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WO1992006185A1 - Procede d'electroporation utilisant des champs electriques bipolaires oscillants - Google Patents

Procede d'electroporation utilisant des champs electriques bipolaires oscillants Download PDF

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Publication number
WO1992006185A1
WO1992006185A1 PCT/US1991/006884 US9106884W WO9206185A1 WO 1992006185 A1 WO1992006185 A1 WO 1992006185A1 US 9106884 W US9106884 W US 9106884W WO 9206185 A1 WO9206185 A1 WO 9206185A1
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WIPO (PCT)
Prior art keywords
bipolar
biological particles
electric field
cells
poration
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PCT/US1991/006884
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English (en)
Inventor
Ephrem Tekle
P. B. Chock
R. D. Astumian
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The United States Of America, Represented By The Secretary, U.S. Department Of Commerce
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Application filed by The United States Of America, Represented By The Secretary, U.S. Department Of Commerce filed Critical The United States Of America, Represented By The Secretary, U.S. Department Of Commerce
Publication of WO1992006185A1 publication Critical patent/WO1992006185A1/fr

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    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the present invention relates to the field of poration and fusion of biological cells. More particularly, the present invention relates to a method of electroporation and fusion which utilizes high freguency bipolar oscillating electric fields to per eabilize biological cells.
  • Electric field induced per eabilization of cell membranes is an important technique for gene transfection and cell hydridization.
  • Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed.
  • Typical parameters reported to have improved the efficiency of transfection in some cell types include lowering of the temperature during and after the electric pulse, use of linearized plasmids, adjustments of the ionic strength of the pulsing medium, addition of carrier DNA, use of actively growing cells, and post-pulse chemical treatment. While some of these optimization techniques have resulted in considerable improvements in the cell lines used, others have, nevertheless, been shown to have either no or the opposite effect when used on other cell types.
  • Electric field of sufficient magnitude and duration has been shown to create transient pores in cellular membranes. This method has been widely used for introducing DNAs, allosteric effectors, antibodies and other macromolecules into living cells either by molecular exchange between intra and extracellular media or by cell to cell fusion. In many instances, these field methods have proved to be superior to the more conventional methods of chemical or viral-induced cell transfection and fusion. Some studies further suggest that electric fields could be used to modulate the activities of membrane enzymes in transducing biological signal and energy.
  • the present invention is an improvement over prior electro-per eabilizations which allows for a number of advantages over prior methods. Disclosure of the Invention
  • Another object of the present invention is to provide a method of electroporation which increases cell survival and transfection efficiency.
  • a further object of the present invention is to provide a method of electroporation and fusion which utilizes bipolar oscillating electric fields having square bipolar waveform, or other bipolar periodic waveforms.
  • a still further object of the present invention is to provide a method of electroporation and fusion which permeabilizes cell membranes symmetrically.
  • a method of causing poration of biological particles comprising the steps of: positioning a plurality of biological particles between electrodes; and applying a bipolar oscillating electric field of sufficient strength to the plurality of biological particles to cause poration, wherein the bipolar oscillating electric field is either a square bipolar waveform, or other bipolar periodic waveform.
  • the present invention further provides a method of causing poration of biological particles, comprising the steps of: positioning a plurality of biological particles between electrodes; and subjecting the plurality of biological particles to an electric field having a sufficient strength to cause fusion of at least a portion of the cells.
  • Figure la is a block diagram illustrating an electro-permeabilization apparatus according to one embodiment of the present invention.
  • Fig. la shows the connections arranged to produce bipolar pulses.
  • Figure lb shows oscilloscope traces of the different waveforms used in the present study.
  • Figure 2 shows results of a typical electro- permeabilization experiment of adherent cells on a culture dish.
  • Figure 3 shows reversible electro- permeabilization of cells utilizing different wave forms including the preferred waveform of the present invention.
  • Figure 4 shows the relationship between transfection efficiency and percent survival for various waveforms including the preferred waveform of the present invention.
