WO2007012377A2

WO2007012377A2 - 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

Info

Publication number: WO2007012377A2
Application number: PCT/EP2006/006388
Authority: WO
Inventors: Dirk Zimmermann; Vladimir Soukhoroukov; Ernst Bamberg; Ulrich Zimmermann
Original assignee: Eppendorf Ag
Priority date: 2005-07-28
Filing date: 2006-06-30
Publication date: 2007-02-01
Also published as: AU2006274294A1; WO2007012377A3; CN101278048A; DE102005035456A1; EP1907541A2; JP2009506756A

Abstract

The invention relates to methods for the treatment of cells with electric fields in order to modify the membrane permeability of said cells with the aid of lipophilic ions as a medium additive. The invention more particularly relates to cell electroporation and electrofusion methods using lipophilic ions as a medium additive.

Description

A method of electromanipulating cells to alter the membrane permeability of these cells using lipophilic ions as a medium supplement

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.

State of the art

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). A wide range of molecules, including hormones, proteins, RNA and DNA, can be introduced into living cells by electroporation. In dielectrophoretically contacted cells, 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).

As a rule, field strengths of a few kV / cm and a pulse duration of a few 10 μs (to ms) are used for the electroporation / fusion of mammalian cells. The applied field leads to an exponential membrane charge, which can be indicated by the induced transmissive voltage V _% :

Fg (O = 1.5 α E ₀ cos 0 (l-exp (-t / τ _m )) (1),

where a is the cell radius, θ the polar angle to the direction of the applied field E ₀ and τ _m the time constant of the membrane charging. The membrane breakthrough takes place when V _g the critical value V _c = 1 volt exceeds.

The time constant τ _{m of} the membrane charging is calculated using the following equation:

_{Tm = aC} (± ₊ -L) (2) ^m ~ {σ, 2σ. ||

where C _m [μF / cm ² ] is the area-specific membrane capacity and σ; and σ _{e are} the specific conductivities [mS / cm] of the cytosol and the outer poration or fusion medium.

If the cell radius is given a typical value of 7 μm in Equation 1, the minimum critical field strength for the breakdown in the membrane regions facing the electrodes (θ = 0) can be calculated (provided that the pulse duration is much longer than the relaxation time of the membrane is t »τ _m ): £ _kr i _t = ^" VV (1.5 ^χ a) "1 kV / cm For larger cells, considerably smaller field strengths are required in accordance with equation 1. The time constant τ _m lies in mammalian cells in the range of 0.1-1 μs (equation 2: C _m = 1 μF / cm ² , σ; 5mS / cm, σ _e = 0.1-10 mS / cm, see below). Therefore, the pulse durations used are from 10 to 100 μs much longer than τ _m , so that equation 1 can be simplified: V _g = 1.5 a EQ COS θ.

It is known in the art that the efficiency of electro-transfection and electro-fusion techniques can be significantly increased by the use of low-conduction, hypotonic media during pulse application [Zimmermann & Neil, 1996; Sukhorukov et al. 1998]. Such media contain as the main osmotic agent various sugars and sugar derivatives. Among other sugars, monomeric carbohydrates such as glucose, sorbitol, inositol or mannitol, as well as disaccharides including sucrose or trehalose are commonly used [Zimmermann & Neil, 1996].

The reduction of osmotic pressure and conductivity serves several purposes in electroporation and fusion:

1) The hypotonic swelling of cells promotes membrane breakthrough because the induced membrane voltage depends linearly on the cell radius (see Eq. 1). 2) Reducing the outer conductivity greatly improves the electroinjection due to the transient electrodefractive force F _D EF on the cell membrane:

F ^r DEF ^X _∞ e - ⁰ O ² / ^σ t '_ ^{~ 2} ^σ e \) 2

If σj = σ _e , the deformation force remains: FD = 0. Under the condition σj <σ _e an elongation force F _D in the field direction is exerted on the cell membrane, the extent of which increases with decreasing σ _e (equation 3). At the same time, the decrease in σ _e leads to a large increase in the amounts of electroinjected molecules [Sukhorukov et al., 1998].

