WO2006018528A1

WO2006018528A1 - Monitoring and control of an electroporation

Info

Publication number: WO2006018528A1
Application number: PCT/FR2005/001914
Authority: WO
Inventors: Lionel Cima; Lluis Mir
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2004-07-23
Filing date: 2005-07-25
Publication date: 2006-02-23
Also published as: FR2873385B1; FR2873385A1

Abstract

The invention relates to an assembly which is intended for the electroporation of a cell structure containing at least one cell. The invention comprises means for monitoring the power supplied to the electroporation electrodes. The invention is characterised in that the monitoring means comprise an estimator which can estimate the effective part of the power supply delivered to one or more determined cell parts of the cell structure, using data which is received by the estimator and which comprises data that are representative of the power supply and data relating to the assessed bioimpedance components of each determined cell part. The invention also relates to methods and uses for the assembly that is intended for electroporation.

Description

MONITORING AND CONTROL OF ELECTROPORATION

The invention relates to an assembly for the electroporation of a cell assembly consisting of at least one cell, comprising means for monitoring and possibly controlling the electrical supply of the electroporation electrodes.

Electroporation is the generation of electropores, within cell membranes, following the application (conventionally between two electrodes) of an electric field greater than a determined threshold value. These electropores are not selective and any chemical species can then penetrate the cell. This technique, used for the transfer of chemical substances of various sizes (small chemical molecules or macromolecules, conventionally RNA or DNA molecules), opens the field to many applications in biomedical fields, such as the non-viral transfer of genes for example. Electroporation is attributed to a reversible destabilization of the membrane causing the penetration of water molecules and a local decrease in electrical impedance. If the electric field continues to grow, the destabilization becomes irreversible and causes the destruction of the cell.

It is therefore important to control the degree of electroporation to overcome cell destruction, while ensuring internalization of chemical substances of various sizes (transfer of macromolecules of DNA, RNA or small therapeutic molecules).

In addition, a control of the size of the electropores would ensure an opening wide enough to internalize the substance, while sustaining the cell (s).

There are known techniques for monitoring and controlling electroporation.

Monitoring methods are based on the measurement of bioimpedance during electroporation, as for example described in WO 99/52589. It will also be possible to control the electroporation in real time by monitoring firstly the impedance variations of the electroporated fabric, and secondly by back-locking the electrical supply of the electrodes to these observed impedance variations. as described in US 2003/0194808 and WO 01/81533.

These control techniques thus seem to be able to detect, in real time, the exceeding of the different electroporation thresholds (electropore appearance thresholds and cell destruction threshold) by measuring a characteristic impedance drop, and then be able to retroactively adapted to the applied electric field.

However, these control techniques using a simple impedance measurement are not satisfactory, electroporation being a dynamic, non-linear phenomenon, and therefore difficult to model.

In particular, the threshold voltages themselves (electropore appearance threshold and cell destruction threshold) are intimately related to the geometry of the electrodes, to the quality of the electrode-tissue contact, to the influence of the extracellular medium. , bioimpedance that depends on many electrical parameters (as many parameters that require control over the shape and amplitude of the electrical quantities applied), etc. On the other hand, these techniques do not really propose methods to follow in real time the state of the electroporated biological cells.

An object of the invention is to improve the real-time control, relative to the state of the art, of the degree of cell electroporation, to maximize the number of electroporated cells and to minimize the number of destroyed cells, by playing in particular on the size of the electropores within the cell membranes.

A second objective of the invention is to overcome all the parameters external to electroporation (electrode geometry, electrode-tissue contact quality, influence of the extracellular medium, etc.). A third objective of the invention is to estimate in real time the state variables specific to the electropores of the tissue (ie the electric field average transmembrane, and the average density of the current passing through the electropores).

A fourth objective of the invention is to control in real time the electric field delivered by the electrodes to the tissue, taking into account the electrical quantities estimated during the monitoring, in order to self-adapt the electroporation to electrochemical phenomena appearing at the level of the treated cells.

A fifth objective of the invention is the implementation of a simple method to implement to achieve the previous objectives.

A sixth objective of the invention is to implement a suitable electroporation device and method irrespective of the biological nature of the sample, whatever the configuration in which this sample is (in vivo, ex vitro or in vitro), and regardless of the environment in which the biological sample is located.

A seventh objective of the invention is to adapt the shape of the pulse to the biological load considered (i.e. biological sample considered), this pulse is not necessarily square, as that conventionally encountered in the state of the art.

For this purpose, the invention proposes, according to a first aspect, an assembly for the electroporation of a cell assembly consisting of at least one cell, comprising means for monitoring the power supply of the electroporation electrodes, characterized in that the monitoring means comprise an estimator capable of estimating the effective portion of the power supply that is delivered to one or more determined cell parts of the cell set, from data received by the estimator comprising representative data. power supply and data relating to evaluated bioimpedance components of each determined cell part.