  • Figure 5 shows selected videoframes of electro- permeabilized cells subjected to electric field of various waveforms according to the present invention. Best Mode for Carrying out the Invention
  • the electroporation method of the present invention involves applying a bipolar oscillating electric field of sufficient strength to biological particles including cells in suspension and cells attached in a cell culture to cause poration of the particles.
  • the bipolar oscillating electric field is unique in that it can easily permeabilize cells at multiple positions and retain high survivability.
  • the bipolar oscillating electric field may have a frequency of about 10 kHz to about 1000 kHz. However, a frequency of about 10 kHz to about 250 kHz is preferred, with afrequency of about 10 kHz to about 100 kHz being more preferred, and a frequency of about 60 kHz being most preferred for present applications.
  • the strength of the bipolar oscillating electric field may be between about 0.8 kV/cm to about 12.5 kV/cm, with a strength of about 2.2 kV/cm being preferred for present applications.
  • the square bipolar waveform may have a peak-to- peak amplitude of up to 5kV and a pulse duration of about 50 ns to about 10ms, with a preferred pulse duration being about 400 ⁇ sec.
  • the electroporation method of the present invention has been found to be particularly advantageous in that the optimum transfection efficiency is obtained at a relatively high cell survival when utilizing a bipolar oscillating electric field having a square bipolar waveform.
  • Cell survival is increased probably because the cell membranes are not polarized for extended periods of time and hence are not susceptible to irreversible damage.
  • Transfection efficiency is increased because it has been discovered that when utilizing a bipolar oscillating electric field having a square bipolar waveform poration occurs symmetrically at the two hemispheres of the cells which face the electrodes.
  • X is the relaxation time
  • f the frequency of the applied field
  • R the radius of the cell
  • E the electric field strength
  • ⁇ is the angle between the field direction and any point on the cell membrane.
  • membrane breakdown is essentially asymmetric with unipolar a.c. or single d.c. electric fields and that symmetrical permeabilization occurs only with bipolar a.c. fields.
  • the electric pulses were produced by serially gating several square wave generators as follows:
  • the total on-time of the bipolar a.c. burst is controlled by a single square pulse generator (HP-8011A) of variable pulse duration (amplitude 5V) whose output gates the pulse frequency generator (Wavetek 183) equipped with a frequency dial adjustable from d.c. to IMHz.
  • the rising edge of each incoming single square pulse from the pulse frequency generator (amplitude 10V) triggers the high power positive pulse generator (amplitude 10V) triggers the high power positive pulse generator (Cober 606P) .
  • the amplitude and duration of the pulse from the Cober is independently adjustable from 0 to +2.5 kV, and 50 ns to 10ms, respectively (for both unipolar and bipolar a.c. pulses, however, the period is limited by the repetition rate selected on the pulse frequency generator) .
  • the falling edge of a synchronous 10V square pulse from the Cober is then allowed to trigger the second high power pulse generator (Cober 606P) with similar adjustable output (but with amplitude of 0 to -2.5kV). Combination of these pulses finally produces the desired bipolar waveform of peak-to-peak amplitude of up to 5kV.
  • the maximum delay in the trigger mechanism between the two high power generators is less than 0.5 ⁇ s.
  • the magnitude of the electric pulses were measured across the electrodes with a 1000:1 probe (Tektronix P6015) and monitored on a storage oscilloscope (Tektronix 7704A) .
  • the electrodes for the instrument were constructed out of 50mm long stainless steel and were held fixed on a Kel-F support in grooves with a separation of either 1 or 2mm.
  • a culture dish containing cells grown in monolayer or suspended in droplets was placed on a platform which could be moved vertically to make contact with the electrodes.
  • horizontal movement of the platform allows for up to twelve experiments (using 2mm electrode separation) to be performed on the same culture dish (8cm diameter) .
  • Another cell holding chamber was constructed using a pair of stainless steel electrodes sandwiched between two microscope slides (electrode separation 0.75mm) with a capacity to hold 25 ⁇ l of cell suspension.
  • the chamber was mounted on a ZEISS (ICM405) inverted low light fluorescence microscope.
  • the increase in fluorescence intensity, monitored at 610nm (excited at 520) that results from the binding of ethidium bromide (Sigma) to cytosolic DNA or RNA was used as indicator of electro-permeabilization.
  • Real time images of these events were acquired with an image intensifier (Videoscope International, KS-1381) attached to a CCD camera (COHU, 4815) and recorded on a videotape (Sony, VO-5600) .
  • the time resolution of the videoscope recordings was 33ms.