3/4) In electrofusion, low-conductivity media are mandatory. Analogous to the deformation force, both the electrical polarization of the cell or the induced cell dipole μc (equation 4) and the positive dielectrophoretic force in the MHz range (1-10 MHz) (equation 5) increase with decreasing σ _e [Jones, 1995]:

μ _c = 4π ε _e ε ₀ a ^{3 gj} ^e E ₀ ( _4)> σ, + zσ "e

F _DEP = ² ^ ε _e ε ₀ a ³ ° ^{ ° ^e VE ₀ ² ₍₅₎ σ _; + zσ _e where ε _e εo = 8O><8.85 10 ^"12 F / m is the permittivity of the aqueous external medium The term VE ₀ ² reflects the inhomogeneity of the field and the reduction of σ _{e also} leads to an increase of the dipole Dipole interactions, which favor the mutual attraction of the fusion partners.

5) In addition to the physical effects described above, hypotonic swelling facilitates electrofusion by dissolving the cytoskeleton, smoothing the cell membranes (retraction of the microvilli) and increasing the mobility of the membrane components.

However, despite widespread use of electrotransfection / electroporation and electrofusion methods, there are still a number of unresolved problems in the current methods, such as: (1) low transfection and fusion rates in particularly "field resistant" cell types,

(2) high mortality rates of cells after electroporation / fusion,

(3) strong batch-to-batch variations in transfection and fusion rates as well

(4) Very often fluctuating reproducibility in the electroinjection or fusion yield.

Object of the invention

It is therefore an object of the present invention to provide novel improved electromanipulation methods for cells having increased efficiency and reproducibility. A related further object is the provision of means for improving conventional methods of electromanipulation, in particular electroporation and electrofusion techniques.

In-depth investigations by the inventors have led to the surprising result that by adding lipophilic ions to the treatment medium, in particular a hypotonic treatment medium, the efficiency and reproducibility of electromanipulation methods for cells, such as e.g. Electroporation and electrofusion process, greatly improved and in particular the yield and viability of the electromanipulated cells can be significantly increased.

Accordingly, the above objects are achieved according to the invention by providing a method for treating cells with electric fields according to claim 1, in which the treatment medium contains lipophilic ions, and the use of these lipophilic ions according to claim 23. Specific and preferred embodiments of the invention are the subject the dependent claims.

Description of the invention

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.

In hypotonic poration and fusion media substituted with an oligosaccharide (eg, trehalose, sucrose or raffinose), a regulatory volume decrease (RVD) 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.

Electrophysiological studies by other authors suggest that initial cell swelling may activate the volume-sensitive chloride channels, resulting in chloride outflow from the cell and cell membrane depolarization. The membrane depolarization causes activation of the voltage-dependent K ⁺ channels and a K ⁺ outflow. The net KCl outflow along with the osmotic water outflow is expected to restore normal (ie, isotonic) cell volume [Fürst et al., 2002; Lang, 1998].

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].

However, even in media without RVD, eg in inositol or sorbitol-substituted media, hypotonic stress leads to significant electrolyte losses and decreases in cytosolic conductivity (as evidenced, for example, by electro-rotation spectra (Figure 7A)). The ion losses from the cytosol reduce the conductivity σ; of the cytosol, whereby the poration and fusion-relevant deformation force (equation 3), cell polarization μc (equation 4) and dielectrophoretic force (equation 5) are greatly reduced. In addition, the ion outflow from the cytosol results in significant loss of vitality in cells.

These effects can completely or partially destroy the positive effects of hypotonic media during electrotherapy / electrofusion.

However, in the literature, the complex volume response of cells, especially mammalian cells, in hypotonic sugar media and the associated ion fluxes have not yet been discussed in terms of their relevance to electroporation and fusion.

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.

The adsorption or incorporation of lipophilic anions into the cell membrane possibly leads to an inhibition of the ion channels involved in the volume regulation. Alternatively, it may be that 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.

Without wishing to be bound by theory as to the mechanism of action, it is presently believed that this inhibition of certain ion channels is a consequence of the alteration, in particular increase, of the membrane capacity of the cell, as a function of the membrane voltage, by the adsorption and / or incorporation of the membrane lipophilic ions is in the membrane. Extensive investigations by the inventors have shown that the change in the membrane capacitance induced by lipophilic ions as a function of the membrane voltage usually follows a certain relationship and preferably the behavior of a symmetrical or asymmetrical bell curve with a voltage V _max at which the capacitance increase C (V _max ) is maximum.

