suspensions during such poration processes has varied from between about 100 V/cm to about 30 KV/cm. Sale and Hamilton, "Effects of High Electric Fields on Micro-Organisms III. Lysis of Erthyrocytes and Protoplasts", Biochimica Et Biophysica Acta, 163 (1968) 37-43.
  • the electric field has been applied for such a length of time and at such a voltage that a set potential (a transmembrane potential between about 0.5 volt and about 2.5 volts) has been created across the membrane for a length of time adequate to create a pore in the membrane, as is well known in the art.
  • the electric field created across the membrane of a vesicle about 100 angstroms at 1 volt transmembrane potential is about 1 megavolt per centimeter.
  • the first is the phenomenon of dielectric breakdown, that is, the ability of a high electric field to create a small hole or pore in a thin membrane. Once a vesicle is porated it can be loaded or unloaded.
  • Zimmerman et al, in U.S. Patent 4,081,340 discloses that the "permeability" of cell membranes of cells in an electrically conductive solution can be increased by pumping them through an aperture separating two discrete volumes of electrolyte solution and two electrodes, wherein the electrodes apply an electric field to the cells as they traverse the aperture.
  • the presenb invention does not "increase the permeability" of cell membranes, but rather puts actual holes or pores in cell membranes as taught by Kinosita and Tsong, "Formation and Resealing of Pores of Controlled Sizes in Human Erythrocyte Membrane", Nature, Vol. 268, pg . 438-441, 4 Aug. 1977.
  • Kinosita and Tsong describe the use of a homogeneous, uniform electric field to create pores in the erythrocyte membrane.
  • At least two metallic electrodes protrude into this area to form a space in which the cells are exposed to an electrical field created by the electrodes. These electrodes are shaped to purposefully generate non-uniform fields. At the same time, the electrodes are connected to a device producing electrical voltage impulses in order to achieve electrical permeation.
  • the apparatuses and methods of the present invention utilize homogeneous, uniform electric field generation for treatment of biological and non- biological vesicles including prealignment of vesicles, poration of vesicles, loading of vesicles (and unloading) and fusion of vesicles.
  • the present invention improves upon the teachings of Stolley and Kinosita by providing: (a) rotational prealignment to increase the uniformity of poration of vesicles and the loading of vesicles over a whole sample providing high yield rates, (b) collection of vesicles in a uniform, homogeneous electric field in conjunction with a fusion pulse also generated uniformly and homogeneously, (c) processing of much greater volumes of vesicles and in much lower conductivity suspensions by switching large power signals at very fast switching times, and (d) an apparatus for magnetically producing uniform, homogeneous electric fields in an electrodeless chamber. None of these features of the present invention are found in any of the prior art references .
  • the present invention therefore, relates to methods and apparatuses for use in vesicle rotational prealignment, in vesicle bunching, in dielectrophoretic electroporation of vesicles, in cell hybridization (fusion), and in vesicle loading and unloading.
  • the present invention provides a solution to the prior art problem of only obtaining low yield rates in the treatment of vesicles and the prior art problem of being able to treat small volumes of vesicles especially in high-conductivity suspensions.
  • the chamber of the present invention is designed to contain large volumes of suspensions of living cells or other suspended living or non-living vesicles. Furthermore, high speed, high power pulses of electric charge or field can be uniformly and homogeneously applied throughout the entire suspension, excepting the actual imhomogeneties to the field created by the cells themselves, to uniformly treat (i.e., to prealign, to bunch, to porate, to fuse, etc.) large numbers of the vesicles.
  • the present apparatus and method is an identifiable departure from the scope and the spirit of the inventions described in Pohl U.S. Patents 4,441,972 and 4,476,004, and from other references which mandate the application of a non-homogeneous, non-uniform electric field to treat vesicles.
  • the present invention improves upon the teachings of Stolley and Kinosita and their limited use of uniform electric fields.
  • the present invention also applies the principle of the attractive polarization of cells which have been placed in uniformly generated high frequency electrical fields, which attraction is due to the fact that such cells produce attractive forces between one another as a result of the reformation of the uniform electric field caused by the presence of the cells themselves .
  • the pulse chamber of the present invention will consist, in general, of two preferred embodiments: (a) two spaced- apart, parallel electrodes and (b) an electrodeless hollow toroidal disposable chamber.
  • the electrodes are preferentially circular in cross-section and cap the ends of a cylindrical chamber, thus defining a cylindrical volume between them.
  • the volume of the cylinder will measure anywhere between about 0.5 milliliters and about 100 milliliters.
