SI22368A - The tip electrode chamber for small volume electroporation, electrofusion and gene transfection - Google Patents

Abstract

Subject of present invention is an apparatus for electroporation with pipetter, multi-electrodes design with minimized inhomogeneous electric field and solid filler for electrode chambers to minimize inhomogeneous electric field. A tip electrode chamber for electroporation according to the invention is characterized in that the opening 5 is in the middle of housing (1) and in the longitudinal direction through the housing (1) and that from two to six electrodes (2) are parallelly and symmetrically mounted into the inner surface of the housing (1) and longitudinally to the opening (5) and partially extending to the said opening (5) and that the said electrodes (2) have electrical connections (3) to exterior. A lso a tip electrode chamber is disclosed, where the solid filler (6) is inserted into the said opening (5) with the hole (7) in the longitudinal direction of the housing 1 and solid filler (6) two to six electrodes (2) are parallelly and symmetrically mounted into the contact of surfaces of the housing (1) and solid filler (6) and that the said electrodes (2) have electrical connections (3) to exterior.

Description

The object of the present invention is a pipette electroporation apparatus, multi-electrode preparation with a minimized inhomogeneous electric field and a compact chamber filler with integrated electrodes for minimizing a non-homogeneous electric field.

The present invention relates to a tapered chamber with built-in electrodes for small-volume electroporation, for electrofusion and gene transfection. The invention relates to the field of electroporation of biological cells in suspension and, more specifically, to the field of techniques and devices for electroporation with the aim of electrically permeabilizing the cell membrane and, consequently, electrically inserting any active pharmaceutical-chemical or biological-active substance, and the aim of gene transfection, and cell fusion. The invention provides electrodes for the fast and efficient electroporation of small and controlled volumes of cell suspensions, which retain their high growth ability due to minimal mechanical measures and the ability to use electrical impulses in different directions, thereby increasing the membrane area of the electroporated cell and thus the electroporation efficiency without significant cell viability losses.

BACKGROUND OF THE INVENTION

Use of electroporation

Electroporation (also called electropermeabilization) of cells is applied for various purposes that utilize the permeabilized state of the cell membrane.

Electrical implantation or electrical extraction represents the introduction or extraction of various molecules, such as drugs, proteins or genes, into cells. The implantation of small molecules is effective

-2 reaches using some short rectangular high voltage pulses. This method is now effectively used in electrochemotherapy to introduce drugs into tumor cells in situ of patients. Electro-extraction is used to extract yeast products from processes in the opposite direction of flow.

Genetic electrotransfection in vitro and in vivo represents the implantation of plasmids containing the appropriate genetic material into cells. DNA is a large molecule that has been found to be successfully implanted using exponentially decaying pulses or a combination of shorter high voltage pulses and longer low voltage pulses.

Another related procedure is cell electrofusion. It consists of two stages. One stage causes the fusogenization of the membranes by electroporation, and through the other, close contacts between the cells are achieved. They can be secured either before electroporation or shortly thereafter (within the first few minutes) as long as the membranes are still in the fusogenic state

Mechanical treatment

In the vast majority of existing methods for electroporation of cells in suspension, either a chamber or veto or flow system with one or more pairs of flat electrodes is used. Place the cell suspension or cells grown on the substrate between the electrodes and release a voltage pulse across them. The cells in suspension are usually pipetted in electrode chambers or cells or outside one of them.

These statements represent mechanical processing that is associated with two problems. The first is significant volume loss, and the second is that pipetting is detrimental to cells and could even be fatal to certain cell types that are more sensitive, especially after being exposed to electroporation.

Electroporated cells have damaged membranes and are significantly more sensitive to pipetting, which forces pressure on the cells only. This comes up as a problem e.g. in electrofusion protocols where intercellular contact is achieved after electroporation. The cells should be moved from the chamber to the centrifuge tube with a micropipette immediately after electroporation.

-3Small volume

Another problem is the size of the existing chambers. Molecules (dyes, plasmids) used in experiments are often expensive and only available in small quantities. Additionally, cells to be bound or transfected are often valuable and only available in small quantities. It is therefore necessary to enable small volume work.