  • the recorded images were later transferred to a digital disk recorder (Panasonic, TQ2028F) and analyzed with a digital image processor (Recognition concepts, 55/48Q) using the RTIPS software library from TAU Inc. Some of the video images in the time series were then displayed on a color monitor and photographed by transferring to a freeze frame recorder (Polaroid Corp.)
  • DMEM Dulbecco's modified Eagle's medium
  • Forma Dulbecco's modified Eagle's medium
  • Adherent cells were removed from the culture dish with 0.25% trypsin solution and washed three times with a pulsing buffer which consisted of 250mM sucrose, lOmM phosphate, ImM MgCl 2 , pH 7.2. The number of harvested cells was determined in at least three readings using a hemocytometer chamber and the concentration was adjusted by appropriate dilutions to be 10 6 cells/ml. Cells were kept at ice-cold temperature just prior to both DNA transfection and permeabilization experiments (using ethidium bromide) . For experiments performed on cells adherent to the culture dish, the growth medium was substituted with 4ml of cold pulsing buffer. The viability of cells suspended in the pulsing buffer was not altered for up to two hours.
  • the circular plasmid DNA, pSV2-neo, ( ⁇ 8kb) was kept frozen prior to use.
  • Powdered antibiotic G-418 sulfate was obtained from GIBCO. Its specific activity was 516.7 ⁇ g per mg of powdered compound.
  • a stock solution of selection media was prepared by first dissolving the antibiotic in serum free media which was then filtered using 0.2 micron filter. The filtered solution was later supplemented with 10% calf serum. The final concentration of G-418 was 400 ⁇ g of active antibiotic per ml of medium.
  • Cells were then diluted with 5ml of growth medium on the same culture dish and distributed in a 96- well flat bed chamber (Costar) at 10 3 cells per well and kept in the incubator for 6-8 hours. After this incubation period, the medium was aspirated off and replaced with selection medium at lOO ⁇ l per well. Every 2 days the selection medium was replaced with fresh medium and after 14 days the number of G-418 resistant colonies was counted in each well.
  • Costar flat bed chamber
  • Permeabilized sites on the membrane were identified using the increase in fluorescence intensity resulting from the binding of ethidium bromide to cytosolic DNA/RNA as described earlier.
  • the concentration of ethidium bromide ranged from 0.01% to 1%.
  • the relative efficiency of transfection of many cell types may depend on a multitude of parameters (i.e., temperature, form of DNA, ionic strength, etc.). However, in most reported experiments involving transfections, the critical parameters have generally been the electric field strength and duration. Therefore, comparative studies of transfection efficiency obtainable between different waveforms were based on these parameters.
  • FIG. 2 A typical representative example of electro- permeabilization of cells grown in a culture dish as a function of electric field is shown in Fig. 2.
  • Fig. 2 the dark stripes show cells that lay between the two parallel electrodes that have been stained with trypan blue.
  • the dye was introduced 1 hour after the termination of the electric pulse and labeling was continued for 30 minutes after which the cells are washed and photographed against a white background.
  • the waveform utilized was a d.c. pulse having a pulse width of 400 ⁇ s.
  • NCBEP Numberer of cells before electric pulse
  • BAC Biter of cells before electric pulse
  • UAC Unipolar A.C
  • DC D. ⁇ e electric field for the bi olar a.c. field is evaluated from the eak-to- eak a lied volta e.
  • Viability (% survival) was quantitated by counting the percentage of cells excluding trypan blue that was introduced 1 hour after the termination of the electric pulses. In a separate experiment, it was found that no further improvements in viability for cells incubated for longer than 1 hour.
  • the viability of cells was found to decrease with increasing electric field. Cells were, however, found to survive more in the oscillating pulses than the d.c. pulse of the same peak amplitude.
  • the survivability data shown in Table 1 corresponds to electric field values computed from the peak-to-peak applied voltage. If these field values are recalculated from the amplitude of the half cycle, the survivability is found to be still better than in the d.c. pulse and lower but comparable to the unipolar a.c. pulse.
  • Fig. 3 The transient behavior of electric field induced permeabilization was monitored through time course measurements. When the electric field is not exceedingly high or the pulse width too long, permeabilization of the cell membrane was found to be reversible. This is demonstrated in Fig. 3 for the three waveforms studied. In Fig. 3 cells were permeabilized using the optimum field strength found for transfection for each waveform shown. Pulse width: 400 ⁇ s. Frequency: 60kHz. a) Bipolar a.c. field: 2.2kV/cm. b) Unipolar a.c. field: 1.6kV/cm. c) d.c. field: 1.2kV/cm. Trypan blue was added after the electric field application at the times shown. Permeabilized cells were quantitated by counting the number of stained cells.