  • the two spaced- apart electrodes will, when subjected to an electric charge, create a uniform, homogeneous electric field across a test (particle-free) electrolyte solution located in the cylindrical gap between them.
  • the cylinder intermediate to the electrodes will be composed of electrically insulating material, such as glass, plastic or other non-conductive material with a low dielectric constant.
  • the exposed surfaces of the conductive electrodes which reside within the chamber cavity will preferably be composed of a material, such as platinum or gold, which is selected to minimize or prevent electrolytic decomposition of the vesicles or suspension on the electrodes in the chamber during electrical treatment. Electrical coupling contact between the electrodes of the pulse chamber and an electrical treating system are made, for example, using clips or clamping devices to each electrode, although permanent connections may also be made .
  • the electrodeless magnetic chamber of the present invention therefore avoids the decomposition problem of the a-for-esaid parallel plate electrode approach and
  • ID- * pr ⁇ vides a convenient disposable chamber.
  • capacitance of the chamber are chosen to match the impedance of the electrical source. This impedance matching will be accomplished, for example, by providing a coaxial or biaxial current feed path to the electrodes .
  • the electric charge source is designed to deliver a large range of currents, anywhere from about 0.1. ampere to about 100,000 amperes, depending upon the 5- conductivity a d amount of the pulsed suspension.
  • the delivery of high voltages also requires sophisticated switching techniques, as taught by the present invention.
  • the high voltage source will be connected to the chamber through a 0 triggered ionization breakdown such as that achieved with a moving spark gap (such as a gas filled relay), gas filled spark gap, vacuum spark gap, ignitron, high current series SCR stacks, or hot or cold cathode hydrogen thyratrons, as will be set forth in greater detail below.
  • the present invention therefore is capable of delivering at high speeds (i.e. , less than 50 nanoseconds) high current and high voltage electric field charges (i.e., at a power rate greater than 1 Megawatt) uniformly and homogeneously over large volumes (i.e. , greater than 1 milliliter) of suspensions containing vesicles to be treated. None of the prior references set forth a system for delivering such power levels, at such speeds, to such quantities of such highly conductive suspensions.
  • the electrical driving mechanism of Kinosita was a Cober 605P pulse generator having a maximum output of 2200 volts at 10 amps, and not being able to drive resistive or capacitive loads with impedances less than 50 ohms while maintaining rise time.
  • the present invention is capable of delivering up to 35,000 volts at currents up to 100,000 amperes in less than 50 nanoseconds.
  • the system of the present invention may optionally be used in conjunction with a high frequency random function generator for producing cell rotational alignment prior to poration.
  • Rotational alignment is the creation of torques on cells or other vesicles in suspension in the pulse chamber by means of high- frequency polarization of the suspended material.
  • rotational alignment will be accomplished utilizing a timer which will allow the controlled connection of a high-frequency electric field at selected voltage and frequency across the pulse chamber prior to poration treatment.
  • the dielectrophoretic bunching voltage will be applied across the pulse chamber long enough, and at a predetermined frequency, to maximize the number of cell pairs that are formed in the chamber.
  • more than one dielectrophoretic high frequency electric field may be applied by use of a random function generator under the teachings of the present invention. Then, after dielectrophoresis treatment is completed, the application of a high voltage pulse to the suspension will create mutual pores between pairs of conjoint vesicles. In a high percentage of such cases each pair of porated cells will fuse into one cell after a short period of time, generally about 1 to 20 minutes ⁇
  • the size of the pores created in cells or other vesicles will be controlled under the teachings of the present invention by varying three parameters of electric field exposure: pulse voltage, pulse duration, and rate of change of electric field rise time and fall time. Since the membrane of each type of cell or other vesicle will have characteristic elements of resistance and capacitance, each type of cell or vesicle will exhibit a characteristic time constant related to the resistance and capacitance of the cell's membrane or the vesicle's skin. The specific time constant of any cell membrane will be governed basically by a multiple of resistance and capacitance':
  • the capacitances of cell membranes are usually on the order of 1 microfarad per square centimeter of membrane.
  • the resistance of the membrane per unit area is likely to decrease. This requires in turn a faster rise of surrounding electric field such that transmembrane potential does not "bleed off" across the transmembrane resistance so quickly as to keep the membrane from reaching poration potential.
  • the smaller a cell is the faster the rise time necessary to reach a specific transmembrane potential.