The local electric field is important for electroporation. For some cells, e.g. small cells, we need high voltage to the distance quotient. This can only be achieved with a small distance between the electrodes because the electrical impulse generators are limited in the voltage they can generate. The problem of small spacer chambers or the wind is the use of them without significant loss of sample volume. It is difficult to fill such chambers without air bubbles, which significantly alter the electrical distribution of the field in the sample. Filling a chamber or cell with a cell suspension without air bubbles and getting out the cell suspension after electroporation is difficult, if not impossible. Cleaning is difficult too. Effective emptying, filling and cleaning of the small space between the electrodes should be possible.

According to US patent US6492175, there is a flow system that allows low volume operation and reduces mechanical manipulation, but only allows the processing of cells with impulses in one or two opposite directions.

Different impulse directions

Most existing electrode systems allow the use of pulses in only one or mostly two opposite directions. Bipolar impulses are described in International Patent Application WO92 / 06185. Over the past few years, it has been demonstrated that the use of pulses in different directions results in more efficient permeabilization and gene electrotransfection. This is also important for cellular electrofusion and protein implantation. The reason for this is the larger surface area of the electroporated membrane that can be achieved by such pulses without significant loss in cell viability.

A standard option for increasing the surface area of an electroporated cell membrane is to increase its electrical power, which invariably leads to a significant reduction in cell viability. Different

-4 impulse directions, however, increase the permeabilized membrane area without significantly affecting cell survival. The possibility of electrofusion of the cell suspension which could be electroporated in different directions should be provided. The possibility of electroporation should be given after cell contact is ensured or before.

US6746441 describes flow through a chamber apparatus with rotating electric fields generated by two or more than two (four or six) electrodes. However, this is a flow through an ex vivo gene therapy system that does not allow for low volume surgery. US2005048651 discloses tapered electrodes that also contain various types of conductive surface electrodes. They are intended for the passage of various substances from the inside of the tip to the targets (cells, etc.) that are outside the tip and are synchronized in such a way that electrical impulses are used. However, the cells are not intended to be implanted into the lumen of the tapered electrodes and electrically treated there.

Simultaneous electroporation for electrofusion

A significant problem is the process for the fusion of two different cell types. This is the case, for example. occurs in hybrid monoclonal antibody production technology, in which B lymphocytes have to fuse with myeloma fusion partners. The latter may be twice as large as lymphocytes. The total electrical treatment of either cell type is not optimal in this case. Separate electroporation and cell contact following electroporation are necessary for the optimum result of the fusion process. The same problem is present with any cell pair that is differently sensitive to the electric field for any reason other than different size.

According to International Patent Application W003 / 050232, the cuvette contains implants supporting structure that holds the porous membrane and facilitates cellular fusion on a membrane basis. In such vets, both cell types are electroporated simultaneously. In this case, smaller cells either sub-optimally permeabilize or larger cells get damaged. The situation is the same with the systems according to DE10359189 and DE10359190. US4882281 and US513470 also describe electroporation apparatus which avoids mechanical treatment because cells are treated in culture vessels, although cell transfer still requires pipetting to further fuse separately electroporated cells.

-5The nature of the fusion process requires that cell contact must be established within a short time after electroporation, when the cell membranes are still in the so-called fusogenic state. Pipetting cells from two separate chambers is in addition to inducing mechanical stress, which causes the cells to be damaged, even slowly, and poses a considerable problem in achieving cell contact in the short time required for efficient cellular electrofusion.

Semiconductor inside electrode tapered chamber

Cell viability has recently been shown to be the lowest near the electrodes. This is due to electrochemical reactions occurring at the interface between the electrode and the medium. In DE20302861U1, it was therefore proposed to cover the electrodes with a thin coating of biodegradable coating, keeping cells away from this boundary.

However, another problem arises regarding the homogeneity and distribution of the electric field between the electrodes. The electric field used (with certain electrical impulses) is not homogeneous over the entire space between the electrodes, but only in the middle part of this space. Only cells in the midbrain are exposed to the desired electric field.