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.

An equal distribution of the lipophilic ions on both sides of the membrane should be present at the membrane voltage, at which the maximum capacity increase is detected. By contrast, the most extreme unequal distribution of the lipophilic ions (either completely on the cytosolic or extracellular membrane side) is present if no increase in capacity by the lipophilic ions can be measured. An uneven distribution of the lipophilic ions should lead to a strong lowering of the surface potential (up to ΔV ~ -400 mV) on one side of the membrane. This will usually lead to a drastic change in the potential profile across the membrane. This "membrane asymmetry" effect may facilitate electromanipulations of treated cells in several ways:

1) the drastic reduction of the surface potential leads to such a steep potential profile across the membrane that local breakthroughs are generated or local breakthroughs are facilitated by superimposed external fields (electroporation, electrofusion).

2) The altered potential course leads to the influence of voltage-dependent transport systems in the membrane (volume regulation).

3) In addition to the "membrane asymmetry", 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 finding allows the selection of particularly suitable lipophilic ions by determining their effects on changing the membrane capacity under given conditions. Such a selection process will typically include the following steps:

Measuring the electrical membrane capacity of the biological cell as a function of a specific membrane voltage, the voltage dependence of the lipophilic ion-induced membrane capacity being measured with a plurality of different lipophilic ions in the treatment medium, and

Detection and selection of the lipophilic ions for which the lipophilic ion-induced membrane capacity of the cell meets a predetermined selection criterion. This selection criterion can be, for example, the shape or position of the bell curve at certain membrane stresses.

Depending on the desired results and applications, e.g. 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 ,

In one embodiment of the invention, 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. In this case, it is expected that 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. An example of this is 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.

After the natural membrane voltage (i.e., without applied field) depends on ion gradients of permeable ions between the external bath medium (treatment medium) and the interior of the cell, this can be adjusted by adding salts to the treatment medium. Suitable soluble salts may include, but are not limited to, sodium, potassium or barium ions.

Since the change in the membrane capacity can be influenced on the one hand by the choice of the lipophilic ions and on the other hand by changing the natural membrane voltage, this means that a desired distribution of lipophilic ions either with unchanged membrane voltage by the choice of a lipophilic ion with the desired shape (eg flank or Maximum) of the bell curve at this voltage or at a particular ion by appropriate displacement of the natural membrane voltage up to the voltage corresponding to the corresponding bell curve section, can be obtained.

Another advantage of adjusting cells to a particular membrane potential (e.g., by using cell-cycle synchronized cells, adjusting membrane potential through appropriate salt conditions) is the better reproducibility of the results.

As already mentioned, 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 σ _\ cytosolic conductivity, which greatly reduces the porosity and fusion relevant deformation force (equation 3), cell polarization μc (equation 4) and the dielectrophoretic force (equation 5). In addition, the ion outflow from the cytosol results in significant loss of vitality in cells.

By eliminating these negative effects, the efficiency and reproducibility of Elektromanipulationsverfahren can be significantly increased.

However, as can be seen from the above, the presence of 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 Elektromanipulationsverfahren membrane breakthrough. Thus, 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. Typically, 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. Typically, 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.

Specific, non-limiting examples of lipophilic ions which can be used according to the invention, in particular lipophilic metal complexes, are the following:

O

Preferably, 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.

Particularly preferred are the lipophilic anions from the group of the following anions

^~ r

selected, wherein: (1) = [C ₁₂ H ₄ N ₇ O _12] ^", 2,2 ', 4,4', 6,6 ^I -Hexanitrodiphenylamid (hexanitrodiphenylamine = DPA), (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) (3) the anion of Et _t N [W ₂ (CO) 10 (μ-S ₂ CH)], tetraethylammonium decacarbonyl-μ-dithioformiato-di-tungstate, MW 725 (hereinafter abbreviated as LTC ("Lipophilic Tungsten Complex") or WW) (4) The anion of Na [B (C ₆ Hs) ₄ ], sodium tetraphenylborate, MW 342.

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.