  • a red blood cell is a very small mammalian cell. To maintain morphology, one maintains it in a low-resistance isotonic solution. It is a good example of a cell needing specialized high-power, fast rise time pulse techniques as taught by the apparatus of the present invention to achieve poration when porating milliliter or greater quantities of these cells.
  • the porated cells will be left in the suspension with open pores and stored at cool to ambient temperatures for a period of time.
  • the present invention loads cells with substances by first creating stable holes or pores in them, and then, much in the manner taught by Kinosita and Tsong, supra, lets intracellular and extracellular fluids intermix by passive diffusion and Brownian motion through the pores. This will result in the equilibration of the mixture within the cells and outside of the cells if the pores are left open long enough.
  • Cells can also be porated in an isotonic solution, centrifuged down, separated, and then resuspended in a solution which is to be loaded into the cells. Pores in cell membranes will then be resealed after a period of time at ambient temperature, or by gently heating them to some non-destructive temperature greater than ambient, most usually 37 degrees C.
  • cells undergoing treatment are not spheres or are not electrically or dielectrically spherical, but are aspheri ⁇ al, ellipsoidal or electrically or dielectrically aspherical in nature, they are generally distributed in random orientation in the suspension. Even where cells are structurally spherical, they may still be electrically aspherical due to cell membrane charge imbalances. Such cells will have a long axis and a short axis or a polarity, and as such, when a porating electric field is applied, differing transmembrane potentials develop, depending on which orientation of the cell is exposed to the electric field. This creates a problem if one wishes to uniformly porate all cells in the suspension.
  • cell membranes may also be porated under the teachings of the present invention by pulsed magnetic field techniques . As detailed below, this will be accomplished by loading an electrolytic cell suspension in a toroidal chamber that is exposed to rapidly changing magnetic flux fields. The flux vector of the magnetic field will be directed through the center of the toroid. This causes an electric gradient field to be generated through the cell suspension loaded in the toroidal chamber. All effects of poration, dielectrophoretic bunching, dielectrophoresis and cell fusion may- be achieved? however, voltages generated would now be impressed on a member which will serve much like the primary of a transformer coupling, rather than by impressing the voltage directly on chamber electrodes .
  • Fig. 1 is a front plan view, partially broken away and partially in cross-section illustrating the electroporation chamber of the present invention
  • Fig. 2 is a side view in elevation taken along
  • Fig. 3 is an enlarged cross-section of the block portion of the electroporation chamber of Figs. 1 and 2;
  • Fig. 4 is a partially schematic, partially diagrammatic representation of the electronic control system of the present invention.
  • Fig. 5a is a cross-sectional diagrammatic representation of spheroidal cells in a random suspension in a pulse chamber
  • Fig. 5h is a cross-sectional diagrammatic representation similar to Fig. 5a, but with the cells bunched by mutual dielectrophoresis into pairs and pearl chains;
  • Fig. 6 is a schematic representation of voltage vs. time during various operations of the apparatus of the present invention
  • Fig. 7a is an exaggerated cross-sectional diagrammatic representation of ellipsoidal cells in a random suspension in pulse chamber 4;
  • Fig. 7b is a cross-sectional diagrammatic representation, similar to Fig. 7a, in which the ellipsoidal cells are being acted upon by a voltage of an orienting frequency
  • Fig. 7c is a cross-sectional diagrammatic representation, similar to Fig. 7a in which the ellipsoidal cells have been oriented along their major axis
  • Fig. 8 is a representation, partially in cross- section, partially in phantom, of a modified system of the present invention for poration and fusion using magnetic fields;
  • Fig. 9 is a cross-sectional view taken along line 9-9 of Fig. 8;
  • Fig. 10 is a perspective view, partially broken away, of a toroidal pulse chamber for use with the system of Fig. 8;
  • Fig . 11 is a graph illustrating the controllable rise time of the uniform, homogeneous electric field of the present invention.
  • Fig. 12 is a schematic showing on alternate circuit for the charge accumulator of the present invention.
  • an electroporation or cell fusion chamber 1 which will apply electric fields uniformly and homogeneously to suspensions containing vesicles or other suspended media.
  • the vesicles in the suspension will create inhomogeneities to the field.
  • the chamber 1 includes a block 2 of dielectric - material such as polacetyl plastic, which is machined or molded to define a cylindrical central pulse chamber 4, bracketed by opposed threaded open ends 6 and 8 and including shoulders 22 and 24.
  • thread-ended electrodes 14 and 16 will each be screwed into threaded open ends 6 and 8, respectively, towards chamber 4 until they rest against shoulders 22 and 24 respectively, with the result that a fluid tight fit is maintained between the electrodes and the openings into which they are fitted.