Other features of the invention are:

said data relating to said evaluated bioimpedance components are independent of the data representative of the power supply, and are stored in memory, the bioimpedance components have been evaluated previously in the absence of electroporation,

each determined cellular part is chosen from the intracellular medium, the extracellular medium, the cell membranes, the pores formed from the cellular membranes of said cell assembly,

the estimator is able to estimate, from data comprising said data that it receives, and relations existing between them, at least one of the following electrical quantities: the mean intensity of the average current I _c delivered to the cells of the cellular set, the average voltage Uj _ntra and the mean intensity of the average current I _mtra applied to the only intracellular medium, the average voltage U _m applied to the single cell membrane, the average intensity of the electric current I _p delivered to the only pores formed from the cell membrane,

the estimator is able to estimate, from data comprising said data that it receives, data relating to the average cell thickness, data relating to the average thickness of the cell membrane, data relating to the distance e between the electroporation electrodes, and the relation existing between these data, the electric field applied solely to the cellular membranes of the cell assembly,

the estimator is able to estimate, from data comprising said data that it receives, data relating to the surface S of the electrodes used for electroporation, and of the relation existing between these data, the average density of the electric current. delivered through the pores formed;

the assembly furthermore comprises the electrodes used for electroporation, a generator supplying electrodes to the electrodes and capable of generating a pulsed U voltage and / or a current / pulse suitable for electroporation, means for measuring the voltage U and / or the intensity of the current / providing at the input of the estimator said data representative of the power supply, and means for controlling the values of U and / or of / supplied by the generator to the data output from the estimator, so as to carry out a control on the electroporation; the assembly furthermore comprises a generator capable of generating a voltage and a current lower respectively than the threshold voltage and the threshold current from which electroporation starts, means for measuring the bioimpedance of the cell assembly subjected to electric field of the electrodes fed by the generator with a voltage and / or a current lower respectively than the threshold voltage, and means for evaluating bioimpedance components of each determined cellular part of the cell assembly, from the data provided output means for measuring bioimpedance;

the assembly further comprises switching means capable of switching the electrical supply of the electrodes of a first of the two generators to the second of the two generators, so that it is thus possible to carry out bioimpedance measurements between two electrical pulses with the same electrodes;

- A use of this set for the electroporation of the cell assembly, for the administration of drugs.

According to a second aspect, the invention proposes a method of monitoring a cellular electroporation comprising the following steps:

(a) receiving data representative of the applied electric field or power supply provided during the electroporation; (b) receiving data relating to evaluated bio¬ impedance components of one or more determined cell parts of the cell assembly;

(c) calculating an estimate of the effective portion of the power supply that is delivered to each determined cell part, from the data received in step (a) and step (b).

Other features of the monitoring process are:

said data relating to said evaluated bioimpedance components are independent of the data representative of the electric field, and are stored, the method furthermore comprises at least one evaluation step, prior to step (a), of the components of bio-impedance, in the absence electroporation, performed before any electroporation and / or between two electrical pulses,

step (c) gives an estimate, from data comprising said data received in (a) and (b), and relationships existing between them, of at least one of the following electrical quantities: the average voltage U _ex t _r applied only to the extracellular medium, the average voltage Uintra applied to the only intracellular medium, the average voltage U _m applied to the single cell membrane, the average electrical current I _p delivered to the only pores formed of the cell membrane,

step (c) gives an estimate, based on data comprising said data received in (a) and (b), on the average cell thickness data, data on the average thickness h of cell membrane, data relating to the distance e between the electrodes used for electroporation, and the relation existing between these data, of the average electric field applied to the cell membranes of the cell assembly alone,

step (c) gives an estimation, from data comprising said data received in (a) and (b), of data relating to the surface S of the electrodes used for electroporation, and of the relationship existing between these data, the average density of the electric current delivered through the pores formed,

the method further comprises, after step (c), a step of controlling the electric field to the estimate made during step (c), the method further comprises, before step (a) ), a step of measuring characteristics of the applied electric field for electroporation, or characteristics representative of the electrode power supply (correlated with the electric field), for providing said data representative of the applied electric field or the power supply. provided the electric power received in step (a), - the method further comprises, before step (a), a measurement of the bio¬ impedance of a cell assembly in the absence of electroporation; evaluation bioimpedance components of each determined cellular part of the cell set is then made from this bioimpedance measurement.

According to a third aspect, the invention proposes a method of electroporation of a cell assembly consisting of at least one cell, comprising the following steps:

(a) applying one or more electric field pulses to the cell assembly, capable of causing electroporation in the cell assembly;

(b) measuring data representative of the applied electric field or power supply supplied to the electrodes for electroporation;

(c) implementing said monitoring method.

