WO2007012377A2 - Procedes d'electromanipulation de cellules pour modifier la permeabilite membranaire de ces cellules par adjonction d'ions lipophiles - Google Patents

Procedes d'electromanipulation de cellules pour modifier la permeabilite membranaire de ces cellules par adjonction d'ions lipophiles Download PDF

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WO2007012377A2
WO2007012377A2 PCT/EP2006/006388 EP2006006388W WO2007012377A2 WO 2007012377 A2 WO2007012377 A2 WO 2007012377A2 EP 2006006388 W EP2006006388 W EP 2006006388W WO 2007012377 A2 WO2007012377 A2 WO 2007012377A2
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lipophilic
cells
anions
membrane
ions
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PCT/EP2006/006388
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German (de)
English (en)
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WO2007012377A3 (fr
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Dirk Zimmermann
Vladimir Soukhoroukov
Ernst Bamberg
Ulrich Zimmermann
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Eppendorf Ag
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Priority to JP2008523158A priority Critical patent/JP2009506756A/ja
Priority to EP06762319A priority patent/EP1907541A2/fr
Priority to AU2006274294A priority patent/AU2006274294A1/en
Publication of WO2007012377A2 publication Critical patent/WO2007012377A2/fr
Publication of WO2007012377A3 publication Critical patent/WO2007012377A3/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
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F11/00—Compounds containing elements of Groups 6 or 16 of the Periodic Table
    • C07F11/005—Compounds containing elements of Groups 6 or 16 of the Periodic Table compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
    • C07F13/005—Compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/06—Aluminium compounds
    • C07F5/061—Aluminium compounds with C-aluminium linkage
    • C07F5/066—Aluminium compounds with C-aluminium linkage compounds with Al linked to an element other than Al, C, H or halogen (this includes Al-cyanide linkage)
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to methods of treating cells with electric fields to alter the membrane permeability of these cells using lipophilic ions as a medium supplement. More particularly, the invention relates to methods of electroporation and electrofusion of cells using lipophilic ions as a medium supplement. This addition of lipophilic ions significantly increases the efficiency and above all the reproducibility and reduces the otherwise relatively high variation of the electroporation or electrofusion process.
  • Electro-transfection and electro-fusion of cells are routinely used in many areas of biotechnology and biomedicine for the manipulation of the genome and cytosol. Both methods are based on the application of high-intensity and very short duration electric field pulses, which lead to a reversible electrical membrane breakdown (so-called electroporation).
  • electroporation A wide range of molecules, including hormones, proteins, RNA and DNA, can be introduced into living cells by electroporation.
  • the membrane breakthrough in the contact zone leads to the fusion of the cells, which can be used for the production of hybrid cells (electrofusion).
  • Tm aC ( ⁇ + -L) (2) m ⁇ ⁇ , 2 ⁇ .
  • C m [ ⁇ F / cm 2 ] is the area-specific membrane capacity and ⁇ ; and ⁇ e are the specific conductivities [mS / cm] of the cytosol and the outer poration or fusion medium.
  • ⁇ c 4 ⁇ ⁇ e ⁇ 0 a 3 gj e E 0 ( 4)> ⁇ , + z ⁇ "e
  • F DEP 2 ⁇ ⁇ e ⁇ 0 a 3 ° ⁇ ° e VE 0 2 (5) ⁇ ; + z ⁇ e
  • ⁇ e ⁇ o 8O> ⁇ 8.85 10
  • 12 F / m is the permittivity of the aqueous external medium
  • VE 0 2 reflects the inhomogeneity of the field and the reduction of ⁇ e also leads to an increase of the dipole Dipole interactions, which favor the mutual attraction of the fusion partners.
  • hypotonic swelling facilitates electrofusion by dissolving the cytoskeleton, smoothing the cell membranes (retraction of the microvilli) and increasing the mobility of the membrane components.
  • a related further object is the provision of means for improving conventional methods of electromanipulation, in particular electroporation and electrofusion techniques.
  • the starting point for the present invention was experimental evidence that many cells, especially mammalian cells, are capable of complex volume regulation, particularly under hypotonic conditions typically present during electroporation / electrofusion.