  • This tight fit provides a fluid seal to prevent fluid suspensions from leaking from pulse chamber 4 through threading in open ends 6 and 8.
  • a specific distance "L" will be defined and maintained between the ends of electrodes 14 and 16 to define a specific size or volume for pulse chamber 4.
  • a fillhole 18 will be provided through block 2 to pulse chamber 4, to allow addition to or removal of suspensions from pulse chamber 4, for example, by the use of a hypodermic syringe.
  • the body of each electrode 14 and 16 will be formed of metal or other electrically conductive material.
  • electrode tip faces 10 and 12 will be coated with a thin film of gold, platinum, or other inert, stable, conductive material 13, in order to prevent electrolytic decomposition of liquid in the chamber or on the electrodes during electrical pulses .
  • the materials for the chamber and for the electrodes will also be chosen so that sterile conditions can be easily maintained, and preferably are composed of materials which can endure autoclave temperatures.
  • suspended cell media 28 are inserted into pulse chamber 4 through fillhole 18 by use, for example, of a hypodermic syringe, not shown. Once within pulse chamber 4, the suspension will be subjected to treatment, as described below.
  • the current feed path to electrodes 14 and 16 is coaxial with chamber block 2.
  • an outer current return path in the form of a cup 32 having a cylindrical conducting shell and closed end 36 is provided. End 36 will be in electrical contact with one electrode, in this case electrode 14, while conducting shell 32 substantially encloses chamber block 2 and electrode 16.
  • the distance "d" between shell 32 and the outer of chamber 2 and the distance "D" which is the chamber- block diameter might be adjusted, for example by selecting a cup having the correct dimensions, so that impedance matching of chamber 2 to various voltage/current generating sources, can be provided or low inductance can be maintained.
  • a high voltage connection will be made to chamber 1, for example, with a moving spark gap or high speed switch.
  • the spark gap 41 will consist, for example, of two spherical electrodes 42 and 44.
  • electrode 42 will be connected to charge accumulator 46, such as a capacitor which will serve as a voltage source to chamber 1 during poration.
  • the accumulator may be any conventional charge storage device.
  • Spherical electrode 42 will be designed for movement toward and away from electrode 44.
  • electrode 42 will be moved under control of trigger 50 toward electrode 44 as shown by arrow 43 until a spark is initiated between electrodes 42 and 44.
  • ionization breakdown between the electrodes quickly occurs and the full power of the charge is delivered to the opposing switch electrode and, thence, to the chamber in less than 50 nanoseconds.
  • This spark serves to connect chamber 1 to charge accumulator 46 through electrode 14.
  • an electronic pulse controller countdown timer 48 is started, as detailed below. Movement 43 of electrode 42 towards and away from electrode 44 will be provided, for example, by the use of a conventional solenoid driver with spring return, not shown.
  • moving spark gap 42 - 44 may be replaced by a high speed switch (e.g., spark gap, thyratrons or ignitr ⁇ ns) so that precision triggering of initial charge is accomplished.
  • a high speed switch e.g., spark gap, thyratrons or ignitr ⁇ ns
  • the system of the present invention is utilized for dielectrophoretic bunching, poration and/or fusion of suspensions in bunching, poration and/or fusion of suspensions in pulse chamber 4.
  • Dielectrophoretic bunching will be accomplished by imposing a high frequency AC electrical field uniformly across the suspension in chamber 4, whereby suspended, randomly distributed vesicles or particles between electrodes 14 and 16, as illustrated in Fig. 5a, attract to one another in a pearl chain, as illustrated in Fig. 5b.
  • This attraction occurs because each vesicle or particle in the suspension will be polarized on each cycle of the applied alternating uniform and homogeneous field.
  • vesicles or other particles suspended in pulse chamber 4 will, by their presence, affect the local electric field within chamber 4, with the result that local field inhomogeneities will be formed in what would otherwise be the homogeneous electric field generated between electrodes 14 and 16.
  • Non-spherical (ellipsoidal) cells will also be subject to being aligned in pulse chamber 4 of the present invention, by the application of the effect known as the Maxwell-Wagner dispersion, su ra.
  • an ellipsoidal cell 28e or particle placed in chamber 4 will experience different rotational forces 29 for different applied frequencies of AC electrical field.
  • such particles will be capable of being rotationally aligned in the presence of a uniform, homogeneous high frequency electric field as taught by the present invention. In operation two frequencies will be of importance for alignment of ellipsoidal cells.