A problem that has not yet been solved is the electroporation of predefined small volumes of cells in suspension. This will reduce costs or allow for gene transfection, protein implantation and cell fusion experiments when the amount of proteins, plasmids or cells is limited.

The object of the present invention lies in the successful electroporation of cells with impulses that reach the cell in different directions, thereby achieving minimal mechanical loading of the cells with the possibility of rapid cell processing.

It is a further object of the present invention to construct an electroporation apparatus to facilitate cellular fusion in vitro. Furthermore, it is an object of the present invention to provide a method for the fusion of two different cell types, which is enabled by separate electroporation with different values of electrical parameters.

-6 According to the present invention, it is also possible to provide a plan for the interior of a tapered chamber with embedded electrodes with conductivity comparable to the conductivity of the cell suspension medium. This ensures that the cells in the central region of the chamber are retained and the desired homogeneous electric field in the area is obtained.

SUMMARY OF THE INVENTION

According to the present invention, cells can be retained in the central lumen region using e.g. semiconductor material or composite conductivity material comparable to the conductivity of the cell suspension medium in the lumen of a chamber with embedded electrodes. In this way, the homogeneity of the field is ensured by conductivity similar to the conductivity of the cell suspension medium. Therefore, the first object of the present invention is to prepare a chamber with built-in electrodes for electroporation of small controlled cell volumes with minimal mechanical processing and consequently minimal mechanical load.

The present invention allows for the rapid treatment of different cell types in different ways by electricity, i.e. with different values of electric field parameters. The invention further provides the possibility of using electrical impulses in several directions, thereby increasing the membrane area of electroporated cell membranes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a cross-section through a tapered chamber with embedded electrodes, on a pipette 4.; electrodes 2 are mounted on the inside of the housing 1 and electrically connected to the outside 3;

FIG. 2 is a cross-sectional view of a tapered chamber with embedded electrodes;

FIG. 3 shows possible electric field directions in the lumen of a chamber with electrodes mounted in one electrode position;

-7Sl. 4 shows a tapered chamber with built-in electrodes partially filled with material 6 and conductivity comparable to the conductivity of the cell suspension medium.

DETAILED DESCRIPTION OF THE INVENTION

The tapered chamber with built-in electrodes consists of a housing 1, electrodes 2 and electrical connectors 3 for connection to the outside. The housing 1 is made of non-conductive material such as polymethyl methacryl (PMMA) or plastic materials. Electrodes 2 and electrical connectors 3 are made of electrically conductive material such as metals or their alloys, or e.g. graphite.

The upper part la of the housing 1 of the tapered chamber with built-in electrodes is designed to fit with the pipette 4. Pipette 4 usually sucks 1 μΐ to 100 μΐ of liquid. However, pipette 4 is not an object of the present invention. The exterior of the lower lb of housing 1 is tapered, making it easier to use with standard laboratory equipment such as centrifugal tubes. Throughout the housing 1 there is an opening 5 in the middle. Through this opening 5, the cell suspension is sucked into the needle electrode chamber and, after electroporation, is blown out of the tapered chamber with pipette electrodes 4. When the cell suspension is sucked in, it is placed in the opening where the electrodes are located 2. The aperture 5 between the electrodes determines the maximum volume of evenly treated cells. The maximum volume of the cell suspension that can be inserted thus lies in a relationship limited by design options. The design permits a maximum volume of 10 μΐ to 2500 μΐ, mostly from 10 μΐ to 1000 μΐ. However, the real volume of evenly treated cells can be selected appropriately for each experiment simply by adjusting the pipette.

The cross-sectional shape of the aperture 5 between the electrodes depends primarily on the number of electrodes 2. If two electrodes 2 are used, the cross-section of the aperture 5 is in principle rectangular. If three or more electrodes 2 are used, the cross-section of the aperture 5 is in principle an equilateral shape with the number of sides such as the number of electrodes 2. Therefore, if three electrodes are used, the cross-sectional shape of the aperture 5 is in principle an equilateral triangle; if four electrodes 2 are used, the cross-sectional shape of the aperture 5 is in principle an equilateral quadrilateral, or. square, etc. If three or more electrodes are used, the sides of the aperture 5 are slightly concave to minimize the inhomogeneity of the electric field in the aperture 5. This concavity can be up to 20% of the length of the side.

-8Multiple electrodes 2 are mounted parallel and symmetrically to the inner surface of the housing 1 and longitudinally to the opening 5. The construction allows the length of the electrodes 2 from 5 mm to 60 mm, mostly from 10 mm to 40 mm. The number of electrodes 2 can vary between two to six electrodes; this number can be odd or even. The number of electrodes 2 affects the number of different directions of the electric field used. For example, four electrodes 2 placed in quadrant angles allow the use of pulses in eight different directions (IB and ET, BI and IE, IT, TI, BE, EB, IE and BT, El and TB). The directions are shown in FIG. 3a, 3b and 3c. With the number of electrodes, the number of different directions of the electric field increases.

The detailed design of the aperture 5 and the electrodes 2 depends largely on the cross-sectional shape of the electrodes 2. There are three main options for the cross-sectional design of the electrodes 2: rectangular, circular, or on the one hand elliptical, parabolic or. hyperbolic shape, and on the other hand, any shape. If only two electrodes are used, the shape is preferably rectangular in order to obtain the most homogeneous electric field in the aperture 5, and the other shapes mentioned previously may be used. When two electrodes are used, the shape of the electrodes is usually circular because it is easier to produce; however, elliptical, parabolic or hyperbolic one-sided designs may also be used. However, a rectangular shape can be used for more than two electrodes, except that the homogeneity of the electric field is very low.

Most of the surface of said electrodes 2 is usually embedded in the material of the housing 1, but a smaller portion of the electrodes 2 is usually viewed from the material of the housing 1. These smaller parts are in direct contact with the cell suspension when sucked into the opening 5 of the housing 1. When using a rectangular electrode shape 2, preferably insert three of the four straight sections of the electrode 2 into the housing material 1 and preferably one of the four planes project out of the housing material 1. The rectangular electrodes 2 may be arranged symmetrically in the centers, or preferably three or more electrodes 2 may be placed at angular points of the edge 5. When using the other electrode shapes 2 mentioned above, the part of the surface embedded in the material of the housing 1 depends on the number of electrodes 2. When two electrodes 2 are circularly shaped, their centers lie symmetrically with the center edge of the cross-section of the aperture 5 , and preferably 180 ° electrodes 2 are embedded in the housing material 1, and preferably 180 ° electrodes 2 are viewed 1. When three or more electrodes 2 of circular shape are used, their centers are positioned at the angular points of the cross-section of the shape of the aperture 5, their parts projecting from the material of the housing 1 being preferably the same as the inner angles. equilateral orifice plan 5. So if three are used

-9electrodes 2 in circular shape, 60 ° electrodes 2 are preferably projected out of the material of the housing 1 and preferably 300 ° electrodes 2 are embedded in the material of the housing 1; if four circular electrodes are used, it preferably views 90 ° electrodes 2 from the housing 1 and preferably 270 ° electrodes 2 are embedded in the housing material 1, etc. When using an elliptical, parabolic or hyperbolic unilateral electrode shape 2, these specific shapes must be directed to the center of the cross-section of the aperture 5. Any form of unilateral elliptical, parabolic or hyperbolic electrode shape 2 is preferably fully embedded in the housing material 1. Thus, this is arbitrary the shape is usually a T-shaped profile to achieve strong contact between the electrodes 2 and the housing 1. Elliptical, parabolic or hyperbolic shapes of one side of the electrodes 2 preferably all look out from the material of the housing 1. When two elliptical, parabolic or hyperbolic one-sided electrode shapes 2 are used are usually positioned at the center of the edge of the transverse aperture 5. If three single-sided electrode shapes 2 are used or multiple elliptical, parabolic or hyperbolic one-sided electrode shapes 2 are used, the former are typically positioned at angular points of the transverse aperture 5.

The diameter of the electrodes 2 and the distance between them affect the homogeneity of the electric field of the aperture 5. This influence is not so obvious when two electrodes 2 of rectangular shape are used. Therefore, for two electrodes of rectangular shape, the diameter-to-distance distance between electrodes 2 is arbitrary. The diameter can vary from 0.5 to 7 mm and the distance between them can vary from 0.5 to 6 mm. The choice depends on the maximum volume of evenly treated cells and the desired voltage-to-distance ratio. As mentioned above, the maximum volume of uniformly treated cells is defined as the volume of openings 5 between the electrodes and, consequently, depends on the length, diameter and distance between the electrodes 2. For two electrodes of rectangular shape, the largest volume of uniformly treated cells is the product of length, diameter and distance between electrodes 2. The green voltage-distance relationship depends primarily on the cell type and electroporation target and is determined experimentally. The desired voltage / distance ratio varies from 200V / cm to 4000 V / cm, mostly from 500 V / cm to 1500 V / cm. Generators for electroporation typically generate electrical pulses up to 100 V. So the distance between electrodes 2 must be designed so that the desired voltage-to-distance ratio can be obtained. As an example, if the electroporation generator used generates pulses up to 200 V and equals the desired voltage to distance ratio of 1000 V / cm, the distance between the electrodes 2 should not exceed 2 mm. However, the generator is not the subject of the invention. For two electrodes of 2 circular shapes, the diameter can vary from 0.5 to 5 mm and the distance between them can vary from 0.5 to 6 mm. Diameter to distance ratio

-10 between electrodes 2 of circular shape should be as large as possible to achieve the most homogeneous electric field in the aperture 5.

When using three or more electrodes 2, to obtain the most homogeneous electric field, the linear conductivity of the shortest force line between the electrically opposite electrodes 2 should be the same as the linear conductivity of the force line between the electrically opposite electrodes 2 running through the center of the cross-section of the aperture 5. Linear conductivity is the integral of the current density divided by the electric field above the line, which in this case is the line of force. Electrode opposite electrodes are electrodes 2 which are charged with different electrical potential during electroporation. The electrodes 2 are usually symmetrically charged with different electrical potential. For three rectangular electrodes, the ratio of the diameter to the distance between the nearest electrodes 2 should be approximately 0.3. The distance between the nearest electrodes 2 can vary between 0.25 and 3 mm. For four rectangular electrodes 2, the ratio of the diameter to the distance between the opposite electrodes 2 should be approximately 0.3. The distance between opposite electrodes 2 can vary between 0.5 and 6 mm. For five rectangular electrodes 2, the ratio of the diameter to the distance between the nearest electrodes 2 should be approximately 0.5. The distance between the nearest electrodes 2 can vary from 0.25 to 3 mm. For six rectangular electrodes 2, the diameter-to-spacing of the opposite electrodes 2 should be approximately 0.2. The distance between opposite electrodes 2 can vary between 0.5 and 6 mm. For three electrodes 2 of circular shape, the ratio of the diameter to the nearest electrodes 2 should be approximately 1.2. The distance between the nearest electrodes may vary from 0.25 to 2.5 mm. For four circular-shaped electrodes, the diameter-to-distance ratio of the opposite electrodes 2 should be approximately 0.7. The distance between opposite electrodes 2 can vary from 0.5 to 5 mm. For five electrodes of 2 circular shapes, the ratio of the diameter to the distance between the nearest electrodes 2 should be 1. The distance between the nearest electrodes may vary from 0.25 to 2.5 mm. For six electrodes 2 of circular shape, the ratio of the diameter to the distance between the opposite electrodes 2 should be approximately 0.3. The distance between opposite electrodes 2 can vary from 0.5 to 5 mm.

When elliptical, parabolic or hyperbolic shapes of one side of electrodes 2 are used, the relationship between the parameters of the shape of one side and the distance between the electrodes 2 is determined numerically. The circular shape of the electrodes 2 is used as a base in the numerical model. An elliptic, parabolic or hyperbolic one-sided shape is then added to the circular shape of the numerical model such that the integral of the Manhattan distance between the two shapes is in polar coordinates above the shape that will be in

-11direct contact with cell suspension, minimum. The housing 1 and the aperture 5 with the cell suspension are added to the numerical model based on the material conductivity. Furthermore, the electric field between electrodes 2 of one side of an elliptical, parabolic or hyperbolic shape is calculated using the finite element method (FEM). The electric field homogeneity estimate is calculated by the absolute error integral (IAE) between the mean electric field value in aperture 5 and the actual electric field value at a given point of the entire aperture 5. The parameters of the elliptic, parabolic or hyperbolic shape of one side are then iteratively modified so that the estimate electric field homogeneity minimized. One side elliptical shape parameters are main axis, short axis and offset, one side parabolic shape parameters are focus and offset and one side hyperbolic shape parameters are half main axis, half shorter axis and offset. The displacement parameter is equal to the displacement of the shape from its initial position before minimizing the electric field homogeneity estimation. In this way, the resulting shapes are then used to make electrodes 2.

According to another embodiment, the design of the three electrodes 2 can also be used with any electrode chamber to minimize the inhomogeneous electric field between the electrodes. In this case, the ratio of the diameter to the distance between the closest or opposite electrodes should be the same as previously described for the rectangular or. circular electrode shape. For elliptical, parabolic or hyperbolic shapes of one side of the electrodes, the same procedures should be used to determine the most suitable electrode shape with respect to the homogeneity of the electric field between the electrodes according to the previous description.

The electrodes are connected above to the exterior of the electrode tapered chamber so that they can be connected to an electrical impulse generator. Generators typically generate square, sinusoidal, and / or exponential electrical pulses up to 1000 V for up to 10 s. As mentioned above, the generator is not an object of the invention. Electrical connections to the exterior are made using curved electrodes and / or additional wire 3.

The tapered chamber with built-in electroporation electrodes according to the invention is characterized in that the aperture 5 is positioned in the center of the housing 5 and longitudinally through the housing 1 and provides two to six electrodes arranged parallel and symmetrical to the inner surface of the housing 1 and longitudinally with the opening 5 and extends partly to said opening 5, and said electrodes 2 have connectors 3 for connection with the outside.

-12 In a further embodiment, in order to minimize the electric field in the cell suspension, the compact charger 6 can be inserted into a tapered chamber with built-in electrodes or any other chamber with built-in electrodes, as shown in FIG. 4. The compact charger 6 consists of a conductive material having a conductivity comparable to that of the cell suspension medium (0.001 S / cm to 10 S / cm) as e.g. certain semiconductor devices (Si, Ge) and their compounds or alloys or compounds with metals or alloys with metals or any other solid material with conductivity over a specified range. The length of the solid charger 6 is preferably the same as the length of the electrodes. The cross-sectional shape of the solid charger 6 is preferably the same as the cross-sectional shape of the opening, so that the compact charger 6 sits in the opening and has good contact with the electrodes. The compact charger 6 is inserted into the opening and positioned between the electrodes. The maximum volume of cell suspension that can be inserted is as small as the opening between the electrodes and is the same as the volume of hole 7 described below. The compact charger 6 must be designed in such a way that it must be cooled before insertion. As a result, it must have good contact with the electrodes so that it does not fail after it has warmed up. In order to prevent the failure of the solid charger 6, it can also be glued to the housing 1. Longitudinally throughout the compact charger 6, there is a hole 7 in the middle, which is separated from the electrodes by said material. The diameter of the hole 7 can vary from 1 mm to the diameter of the circle, which is inserted into the cross-sectional shape of the solid filler 6, reduced by 2 mm. In this case, the electric field in hole 7 is almost completely homogeneous because almost all the inhomogeneity is distributed within the solid charger 6 located on the outer parts of the space between the electrodes. For a tapered chamber with built-in electrodes or any other chamber with built-in electrodes and with a compact charger, the rectangular electrode shape is preferably used to achieve good electrical contact between the electrodes and the compact charger.

The tapered chamber with built-in electrodes according to the invention is characterized in that the aperture 5 is positioned in the center of the housing 1, while the compact charger 6 is inserted into said aperture 5 with a hole 7 in the longitudinal direction of the housing 1 and two to six electrodes 2 are arranged in parallel and symmetrically in contact with the surfaces of the housing 1 and the solid charger 6, and the electrodes 2 have electrical elements 3 for the junction with the outside.