Other characteristics of the electroporation process are:

the method further comprises, before step (a) and / or during step (a) between two electrical pulses, the following steps: applying an electric field to the cell assembly, the amplitude of which is less than to that of the electric threshold field beyond which the membrane pores are formed by electroporation; measure the bio-impedance of the whole cell; evaluating bioimpedance components of one or more determined cell parts of the cell set, from the measurement results of step (b), so as to provide said data relating to the evaluated components of the bioimpedance of each determined cellular part of the cell set,

a use of the electroporation method for the administration of medicaments.

Other characteristics, objects and advantages of the invention will emerge from the description which follows, which is purely illustrative and nonlimiting, and which will be read with reference to the appended drawings in which:

Figure 1 schematically shows an assembly for electroporation according to the invention.

FIG. 2 represents an estimator according to the invention. FIG. 3 represents an electrical assembly modeling the electrical characteristics of a cell or a set of several cells in the absence of electroporation.

FIG. 4 represents an electrical assembly modeling the electrical characteristics of a cell or a set of several electroporated cells.

FIG. 5 is a graph showing the evolution of a mean membrane tension U _1n as a function of the time of application of a biological tissue to an electroporation electric field, calculated according to the invention.

FIG. 6 is a graph showing the evolution of an average intensity I _p passing through the electropores of an electroporated membrane as a function of the time of application of a biological tissue to an electroporation electric field, calculated according to FIG. invention.

With reference to FIG. 1, a set 1 intended for cellular electroporation according to the invention comprises electrodes 51 and 52 placed around the cell assembly 100 (with one or more cells) fed by a generator 30 able to generate a electrical power adapted to the electroporation of the cell assembly 100, namely classically a pulse voltage U and a current / pulse suitable for electroporation. The assembly 1 also comprises means for measuring the electrical quantities, such as an ammeter 61 and a voltmeter 62, representative of the power supply of the electrodes 51-52 by the generator 30, these means then providing at output 65 and 66 data representative of the power supply, such as data relating to U and /.

Upstream of the generator 30, a control unit 20 controls, via the link 25, the generator 30 to deliver to the electrodes 51-52 a voltage profile U and / or electric / desired current. Upstream of the control unit 20, setpoints (here transmembrane field E _1n and average density J _p of the electric current passing through the pores) are sent automatically or after manual input (by a user via an interface by example of type keyboard or computer mouse) to the control unit 20 which takes them into account then correcting the general command to send to the generator 30. According to the invention, these instructions are modified in 15 and

16 according to the data (here _{m m} and ^) provided at the output 11 and 12 of an estimator 10.

This estimator 10 is able to estimate the effective portion of the power supply that is delivered to one or more determined cellular parts of the cell assembly 100, a cell part being typically the intracellular medium, the extracellular medium, the cell membrane, or the pores formed of the cell membrane, of the cell assembly 100. This estimator 10 mainly bases its estimation on the data (U, I) that it receives (via the links 65 and 66) from the measuring means 61-62, and on data relating to evaluated bioimpedance components of each determined cell part (initially stored in memory and received via the link 95), as well as relationships existing between them.

The set 1 may further comprise an apparatus for evaluating the latter data relating to bioimpedance components of each determined cell part. The latter apparatus comprises a second generator 40 capable of generating a voltage and a current lower respectively than the threshold voltage and the threshold current above which the electroporation begins. The assembly 1 can in this case be provided with switching means 81-82 and 71-72 able to switch the power supply of the electrodes 51-52 of the second generator 40 to the first generator 30, so that the electrodes 51- 52 first provide an electric field capable of not electropore the cell assembly 100 and an electric field capable of electroporation.

A measurement unit 90 is connected upstream of the second generator 40, and comprises: means for measuring the bio-impedance of the cell assembly 100 in the absence of electroporation, when the second generator 40 delivers its low voltage,

Means for evaluating the bio-impedance components of the determined cellular part (s) (ie extracellular, intracellular, membrane, and / or other) of the cell set, on the basis of the data provided by the means for measuring bioimpedance, Means for storing the data relating to the evaluated cellular bio-impedance evaluation components, so that these data can be used (via link 95) by the estimator 10 during F electroporation. Optionally, this measurement unit 90 also makes it possible to evaluate parameters other than electrical components, such as parameters related to the configuration of the electrodes (surface S, inter-electrode distance), and / or parameters relating to the cell assembly 100 to be electroporated (average thickness h of cell membranes, average thickness / biological cells). With reference to FIG. 2, the present invention may also concern, not a complete set 1 intended for electroporation as represented in FIG. 1, but only a monitoring device comprising an estimator 10, able to estimate the effective part. of the power supply which is delivered to one or more determined cellular parts of the electroporated cell assembly 100. This monitoring device can typically comprise a microprocessor making it possible, in particular to perform the calculations according to the invention, a memory for storing data such as electrical data and mathematical formulas, and ports or input links (65, 66, 95 ) that allow input of the payload and ports or output links (11, 12) that allow output of the estimated data. The estimator 10 receives for this purpose (via links 65 and 66) data representative of the power supply delivered to electrodes 51-52 for electroporation of a cell assembly 100 or data representative of the applied electric field (such as eg amplitudes of pulsed electric fields, pulse voltages, pulse currents, pulse frequencies, pulse durations, and / or others).

The estimator 10 further receives (via the link 95) data relating to evaluated cell-specific bioimpedance components (s), and optionally information relating to the configuration of the electrodes and to biological characteristics of the electroporated cell assembly 100. To fully understand the electrical characteristics of a biological cell, it may be necessary, first, to know that biological tissues (or a set of any cell 100) are composed of living cells bathed in an extra-cellular medium. The intracellular medium is protected by a plasma membrane that selects the passage of certain chemical species, the cell membrane is a lipid bilayer with a high power of electrical isolation. With reference to FIG. 3, it would then be possible to model the electrical characteristics of a cell 100 (or of a multi-cell assembly 100) by an electrical circuit whose extracellular portion 130 would be represented by a resistor R _e χ _tra and a capacitor C _ex tra in parallel, the intracellular portion 120 by a resistor Rintra, and the cell membrane 110 by a resistor R _m and a capacitor C _n , in parallel. The intracellular portion 110 is in series with the intracellular portion 120, and all of these two cell parts is in parallel with the extracellular portion 130.

However, it would be wrong to consider the membrane 110 as a simple RC circuit. Indeed, a coefficient called "Cole-Cole coefficient" (see in particular J. Chem Physics 9 341, (1941) of KS CoIe and RH CoIe) is involved in the expression of the electrical parameters of this membrane 110, this coefficient has in particular taken into account the influence of the mode of distribution of the cells and their dispersion in sizes (in the case of a 100 cell multi-cell set) on bioimpedance. The electrical characteristics therefore further include this Cole-Cole coefficient a. All these electrical characteristics form as many bio¬ impedance components that may have been identified for example, from a bioimpedance measurement, by a least squares method.

For a sinusoidal electric field delivered, at a frequency / = ω / 27r, to the cell assembly 100 at an amplitude less than that beyond which the membrane is likely to have electropores, the bioimpedance Z _t (ω ) of the cellular set 100 can then be written according to the following formula:

This bioimpedance Z, (ω), measured for a weak applied electric field (ie not likely to cause electroporation of the cell assembly 100), thus has a linear behavior. From the measurement of Z _t (oi), and ω, we can evaluate the components Rextr _a , C _ex tra, Rfatra, R _m , Cm and a of the cell set 100.

These bioimpedance components are then advantageously stored in memory. When an electric field (or a voltage U and / or a current / to the electrodes used for electroporation) is then applied, capable of creating electropores in the cell assembly 100, electropores are formed in the membranes 110 which thus become permeable to molecules, thus showing, this time, a non-linear behavior of the electroporated cells.

The Applicant proposes, with reference to FIG. 4, an electrical assembly equivalent to the modeled electrical characteristics of a cell assembly 100 thus electroporated. The Applicant proposes to model the electrical part linked to the electropores by a shunt 112 of the unaltered membrane portion 111, the charge of which is a nonlinear dipole D _p . Part of the electric current I _c which feeds the cells directly will therefore go to the unaltered membrane portion 111, and another portion of / _c , denoted I _p , will be only the current flowing through the altered membrane portion 112 through the electropores.

In this context, the estimator 10 is then able to estimate the effective portion of the power supply (UJ) that is supplied to the electrodes or the electric field applied to the determined cellular parts of the cell assembly 100.

It should be noted that, if the data received via links 65-66 come from measurement of voltage U and / or current / made upstream of electrodes 51-52, it is preferable that the frequency of the sinusoidal signal then chosen for this voltage and / or this current is not too low, so that the effects of the electrode-tissue contact tissue do not significantly disturb the results of the estimation calculations to be performed (listed below). Typically, the frequency is chosen greater than 50 Hz.

Thus, the average voltage O ext _ra applied to the extracellular medium 130 alone can be calculated as follows:

Uexra ^≈ U (2) Thus, the average electric current / _c delivered to the cells of the cell assembly 100 alone can be calculated as follows:

¹ C - ¹ _p 'extra, tJ

The formula (3) eliminates the effects related to the extracellular medium. Thus, the average voltage _{U i ntra} and the average current I _{i will be} delivered to the only intracellular medium of the cell assembly 100, can be calculated as follows:

Mntra - * c> Uintra K intra intra vv

Thus, the average voltage U _m applied to the only cell membrane of the cell assembly 100 can be calculated as follows:

U _n = U-Ru _n I ₀ (5)

The formula (5) eliminates the effects related to the intracellular medium 120.

The average current I _p delivered to the only pores formed of the cell membrane of the cell assembly 100, can be calculated by modeling in the firequential domain the unaltered membrane portion 111 by a constant phase element admittance (also known as "admittance"). CPE ", from the English acronym

"Constant Phase Element"). This model takes into account a distribution of the electrical time constants of the membrane and can be used at the level of a cellular tissue (in vivo, ex vivo), or cell (s) in artificial medium (in vitro). In the time domain, the unaltered membrane can then be characterized by means of its impulse response ycpε (t) calculated by the inverse Laplace transform of this admittance, which can be expressed as follows:

y _CPE (t) ≈ Tr \ ~] (6)

where: TL ^'1 represents the inverse Laplace Transform, τ = R _m C _m , p = / ω, where "y" indexes the imaginary part of a complex number. We then obtain, for _p , the following expression:

I _p (t) = I _c (t) -y _CPE (t) * U _m (t) (7) where the product symbolized by "*" is a product of convolution, between the average transmembrane voltage U _m (t) and its impulse response ycpεit).

The formula (7) makes it possible to eliminate the influence of the unaltered membrane 111, so as to retain only the average intensity of the current linked to the electropores (altered membrane 112).

It should be noted that all the previous electrical quantities (JJex _t m, Ic, Ui _ntra , Ii _ntm , U _m , I _p ) correspond to macroscopic quantities. Thus, for example, U _1n corresponds to the average estimated voltage applied to all the membranes of the cells making up the cell assembly 100. In the absence of electroporation, I _c (t) is naturally equal to yc _PE ( ή * U _m (t), and

I _p (t) then becomes zero, as if the nonlinear dipole D _p then had an infinite impedance. The model established by the Applicant (with reference to FIG. 4) thus functions as well for a cell assembly 100 undergoing electroporation as for a cell assembly 100 not undergoing any electroporation.

Optionally, the estimator 10 also receives, via the link 95, information relating to the configuration of the electrodes and to the biological characteristics of the electroporated cell assembly 100, in order to estimate other electrical quantities representative of the distribution of the diet in the cellular set.

Thus, if the estimator 10 receives average cell thickness data, data relating to the average cell membrane thickness h (typically between about 5 and about 6 nanometers), and data relating to the interelectrode distance e, the estimator 10 can calculate the estimated average electric field _{m m} applied to only the cell membranes 110 of the cell assembly 100 by means of the following formula:

The formula (8) makes it possible to normalize the average transmembrane voltage with respect to the cell sizes and the inter-electrode distance in order to define the average transmembrane electric field. This estimated average electric field E _M therefore represents the average electric field applied to each cell membrane of the cell assembly 100.

Thus, if the estimator 10 receives data relating to the surface S of the electrodes used for electroporation, the estimator 10 can calculate the estimated average density J _p of the electric current delivered through the pores formed of the cell assembly 100. using the following formula:

Λ ^p = ~ S (9)

J _p is a macroscopic quantity.

The estimator 10 can thus output one or more of these electrical quantities (estimated using the preceding formulas), and / or other electrical quantities participating in the same concept.

For example, the estimator 10 can provide on the outputs 11 and 12 one or more of the following pairs of values: (Ui _ntra , Iintra), (U _m Jp), ( _{m m} , J _p ).

The output (here 11 and 12) of the estimator 10 can then be connected to a screen-type terminal, or to other calculation means, or to a memory in which the data it provides are stored, or to another system such as a device for controlling the power supply of the electroporation electrodes.

This estimator 10 thus allows a real-time monitoring of the degree of electroporation of the cell assembly 100, since it provides data to adapt (manually or automatically by a servo system allowing self-adaptation of the system) the electric field of electroporation according to the electrochemical phenomena observed. It will thus be possible to maximize the number of electroporated cells and minimize the number of cells destroyed, by varying the size of the electropores within the cell membranes.

This estimator 10 can also make it possible to dispense with all the parameters external to the electroporation (electrode geometry, quality of the electrode-tissue contact, influence of the extracellular medium, etc.), if it provides, for example at the output, a mean membrane voltage estimate U _m and / or an estimate of the average intensity I _p of the pore stream.

This estimator 10 also gives, in real time, state variables specific to the electropores of the cell assembly 100 (ie the estimated average transmembrane electric field _{m m} , and the estimated average density J _p of the current passing through the electropores).

This estimator 10 can function regardless of the biological nature of the cell assembly 100 to be electroporated, regardless of the configuration in which the cell assembly 100 is (in vivo, ex vivo, or in vitro), and whatever the extracellular environment in which it is bathed.

According to a first variant of the invention, the monitoring device comprising the estimator 10 (and possibly a screen and / or others) is part of a device for controlling an electroporation of a cell assembly 100. control then comprises, with reference to Figure 1, the estimator 10 and said control unit 20. It further comprises means for generating instructions upstream of the control unit (at 17 and 18), these means may be a user interface by means of which it enters the desired instructions, or means to automatically generate these instructions according to specific parameters, or means able to fetch in memory, at a certain frequency, the pre-recorded instructions . In addition, this control device comprises means 15 and 16 for modifying the desired setpoints (coming from 17 and 18) according to the estimate data supplied at the output (11 and 12) of the estimator 10.

This control device then makes it possible to enslave the electroporation to the estimate made by F estimator 10.

According to a second variant of the invention, the control device is part of an assembly for electroporation further comprising said voltage generator U and / or pulsed current I suitable for electroporation, the electrodes 51-52 of the electrodes. electroporation, as well as the measuring means 61-62 of electrical quantities of the supply delivered by the generator 30 or by the electrodes 51-52, these measuring means providing information representative of the applied electric field or the electrical supply supplied to the electrodes for electroporation, at the input of the estimator 10 (via links 65 and 66).

The generator 30 may be a controlled power generator for delivering voltage and / or current levels (possibly high) to initiate electroporation of the "biological charge" (i.e., the cell assembly 100). This generator 30 can deliver pulses of any shape, since the estimator 10 can be adapted (by playing on the instruction sent to the control unit 20) the shape of the pulse to the biological load considered (ie cell assembly 100 ). This impulse is not necessarily square.

The electrodes 51-52 may be a pair of electrodes making it possible to impose an electric field of electroporation on a "biological charge" (ie the cell assembly 100) which may be a cellular tissue in vivo or ex vivo, or cells in vitro (or even a single cell), or a cell tissue placed in a bio-chip. The measuring means 61-62 may comprise means for measuring electrical quantities representative of the electrical supply supplied to the electrodes for electroporation, thus including, for example, an ammeter 61 measuring the intensity of the current absorbed by the cell assembly 100. and a voltmeter 62 measuring the inter-electrode voltage. The measuring means 61-62 may also comprise measurement electrodes, arranged on the cell assembly 100, for directly measuring the electric field applied to the cell assembly, and thereby avoiding the influence on the result of the measurements. measurements, of the electrode-tissue contact.

This set makes it possible in particular to control the quantities specific to electropores on a particular instruction, making it possible to control the degree of electroporation of the cells without destroying them. The control of a mean transmembrane electric field E _n , makes it possible to trigger the electroporation phenomenon without destroying the membranes. The control of the average density J _p of the current passing through the electropores makes it possible to control the average surface area of the electropores. Prior to the implementation of electroporation, an evaluation of bioimpedance components can be implemented (Rextra, C _e χtra ₃ Rintra, Cintra _» Rm, C _m, a, ...) of each cell fixed portion (extracellular medium 130, intracellular medium 120, membrane 110) of the cell assembly 100, from a measurement of bio-impedance of the cell assembly 100 in the absence of electroporation. For this purpose, a device according to the invention for performing such functions is provided, incorporated or not incorporated in said monitoring device (with reference to FIG. 2), or said control device according to the first variant of the invention, or audit set for electroporation according to the second variant of the invention.

The latter device requires a second generator 40, with low-voltage electronics, to determine the linear behavior of the "biological charge" (the cell set 100) and the electrodes (51-52) for measuring the bioimpedance Z, (ω ) in the absence of any electroporation.

In addition, this device comprises the measurement unit 90 previously described (with reference to FIG. 1). The treated biological tissue and its electrodes are therefore previously characterized by a small signal impedance measurement. This measurement implements a low voltage electronic electronics, which does not cause electroporation.

In the case where this impedance component evaluation device is incorporated in the rest of the assembly intended for electroporation, then the assembly 1 of FIG. 1 is obtained.

Optionally, switching means 71-72 and 81-82 are provided between the generators 30 and 40 and the electrodes 51-52, for switching the supply of the electrodes of a generator 30 or 40 to the other generator 40 or 30.

This arrangement can then be used to supply low voltage electrodes 51-52 with the generator 40 between two high voltage electric pulses delivered by the generator 30. It is thus possible to carry out bioimpedance measurements between two electrical pulses. electroporation, with the same electrodes 51-

52, without intervening on the latter.

This arrangement therefore makes it possible to observe the possible modifications that the electrical components of bioimpedance (in the absence of electroporation) can undergo as the number of pulses given to the tissue increases. Cellular 100 increases, thus updating the respective values of these components. This assembly may be particularly useful during electroporation in vivo, the cells may then possibly swell under the effect of electroporation and thus see their electrical components slightly change over time).

The cell assembly 100 to be electroporated according to the invention may be a living tissue in vivo around which the electrodes 51-52 have been placed, or a tissue taken (ex vivo), or a tissue placed in an artificial medium (in vitro). , or a cellular tissue placed in a bio¬ chip. It can be vegetal, animal or human. Electroporation can bring into play active products such as mineral drugs, proteins or peptides (nucleic acid, DNA or RNA molecule having sequences encoding therapeutic genes or genes that are of interest to health or for biotechnological purposes, natural oligonucleotide or modified, ...) to administer them to the cell assembly 100. Experimental study: The Applicant presents hereinafter results obtained after the implementation of an electroporation method according to the invention, made on the tissue 100 of a potato.

The fabric 100 is placed between two electrodes 51 and 52 of stainless steel, having a surface S of 30 mm ² and separated by a distance e of about 6 mm. The bioimpedance of the tissue is measured using an impedance analyzer

HP4192A, and a sinusoidal low voltage generator (that is to say a generator capable of delivering sinusoidal voltages below the threshold voltage beyond which electropores would appear) at 1 V amplitude and at frequencies between 50Hz and 10MHz. This latter frequency range is preferably chosen because it makes it possible to detect the components of bio¬ impedance without these being significantly disturbed by the effects of the electrode-tissue contact tissue.

The linear components of bioimpedance are then evaluated by means of a calculation using a least squares identification of the measured bioimpedance module, and are as follows:

R _extm = 18 kΩ; i _{ra Ra} = 151 Ω; R _m = l, l kΩ; C _m ≈ 7.2 nF; a ≈ 0.1677 Good agreement was found between the impedance measured and the impedance reconstituted according to formula (1) from the identified components.

Once these components are known, unipolar pulses and currents of length of about 150 μs each, by varying the amplitudes between 0 V and 300 V, are applied to the electrodes 51-52 by a suitable generator 30.

The shapes of the wave intensities I of the current supplied to electrodes 51-52, when measured, then revealed linear behavior for voltages below 100V ₅ while a large nonlinearity appeared beyond 125 V.

The threshold voltage from which the electropores begin to appear would therefore be between 100 V and 125 V under the conditions of the experiment.

FIG. 5 represents the mean macroscopic transmembrane voltage U _m as a function of the time of application of the fabric 100 to the pulsed electric field, calculated using formula (5).

FIG. 6 represents the average macroscopic intensity I _p passing through the pores as a function of the time of application of the tissue 100 to the pulsed electric field, calculated using formula (7).

It can be seen that U _m (t) increases with U (t), and that I _p (t) appears from the threshold voltage (located between 100 V and 125 V) and that it becomes important as soon as it is applied. a voltage U (t) much higher than this threshold voltage (here U (t) = 200V during the pulse).

It can be observed that the mean macroscopic membrane tension U _m (t) does not vary linearly with the voltage U (t). These measurements are in adequacy with results of theoretical analytical modelings giving dynamic evolutions of electropores starting from transmembrane voltage values, in particular given by JC Neu and W. Krassowska (Phys Rev. E. 59, 3471 (1999)) and by KA DeBruin and W. Krassowska (Ann Biomed Eng 26, 584 (1998)).

Claims

An assembly for the electroporation of a cell assembly consisting of at least one cell, comprising means for monitoring the electrical supply of the electroporation electrodes, characterized in that the monitoring means comprise an estimator capable of estimating the effective portion of the power supply that is delivered to one or more determined cellular parts of the cell assembly, from data received by the estimator including data representative of the power supply and data relating to components evaluated bioimpedance of each determined cell part.

2. An assembly according to the preceding claim, characterized in that it further comprises a memory storing said data relating to said evaluated bioimpedance components, these being independent of data representative of the power supply.

3. An assembly according to the preceding claim, characterized in that it further comprises means for evaluating the bioimpedance components in the absence of electroporation.

4. The kit as claimed in claim 1, wherein each determined cellular part is chosen from the intracellular medium, the extracellular medium, the cell membranes and the pores formed from the cell membranes of said cell assembly.

5. An assembly according to the preceding claim, characterized in that the estimator is arranged to estimate, from data comprising said data it receives and relationships existing between them, at least one of the following electrical quantities: the average intensity of the current I _c delivered to the cells of the whole cell only, the average voltage Uj _n t _ra and the average intensity of the current Iintr _has applied to only intracellularly, the mean voltage _Um applied to the. Only cell membrane, the average intensity of the electric current I _p delivered to the only pores formed of the cell membrane.

6. An assembly according to one of the preceding claims, characterized in that the estimator is arranged to estimate the average electric field applied to each cell membrane of the cell assembly, from data comprising said data it receives, from mean cell thickness data; data relating to average cell membrane thickness; data relating to the distance e between the electroporation electrodes; and the relationship between these data.

7. Assembly according to one of the preceding claims, characterized in that the estimator is arranged to estimate the average density of the electric current delivered through the pores formed, from data comprising said data it receives, data relating to to the surface S of the electrodes used for electroporation, and of the relation existing between these data.

8. Assembly according to one of the preceding claims, characterized in that it further comprises:

The electrodes used for electroporation,

A generator electrically supplying the electrodes and capable of generating a pulsed U voltage and / or a current / pulse suitable for electroporation; means for measuring the voltage U and / or the intensity of the current / supplying the input from the estimator said data representative of the power supply; and

Means for controlling the values of U and / or I supplied by the generator to the data delivered at the output of the estimator, so as to produce. thus a control on the electroporation. 9. Assembly according to one of claims 3 to 8, characterized in that said means for evaluating the bioimpedance components in the absence of electroporation include:

A generator capable of generating a voltage and / or a current lower respectively than the threshold voltage and the threshold current from which the electroporation starts;

Means for measuring the bio-impedance of the cell assembly subjected to the electric field of the electrodes supplied by the generator with a voltage and / or a current lower respectively than the threshold voltage and the threshold current; and

Means for estimating bioimpedance components of each determined cellular part of the cell assembly, from the data supplied at the output of the means for measuring the bioimpedance.

10.A set according to the two preceding claims, characterized in that it further comprises switching means adapted to switch the power supply of the electrodes of a first of the two generators to the second of the two generators, so as to be able to achieve bioimpedance measurements between two electrical pulses with the same electrodes.

11. Assembly according to one of the preceding claims, characterized in that it is intended for the administration of medicaments in the cell assembly.

H.A method of monitoring cellular electroporation comprising the steps of:

(a) receiving data representative of the applied electric field or the power supply supplied to the electrodes during electroporation;

(b) receiving data relating to evaluated bioimpedance components of one or more determined cellular ensemble cell parts; (c) calculating an estimate of the effective portion of the power supply that is delivered to each determined cell part, from the data received in step (a) and step (b).

13. Monitoring method according to the preceding claim, characterized in that said data relating to said evaluated bioimpedance components are independent of the data representative of the electric field, and are stored.

14.Procédé monitoring according to the preceding claim, characterized in that it further comprises at least one step of evaluation, prior to step (a), bioimpedance components, in the absence of electroporation .

15.The monitoring method according to the preceding claim, characterized in that a step of evaluation of bioimpedance components is performed before any electroporation.

Monitoring method according to one of the two preceding claims, characterized in that the data received during step (a) are representative of the application of an applied pulsed electric field or a pulsed power supply provided. to perform the electroporation, and in that a step of evaluating bioimpedance components is performed between two electrical pulses.

17.The surveillance method according to one of claims 12 to 16, characterized in that each determined cell part is selected from the intracellular medium, the extracellular medium, cell membranes, pores formed cell membranes of said cell assembly.

18.A surveillance method according to the preceding claim, characterized in that step (c) gives an estimate, from data comprising said data received in (a) and (b) and relations existing between them, of at least one of the following electrical quantities: the average intensity of the current I ₀ delivered to the single cell (s) of the electroporated cell assembly, the average voltage U _mtra applied to the extracellular medium only, the average voltage U _m applied to the single cell membrane, the average intensity of the electric current I _p delivered to the only pores formed of the cell membrane.

19.Surveillance method according to one of the two preceding claims, characterized in that step (c) gives an estimate of the average electric field applied to each cell membrane of the cell assembly, from data comprising said received data. in (a) and (b), data on average cell thickness /, data on average cell membrane thickness h, data on distance e between electrodes used for electroporation, and the relationship between these data.

20.Surveillance method according to one of the three preceding claims, characterized in that step (c) gives an estimate of the average density of the electric current delivered through the formed pores, from data comprising said data received in (a) and (b), data relating to the surface S of the electrodes used for electroporation, and the relationship between these data.

21.Surveillance method according to one of claims 12 to 20, characterized in that it further comprises, after step (c), a servocontrol step of the electric field applied to the estimate made during the step (c).

22.Surveillance method according to one of claims 12 to 21, characterized in that it further comprises, before step (a), a step of measuring characteristics of the applied electric field for electroporation, or of Representative characteristics of the electrical power supply of the electrodes (correlated with the electric field), to provide said data representative of the field applied electrical power or power supplied to the electrodes received in step (a).

23.Surveillance method according to one of claims 14 to 22, characterized in that it further comprises a measurement of the bioimpedance of a cell assembly in the absence of electroporation; said step of evaluating the bioimpedance components of each determined cell part of the cell assembly is then performed from this bioimpedance measurement.

24.A method of electroporation of a cell assembly consisting of at least one cell, comprising the following steps:

(a) applying one or more electric field pulses to the cell assembly, capable of causing electroporation in the cell assembly; (b) measuring data representative of the applied electric field or power supply supplied to the electrodes for electroporation; (c) implementing the monitoring method according to one of claims 12 to 21.

25. Electroporation method according to the preceding claim, characterized in that it further comprises, before step (a) and / or during step (a) between two electrical pulses, the following steps:

Applying an electric field to the cell assembly, the amplitude of which is smaller than that of the threshold electric field beyond which the membrane pores are formed by electroporation;

- measure the bio-impedance of the whole cell;

Evaluating bioimpedance components of one or more determined cellular parts of the cell set from the measurement results of step (b) so as to provide said data relating to the evaluated components of the bioimpedance of each determined cell part of the cell set.

26.Use of the electroporation method according to one of the two preceding claims for the administration of medicaments.