  • At least one frequency that will align such cells between parallel electrode faces 22 and 24 with their major semi-axis orthogonal (90 degrees) to the electrode surfaces.
  • At least one second frequency will align such cells' minor axis orthogonal to the electrode surfaces 22 and 24.
  • erythrocytes rather than for inducing cell fusion when used only for times short enough for rotational alignment, but not long enough to produce mutual "dielectrophoretic bunching" attraction as discussed above.
  • extremely high voltage, high current pulses of specific short duration will be passed through electrodes 14 and 16.
  • Such current pulses of a specific length will be created using the high speed switching such as provided by spark gaps, thyratrons, ignitrons, etc. as set forth above.
  • Countdown timer 48 will initiate its operation in response to the flow of current through the spark gap between electrodes 42 and 44. When countdown timer 48 reaches zero it will activate high speed shorting switch 60. Shorting switch 60 will instantaneously impose a short circuit across chamber 1. This short circuit will cause even larger currents to flow from accumulator 46, but will "dump" its entire potential, and dissipate it as heat energy or discharge it to ground 70, to terminate the flow of current through chamber 1. However, at the time that current flow through chamber 1 is initiated, shorting switch 60 must be open in order to allow the current flow through chamber I. High speed switch 60 may also be a spark gap, ignitron or hydrogen thyratron. c. Electro-fusion
  • the operator will select one or more frequencies at frequency set 62, through frequency generator 64.
  • the frequency or frequencies will be selected to cause the randomly distributed cells, as shown in Fig. 5a, to dielectrophoretically bunch, attract one another, and to even form pearl chains after a period of time, as shown in Fig. 5b. 5
  • the selected frequency or frequencies will pass through closed high voltage isolation switch 66.
  • switch 66 When switch 66 is closed, one or more bursts of high frequency alternating current, each one or more cycle in duration, will be applied to the cell suspension in chamber 1.
  • isolation switch 66 will be opened to protect frequency generator 64 from the high voltage poration/fusion pulse.
  • chamber 1 has "to-be-treated" cells injected into pulse chamber 4 through fillhole 18.
  • Chamber 1, possibly including shell 32 is then connected to the apparatus via. electrodes 14 and 16.
  • Shell 32 is designed for ' insertion into, and easy high current electrical connection with the treating apparatus by means of low resistance mating connectors, not shown.
  • the "to-be- applied" voltage will be selected at voltage set 52 and accumulated in charge accumulator 46.
  • the pulse length is selected at pulse length set 58, but high speed shorting switch 60 is open in order to avoid short circuiting of the current which is applied to the chamber. Also, high voltage isolation switch is opened in order to protect frequency generator 64.
  • trigger control 50 is activated and electrode 42 begins to move towards electrode 44.
  • Trigger control 50 is connected over lines 51 and 53 to spark gap 41 and to isolation switch 66, respectively.
  • the electric potential in accumulator 6 will be sufficient to cause current to jump from electrode 42 to electrode 44.
  • treatment of suspended material in chamber 1, and countdown of timer 48 will be simultaneously initiated, and the cells within pulse chamber 4 will undergo poration treatment.
  • the start of this treatment will correspond to the initial vertical line 600 of t4 in Fig. 6.
  • high speed shorting switch 60 will then be closed, with the result that a short circuit will be imposed across chamber 1.
  • the short circuit will terminate the flow of current through the chamber and stop cell treatment at a time which will correspond to the second vertical line 610 of t4.
  • the same cells in the same chamber can be subjected to a second electroporation treatment, as at t6 of Fig. 6, in much the same manner and sequence as set forth above.
  • the cells are then removed from pulse chamber 4 by a hypodermic syringe inserted through fillhole 18, and the preponderance of the cells contain stable pores through their cell wall membranes.
  • the porated cells can then be treated, as set forth in the EXAMPLES, below.
  • the high speed, high current and high voltage system of the present invention can charge up a large amount of aqueous solution with a rise time of less than 50 nanoseconds to produce homogeneous poration in vesicles contained in the suspension.
  • ID frequencies' at frequency set 62 which frequencies will be selected to orient and/or cause dielectrophoretic couplin and pearl chaining of cells, as shown diagrammatically in Fig. 5b.
  • isolation switch 66 will be closed, thus allowing one or more high frequency bursts of alternating current (i.e., waveforms shown at times tl, t2 and t3 in Fig. 6) to be applied to the cells in chamber 1. During these bursts randomly distributed cells as shown in
  • Figure.5(a) will experience mutual dielectrophoresis or bunching. Nearest neighboring cells will attract one another forming pairs and then, if dielectrophoratic bunching treatment is applied longer, will form chains as shown in Figure 5(b) and as discussed above.