  • Recent studies [Reuss et al., 2004] have shown that after the rapid initial swelling of the cells in hypotonic sugar media, depending on the sugars used, both slower secondary volume increase and shrinkage of the cells may occur. Both can lead to rapid outflow of the electrolyte through so-called volume-sensitive channels from the cytosol.
  • a regulatory volume decrease occurs in all human and mammalian cells studied so far After a brief period of swelling (1-3 min) in hypotonic medium, cells can adjust their original volume (despite persistent hypotensive stress) within 15-20 min.
  • an oligosaccharide eg, trehalose, sucrose or raffinose
  • RVD Unlike oligosaccharides, RVD does not occur in hypotonic solutions of monomeric sugar derivatives (e.g., sorbitol, inositol, or glucose) [Reuss et al., 2004]. In some cell types, even a secondary, slower swelling of the cells is observed. The difference between oligosaccharides and monosaccharides could be explained by the size selectivity of volume-sensitive channels (VSC), which are ubiquitous in the membranes of mammalian cells [Kirk, 1997, Strange, 1994].
  • VSC volume-sensitive channels
  • the present invention is now based u. a. on the surprising experimental finding that the addition of lipophilic ions, in particular anions, to the treatment medium inter alia prevent this volume regulation and thus can significantly increase the efficiency of electroporation or electro-fusion.
  • lipophilic anions possibly leads to an inhibition of the ion channels involved in the volume regulation.
  • the lipophilic ions directly or indirectly regulate the likelihood of the channels or their other transport properties. From both models, it can be deduced that the use of lipophilic ions greatly reduces the ion losses from the cytosol during the hypotonic stress. On the one hand this stabilizes the cell volume, on the other hand the ion content and the conductivity of the cytosol are kept at high levels.
  • the parameters of the bell curve depend on the chemical nature of the lipophilic ions and on the nature of the membrane (or cell type). On the basis of the voltage dependence of the bell curve of a lipophilic ion conclusions can be drawn on the distribution of the lipophilic ions in the membrane.
  • a “hemispheric asymmetry” of the cell as a whole will occur. This facilitates the electrical breakdown in two ways. On one side of the cell, the potential for breakdown is already almost reached and the externally applied breakdown voltage can be chosen to be lower than normal. On the other side of the cell, the applied potential gradient is "unfavorable" for the externally applied breakdown voltage, so this cell side is “protected” by the lipophilic ions. Alternatively one can do this under the external field amplitude or in the length / brevity of the applied field pulse use. Both hemispheres will have different charging times. As a result, this "asymmetric" breakthrough is gentler on the cell than the normal breakthrough.
  • This selection criterion can be, for example, the shape or position of the bell curve at certain membrane stresses.
  • those lipophilic ions are selected for which the membrane capacitance has a minimum at the natural membrane voltage of the cell (ie lies on the hypo- or hyperpolarized flank of the bell curve), or those for which the membrane capacitance has a maximum at the natural membrane voltage of the cell ,
  • those lipophilic ions are selected for which the membrane capacity has little or no increased value in the natural membrane voltage of the cell, ie those lipophilic ions whose characteristic bell curve is such that it falls off at the natural membrane voltage Flank has.
  • the lipophilic ions will be distributed unevenly across the membrane at natural membrane stress and cause the above useful asymmetry effect. This concerns, for example, hyperpolarized cells which are exposed to an extremely weakly conducting fusion medium. Lipophilic ions, for which the membrane capacitance has a maximum in the natural membrane voltage of the cell, could be used for applications where high membrane capacity or uniform distribution of the lipophilic ions is useful.
  • cells with inhomogeneous membrane structure which in this way obtain a more homogeneous membrane charge, such as most mammalian cells, in which the negatively charged phosphatidylserine is located mainly on the cytosolic side of the plasma membrane.
  • Suitable soluble salts may include, but are not limited to, sodium, potassium or barium ions.
  • Another advantage of adjusting cells to a particular membrane potential is the better reproducibility of the results.
  • the lipophilic ion-induced change in membrane capacity can be used to completely or partially eliminate the volume regulation of cells in hypotonic media. As discussed above, this leads to significantly reduced electrolyte losses and thus to an improvement of the basic conditions for electromanipulation processes such as electroporation or electrofusion. Loss of ions the cytosol reduces the ⁇ ⁇ cytosolic conductivity, which greatly reduces the porosity and fusion relevant deformation force (equation 3), cell polarization ⁇ c (equation 4) and the dielectrophoretic force (equation 5). In addition, the ion outflow from the cytosol results in significant loss of vitality in cells.
  • lipophilic ions or the change in membrane capacity induced thereby may also occur under non-hypotonic, e.g. Isotonic conditions lead to a membrane charging, which greatly facilitates the required or required for Elektromanipulationsvon membrane breakthrough.
  • the use according to the invention of lipophilic ions also makes it easier and more efficient to carry out methods of electromanipulation, such as electroporation or electrofusion in non-hypotonic media.
  • the avoidance of hypotonic stress for the cells would be of great advantage and in any case has a favorable effect on the survival rates of the manipulated cells.
  • Suitable cells for the application of the method according to the invention or the use according to the invention of lipophilic ions are basically all cells of natural or synthetic origin.
  • Synthetic cells may be, for example, artificial membrane-enveloped vesicles, liposomes or micelles.
  • Natural cells include prokaryotic cells, e.g. Bacteria or yeasts, or eukaryotic cells, e.g. animal or plant cells.
  • Preferred animal cells are mammalian cells, especially human cells. In a preferred embodiment, the cells are living cells.
  • the lipophilic ions used according to the invention can be cations, anions or a mixture of cations and anions.
  • Cations are especially preferred for use with plant cells, while anions are particularly preferred in animal cells, especially mammalian cells.
  • the cations used in the invention may consist of one species or comprise several different cations.
  • the lipophilic cations used will contain at least one of the group of cations comprising cations of lipophilic metal complexes, lipophilic organometallic compounds, and lipophilic organic compounds having at least one positively charged functional group.
  • Specific examples are rhenium hexacarbonyl (Re (CO) 6 ) "1" , tetraphenylphosphonium or tetraphenylarsenium compounds.
  • Other suitable compounds will be readily apparent to one skilled in the art through routine experimentation.
  • the anions used in the invention may consist of one species or comprise several different anions.
  • the lipophilic anions used will contain at least one of the group of anions comprising anions of lipophilic metal complexes, lipophilic organometallic compounds and lipophilic organic compounds having at least one deprotonatable functional group.
  • lipophilic ions which can be used according to the invention, in particular lipophilic metal complexes, are the following:
  • the lipophilic anions contain at least one of the group of anions, the anions of lipophilic tungsten complexes (LTC), especially lipophilic tungsten carbonyl complexes, dipicrylamine (DPA) and derivatives thereof, anions of lipophilic organic compounds containing at least one carboxylate, sulfonate , Sulphate, thiosulphate or thiocyanate group, in particular arachidonic acid, or anions of an optionally substituted tetraphenylborate or Phenyldicarbaundecandecaborans (PCB) or a Tetracyanoborats or Tetratrifluormethylborats comprises.
  • LTC lipophilic tungsten complexes
  • DPA dipicrylamine
  • anions of lipophilic organic compounds containing at least one carboxylate, sulfonate , Sulphate, thiosulphate or thiocyanate group in particular arachidonic acid
  • lipophilic anions from the group of the following anions
  • (2) the anion of Et 4 N [W (CO) 5 (SCNOC 6 H 4 )], tetraethylammoniumbenzoxazolidine-2-thion-1-yl-pentacarbonyl-tungstate, MW 604 (abbreviated to WO)
  • LTC Lipophilic Tungsten Complex
  • lipophilic ions of different nature By using several lipophilic ions of different nature in equal or different concentrations at the same time or in time, an "overlap" of the individual properties of the lipophilic ions can be achieved, which can be used for different purposes, eg to ensure more robust, ie reproducible and less susceptible to interference Procedures be beneficial.
  • the lipophilic ions useful in this invention are compounds well known in the art and are commercially available or can be prepared by known routine methods.
  • the lipophilic ions in this case should be physiologically compatible with living cells.
  • the lipophilic ions used are degradable by UV light and yield physiologically acceptable degradation products for living cells. Both conditions are e.g. in the lipophilic anionic tungsten complex of the above formula 3 is ideally fulfilled. It will be apparent to those skilled in the art that similar compounds with similar efficacy, compatibility and similar degradation behavior can be readily prepared.
  • the lipophilic ions are typically used in a method for treating cells with electric fields for changing the membrane permeability of these cells, which comprises at least the following steps:
  • Such a method generally comprises the various known or conceivable electro-manipulation methods for altering the membrane permeability of cells in which the cells are exposed to an electrical voltage pulse, and in particular methods of electrotransfection / electroporation or electrofusion.
  • the lipophilic ions are as a rule added to the treatment medium (2) in a concentration in the range from 1 nM to 100 ⁇ M, preferably 0.1 ⁇ M to 60 ⁇ M, particularly preferably 1 ⁇ M to 50 ⁇ M.
  • an osmotically active substance for generating hypotonic stress for the at least one biological cell (1) is also added to the treatment medium (2).
  • Such substances include all substances known for this purpose, preferably mono- and / or disaccharides, e.g. Sucrose, trehalose, glucose, mannitol, inositol, sorbitol, etc.
  • the application of the electrical voltage pulse to the biological cell (1) takes place with a delay time after the addition of the osmotically active substance, the delay time preferably being selected in the range from 15 s to 10 min.
  • hypotonic poration and fusion media were used, the composition of which is given in Tables 1 and 2.
  • isotonic media were also used.
  • the addition of the lipophilic ions to the treatment medium (2) takes place in a sufficient concentration and for a sufficient period of time before the voltage pulse that doping of the membranes of the cells to be treated with these lipophilic ions takes place.
  • doping in this context includes the adsorption of the ions on the membrane and / or the incorporation of the ions into the membrane Suitable concentrations and time periods (usually of the order of minutes) may be readily measured by those skilled in the art in light of the present disclosure be determined. figure description
  • FIG. 1 Schematic representation of the method according to the invention
  • FIG. 2 Schematic representation of a test arrangement for carrying out the method according to the invention
  • FIG. 3 Schematic representation of the method for selecting suitable lipophilic ions
  • Fig. 4 shows "bell curves" showing the capacity change of the cell membrane of oocytes as a function of the voltage applied to the membrane for three different lipophilic anions.
  • the lower two curves represent the results of the control measurements in the absence of the lipophilic ion DPA at the beginning (squares) and at the end (triangle with the top down) of the measurement series.
  • the top curve (filled circles) shows the results in the presence of extracellular 50 ⁇ M DPA and the underlying curve (triangle topped) in the presence of extracellular 50 ⁇ M DPA and 2.8 mM BaCl 2 .
  • the lower two curves represent the results of the control measurements in the absence of the lipophilic ion WO at the beginning (squares) and at the end (triangle with the top down) of the measurement series.
  • the top curve shows the results in the presence of extracellular 10 ⁇ M WO and 2.8 mM BaCl 2 and the underlying curve (solid circles) in the presence of extracellular 10 ⁇ M WO.
  • Fig. 5 shows time lapses of the relative volumes of activated B lymphocytes (A) and Jurkat cells (B and C) in highly hypotonic fusion media (75 and 100 mOsm, respectively) containing either sorbitol or trehalose as the major osmotic agent.
  • hypotonic trehalose medium filled circles
  • both cell types showed RVD after the first rapid swelling.
  • hypotonic sorbitol eliminated RVD in Jurkat cells (data not shown) or even induced a secondary swelling (A, open circles).
  • the tungsten complex LTC partially or completely inhibited RVD in B lymphocytes in Jurkat cells (squares in A and B, respectively).
  • FIG. 6 Complete or partial inhibition of RVD in mammalian cells by various structurally unrelated lipophilic anions (compounds (1) (DPA), (2) (WO) and (4) TPB) (A-C).
  • DPA lipophilic cation tetraphenylphosphonium
  • TPP lipophilic cation tetraphenylphosphonium
  • Fig. 7 Typical rotation spectra of H7 and Jurkat cells (A and B, respectively).
  • H7 cells were incubated for 15-20 min in isotonic or hypotonic sorbitol media (filled or empty circles) of the same conductivity of 120 ⁇ S / cm. Compared to isotonic conditions, hypotension resulted in a marked shift of the cytosolic peak f c2 to a lower frequency, indicating a significant reduction in cytosolic ⁇ .
  • Fig. 8 Effect of LTC on the electroinjection of propidium iodide Dependence of the electrically induced PI uptake on the applied field strength Eu with a pulse duration of 40 ⁇ s.
  • the data points are the mean PI acquisition values obtained from three independent flow cytometric determinations. Within the range of field strength examined, PI uptake in cells treated with 10 ⁇ M LTC " (filled circles) was greater than control (open circles).
  • Table 1 Composition of washing and pulse solutions used for electrotransfection
  • Sorbitol 75 1.3 ⁇ 0.1 2.2 ⁇ 0.2 58 ⁇ 4 0.5 ⁇ 0.1
  • Sorbitol 300 1 11.0 ⁇ l. (1.3 ⁇ 0.1 12
  • Trehalose 300 1 8.5 ⁇ 0.7 HOttlO 1.3 ⁇ 0.1 9
  • Trehalose 100 1 4.1 ⁇ 0.2 120 ⁇ 20 l.l ⁇ 0.1 8
  • Trehalose 100 10 1 .5 ⁇ 0. 1 5.7 ⁇ 0.2 110 ⁇ 12 0.7 ⁇ 0.1 12
  • Trehalose 100 10 1.3 ⁇ 0.1 60 ⁇ 5 133 ⁇ 28 28 ⁇ 2 16.8
  • a T is the duration of hypotonic treatment before the first electropulse
  • v (7) is the relative
  • the TA and GE values represent the percentage or geometric mean fluorescence intensity of GFP positive cells, as determined from GF histograms.
  • c PF is the number of viable cells 48 h after electro-transfection, expressed as a percentage of the original number of cells.
  • the transfection license TE is defined as TA x PF in percent. TE is proportional to
  • CGM Complete Growth Medium
  • fetal calf serum 10% (v / v).
  • the adherent cells ie, HEK 293 and L929 cells
  • the enzyme was removed by washing with CGM.
  • the electrotransfection / poration protocols included the following steps:
  • L929 cells were electrotransfected with a pulse of 3 kV / cm field strength and 100 ⁇ s duration in 100 mOsm pulse media (HPS, Table 1).
  • Jurkat cells were transfected at 1.2 kV / cm and 40 ⁇ s in 100 mOsm HPS.
  • HEK cells were treated at 1.5 kV / cm and 70 ⁇ s in 150 mOsm HPS pulsed media.
  • LTC lipophilic tungsten complex
  • Table 4 shows the results obtained at various medium compositions and time conditions for hypotensive shock.
  • the improved transfection yields could also be confirmed in additional experiments with electroinjection of the marker propidium iodide (FIG. 8).
  • the pulsed media contained 25 ⁇ g / ml propidium iodide (PI), a cationic, normally non-membrane permeable, dye which shows strong fluorescence upon binding to nucleic acids.
  • PI performed two tasks, first as a dye that allows live cells to be distinguished from dead (short term vitality test) and secondly as an indicator of transient electrically induced membrane permeability. The PI staining of the cells was analyzed by flow cytometry within 10-15 minutes after pulse application.
  • human B lymphocytes were isolated from the peripheral blood of healthy donors and activated with 2.4 ⁇ g / ml phytohemagglutinin (PHA-L) as known.
  • the activated cells were cultured at 37 ° C in a 5% CO 2 enriched atmosphere. When the cell density exceeded ⁇ 5x10 6 cells / ml, the cells were diluted to a density of 1x10 6 cells / ml.
  • the activated B lymphocytes were fused to the human mouse heteromyeloma cell line H73C11 (hereinafter referred to as H7 cells) grown under standard conditions.
  • B lymphocytes and H7 cells were mixed in CGM in a 1: 1 ratio, pelleted by centrifugation at 200 xg for 10 min, and in a hypotonic, sugar-supplemented fusion solution (HFS) with an osmolality of 75 or 100 mOsm (see Tab. 2) resuspended.
  • HFS sugar-supplemented fusion solution
  • the cells were then washed twice with HFS (re-centrifugation at 200xg for 10 min and resuspension).
  • 200 ⁇ l of the cell suspension in HFS were pipetted into a helical chamber consisting of a Perspex tube around which two platinum wires were wound at a distance of 200 ⁇ m.
  • the final cell density was 3 ⁇ 10 5 cells / ml.
  • the cells were exposed to hypotonic stress for at least twenty minutes during the washing steps before the first fusion pulse was applied.
  • the duration of hypotensive stress was significantly reduced by using isotonic wash solutions (IWS-F, Table 2).
  • the Eppendorf Multiporator was used for electrofusion.
  • the cells were first approximated dielectrophoretically by an alternating field of 45 Vpp amplitude and 2 MHz frequency for 30 s. Thereafter, the RF field was turned off and fusion initiated by three rectangular DC pulses of 30 V amplitude and 15 ⁇ s duration. Then again a 2 MHz field of 5 Vpp amplitude was applied to hold the cells in place during the ensuing fusion process.
  • the helical chamber was then held at RT without disturbance for 10 min.
  • the chamber was then rinsed with 1 ml of isotonic CGM and cells were placed in 4 wells of a 24-well plate filled with ImI isotonic CGM, respectively. After 24 hours, the selection HAT medium in which only the hybridoma cells proliferated was added to the wells. Hybridoma colonies were counted 1-3 weeks after electrofusion.
  • the ND96 solution contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, and was adjusted to pH 7.4 with NaOH.
  • ND96 + 2.8 mM BaCl 2 1.8 mM CaCl 2 and 1 mM MgCl 2 were replaced by 2.8 mM BaCl 2 .
  • Collagenase-treated stage IV-VI oocytes were used for the measurements.
  • bell curve The determination of the voltage dependence of the capacitance increase ("bell curve") for ions 1 to 3 shows that DPA and WO have nearly identical bell curves, whereas WW (LTC) has a strongly shifted bell curve, with a maximum of -130 mV (in comparison to -70 mV and -80 mV at DPA or WO).

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Abstract

L'invention concerne des procédés pour traiter des cellules au moyen de champs électriques pour modifier la perméabilité membranaire de ces cellules par adjonction d'ions lipophiles. L'invention concerne en particulier des procédés électroporation et d'électrofusion de cellules par adjonction d'ions lipophiles.
PCT/EP2006/006388 2005-07-28 2006-06-30 Procedes d'electromanipulation de cellules pour modifier la permeabilite membranaire de ces cellules par adjonction d'ions lipophiles WO2007012377A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2008523158A JP2009506756A (ja) 2005-07-28 2006-06-30 親油性イオンを培地添加物として用いる、細胞の膜透過性を変化させるための細胞の電気的操作法
EP06762319A EP1907541A2 (fr) 2005-07-28 2006-06-30 Procedes d'electromanipulation de cellules pour modifier la permeabilite membranaire de ces cellules par adjonction d'ions lipophiles
AU2006274294A AU2006274294A1 (en) 2005-07-28 2006-06-30 Methods for the electromanipulation of cells in order to modify the membrane permeability of said cells with the aid of lipophilic ions as a medium additive

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DE102005035456.4 2005-07-28
DE200510035456 DE102005035456A1 (de) 2005-07-28 2005-07-28 Verfahren zur Elektromanipulation von Zellen zur Veränderung der Membranpermeabilität dieser Zellen unter Verwendung von lipophilen Ionen als Mediumzusatz

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WO2007012377A2 true WO2007012377A2 (fr) 2007-02-01
WO2007012377A3 WO2007012377A3 (fr) 2007-04-12

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CN107841462B (zh) * 2017-10-11 2021-07-16 山东第一医科大学(山东省医学科学院) 电化学可控细菌粘附界面的构建及使用方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022147A1 (fr) * 1998-10-09 2000-04-20 Stratagene Procede ameliore pour une electroporation plus efficace
WO2001066694A2 (fr) * 2000-03-06 2001-09-13 Eppendorf Ag Procede pour transferer de la matiere dans un systeme cellulaire
WO2002086134A2 (fr) * 2001-04-23 2002-10-31 Amaxa Gmbh Solution tampon destinee a l'electroporation et procede utilisant cette solution

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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