In principle, all ions capable of increasing the membrane capacity such that the increase in capacitance has a symmetrical bell curve as a function of the trans-membrane voltage are suitable. As an example, this is shown for some ions (tungsten compounds, DPA, etc.).

Since a main application of the present invention will be the electromanipulation of living cells, the lipophilic ions in this case should be physiologically compatible with living cells. For some applications, it is particularly preferred that 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.

According to the invention, 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:

- Providing at least one cell (1) in a treatment medium (2), and

- Acting on the cell (1) with at least one electrical voltage pulse, characterized by the step:

- Addition of lipophilic ions to the treatment medium (2).

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 μM, preferably 0.1 μM to 60 μM, particularly preferably 1 μM to 50 μM.

In a special embodiment of the method according to the invention, 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.

Preferably, 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.

Investigations by the inventors have shown that in the case of electrotransfection / poration and electrofusion methods, the duration of the hypotonic stress before the field pulse application has a considerable influence on the efficiency of these methods.

Based on biophysical and volumetric experiments, the duration of hypotonic treatment in the electromanipulation protocols could be significantly optimized. In these experiments hypotonic poration and fusion media were used, the composition of which is given in Tables 1 and 2. For washing the cells, corresponding isotonic media were also used.

a) Electrofusion:

Significantly higher yields of living hybrid cells could be achieved by optimizing the timing of the pulse application. The hybrid yields depended strongly on the duration of the hypotonic stress before the pulse application. A short hypotonic treatment of 2 min, which corresponded to the end of the fast swelling phase, gave the best results. Longer incubation times (eg 20 or 40 min) reduced the fusion yields (data not shown). b) Electro-transfection with the GFP plasmid:

Analogous to electrofusion, brief hypotonic treatment (i.e., 2 min prior to pulse application) usually gave significantly higher yields of GFP-transfected cells than a longer incubation of 10 min (Table 4). In general, the incubation-time-dependent transfection yields (TY) correlated well with the transient changes in cell size (see equation 1) in different cell types.

Prolonged incubation always resulted in a decrease in cell proliferation (PF, e.g., from 47 to 33% for HEK cells in inositol 150), indicating decreased cell viability (due to electrolyte loss).

This strong dependence of electroporation / transfection / electrofusion efficiency on the duration of the hypotonic stress can be exploited to further improve the above-described electromanipulation processes using lipophilic ions by optimizing the periods of hypotonic stress. It should be noted that in some cases, even without the use of lipophilic ions, very high efficiencies were already achieved by this temporal optimization, which could only be increased slightly with lipophilic ions. However, the use of lipophilic ions in any case provides the added advantage of a more robust protocol, i. H. a protocol that does not rely on the precise setting of optimal time values to achieve high efficiencies. This represents a great practical advantage, especially in the commercial application of these methods.

Preferably, 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. The term "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.

A: DPA (compound 1)

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 μM DPA and the underlying curve (triangle topped) in the presence of extracellular 50 μM DPA and 2.8 mM BaCl ₂ .

B: WO (compound 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 (triangle with the tip up) shows the results in the presence of extracellular 10 μM WO and 2.8 mM BaCl ₂ and the underlying curve (solid circles) in the presence of extracellular 10 μM WO.

C: WW (Compound 3, LTC) The lower two curves represent the results of the control measurements in the absence of the lipophilic ion WW at the beginning (squares) and at the end (triangle with the tips down) of the measurement series. The top curve (filled Circles) shows the results in the presence of extracellular 10 μM WW and the underlying curve (triangle topped) in the presence of extracellular 10 μM WW and 2.8 mM BaCl ₂ .

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. In hypotonic trehalose medium (filled circles), both cell types showed RVD after the first rapid swelling. In contrast, 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). Arachidonic acid picked up the RVD in Jurkat cells (C, squares).

Figure 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). In contrast, the lipophilic cation tetraphenylphosphonium (TPP, 10-50 μM) did not affect the RVD (C, squares).

Fig. 7 Typical rotation spectra of H7 and Jurkat cells (A and B, respectively).

A: H7 cells were incubated for 15-20 min in isotonic or hypotonic sorbitol media (filled or empty circles) of the same conductivity of 120 μS / 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 σ.