The tapered chamber with built-in electrodes is simply attached to or removed from the pipette 4 in the same manner as the needles used for pipetting. After mounting the tapered chamber with embedded electrodes, the cell suspension is sucked into the tapered chamber with embedded electrodes and

-13The electrical impulse generator is connected. Electroporation is established during the pulse. After the pulsation is complete, the cell suspension is pipetted out of the tapered chamber with the electrodes mounted. The present invention provides electroporation to achieve cell electrical fusion, cell gene transfection, and cell electroporation.

The process time for pulsing one volume of cell suspension with said tapered chamber with built-in electrodes is minimized, since much less pipetting is required than for pulsing cells in chambers that need to be filled with a separate pipette. Filling the tapered chamber with built-in electrodes is easy, with no bubbles in the suspension. Mechanical processing is therefore also minimized. Cleaning the chamber with electrodes is also easy by pushing the cleaning solution up and down several times. The tip can be sterilized with an autoclave or other means, depending on the material used.

Claims (14)

1. A tapered chamber with built-in electroporation electrodes for the purpose of electrically fusing cells in suspension, gene transfection of cells in suspension or electroporation of cells in suspension, in which a precision insertion chamber is provided in the pipette, characterized in that the opening (5) is positioned in the center of the housing (1) and in a longitudinal direction through the housing (1), and affords from two to six electrodes (2) arranged parallel and symmetrical to the inner surface of the housing (1) and longitudinally with the opening (5) and partially extending into said opening (5). 5), and said electrodes (2) having connectors (3) for connection to the outside. A tapered chamber with built-in electrodes according to claim 1, characterized in that the housing (1) has a cylindrical upper part (1a) and a conical shaped lower part (1b), with an opening ((1) provided in the axis of said housing (1)). 5), which is designed on the upper side to fit the pipette. A tapered chamber with built-in electrodes according to claim 1, characterized in that the housing (1) is made of non-conducting material. A tapered chamber with electrodes according to claim 1, characterized in that the electrodes (2) are inserted into the housing material (1). 5. A tapered chamber with embedded electrodes according to claim 1, characterized in that the electrode shape is rectangular, circular or elliptical, parabolic or hyperbolic on one side. A tapered chamber with embedded electrodes according to claim 1, characterized in that said electrodes (2) are made of conductive material. 7. A tapered chamber with built-in electrodes according to claim 1, characterized in that the connectors (3) for connection with the outside are provided on the upper part of the electrodes (2). 8. A tapered chamber with built-in electroporation electrodes to achieve electrical fusion of cells in suspension, gene transfection of cells in suspension, or electroporation of cells in suspension, wherein said installation chamber is fitted to the pipette (4), characterized in that the opening is (5) positioned in the middle of the housing (1), and that the compact charger (6) is inserted into said opening (5) with a hole (7) and is positioned from two to six in the longitudinal direction of the housing (1) and the compact filter (6) electrodes (2) in parallel and symmetrically to the intersection of the junction surfaces of the housing (1) and the compact charger (6), and said electrodes (2) having connectors (3) for connection with the outside. -159. A tapered chamber with embedded electrodes according to claim 8, characterized in that the housing (1) has a cylindrical upper part (1a) and a conical shaped lower part (1b), with an opening (5) in the axis of said housing (1), provided on the upper side by fitting to the pipette (4). 10. A tapered chamber with integrated electrodes according to claim 8, characterized in that the housing (1) is made of non-conducting material. 11. A tapered chamber with embedded electrodes according to claim 8, characterized in that it is a compact charger of conductive material comparable to the conductivity of the cell suspension medium. 12. A tapered chamber with built-in electrodes according to claim 8, characterized in that the electrodes (2) are inserted into the housing material (1) at the junction of the surfaces of the housing (1) and the compact charger (6). 13. A tapered chamber with embedded electrodes according to claim 8, characterized in that the electrode shape is rectangular, circular or elliptical, parabolic or hyperbolic on one side. A tapered chamber with embedded electrodes according to claim 8, characterized in that said electrodes (2) are made of conductive material. 15. A tapered chamber with integrated electrodes according to claim 8, characterized in that the electrical contacts (3) for connection with the outside are made on top of the electrodes (2).