  • poration treatment will be carried out by the waveform shown at time t4 when the charge crosses from electrode 42 to electrode 44, as described above. If a second poration treatment is to be carried out by the waveform shown at time t6, then one or more 0 intervening dielectrophoretic bunching bursts may be applied, at time t5. When this operation is completed, a current rampdown may be allowed to occur as shown by waveform at time t7. After this operation is completed, then a substantial number of paired, porated cells with mutual pores will fuse into single cells, as described below in the Examples.
  • the circuit of the present invention will permit very high voltage, high current pulses (i.e., greater than 1 Megawatt in power rate) to be applied for accurately controlled lengths of time (i.e. , less than 50 nanoseconds) to samples in the pulse chamber 4.
  • the apparatus of the present invention can be scaled up to process vast amounts of solution as long as one uses the aforesaid high voltage, high current, high speed switching techniques, i.e., switches that operate on a spark or fast ionization breakdown and maintains a source impedance that is reasonably matched to load impedance.
  • the current source including switching must have a very low impedance at these high currents such that all the field drop does not happen in the source. At 16,000 amperes, just 0.1 ohm of impedance would produce a voltage drop of 1600 volts, an unacceptable loss in volume processing. In scaling this further, it can be seen that a volume of 650 ml of isotonic suspension in chamber 4 requires a current of 100,000 amperes to bring the suspension to an electric field stress of 10,000 volts/cm in a chamber with .33 cm gap.
  • the charge accumulator was a low inductance capacitor. The capacitor may also be replaced by a pulse forming network as detailed in Figure 12.
  • a pulse forming network is a set of equal valued inductors 1200 and capacitors 1210 configured such that when charged to a voltage V by supply 56 and then connected to a load (such as a pulse chamber 4) the network will deliver a fixed length pulse of amplitude V/2.
  • n the number of capacitors
  • L inductance in micro-Henries
  • C capacitance in micro-Farads
  • the re s i s tance o f the chamber 4 and the resistance Rl should be approximately equal .
  • a small capacitance Cx may be added and should approximate the capacitance of the chamber.
  • the rise time in a high power system of this sort can then be controlled by the pulse shaper 49 as shown in Figure 11 and discussed later.
  • Cx is a high power vacuum capacitor.
  • Lx is a single turn of bus wire where the inductance varies by moving a piece of magnetic core material in or out of the one-turn inductor.
  • controllable rise time is an important factor for the uniform poration of the vesicles in the suspension.
  • the special switching techniques of the present invention are necessitated.
  • the water which is molecularly very polar molecule begins to line up.
  • the water molecules take a set time at a set temperature to line up and that time is about 250 nanoseconds at room temperature (20 degrees Celsius). Therefore, the capacitance "C", of a parallel plate pulse chamber filled with water where the electrode discs have a radius of 3.1 cm and are spaced apart by .33cm (this chamber being one that holds 10 ml) .
  • SUBSTITUTESHEET charging duration The necessary current to charge the capacitance is then in the order of 2500 - 3000 amperes over the first 30 or 40 nanoseconds.
  • the general resistance of the chamber with this volume of isotonically suspended cells is 2 ohms.
  • Toroidal Chamber In an alternative embodiment of the present invention, poration and fusion of cells will be energized by magnetic means in a toroidal chamber. Referring to Figs. 8, 9, and 10 there is shown magnetic core 82, including C-shaped conductive member 84, to which closure arm 86 will be swingably connected at pivot 88 and disconne ⁇ table at connecting point 92. When arm 86 is disconnected at connecting point 92, hollow toroidal chamber 94 will be capable of being slid onto or off of arm 86. Chamber 94 will be designed for the insertion and removal of "to-be- treated" electrolytic suspensions through fillhole 96.
  • Half-wind current sheaths 98 and 102 will be wound on core 82, which sheaths, when current is applied with arm 86 closed, will induce magnetic flux around core 84 and arm 86. It is to be expressly understood, that the use of two sheaths is a preferred embodiment and that a single sheath by one skilled in the art could be utilized.
  • Wires 104 and 106 may be connected to a power system, such as that shown in Fig. 4, in place of chamber 1, with wires 104 and 106 electrically in series with high voltage electrode 44, and also to ground 70.
  • the interconnection for the sheaths 98 and 102 are shown in Figure 9.