B: In comparison to the untreated control (open circles), 10 μM LTC caused an additional anti-field peak (furc) and also shifted the f _c2 to a higher frequency. The latter effect is due to decreased electrolyte loss from the cytosol.

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 μs. 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 μM LTC ^" (filled circles) was greater than control (open circles). Table 1. Composition of washing and pulse solutions used for electrotransfection

Isotone washing solution Hypotone for poration pulse solution IWS-P HPS

Inositol or trehalose 24OmM 50 or 10OmM

KCl 25mM 25mM

KH ₂ PO ₄ 0.3 mM 0.3 mM

K ₂ HPO ₄ 0.85 mM 0.85 mM pH 7.4 7.4

Conductivity at 22-24 ° C ~ 3.5 mS / cm -3.5 mS / cm

Osmolality -290 mOsm -100 or 150 mOsm

Table 2. Composition of media used in the improved electrofusion protocol

Isotone washing solution Hypotone for fusion fusion solution IWS-F HFS-75 / HFS-100

Ca-acetate 0.1 mM 0.1 mM

Mg acetate 0.5 mM 0.5 mM

Bovine serum albumin 1 mg / ml 1 mg / ml

Sorbitol 29OmM 75 or 10OmM pH 7.0 7.0

Conductivity at 22-24 ° C -120 μS / cm -120 μS / cm

Osmolality -290 mOsm -75 or 100 mOsm Table 3. Electrical properties of H7 and Jurkat cells according to ROT spectra measured in isotonic and hypotonic media of different sugar composition

H7 cells

Sorbitol 270 5.5 ± 0.2 88 ± 4 1.0 ± 0.1

Sorbitol 75 1.3 ± 0.1 2.2 ± 0.2 58 ± 4 0.5 ± 0.1

Jurkat cells

Sorbitol 300 1 11.0 ± l. (1.3 ± 0.1 12

Sorbitol 100 1 .8 ± 0. 1 2.5 ± 0.1 140 ± 5 0.7 ± 0.1 8

Sorbitol 100 10 1 .8 ± 0. 1 5.3 ± 0.3 88 ± 5 0.7 ± 0.1 7

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

^a The mean C _m , σ, and ε, values (± SA) were obtained by fitting the shell model to the ROT spectra of the cells shown in Figure 6 (not treated with LTC ^" ). See legend for further details to Fig. 6. ^b The v-values were obtained by volume measurements and represent the relative cell volume measured 20-25 min after the hypotonic shock.

Table 4. Effects of duration of hypotension and LTC on transfection yield (TA), GFP gene expression (GE) and proliferation factor (PF) of HEK and L929 cells

time of

Sugar and Relative Hypotonic LTC Osmolality Cell Volume TA ^b GE ^b PF ^c TE ^d Stresses μM% au mOsm%% r, min ^a v (7) ^a

HEK cells

Inositol 150 2 1.5 ± 0.1 44 ± 4 80 ± 7 47 ± 8 20.5

Inositol 150 10 1.7 ± 0.1 58 ± 9 158 ± 34 33 ± 4 19.1

Inositol 150 10 10 1.9 ± 0.2 44-fcl 77 ± 8 47 ± 3 20.7

Trehalose 150 2 1.4 ± 0.1 35 ± 3 83 ± 5 68 ± 14 23.9

Trehalose 150 10 l.l ± 0.1 20 ± 5 58 ± 7 56 ± 6 11.4

Trehalose 150 10 10 1.4 ± 0.1 38 ± 3 100 ± 12 59 ± 8 22.4

L929 cells

Inositol 100 2 1.9 ± 0.1 85 ± 1 182 ± 10 16 ± 2 13.7

Inositol 100 10 1.5 ± 0.1 75 ± 4 104 ± 19 12 ± 1 9.1

Inositol 100 10 10 1.8 ± 0.1 87 ± 1 355 ± 22 16 ± 2 14.1

Trehalose 100 2 l. & FcO.l 71 ± 1 177 ± 21 31 ± 4 21.9

Trehalose 100 10 1.3 ± 0.1 60 ± 5 133 ± 28 28 ± 2 16.8

Trehalose 100 10 10 1.5 ± 0.2 69 ± 2 188 = 1 = 10 45 ± 9 31.0

Legend to Table 4: ^a T is the duration of hypotonic treatment before the first electropulse; v (7) is the relative

Cell volume at time T of pulse application after hypotonic shock. ^b The TA and GE values represent the percentage or geometric mean fluorescence intensity of GFP positive cells, as determined from GF histograms.