  • the optimum transfection were found at electric field strength of 1.2, 1.6 and 2.2kV/cm for d.c, unipolar and bipolar pulses respectively. At these field strengths, about 60% of the cells were viable for both unipolar and bipolar pulses but only about 40% were viable for the d.c. pulse. Under these optimal conditions for each waveform, the transfection efficiency with the bipolar a.c. pulse was 1.7 and 5.5 fold greater than the unipolar and d.c. pulses respectively. When the frequency (60KHz) and the pulse width
  • the efficiency was found to very low if either cell survival was above 80% or below 20% for all three waveforms. It is likely that at low electric field strength (even though the membrane is permeabilized (e.g., at 0.8kV/cm)) the pore sizes may not be large enough to allow passage of the plasmid thus decreasing the efficiency. On the other hand, cell death may be the factor for the decreased efficiency at higher electric field strengths.
  • the transfection efficiency was tested as a function of the plasmid concentration at a field strength of 1.6kV/cm with the frequency and pulse width set at 60KHz and 400 ⁇ s, respectively (Table 1) .
  • Fig. 5 depicts selected video frames of both the location and time course of electro-permeabilized NIH3T3 cells subjected to various waveforms of different amplitude and frequency.
  • the positive electrode is at the bottom and the negative electrode is at the top of each frame displayed.
  • the first row for each waveform shows the prepulse image where the nucleus is relatively bright due to the slow uptake of the ethidium bromide during sample preparation.
  • the prepulse image has been subtracted.
  • the fluorescence is color coded in increasing intensity in the following order: blue, purple, gray, yellow, red, and white.
  • Pulse duration 0.400 msec.
  • the data clearly show that the cell membrane is permeabilized at either one hemisphere (that facing the positive electrode) or asymmetrically when the applied electric pulse is unipolar or d.c.
  • Fig. 5 shows results from an unipolar a.c. field with a frequency of 250KHz and electric field magnitude of l.lkV/cm, which is near the field strength required for membrane breakdown (about 0.8 - l.OkV/cm).
  • l.lkV/cm the field strength required for membrane breakdown
  • the entry positions on the cell surface remain unchanged, the permeabilized area increases with increasing electric field.
  • the applied electric field is greatly enhanced, significant permeabilization is observed at both hemispheres facing the electrodes.
  • the permeabilization however, remains asymmetric with the effect being more pronounced at the site facing the positive electrode than the other. This was demonstrated using either d.c. or unipolar a.c. fields.
  • a negative resting potential means that the equilibrium electric field in the membrane is directed inwards.
  • the sign of the imposed membrane potential is (+) inside and (-) outside on the pole of the cell membrane facing the negative electrode and the opposite is true on the other pole facing the positive electrode.
  • the field vectors are addictive and greater than V c at the membrane site facing the positive electrode, while they cancel on the membrane site facing the negative electrode and resulted with a net potential being less than V c .
  • Electro-permeabilization of cell membranes for gene transfection is increasingly being preferred over other chemical techniques partly because of its ease of operation and increased yields.
  • the present invention provides a new and improved method of electro- permeabilization using bipolar a.c. pulses.
  • the efficiency of transfection using the present method provided improvements of 1.7 and 5.5 fold over the conventional unipolar a.c. and the d.c. square pulse techniques, respectively under optimal conditions.
  • the electric field strength and to a relatively lesser extent the pulse width and frequency, were found to be the critical factors with the greatest influence on the transfection efficiency.
  • the transfection efficiency was sensitive in a narrow window of electric field strength (about 1 to 1.6kV/cm; bipolar pulse expressed from half height) .
  • transfection data 60KHz and 400 ⁇ s at this critical electric field region appears to correlate with cell survivability. This situation was observed when the pulse width was increased to 5ms.
  • the transfection efficiency is proportional to the amount of plasmid DNA introduced into the cell when comparable number of transfectable cells are available, then it is reasonable to expect the bipolar a.c. pulse to be more effective than the unipolar a.c. or d.c. method since it has been shown that the present method permeabilizes the membrane symmetrically. Together with the qualitative and quantitative results presented in the course of the present invention, it was concluded that permeabilization of the membrane at multiple sites without affecting cell viability may account for the observed improvements in the transfection efficiency (i.e., the number of cells transformed per ⁇ g of DNA used) of electro-permeabilized NIH3T3 cells with bipolar a.c. fields compared to single d.c. or unipolar a.c. fields or other waveforms.

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Abstract

Procédé d'électroporation de particules biologiques qui consiste à appliquer un champ électrique bipolaire oscillant d'une intensité suffisante aux particules biologiques pour causer la poration des particules, le champ électrique bipolaire oscillant présentant soit une forme d'ondes bipolaire carrée soit d'autres formes d'ondes périodiques bipolaires. Le procédé d'électroporation augmente la survie des cellules et l'efficacité de transfection. Le procédé produit la poration symétrique aux deux hémisphères de cellules qui font face aux électrodes appliquant le champ électrique. Ce procédé présente aussi des avantages analogues relatifs au fusionnement des cellules.
PCT/US1991/006884 1990-09-27 1991-09-26 Procede d'electroporation utilisant des champs electriques bipolaires oscillants WO1992006185A1 (fr)

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US588,998 1990-09-27

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000871A3 (fr) * 2000-06-27 2002-10-17 Amaxa Gmbh Procede permettant d'introduire des acides nucleiques et d'autres molecules actives d'un point de vue biologique, dans le noyau de cellules eucaryotes superieures, grace a un courant electrique
WO2006059084A1 (fr) * 2004-11-30 2006-06-08 The University Court Of The University Of St Andrews Photoporation de cellules
US7704743B2 (en) * 2005-03-30 2010-04-27 Georgia Tech Research Corporation Electrosonic cell manipulation device and method of use thereof
US7732175B2 (en) 2004-06-14 2010-06-08 Lonza Cologne Ag Method and circuit arrangement for treating biomaterial
US8173416B2 (en) 2001-04-23 2012-05-08 Lonza Cologne Gmbh Circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryotic cells using electrical current

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5007995A (en) * 1989-05-11 1991-04-16 Olympus Optical Co., Ltd. Device for electrofusion of cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5007995A (en) * 1989-05-11 1991-04-16 Olympus Optical Co., Ltd. Device for electrofusion of cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 114, No. 3 issued, 21 January 1991, XIE et al., "Study of Mechanisms of Electric Field-Induced DNA Transfection II. Transfection by low Amplitude, Low Frequency Alternating Electric Fields" see page 210, column 1, abstract No. 18900; & BIOPHYS J., 58(4), 897-903. *
PLANT CELL REP., Volume 8, issued May 1990, JOERSBO et al., "Direct Gene Transfer to Plant Protoplasts by Electroporation by Alternating, Rectangular and Exponentially Decaying Pulses", pages 701-705. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000871A3 (fr) * 2000-06-27 2002-10-17 Amaxa Gmbh Procede permettant d'introduire des acides nucleiques et d'autres molecules actives d'un point de vue biologique, dans le noyau de cellules eucaryotes superieures, grace a un courant electrique
US8173416B2 (en) 2001-04-23 2012-05-08 Lonza Cologne Gmbh Circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryotic cells using electrical current
US7732175B2 (en) 2004-06-14 2010-06-08 Lonza Cologne Ag Method and circuit arrangement for treating biomaterial
US8058042B2 (en) 2004-06-14 2011-11-15 Lonza Cologne Gmbh Method and circuit arrangement for treating biomaterial
WO2006059084A1 (fr) * 2004-11-30 2006-06-08 The University Court Of The University Of St Andrews Photoporation de cellules
US8080399B2 (en) 2004-11-30 2011-12-20 The University of Court of the University of St. Andrews Photoporation of cells
US7704743B2 (en) * 2005-03-30 2010-04-27 Georgia Tech Research Corporation Electrosonic cell manipulation device and method of use thereof

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