WO2008051169A1

WO2008051169A1 - Tip electrode chamber for small volume electroporation

Info

Publication number: WO2008051169A1
Application number: PCT/SI2007/000036
Authority: WO
Inventors: Katja Trontelj; Matej Rebersek; Damjan Miklavcic
Original assignee: Univerza V Ljubljani, Fakulteta Za Elektrotehniko
Priority date: 2006-10-25
Filing date: 2007-10-03
Publication date: 2008-05-02
Also published as: SI22368A

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

TIP ELECTRODE CHAMBER FOR SMALL VOLUME ELECTROPORATION

FIELD OF THE INVENTION

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.

The present invention relates to the tip electrodes for small volume electroporation, electrofusion and gene transfection. The invention resides in the field of electroporation of biological cells in suspension and more precisely in the field of techniques and devices for electroporation with the aim of cell membrane electropermeabilization and consequently electro insertion of any active pharmaceutical N chemical or biological active substances, gene transfection and cell fusion. The issue of the invention are the electrodes for quick and efficient electroporation of small and controlled volumes of cell suspensions, which retain their high viability because of minimal mechanical manipulation and the possibility of application of electric pulses in different directions thus increasing the membrane area of cell being electroporated and therefore efficiency of electroporation without a significant loss in cell survival.

BACKGROUND OF THE INVENTION

Use of electroporation

Electroporation (also termed as electropermeabilization) of cells is used for different purposes that employ the permeabilized state of the cell membrane.

Electro insertion or extraction represents introducing or extracting different molecules like drugs, proteins or genes into the cells. The insertion of small molecules is efficiently achieved by application of several short rectangular high voltage pulses. This is now efficiently used in electrochemotherapy for introducing drugs into the tumor cells of the patients in situ. Electroextraction is used for extracting products from yeast in downstream processes.

Gene electrotransfection in vitro and in vivo represents the insertion of plasmids containing appropriate genetic material into cells. DNA is a large molecule and was found to be efficiently introduced with the use of exponentially decaying pulses or with the combination of short high voltage and longer low voltage pulses.

Another related procedure is electrofusion of cells. It consists of two steps. One is making cell membranes fusogene by means of electroporation and another is achieving close contacts between the cells. It can be achieved either before or very soon (in first couple of minutes) after the electroporation until membranes are still in the fusogene state.

Mechanical manipulation

Large part of existing methods for electroporating cells in suspension use a chamber, or cuvette, or a flow system with a pair or pairs of plate electrodes. A cell suspension or cells growing on a support are placed between the electrodes and a voltage pulse is given across them. Cells in suspension are usually pipetted in and out of the electrode chambers or cuvettes.

This represents mechanical manipulation that is connected with two problems. First is a significant loss of volume and the second is that pipetting is harmful for cells and could even be lethal for certain types of cells that are more sensitive especially after being exposed to electroporation.

Electroporated cells have damaged membranes and are substantially more sensitive to pipetting that exerts shear forces to the cells. This appears as a problem for example at the electrofusion protocols, where contacts between cells are achieved after the electroporation. Cells have to be moved from chamber to the centrifuge tube with micropipette immediately after the electroporation. Small volume

Another problem is the size of the existing chambers. Molecules (dyes, plasmids) that are used for experiments are often expensive and available only in small quantities. In addition, the cells to be fused or transfected are often valuable and available only in small quantities. Operating with small volume is thus to be enabled.

For electroporation, local electric field is important. For certain cells, i.e. for small cells, we need high voltage to distance ratio. This can be achieved only with small distance between the electrodes since generators of electric pulses are limited in the voltages they can generate. The problem with small distance chambers or cuvettes is using them without considerable loss of sample volume. It is difficult to fill such chambers without air bubbles, which considerably change electric field distribution in the sample. Filling the chamber or cuvette with cell suspension without air bubbles and getting the cell suspension out after the electroporation is difficult if not impossible. The cleaning is also difficult. Efficient emptying, filling and cleaning of the small space between electrodes are to be enabled.

Flow system of micro electrodes exists, described in US6492175, that allows working with small volumes and reduce the mechanical manipulations however only allows treatment of cells with pulses in one or two opposite directions.

Different pulse directions

The majority of the existing electrode systems allow application of pulses in only one or mostly in two opposite directions. Bipolar pulses are described in WO92/06185. In last couple of years it was demonstrated that use of pulses in different directions cause more effective electropermeabilization and gene electrotransfection. This is also important in cell electrofusion and protein insertion in the membrane. The reason for this is larger area of electroporated membrane that can be achieved with such pulses without a substantial reduction of the cell survival. Standard possibility for increasing the area of the electroporated membrane of the cells is increasing the electric field strength, which inevitably also leads to significant reduction in cell survival. Different pulse directions however increase the area of the permeabilized membrane without considerably affecting the cell survival. Possibility for electrofusion of cell suspension electroporated in different directions is to be enabled. Possibility for electroporation should be allowed for after or before cell contact achievement.

Patent US6746441 describes a flow through chamber apparatus with rotating electric fields produced by more than two (four or six) electrodes. Yet this is a flow through system for ex vivo gene therapy and does not allow for small volume operation. Patent US2005048651 describes the tip electrodes that also include any different kinds of conductive surface N electrodes. It is designed for the delivery of different substances from inside the tip to the targets (cells, etc.) that are outside of the tip synchronized with application of the electric pulses. It does not foresee however cells to be inserted into the lumen of the tip electrodes and electrically manipulated there.

Parallel electroporation for electrofusion

A significant problem is the process of fusion of two different types of cells. This is the case for example in hybridoma technology for monoclonal antibodies production where lymphocytes B are to be fused with myeloma fusion partners. The later can be almost twice as large as lymphocytes. Common electrical treatment of both cell types is not optimal in this case. Separate electroporation and contacting cells after the electroporation is though necessary for optimal result of fusion process. The same problem exists with any pair of cells that are differently sensitive to electric field for any other reason but different sizes.

Cuvette as described in WO03/050232 includes the inserts of support structure that holds a porous membrane and facilitates membrane-based fusion of cells. In such a cuvette, both types of cells are electroporated at the same time. In that case, either cells with smaller size are sub optimally permeabilized or larger cells are damaged. The same is with the systems described in DE10359189 and DE10359190. Also in US4882281 and US5134070 the devices for electroporation are described that avoid mechanical manipulation since cells are treated in the culture dishes, though for subsequent fusion of separately electroporated cells transferring of cells still has to be performed by pipetting.

The nature of the fusion process requires that cell contact be established in short time after electroporation, when the cell membranes are still in so-called fusogene state. The pipetting of cells from two separate chambers in addition to inducing mechanical stress thus damaging cells is also slow and represents considerable problem in achieving cell contact in the short time needed for effective cell electrofusion.

Semi-conductive interior of the tip electrode chamber

It was demonstrated recently that cell survival is the lowest near the electrodes. The reason for that are electrochemical reactions that arise on the boundary between the electrode and the medium. It was therefore proposed in DE 20302861U1 to cover the electrodes with thin layer of biocompatible coating in order to retain cells away from that boundary.

However, another problem exists that concerns the homogeneity and the distribution of the electric field between the electrodes. Electric field that is applied (with defined electric pulses) is not homogeneous in the whole space between the electrodes but only in the middle part of that space. Only the cells that are in that middle part are exposed to desired electric field.

The problem not yet solved is electroporation of predefined small volumes of cells in suspension. This will reduce costs or enable gene transfection, protein insertion and cell fusion experiments when the quantity of the proteins, plasmids or the cells is limited.

The object of this invention is an effective electroporation of cells with the pulses delivered to cells in different directions is also to be enabled at the same time with the possibility of fast manipulation of cells with minimal mechanical stress.

Another object of the present invention is to provide an apparatus for electroporation mediated, in vitro, cell fusion. It is a further object of the present invention to provide a method for fusion of two different types of cells, mediated by distinct electroporation with different values of electrical parameters.

The present invention provides also an interior of the tip electrode chamber made of material with the conductance that is comparable to the conductance of the medium of the cell suspension. This assures the retention of cells in the central area of the chamber and the achievement of a desired homogeneous electric field in that area.

SUMMARY OF THE INVENTION

The present invention allows cells to be confined to that middle part of the lumen by incorporating for example a semi conductive material or composite material of the conductance that is comparable to the conductance of the medium of the cell suspension in the lumen of the tip electrode chamber. It provides the homogeneity of the field because of the conductance that is similar to the conductance of the medium of the cell suspension. Accordingly, it is the primary object of the present invention to provide tip electrode chamber for electroporation of small and controlled volumes of cells with minimal mechanical manipulation and consecutively minimal mechanical stress.

The described invention allows for fast treatment of different types of cells with different electrical treatments, i.e. different values of electric field parameters. The invention further provides the possibility of application of electric pulses in several different directions thus increasing the membrane area of cell membranes being electroporated.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic showing a sectional view through a tip electrode chamber on a pipetter 4. Electrodes 2 are mounted into the inner side of a housing 1 and are electrically connected to exterior 3. Fig. 2 is a cross sectional view of the same tip electrode chamber.

Fig.3 shows possible directions of the electric field in the lumen of the tip electrode chamber in one case of the position of the electrodes.

Fig.4 shows the tip electrode chamber partially filled with a material 6 of the conductance that is comparable to the conductance of the medium of the cell suspension.

DETAILED DESCRIPTION OF INVENTION

Tip electrode chamber is designed from housing 1, electrodes 2 and electrical connection 3 to exterior. The housing 1 is made of a non conducting material such as PolyMethylMethAcrylate (PMMA) or other plastic materials. Electrodes 2 and electrical connections 3 are made of electrically conductive material such as metals or their alloys or graphite for example.

Tip electrode chamber has his upper part Ia of housing 1 designed so that it fits to pipetter 4. Pipetter 4 usually sucks from 1 μl to 1000 μl of fluid. Pipetter 4 is however not an object of this invention. The outer form of lower part Ib of housing 1 is conical, so that it is easier to use with standard laboratory equipment such as centrifuge tubes. Through whole housing 1 in the middle there is an opening 5. Through this opening 5 cell suspension is sucked into the tip electrode chamber and after electroporation blown out from the tip electrode chamber with pipetter 4. When cell suspension is sucked in it is placed in the opening where electrodes 2 are present. Opening 5 between the electrodes defines the maximum volume of uniformly treated cells. The maximum volume of cell suspension that can be inserted is thus in a range limited by possibilities of construction. The construction allows maximum volumes from 10 μl to 2500 μl, predominately from 10 μl to 1000 μl. However, actual volume of uniformly treated cells can be chosen adequately for every experiment simply by adjusting the pipetter.

Cross sectional shape of the opening 5 between the electrodes above all depends on the number of electrodes 2. If 2 electrodes 2 are used cross sectional shape of the opening 5 is in principle rectangular. If 3 or more electrodes 2 are used cross, sectional shape of the opening 5 is in principle equilateral figure with the same number of sides as is the number of electrodes 2. Thus, if 3 electrodes 2 are used, cross sectional shape of the opening 5 is in principle equilateral triangle, if 4 electrodes 2 are used cross sectional shape of the opening 5 is in principle equilateral rectangle or square and so on. When 3 or more electrodes are used sides of the opening 5 can be a little concave to minimize inhomogeneous electric field in the opening 5. This concavity can be up to 20 % of the side's length.

Several electrodes 2 are parallelly and symmetrically mounted into the inner surface of the housing 1 and longitudinally to the opening 5. The construction allows length of the electrodes 2 from 5 mm to 60 mm, predominately from 10 mm to 40 mm. Number of electrodes 2 can vary from 2 to 6 electrodes; the said number may be pair or odd. The number of electrodes 2 influence on the number of different directions of electric field that can be used. For example, four electrodes 2 positioned in corners of a quadrant allows for application of pulses in eight different directions (AB and DC, BA and CD, AC, CA, BD, DB, AD and BC, DA and CB). The directions are shown in figure 3a, 3b and 3c. With the number of electrodes the number of different directions of electric field increases.

Detailed design of the opening 5 and the electrodes 2 is very dependent on a cross sectional shape of the electrodes 2. There are 3 main options of cross sectional shape of the electrodes 2: rectangular, circular or from one side elliptical, parabolic or hyperbolic and from the other side arbitrary shape. When only 2 electrodes are used the shape of the electrodes is preferably rectangular to get the most homogeneous electric field in the opening 5, but also other aforementioned shapes can be used. When more than 2 electrodes are used the shape of the electrodes is usually circular, because they are easier to be produced, but also elliptical, parabolic or hyperbolic one-side shapes can be used, to get more homogeneous electric field in the opening 5, predominately hyperbolic one-side shape. However, for more than 2 electrodes also rectangular shape can be used, only that homogeneity of the electric field is very low.

Most of the surface of the said electrodes 2 is usually molded into the material of the housing 1, but a smaller part of the electrodes 2 is usually projected out of the material of the housing 1. The said smaller parts are in direct electrical contact with the cell suspension, when it is sucked into the opening 5 of the housing 1. When rectangular shape of the electrodes 2 is used, preferably 3 of 4 planes of the electrode 2 are molded into the material of the housing 1 and preferably 1 of 4 planes is projected out of the material of the housing 1. Electrodes 2 of rectangular shape can be positioned symmetrically into the centers or preferably for 3 or more electrodes 2 into the corner points of the edge of the equilateral figure of the opening 5. When other aforementioned shapes of the electrodes 2 are used portion of molded surface into the material of the housing 1 depends on the number of electrodes 2. When 2 circular electrodes 2 are used, their centers are positioned symmetrically in the center of the edge of the cross sectional shape of the opening 5 and preferably 180° of the electrodes 2 is molded into the material of the housing 1 and preferably 180° of the electrodes 2 is projected out of the material of the housing 1. When 3 or more circular electrodes 2 are used, their centers are positioned in the corner points of the cross sectional shape of the opening 5 and their parts, which are projected out of the material of the housing 1, are preferably the same as the inner angles of equilateral figure of the opening 5. Thus, if 3 electrodes 2 of circular shape are used, preferably 60° of the electrodes 2 is projected out of the material of the housing 1 and preferably 300° of the electrodes 2 is molded into the material of the housing 1, if 4 electrodes 2 of circular shape are used, preferably 90° of the electrodes 2 is projected out of the material of the housing 1 and preferably 270° of the electrodes 2 is molded into the material of the housing 1 and so on. When elliptical, parabolic or hyperbolic one-side shapes are used, these specific shapes must be oriented into the center of cross sectional shape of the opening 5. The arbitrary shape of elliptical, parabolic or hyperbolic one-side shapes of electrode 2 is preferably completely molded into the material of the housing 1. Therefore this arbitrary shape is usually a T profile to get a strong contact between the electrodes 2 and housing 1. Elliptical, parabolic or hyperbolic one-side shapes of the electrodes 2 are preferably all projected out of the material of the housing 1. When 2 elliptical, parabolic or hyperbolic one-side shapes of electrodes 2 are used, they are usually positioned symmetrically in the center of the edge of the cross sectional shape of the opening 5. When 3 or more elliptical, parabolic or hyperbolic one-side shapes of electrodes 2 are used, they are usually positioned in the corner points of the cross sectional shape of the opening 5.

The diameter of the electrodes 2 and the distance between them influence on the homogeneity of the electric field in the opening 5. This influence is not so obvious when 2 electrodes 2 of rectangular shape are used. Thus for 2 electrodes 2 of rectangular shape the ratio between the diameter and the distance between the electrodes 2 is optional. 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 uniformly treated cells and on the desired voltage to distance ratio. As aforementioned, the maximum volume of uniformly treated cells is defined as the volume of opening 5 between the electrodes and it is so depended on the length, the diameter and the distance between the electrodes 2. At 2 electrodes 2 of rectangular shape the maximum volume of uniformly treated cells is a product of the length, the diameter and the distance between the electrodes 2. Desired voltage to distance ratio above all depends on cell type and the aim of electroporation and it is usually determined experimentally. Desired voltage to distance ratio vary from 200 V/cm to 4000 V/cm, predominately from 500 V/cm to 1500 V/cm. Generators for electroporation usually generate electrical pulses up to 1000 V. Thus, the distance between the electrodes 2 must be designed so that desired voltage to distance ratio can be achieved. As an example, if used generator for electroporation generates electrical pulses up to 200 V and the desired voltage to distance ratio is 1000 V/cm, the distance between the electrodes 2 must not be greater than 2 mm. Generator is however not an object of the invention. For 2 electrodes 2 of circular shape the diameter can vary from 0.5 to 5 mm and the distance between them can vary from 0.5 to 6 mm. Ratio between the diameter and the distance between the electrodes 2 of circular shape should be as large as possible to get the most homogeneous electric field in the opening 5.

When 3 or more electrodes 2 are used, linear conductivity of the shortest line of force between electrically opposite electrodes 2 should be the same as linear conductivity of the line of force between electrically opposite electrodes 2, which goes through the centre of the cross sectional shape of the opening 5, to get the most homogeneous electric field. Linear conductivity is an integral of the current density divided by electric field over the line, which is in our case the line of force. Electrically opposite electrodes are the electrodes 2 which are during electroporation charged with different electrical potential. Electrodes 2 are usually symmetrically charged with different electrical potential. For 3 electrodes 2 of rectangular shape the ratio between the diameter and the distance between the nearest electrodes 2 should be approximately 0.3. The distance between the nearest electrodes 2 can vary from 0.25 to 3 mm. For 4 electrodes 2 of rectangular shape the ratio between the diameter and the distance between the opposite electrodes 2 should be approximately 0.3. The distance between the opposite electrodes 2 can vary from 0.5 to 6 mm. For 5 electrodes 2 of rectangular shape the ratio between the diameter and 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 6 electrodes 2 of rectangular shape the ratio between the diameter and the distance between the opposite electrodes 2 should be approximately 0.2. The distance between the opposite electrodes 2 can vary from 0.5 to 6 mm. For 3 electrodes 2 of circular shape the ratio between -li¬

the diameter and the distance between the nearest electrodes 2 should be approximately 1.2. The distance between the nearest electrodes 2 can vary from 0.25 to 2.5 mm. For 4 electrodes 2 of circular shape the ratio between the diameter and the distance between the opposite electrodes 2 should be approximately 0.7. The distance between the opposite electrodes 2 can vary from 0.5 to 5 mm. For 5 electrodes 2 of circular shape the ratio between the diameter and the distance between the nearest electrodes 2 should be approximately 1. The distance between the nearest electrodes 2 can vary from 0.25 to 2.5 mm. For 6 electrodes 2 of circular shape the ratio between the diameter and the distance between the opposite electrodes 2 should be approximately 0.3. The distance between the opposite electrodes 2 can vary from 0.5 to 5 mm.

When elliptical, parabolic or hyperbolic one-side shapes of electrodes 2 are used, the ratio between the parameters of one-side shape and the distance between the electrodes 2 is determined numerically. Circular shape of the electrodes 2 is used for the base in the numerical model. Elliptical, parabolic or hyperbolic one-side shape is then appended on the circular shape of numerical model so that the integral of ^"Manhattan distance" between the two shapes in polar coordinates over the shape, which will be in the direct contact with the cell suspension, is the smallest. Housing 1 and opening 5 with cell suspension is with material conductivity added into the numerical model. Then the electric field between the electrodes 2 of elliptical, parabolic or hyperbolic one-side shape is calculated by finite element method (FEM). Estimation of homogeneity of electric field is calculated by integral absolute error (IAE) between the mean value of electric field in the opening 5 and actual value of electric field at given point over all opening 5. Parameters of the elliptical, parabolic or hyperbolic one-side shape are then iteratively changed so that the estimation of homogeneity of electric field is minimized. Parameters of the elliptical one-side shape are major axis, minor axis and displacement, parameters of the parabolic one-side shape are focus and displacement and parameters of the hyperbolic one-side shape are semi-major axis, semi-minor axis and displacement. Displacement parameter equals the displacement of the shape from initial position of the shape before minimization of the estimation of homogeneity of electric field. In such a manner obtained shapes are then used for manufacturing of the electrodes 2.

In another aspect, design of 3 or more electrodes 2 can also be used in any other electrode chamber for the minimization of inhomogeneous electric field between the electrodes. In this case, the ratio between the diameter and the distance between the nearest or opposite electrodes should be the same as previously described for rectangular or circular shape of the electrodes. And for elliptical, parabolic or hyperbolic one-side shapes of the electrodes the same procedure as previously described should be used to determined the most suitable shape of the electrodes regarding the homogeneity of the electrical field between the electrodes.

Electrodes are on the top electrically connected to exterior of the tip electrode chamber, so that they can be connected to a generator of electric pulses. Generators usually generate square, sinusoidal and/or exponential electrical pulses up to 1000 V, duration up to 10 s. As aforementioned, generator is not an object of the invention. Electrical connections to exterior are made by curved electrode and/or by additional wire 3.

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.

In another aspect, a solid filler 6 can be inserted into the tip electrode chamber or any other electrode chambers as presented on Fig. 4, to minimize inhomogeneous electric field in cell suspension. Solid filler 6 is made from the material of the conductance that is comparable to the conductance of the medium of the cell suspension (0.001 S/cm to 10 S/cm), such as for example certain semiconductor elements (Si, Ge) and their composites or alloys or metal composites or alloys or any other solid materials with the conductance in defined range. Length of the solid filler 6 is preferably the same as the length of the electrodes. Cross sectional shape of the solid filler 6 is preferably the same as the cross sectional shape of the opening, so that solid filler 6 fits into the opening and has a good contact with the electrodes. Solid filler 6 is inserted into the opening and placed between the electrodes. The maximum volume of cell suspension that can be inserted is thus smaller than the opening between the electrodes and it is the same as the volume of the hole 7 described below. Solid filler 6 should be designed so precisely, that it has to be cooled before the insertion. So that it has a good contact with the electrodes and does not fall out after it warms up. To prevent the fall out, solid filler 6 can also be glued on to the housing 1. Longitudinal trough whole solid filler 6 in the middle there is a hole 7, which is separated from the electrodes with that material. The diameter of the hole 7 can vary from 1 mm to the diameter of inscribed circle into the cross sectional shape of the solid filler 6 minus 2 mm. Electric field in the hole 7 is in this case almost completely homogeneous since almost all non-homogeneity is distributed inside the solid filler 6 that is on the outer parts of the space between the electrodes. For tip electrode chamber or any other electrode chamber with solid filler the rectangular shape of electrodes is preferably used, to get a good electrical contact between the electrodes and solid filler.

A tip electrode chamber according to the invention is characterized in that the opening 5 is in the middle of housing 1, whereas 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.

The tip electrode chamber is simply attached and detached to the pipetter 4 in the same way as tips used for pipetting. After attaching the tip electrode chamber cell suspension is sucked into the tip electrode chamber and generator is started to generate electrical pulses. During this pulsation electroporation occurs. After the pulsation cell suspension is blown from the tip electrode chamber with the pipetter. The present invention allows electroporation with the aim of electrofusion of cells, gene transfection of cells or electroporation of cells.

The operating time for pulsing one volume of cell suspension by this tip electron chamber is minimized because there is much less pipetting needed than with pulsing cells in chambers which have to be filled with a separated pipetter. The filling of the tip electrode chamber is simple with no bubbles in the suspension inserted. Mechanical manipulation is therefore also minimized. The cleaning of the electrode chamber can also be simply done by pulling the cleaning solution up and down several times. The tip can be sterilized by autoclaving or other means depending on the materials used.

Claims

1. A tip electrode chamber for electroporation with the aim of electrofusion of cells in suspension, gene transfection of cells in suspension or electroporation of cells in suspension, whereas a chamber is designed to fit to pipetter, 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.

2. A tip electrode chamber according to claim 1, characterized in that the housing (1) has an upper part (Ia) of cylindrical shape and a lower part (Ib) of conical shape, whereas in the axis of the said housing there is an opening (5), at the upper part designed to fit to pipetter.

3. A tip electrode chamber according to claim 1, characterized in that the housing (1) is made of a non conducting material.

4. A tip electrode chamber according to claim 1, characterized in that the electrodes (2) are placed into the material of the housing (1).

5. A tip electrode chamber according to claim 1, characterized in that the shape of the electrodes is rectangular, circular or from one side elliptical, parabolic or hyperbolic.

6. A tip electrode chamber according to claim 1, characterized in that the said electrodes are made of conductive material.

7. A tip electrode chamber according to claim 1, characterized in that the electrical connections (3) to exterior are made on the top of the electrodes (2).

8. A tip electrode chamber for electroporation with the aim of electrofusion of cells in suspension, gene transfection of cells in suspension or electroporation of cells in suspension, whereas a chamber is designed to fit to pipetter, characterized in that the opening (5) is in the middle of housing (1), whereas 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.

9. A tip electrode chamber according to claim 8, characterized in that the housing (1) has an upper part (Ia) of cylindrical shape and a lower part (Ib) of conical shape, whereas in the axis of the said housing there is an opening (5), at the upper part designed to fit to pipetter.

10. A tip electrode chamber according to claim 8, characterized in that the housing (1) is made of a non conducting material.

11. A tip electrode chamber according to claim 8, characterized in that the solid filler (6) is of the material of the conductance that is comparable to the conductance of the medium of the cell suspension.

12. A tip electrode chamber according to claim 8, characterized in that the electrodes (2) are placed into the material of the housing (1) in the contact of surfaces of the housing (1) and solid filler (6).

13. A tip electrode chamber according to claim 8, characterized in that the shape of the electrodes is rectangular, circular or from one side elliptical, parabolic or hyperbolic.

14. A tip electrode chamber according to claim 8, characterized in that the said electrodes are made of conductive material.

15. A tip electrode chamber according to claim 8, characterized in that the electrical connections (3) to exterior are made on the top of the electrodes (2).