GE geometric mean values of untransfected cells (i.e., the

Autofluorescence) were 3 ± 0.3 a.u. The data represent the mean ± SA of

3-9 experiments. ^c PF is the number of viable cells 48 h after electro-transfection, expressed as a percentage of the original number of cells. ^d The transfection license TE is defined as TA x PF in percent. TE is proportional to

Number of live cells expressing the reporter gene.

The following examples illustrate specific embodiments of the present invention without, however, limiting it thereto.

EXAMPLE 1

Electroporation / transfection of cells whose plasma membrane has been doped with lipophilic anions

For electrotransfection, Jurkat T lymphocyte cells, HEK 293 cells, and mouse fibroblast L929 cells were incubated in Complete Growth Medium (CGM) supplemented with 10% (v / v). fetal calf serum, cultured at 37 ° C under 5% CO _2. Before electro-transfection, the adherent cells (ie, HEK 293 and L929 cells) were detached with 0.5 mg / ml trypsin without EDTA to prevent contamination of the pulse medium with this chelator The enzyme was removed by washing with CGM.

The electrotransfection / poration protocols included the following steps:

1. washing the cells with an isotonic wash solution (IWS-P, see Table 1);

2. suspension of cells (10 ⁶ cells / ml) in a hypotonic pulse solution (HPS, Table 1) containing 10 μg / ml pEGFP-Cl (Clontech, Heidelberg, Germany) encoding a green fluorescent protein (GFP);

3. Incubate the cells for 2 or 10 min. in HPS;

4. Apply a single exponentially decreasing pulse to the cell samples (-800 μl) at room temperature (RT about 22-24 ° C) using the Multiporator (Eppendorf, Hamburg, Germany) and sterile cuvettes with planar aluminum electrodes at a distance of 4 mm ;

5. re-incubation of the cells in pulse medium for 10 min at RT;

6. Gentle transfer of the cells to 5 ml prewarmed CGM and 2 days of culture to achieve maximal GFP expression;

7. Analysis by flow cytometry and electronic cell counting.

L929 cells were electrotransfected with a pulse of 3 kV / cm field strength and 100 μs duration in 100 mOsm pulse media (HPS, Table 1). Jurkat cells were transfected at 1.2 kV / cm and 40 μs in 100 mOsm HPS. HEK cells were treated at 1.5 kV / cm and 70 μs in 150 mOsm HPS pulsed media. To investigate the effect of a lipophilic anion herichtlich the avoidance or minimization of unwanted electrolyte losses and the stabilization of the cell volume various Porationsmedien with 10 .mu.M of the lipophilic tungsten complex of the above formula 3, abbreviated LTC ("lipophilic tungsten complex") were added.

Table 4 shows the results obtained at various medium compositions and time conditions for hypotensive shock.

The addition of LTC resulted, especially in the longer incubation times in hypotonic media, to a significant increase in transfection efficiencies.

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 μg / 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.

EXAMPLE 2 Electrofusion of cells

For the electrofusion, human B lymphocytes were isolated from the peripheral blood of healthy donors and activated with 2.4 μg / 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. For electrofusion, 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. The cells were then washed twice with HFS (re-centrifugation at 200xg for 10 min and resuspension). 200 μl 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 μm. The final cell density was 3 × 10 ⁵ cells / ml. According to this standard protocol, the cells were exposed to hypotonic stress for at least twenty minutes during the washing steps before the first fusion pulse was applied. In the improved protocols, 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 μs 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 presence of lipophilic anions, eg WW, markedly increased the efficiency and reproducibility of the electrofusion, especially with prolonged duration of hypotonic stress. In particular, a high yield of viable hybrid cells was reproducibly obtained. EXAMPLE 3

Determination of the lipophilic anions-induced membrane capacity course in oocytes as a function of the membrane voltage

Measurements were made on Xenopus laevis oocytes using the two-electrode voltage clamp technique in ND96 solution. To calculate the membrane capacity Cm (U _H ) in the presence and absence of the lipophilic anions, voltage ramp protocols were programmed with the Clampex 9.2 ^® software. In these, the potential was first clamped to a certain value and voltage ramps (dU / dt) were driven to defined voltages. (Measurements were also made on the natural membrane voltage without voltage clamp).