  • the operation of this magnetic system will be much like that set forth with regard to Figure 4. In the operation of this magnetic system, current pulses passing through the two half- sheaths 98 and 102 around core 84, will induce a large magnetic flux 103 in the conductive material of core 84 and arm 86.
  • This magnetic flux flowing through arm 86 will pass through the annulus of toroid 94 and induce a large electric field along the length of the solution contained in toroidal chamber 94 which is orthogonal to the core 84.
  • This voltage pulse will be controlled, as in Fig. 6, and will be large enough to produce poration and/or fusion in the cells of the solution.
  • This . modified embodiment has the benefit of allowing the use of simple, inexpensive glass or plastic toroidal chambers 94, which chambers can be used once and then disposed of, without the need for electrodes or electrical wiring directly to the chambers. Hence, this embodiment provides an inexpensive chamber having many applications both in the laboratory and for industrial and commercial uses.
  • Intact erythrocytes are isolated from freshly drawn heparinized blood by centrifugation at 1000 x g for 10 minutes at 4 degrees C. The serum and buffy coat (other blood components) are then decanted from the top of the centrifuge tube. The erythrocytes or red blood cells are resuspended and recentrifuged in ten volumes of physiological saline. The foregoing procedure is repeated three times to wash away all other blood constituents. The washed erythrocytes are then resuspended in physiological saline, with the red blood cells occupying about one-tenth to one-half of the total volume.
  • the resulting suspension is then put in electrical pulse chamber 4 by means of a syringe through fillhole 18.
  • the temperature of the suspension may be from about 4 degrees C. to about ambient temperature for poration, but the higher the temperature of the suspension, the shorter will be the time that the pores which will be electrically induced in the cells remain open.
  • the pulse chamber is connected to a plug-in pulse module, not shown.
  • the module is designed to be slid into a port in the front of the pulse apparatus to complete electrical connection to the module.
  • "poration and loading” 2 ml of packed, washed human red blood cells are suspended in 8 ml phosphate buffered saline solution (PBS). A physiological saline well suited for maintaining the morphology of red blood cells.
  • PBS phosphate buffered saline solution
  • a physiological saline well suited for maintaining the morphology of red blood cells.
  • the resulting 10 milliliters of suspension is then injected into electrical pulse chamber 4 and chamber
  • the chamber containing the module is removed from the pulse apparatus port, and the pulse chamber is removed from the module.
  • the suspended red blood cells are then removed from the chamber with a syringe and are found to be porated.
  • the porated red blood cells are separated by centrifugation, and are then suspended in a media containing methotrexate, a 455 molecular weight chemotherapy drug. After such suspension a time of about 15 to 30 minutes is allowed to elapse.
  • the suspension is heated to a temperature of 37 degrees C. to close or anneal the pores in the cell membrane.
  • the cells are then washed by centrifuging and resuspending them three times in buffered physiological saline and are ready for use as a carrier for in-vivo injection and timed release of encapsulated drug into a patient's blood stream.
  • Typical loadings have injected about 5 x 10 ⁇ molecules of methotrexate per red blood cell about twice the loadings obtained with poration without rotational pore-alignment.
  • High frequency generator 64 is connected to the chamber by isolation switch 50.
  • the high frequency voltage is applied at 33 volts for .1 seconds at a frequency of 2 MHz.
  • the suspended cells align rotationally as shown in Figure 7.
  • Isolation switch 50 is then opened and as soon as the switch contacts have moved far enough apart to isolate the RF generator 64 the HV pulse switch 41 is closed. The chosen pulse is then applied.
  • No count down timer 48 or shorting switch 60 is necessary when using a pulse forming network charge accumulator of Figure 12. Identical results to previous example are realized in loading of red blood cells.
  • the upper epidermis of leaves from light-grown, 6-day-old oat seedlings (Avena sativa, cv Victory) and 8-day-old corn seedlings (Zea mays, cv Bear Hybrid) are removed with fine forceps.
  • the peeled leaves are then floated, upside down, on a solution containing 2% (w/v) Cellulysin (Calbiochem) , 0.5 M containing 2% (w/v) Cellulysin (Calbipchem) , 0.5 M mannitol, 3mM CaC12, ImM KC1, and 3mM Mes (morpholinoethanesulfoni ⁇ acid) at pH 5.6.
  • the protoplasts are then filtered through a nylon screen (pore diameter 80 urn), layered onto a 17% (w/v) sucrose pad, and centrifuged 10 min at lOOg.
  • the protoplasts at the interface are collected, resuspended in 12 ml of 0.5 M mannitol and centrifuged for 3 minutes at 70g. The pellet is then washed once with 0.5 M mannitol.
  • the oat protoplasts are then suspended in a 0.5 molar sucrose solution and are administered into the fusion/poration chamber with a syringe.
  • the chamber is then connected into the pulse/fusion instrument as described above.
  • the protoplasts are then suspended with 10 x cells per ml. and are treated at a frequency of 2 MHz applied at 70% duty cycle for 10 seconds at a field strength of 30 volts/cm to provide "bunching" or dielectrophoretic voltage.
  • the duty cycle, amplitude time of the alignment voltage may be changed, but the net desired effect is to maximize the creation of cell pairs.
  • the isolation switch After the application of the bunching voltage, the isolation switch is allowed to open, then the poration/fusion pulse is applied. Poration of cells occurs and fusion occurs between cell pairs.
  • the bunching voltage is then reapplied and slowly ramped down over the next two minutes to hold fused cells in close contact and to keep lightly fused cells from coming hack apart due to thermal, mechanical and other naturally occurring perturbations.

Abstract

Dispositif à haute vitesse et à haute tension utilisant des champs électriques homogènes et uniformes pour traiter des vésicules dans une suspension. Dans une forme d'exécution, des électrodes parallèles (14 et 16) sont utilisées pour effectuer le groupement diélectrophorétique, le préalignement rotatif et l'électrofusion des vésicules ainsi que la création de pores dans lesdites vésicules. Dans une autre forme d'exécution, un dispositif magnétique sans électrode est utilisé pour effectuer le traitement. Les deux formes d'exécution sont alimentées par un système d'alimentation électronique à haute vitesse et haute tension utilisant un système (41) de distribution de rupture à ionisation déclenchée.
EP19870903578 1986-05-09 1987-05-08 Dispositif et procede de formation de pores dans des vesicules, ainsi que de chargement et de fusion desdites vesicules. Withdrawn EP0266418A4 (fr)

Applications Claiming Priority (2)

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US86153486A 1986-05-09 1986-05-09
US861534 1986-05-09

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EP0266418A4 EP0266418A4 (fr) 1989-02-13

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WO (1) WO1987006851A1 (fr)

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Publication number Priority date Publication date Assignee Title
GB8807271D0 (en) * 1988-03-26 1988-04-27 Preece A W Cell fusion apparatus
EP0866123B1 (fr) * 1997-03-21 2005-03-16 Eppendorf Ag Procédé et dispositif de circuit pour l'electropermeation ou l'electroporation de cellules vivantes

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Publication number Priority date Publication date Assignee Title
GB1481480A (en) * 1974-02-02 1977-07-27 Kernforschungsanlage Juelich Process and apparatus for increasing the permeability of the membrane of cells of organisms
DE2656746C2 (de) * 1976-12-15 1986-06-26 Kernforschungsanlage Jülich GmbH, 5170 Jülich Verwendung von beladenen Erythrozyten
US4326934A (en) * 1979-12-31 1982-04-27 Pohl Herbert A Continuous dielectrophoretic cell classification method
US4578167A (en) * 1982-09-28 1986-03-25 Biofusion, Inc. Cell fusion
DD213360A1 (de) * 1983-02-02 1984-09-12 Adw Ddr Vorrichtung zur dielektrophoretischen sammlung suspendierter teilchen
US4441972A (en) * 1983-04-08 1984-04-10 D.E.P. Systems, Inc. Apparatus for electrofusion of biological particles
US4476004A (en) * 1983-04-08 1984-10-09 D.E.P. Systems, Inc. Apparatus for electrofusion of biological particles
US4578168A (en) * 1984-07-27 1986-03-25 Biotronics Apparatus for fusing live cells with electric fields

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Title
BIBLIOTHECA HAEMATOLOGICA, no. 51, 1985, pages 108-114, Karger, Basel, CH; T.Y. TSONG et al.: "Use of voltage pulses for the pore opening and drug loading, and the subsequent resealing of red blood cells" *
NATURE, vol. 268, no. 5619, 4th August 1977, pages 438-441, London, GB; K. KINOSITA et al.: "Formation and resealing of pores of controlled sizes in human erythrocyte membrane" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 74, no. 5, May 1977, pages 1923-1927, Washington, D.C., US; K. KINOSITA et al.: "Hemolysis of human erythrocytes by a transient electric field" *
See also references of WO8706851A1 *

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WO1987006851A1 (fr) 1987-11-19
AU7433787A (en) 1987-12-01

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