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. For 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.

The results of measurements with three different lipophilic anions (compounds 1 to 3) are shown in FIG.

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).

It is therefore to be assumed that at normal cell potentials in the case of WO and DPA there is a distribution of the lipophilic ions (V _M = -30-70 mV), which is close to the uniform distribution. On the other hand, under normal cell conditions, LTC (WW) should be unevenly accumulated on the side of the cytosol. Quoted literature

Fürst, J., Gschwentner, M., Ritter, M., Botta, G., Jakab, M., Mayer, M., Garavaglia, L.,

Bazzini, C, Rodighiero, S., Meyer, G., Eichmüller, S., Woll, E., Paulmichl, M.

2002. Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflügers Arch. -Eur. J. Physiol.

444: 1-25

Jones, T.B. 1995. Electromechanics of Particles. Cambridge University Press, New York Kirk, K. 1997. Swelling-activated organic osmolyte channels. J. Membr. Biol. 158: 1-16 Lang, F. 1998. Cell Volume Regulation, Karger, Basel Muller, KJ, Sukhorukov, VL, Zimmermann U. 2001. Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of sub-microsecond duration , J. Membr. Biol. 184: 161-170 Reuss, R., Ludwig, J., Shirakashi, R., Ehrhart, F., Zimmermann, H., Schneider, S., Weber,

M.M., Zimmermann, U., Schneider, H., Sukhorukov, V.L. 2004. Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways. J. Membr. Biol. 200: 67-81 Strange, K., 1994. Cellular and Molecular Physiology of Cell Volume Regulation, CRC.

Boca Raton, FL Sukhorukov, V.L., Mussauer, H., Zimmermann, U., 1998. The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low and high conductivity media. J. Membrane Biol. 163: 235-245 Zimmermann, U., Friedrich, U., Mussauer, H., Gessner, P., Hämel, K., Sukhorukov, V.L.

2000. Electromanipulation of mammalian cells: fundamental and application. IEEE

Trans. Plasma Be. 28: 72-82 Zimmermann, U., Neil, G.A. 1996. Electromanipulation of Cells, CRC, Boca Raton, FL

Claims

claims

A method of treating cells with electric fields to alter the membrane permeability of said cells, comprising the steps of:

- Providing at least one cell (1) in a treatment medium (2), and

- Addition of lipophilic ions to the treatment medium (2).

2. Method according to claim 1, which represents or comprises an electromanipulation, in particular electroporation or electrofusion, of the cell (s).

3. The method of claim 1 or 2, wherein the cells are prokaryotic or eukaryotic cells.

The method of claim 3, wherein the cells are living cells.

5. The method of claim 1 or 2, wherein the cells are natural or artificial membrane-coated vesicles, liposomes or micelles.

6. The method according to at least one of claims 1 to 5, wherein the lipophilic ions are added to the treatment medium (2) in a concentration in the range of 1 nM to 100 uM.

7. The method according to at least one of claims 1 to 6, wherein the addition of the lipophilic ions to the treatment medium (2) leads to a doping of the membranes of the cells to be treated with these lipophilic ions.

8. The method according to at least one of claims 1 to 7, wherein the lipophilic ions are cations, anions or a mixture of cations and anions.

9. The method according to at least one of claims 1 to 8, wherein the lipophilic ions are anions.

10. The method of claim 9, wherein the lipophilic anions contain at least one substance from the group of anions comprising anions of lipophilic metal complexes, lipophilic organometallic compounds and lipophilic organic compounds having at least one deprotonatable functional group.

11. The method of claim 10, wherein the lipophilic anions contain at least one substance from the group of anions, the anions of lipophilic tungsten complexes (LTC), in particular lipophilic tungsten carbonyl complexes, dipicrylamine (DPA) and derivatives thereof, derivatives of lipophilic organic compounds, which contain at least one carboxylate, sulfonate, sulfate, thiosulfate or thiocyanate, particularly arachidonic acid, or anions of a substituted or unsubstituted tetraphenylborate or Phenyldicarbaundecandecaborans (PCB) or a Tetracyanoborats [B (CN) _4] ^'or Tetratrifiuormethylborats [B (CF ₃ ) ₄ ] ^" .

12. The method of claim 11, wherein the lipophilic anions from the group of the following anions

are selected.

13. The method according to at least one of the preceding claims, wherein the lipophilic ions are physiologically compatible for living cells.

14. The method according to at least one of the preceding claims, wherein the lipophilic ions are degradable by UV light and yield for living cells physiologically acceptable degradation products.

15. The method according to at least one of claims 1 to 14, wherein the lipophilic ions to increase the membrane capacitance C of the cell in response to the membrane voltage of the cell.

16. The method of claim 15, wherein the dependence on the membrane voltage, the behavior of a symmetrical or asymmetric bell curve with a voltage Vm _ax , at which the increase in capacitance C (V _max ) is maximum, having.

17. The method according to at least one of the preceding claims, wherein a selection of the lipophilic ions is provided in a preliminary test, comprising the steps:

Measuring the electrical membrane capacity of the biological cell as a function of an applied membrane voltage, the voltage dependence of the lipophilic ion-induced membrane capacity being measured with a plurality of different lipophilic ions in the treatment medium (2), and

Detection and selection of the lipophilic ions for which the lipophilic ion-induced membrane capacity of the cell (1) fulfills a predetermined selection criterion.

18. The method of claim 17, wherein the lipophilic ions are selected for which the membrane capacitance at the natural membrane voltage of the cell (1) is minimal or at one of the falling edges of the bell curve defined in claim 16.

19. The method of claim 17, wherein the lipophilic ions are selected for which the membrane capacity at the natural membrane voltage of the cell (1) has a maximum.

20. The method according to at least one of claims 17 to 19, with the step:

- Addition of a salt in the treatment medium (2) for adjusting the natural membrane voltage of the cell (1).

21. The method according to at least one of the preceding claims, with the step:

- Addition of an osmotically active substance in the treatment medium (2) for generating a hypotonic stress for the at least one biological cell (1).

22. The method of claim 21, wherein the loading of the biological cell (1) takes place with the electrical voltage pulse with a delay time after the addition of the osmotically active substance, wherein the delay time is selected in the range of 15 s to 10 min.

23. Use of lipophilic ions for addition to a treatment medium (2), in which cells are subjected to a treatment with electric fields, which includes an electrical voltage pulse.

Use according to claim 23, wherein the cells are prokaryotic or eukaryotic cells.

Use according to claim 24, wherein the cells are living cells.

Use according to claim 25, wherein the cells are natural or artificial membrane-enveloped vesicles, liposomes or micelles.

27. Use according to any one of claims 23 to 26, wherein the treatment with electric fields is or comprises electroporation or electrofusion.

28. Use according to any one of claims 23 to 27, wherein the lipophilic ions are anions.

29. Use according to claim 28, wherein the lipophilic anions contain at least one substance from the group of anions comprising anions of lipophilic metal complexes, lipophilic organometallic compounds and lipophilic organic compounds having at least one deprotonatable functional group.

30. Use according to claim 29, wherein the lipophilic anions comprise at least one substance from the group of anions, the anions of lipophilic tungsten complexes (LTC), in particular lipophilic tungsten carbonyl complexes, dipicrylamine (DPA) and derivatives thereof, derivatives of lipophilic organic compounds, containing at least one carboxylate, sulfonate, sulfate, thiosulfate or thiocyanate, in particular arachidonic acid, or anions of an optionally substituted tetraphenylborate or Phenyldicarbaundecandecaborans (PCB) or a Tetracyanoborats [B (CN) ₄ ] ^' or Tetratrifluormethylborats [B (CF ₃ ) ₄ ] ^" .

31. Use according to claim 30, wherein the lipophilic anions from the group of the following anions

1

are selected.

32. The method according to at least one of the preceding claims, in which a plurality of lipophilic ions of different nature are used in equal or different concentrations at the same time or in chronological order.

33. Lipophilic metal complexes selected from the following group: