WO2013167185A1

WO2013167185A1 - Electrode assembly for generating electric field pulses to perform electroporation to a biological sample

Info

Publication number: WO2013167185A1
Application number: PCT/EP2012/058587
Authority: WO
Inventors: Tomàs GARCÍA SÁNCHEZ; Xavier Rosell Ferrer; Ramón BRAGÓS BARDIA; Ana M. GÓMEZ FOIX; Maria GUITART DE LA ROSA; Beatriz SÁNCHEZ ORTIZ
Original assignee: Universitat Politècnica De Catalunya; Universitat De Barcelona
Priority date: 2012-05-09
Filing date: 2012-05-09
Publication date: 2013-11-14

Abstract

The invention relates to an electrode assembly for generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, the electrode assembly being adapted to be positioned on the top of the biological sample avoiding direct contact between the electrode assembly and the biological sample, the electrode assembly comprising a substrate of a non-conductive material; an electrode array placed on a first side of the substrate, the electrode array comprising at least two independent closely spaced electrodes; and one electrical connector associated to each electrode, the connectors being placed on a second side of the substrate and being connected to the electrodes through the substrate.

Description

Electrode assembly for generating electric field pulses to perform

electroporation to a biological sample

The present invention relates to an electrode assembly for generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate adapted to be positioned on the top of the biological sample, avoiding direct contact between the electrode assembly and the biological sample. Further, the invention also relates to a method of performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly. More specifically, the invention, for example, relates to an in situ electroporation method by which different molecules, for example, molecules of DNA, oligonucleotides, or molecules of RNA, such as mRNAs, siRNAs and miRNAs, proteins, peptides, chemicals and drugs, are inserted into cells using high electric field pulses generated by an electrode assembly, particularly mammalian adherent cells growing on standard multi-well culture plates. The invention also relates to a computer system and a computer program product suitable for carrying out such a method.

The application of the present invention is focused on biological samples growing in standard surfaces.

BACKGROUND ART

The introduction of foreign molecular species into cells in vitro is a widely used technique in biological research for many different purposes. Mostly the transference of nucleic acid molecules DNA/RNA attracts the main interest. There are several chemical methods to facilitate this transference into the cells, including calcium phosphate co-precipitation [Graham, F. L, et al. (1973), Virology 52, 456- 467], lipofection [Feigner PL et al. (1987) Proc Natl Acad Sci U S A 84: 7413- 7417], viral vectors [Goff S.P, Berg P (1976). Cell 9 (4 PT 2): 695-705] and others. Nonetheless, there have been reports suggesting that these chemical methods may contribute to alterations in gene expression and other phenotypic changes [Y. Fedorov, et al, Nat. Methods 2 (2005) 241]. The main distinctive property of electroporation is the fact that this technique is based only on a physical phenomenon preventing the use of chemical agents.

Traditional in vitro equipment performs electroporation in cuvettes where cells are suspended in order to apply electric field pulses [Leda Raptis and Kevin L. Firth. Electroporation Protocols Preclinical and Clinical Gene Medicine. Humana Press 2008. p. 61]. Particularly, when adherent cells are electroporated it is necessary to remove them from the growing surface usually by trypsinization. However, trypsinizing adherent cells causes an additional stress to the cells that may affect both the electroporation efficiency and the invasiveness of the operation. As explained in [D.C. Chang, B.M. Chassy, J. A. Saunders, A.E. Sowers Eds., Guide to Electroporation and Electrofusion, Academic Press, New York, 1992, Chapter 13, pp. 201-207] there are several reasons to believe that in situ electroporation is more suitable for high efficacy transfection in adherent cells maintaining a reasonable viability.

Instrumentation specifically designed for the application of electroporation into adherent cells has been previously proposed for a small number of cells grown on a substratum [Zheng, Biochim. Biophys. Acta 1088:104-110, 1991], or cells growing directly attached to electrodes [Lin, Y.-C, et al., Sensors and Actuators A:

Physical, 2003. 108(1-3): p. 12-19]. In some of these devices cells have to be grown in specific surfaces such as porous membranes [Iwata et al, U.S. Pat., No. 7,846, 731 B2]. However, those systems require non-standard laboratory techniques in cells culture and treatment processes and one way or another cells become in direct contact with foreign materials. There have been also proposals where electroporation is applied directly to adherent cells growing in standard wells [Hilliard et al., U.S. Pat, No. 4,695,547]. Nevertheless, highly-spaced electrodes used in these setups need high voltage sources in order to provide the necessary electric fields in electroporation, usually several thousands of volts per centimetre as described by Ragsdale on Jun. 7, 1988 in U. S. Pat. No. 4, 750, 100.

Another issue related to those electrodes is the non-uniformity in the electric field created in the proximity of cells substrate resulting in a variety of different effects depending on the area where cells are growing. In some of these areas the electric field intensity exceeds the range where cells remain viable. On the contrary, other areas are under the electroporation threshold and cells are not electroporated. The principal inconvenient of the described systems is the lack of control in the uniformity of the current density through the cells and so the decrease in efficiency achieved.

Some attempts to increase the electroporation efficiency when charged molecules as DNA plasmids are the active compounds to be introduced, is the combination of short high-intensity and long low-intensity electric field pulses inducing the electrophoretic movement of charged particles in the presence of the second type of pulses [Heller et al., U.S. Pat. No., 7,668,592 B2]. These systems make use of the electrophoretic force to attract DNA to the positive electrode where cells are always between the positive and the negative electrodes so DNA is forced to cross through the cells suspension or monolayer in case of adherent cells [Lin, Y. C., M. Li, and C. C. Wu, Lab Chip, 2004. 4(2): p. 104-8]. Consequently, systems designed for adherent cells can only make use of electrophoretic force if one part of the electrodes is on the bottom surface where cells are attached.

As exposed above, there are many limitations to overcome in actual electroporation setups. Herein, we focus on the standardization and automation of the technique for its use in usual laboratory conditions and also on the increase in the uniformity of the results obtained. On the other hand, as also known for those skilled in the art, cell exposure to high electric fields leads to cell-cell electrofusion under certain circumstances. Electrofusion was first presented by Zimmerman in the early 1980's [Zimmermann U. (1982). Biochim Biophys Acta 694(3):227-277] and involves the fusion of membranes of different cells after their membranes have been electroporated. After this process contents of both cells reside within a single membrane forming a cell with the characteristics of both.

There are at least three main applications for cell fusion:

1 . Immuno-therapy - producing therapeutic hybrids by combining a tumor cell with a dendritic cell;

2. Hybridoma Production - to manufacture antibodies;

3. Nuclear Transfer - for fertility treatment and animal propagation.

Parameters for electrofusion are very similar to those used for electroporation, the main distinctive property is the necessity of cell-cell contact during electrofusion. There are several methods to achieve physical contact between cells like chemical methods, the use of Alternating Current (AC) fields to move particles in suspension by dielectrophoresis and in the case of adherent cell lines adherence methods exploit the regions of contact between cells cultured to confluence. Plated cells consisting of the same and different cell lines have been used.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an electrode assembly for generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, which avoid the disadvantages of the prior art.

To achieve the above, according to a first aspect, the invention provides an electrode assembly for generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, the electrode assembly being adapted to be positioned on the top of the biological sample avoiding direct contact between the electrode assembly and the biological sample, the electrode assembly comprising a substrate of a non- conductive material; an electrode array placed on a first side of the substrate, the electrode array comprising at least two independent closely spaced electrodes; one electrical connector associated to each electrode, the connectors being placed on a second side of the substrate and being connected to the electrodes through the substrate.

Basically, the described electrode assembly and its position with respect to the biological sample reduces the electric current needed to "porate" the biological sample membranes and, on the other hand, increases the uniformity in the effect performed in the whole surface where the biological sample are attached. Thus, a preferred way to accomplish the described object is applying electric field pulses directly in situ, and as biological samples are attached to a non-conductive material, pulses need to be applied from the top of the biological sample. When using such a small dimensions in the construction of the electrode array, electrode assembly is needed to be placed closely to the biological sample so that the biological sample is exposed to enough high intensity electric fields.

This way, the provision of an electrode array with at least two independent electrodes closely spaced between them allows using low voltage sources in order to provide the required electric field pulses to produce electroporation in the biological sample membrane. Further, due to the two electrodes are placed on the substrate, the electrode assembly can be positioned on the top of the surface where biological sample is attached, parallel to it and close enough to ensure that, in use, the electric field pulses are crossing through the biological sample. It is not required an electrode placed on the bottom surface where biological sample is attached and consequently it is possible to use standard multi-well plates. It is important to highlight that each electrode of the at least two independent electrodes may comprise a set of microelectrodes that may be connected to the same potential. Further, because the independent electrodes may cover the whole surface of the substrate and the substrate geometry may be in accordance to the geometry of the receptacle where the biological sample is growing, it is possible to perform high uniformity electroporation to the sample, that is, it is possible to create uniform electric field pulses in the proximity of all the surface of the biological sample.

It is important to highlight that the electrode assembly may comprise an even number of independent electrodes forming active pairs in the area covered by each pair.

According to a preferred embodiment of the invention, the non-conductive material of the substrate may be a dielectric material. In case the technology selected for the construction of the electrode assembly was Printed Circuit Board (PCB) technology, the dielectric material may be selected, for example, from FR-2, FR-3 or FR-4. The thickness of the substrate may be in the range of 500 μηι to 2000 m.

As described above, the at least two independent closely spaced electrodes may be made from a conductive material, said material being preferably copper, platinum, gold, stainless steel, titanium, aluminium or alloys thereof. Further, although electrodes are not supposed be in direct contact with the biological sample, it is necessary to use non-cytotoxic materials in their fabrication. Thus, in case the electrodes are made of a cytotoxic material, a final non-cytotoxic conductive layer covering the conductive lines (that is, the independent electrodes) must be added. Consequently, the at least two independent closely spaced electrodes comprised in the electrode array may be covered with one or more non- cytotoxic materials, for example a material selected from gold or platinum. As an example, if copper is the conductive material used in the fabrication of the conductive lines, a thin film of gold may be deposited with standard electrolytic processes. The dimensional parameters of the electrode assembly are for example the following:

• the spacing between adjacent electrodes of the electrode array may be in the range of 50 μηι to 150 μηι.

• the width of each electrode of the electrode array may be in the range of 50 m to 150 m.

• the thickness of each electrode of the electrode array may be in the range of

10 m to 30 Mm.

These dimensions of the electrodes are small enough to increase the electric field intensity using relative low electric currents and are limited by the technology used in the fabrication of the electrode assembly. According to a preferred embodiment of the present invention, in order to reduce costs and even to create disposable or single-use electrodes, the used technology is Printed Circuit Board (PCB) but other technologies could apply.

As previously described, the present invention allows reducing the electric current needed to cause electroporation of biological sample membranes and increasing the uniformity in the effect performed in the surface where biological sample is attached. For improving these features the spacing between adjacent electrodes may be filled with a non-conductive material, such as non-cytotoxic epoxy resins.

The use of this non-conductive material avoids current density to concentrate in this area (that is, the spacing between adjacent electrodes) and to force the maximum current flowing through electrode surfaces exposed directly to the biological sample, that is, the effect in the current density distribution if compared with the absence of any material is an increase in such current density in the region below the conductive lines of the electrode array, vertically expanding the area where this current density is high enough to create an electric field with the proper intensity to perform electroporation.

These covering materials may be conductive or non conductive and the electric current lines distribution during the application of the electroporation pulses is changed by varying their conductivity and thickness. Taking into account that the electrode array is positioned relatively close but not in contact, the increment in vertical direction optimizes the current passing through the biological sample. Additionally, due to the relative distance, the undesirable current density accumulation caused by border effect is reduced, improving subsequently the uniformity of the electric field applied.

The at least two independent closely spaced electrodes may comprise parallel, concentric or uniform ly-spaced-along lines. Irrespective of the chosen configuration, it is important that the lines cover all the surface of the substrate with the intention of increasing the uniformity of the electric field.

According to a preferred embodiment of the invention, the electrode assembly may also comprise a complementary electrode placed relatively spaced from the electrode array. This complementary electrode may be used to create an electrophoretic force to attract or repulse charged molecules as nucleic acids. This way, using the appropriate sequence of electrophoresis and electroporation pulses an enhancement in the transfection efficiency is achieved when large charged molecules are pretended to be introduced into the biological sample (e.g. into cells).

This complementary electrode may be placed in a third side of the substrate. Normally, the second and the third side are the same one and correspond to the opposite side of the electrode array.

In general, it can be understood that the electrode array and the complementary electrode may be of the same composition to avoid generation of redox or electrolysis or electrochemical reactions that may occur when dissimilar electrode material is used. The geometry of such complementary electrode may be designed with the same shape than the substrate of the electrode assembly, covering a large part of the surface of the well plate and may be positioned on the opposite side where the electrode array is placed. The distance between the complementary electrode and the electrode array may be about one order of magnitude bigger is compared with the inter-electrode distance of the electrode array. Such distance is long enough to contain, between the complementary electrode and the bottom of the electrode array, all the volume of buffer solution where molecular species are dissolved.

According to an embodiment of the invention, the complementary electrode may be made from a conductive material selected, for example, from copper, platinum, gold, stainless steel, titanium, aluminium or alloys thereof.

Furthermore, the electrode assembly may also comprise a plug (that may be a standard connector) comprising the electrical connectors associated to each independent electrode. For this reason, the plug may be used for supplying the electric field pulses to the different electrodes. If the plug is a standard connector, the electrode assembly may be a disposable electrode assembly, which improves sterility in laboratory conditions and reduces the cost and the time spent in sterilization processes.

According to another embodiment of the invention, the electrode assembly may also comprise a plurality of separators placed on the first side of the substrate.

These separators avoid direct contact between any part of the electrode assembly and the biological sample in order to reduce the invasiveness of the operation and to avoid causing any physical stress to the biological sample. Further, the separators allow positioning the electrode assembly closely parallel to the biological sample (or the surface where it is growing). Thickness of the separators may be in the range of several microns, for example, thickness may be between 2 to 15 μηι. Basically, thickness of the separators must be sufficient to prevent contact of the electrode with the biological sample, but to keep it close enough.

Alternatively, if separators are or not included, the electrode assembly may comprise an electrical spectroscopy bioimpedance measurement device. Performing these measurements in a proper way and taking the advantage of small inter-electrode dimensions, a precise estimation of the electrode assembly position respect to the biological sample can be calculated with about 1 μηι precision.

According to another aspect, the invention provides a method of performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly as described above, the method comprising:

o Positioning the electrode assembly on the top of the biological sample growing as a monolayer on the surface of the culture plate, avoiding direct contact between the electrode assembly and the biological sample;

o Connecting at least one of the independent electrodes of the electrode array to a positive polarity;

o Connecting at least one other of the independent electrodes of the electrode array to a negative polarity;

o Applying short duration and high amplitude pulses between the independent electrode connected to the positive polarity and the independent electrode connected to the negative polarity in order to generate electric field pulses on the biological sample. This way, the described method allows performing electroporation to a biological sample positioning the electrode assembly on the top of the biological sample. According to an embodiment of the present invention, the method may also comprise:

o Short-circuiting all the independent electrodes of the electrode array; o Connecting the short-circuited electrodes to a positive polarity;

o Connecting the complementary electrode to a negative polarity;

o Applying at least one long duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to increase the negatively charged molecule concentration nearby the biological sample.

According to these steps, a first electrophoretic treatment is applied. This way, a movement of the charged molecules to be introduced in the biological sample is obtained, said movement causing a concentration of charged molecules in the space between the biological sample and the electrode array of the electrode assembly. This increase in concentration around the biological sample contributes to improve the entrance of said molecules in the biological sample during the electroporation treatment previously described. Consequently, the execution of the described steps improves the electroporation method according to the invention. On the other hand, the method may also comprise:

o Short-circuiting all independent electrodes of the electrode array; o Connecting the short-circuited electrodes of the electrode array to a negative polarity;

o Connecting the complementary electrode to a positive polarity;

o Applying at least one medium duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to push negatively charged molecules against biological sample membranes. According to these steps, a second electrophoretic treatment is applied. In this case, a repulsing electrophoretic pulse is applied to move charged molecules in direction to the biological sample. When applying electroporation pulses to the biological sample, the membranes remain opened for a variable period of time and the execution of said steps exploits this state of the membrane to "push" charged molecules through the remaining pores. The parameters cited above may be for example in the following ranges:

• low amplitudes of the pulses may be in the range of 50 to 500 V/cm.

• high amplitudes of the pulses may be in the range of 1000 to 3000 V/cm.

• long durations of the pulses may be in the range of 100 to 1000 ms.

• medium durations of the pulses may be in the range of 50 to 100 ms.

· short durations of the pulses may be in the range of 1 με to 50 ms.

According to another aspect, the invention provides a computer system for performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly as described above, the computer system comprising:

o Computer means for positioning the electrode assembly on the top of the biological sample growing as a monolayer on the surface of the culture plate, avoiding direct contact between the electrode assembly and the biological sample;

o Computer means for connecting at least one of the independent electrodes of the electrode array to a positive polarity;

o Computer means for connecting at least one other of the independent electrodes of the electrode array to a negative polarity; o Computer means for applying short duration and high amplitude pulses between the independent electrode connected to the positive polarity and the independent electrode connected to the negative polarity in order to generate electric field pulses on the biological sample.

The computer system may also comprise:

o Computer means for short-circuiting all the independent electrodes of the electrode array; Computer means for connecting the short-circuited electrodes to a positive polarity;

Computer means for connecting the complementary electrode to a negative polarity;

Computer means for applying at least one long duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to increase the negatively charged molecule concentration nearby the biological sample. On the other hand, the computer system may comprise:

o Computer means for short-circuiting all the independent electrodes of the electrode array;

o Computer means for connecting the short-circuited electrodes to a negative polarity;

o Computer means for connecting the complementary electrode to a positive polarity;

o Computer means for applying at least one medium duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to push negatively charged molecules against biological sample membranes.

According to yet another aspect of the invention, it is provided an apparatus for performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, the apparatus may comprise:

- A pulse generator;

- The electrode assembly previously described;

- An arm comprising a complementary plug to the plug of the electrode assembly for attaching and electrically interconnecting the electrode assembly with the pulse generator;

- An arm positioning system for positioning the electrode assembly in the correct position according to the biological sample;

- The computer system previously described. The computer system may be implemented by software (e.g. a computer program product), by hardware (e.g. logic gates) or by a combination of software and hardware. Thus, for example, the computer system may be implemented by a memory and a processor, embodying instructions stored in the memory and executable by the processor, the instructions comprising functionality to perform the steps of the method previously described.

Alternatively, the electrode assembly may be manually positioned in the correct position according to the biological sample (that is, on the top of the surface where the biological sample is attached, parallel to it and close enough to ensure that, in use, the electric field pulses are crossing through the biological sample) without the intervention of the apparatus described above. In another aspect of the present invention, it is provided a computer program product comprising program instructions for causing a computer to perform the method of generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly as described above. The invention also relates to such a computer program product embodied on a storage medium (for example, a CD-ROM, a DVD, a USB drive, on a computer memory or on a read-only memory) or carried on a carrier signal (for example, on an electrical or optical carrier signal).

The invention also provides a method of performing electrofusion to cells of a biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly described above, cells of the biological sample being in physical contact, the method comprising executing the method of performing electroporation described above. This way, the cells in physical contact get their membranes fused after the membranes have been exposed to high electric fields by executing the method of performing electroporation described above, achieving electrofusion. After this method, contents of both cells reside within a single membrane forming a cell with the characteristics of both.

According to an embodiment, the method may further comprise the step of providing cells of the biological sample in physical contact.

There are several methods to achieve physical contact between cells:

- Chemical methods;

- The use of AC fields to move particles in suspension by dielectrophoresis; or

- In the case of adherent cell lines, adherence methods exploit the regions of contact between cells cultured to confluence.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Particular embodiments of the present invention will be described in the following by way of non-limiting examples, with reference to the appended drawings, in which:

Figure 1 is a perspective view of a schematic representation of an embodiment of an electrode assembly according to the invention;

Figure 2 is a perspective view of a schematic representation of the electrode assembly of Figure 1 where metalized microvias proposed to make connections between the bottom and the top surfaces of the electrode assembly and the connector used therein are shown;

Figure 3 is a perspective view of a schematic representation of an electrode array comprising two independent electrodes with a circular arrangement constructed on a circular non-conductive substrate;

Figure 4 is a perspective view of a schematic representation of an electrode array comprising two independent electrodes with a straight arrangement constructed on a circular non-conductive substrate;

Figure 5 is a perspective view of a schematic representation of an electrode array comprising six independent electrodes with a spiral arrangement constructed on a circular non-conductive substrate;

Figure 6 is a perspective view of a schematic representation of an electrode array comprising six independent electrodes with a straight arrangement with three active areas constructed on a circular non-conductive substrate;

Figure 7 is a cross section view of a schematic representation of the embodiment of the electrode assembly of Figure 1 where the use of variable conductivity materials in the spacing between lines is shown;

Figure 8 is a perspective view of a schematic representation of an electrode assembly comprising a complementary electrode;

Figure 9 is a schematic representation of the method of performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate according to the invention;

Figure 10 is a diagram representing the sequence of electrical field pulses when electrophoresis is used in the method according to the invention;

Figure 1 1 is a block diagram of the system for generating electric field pulses to perform electroporation to a biological sample, according to the invention;

Figure 12 is a schematic representation of a possible embodiment of an apparatus comprising the system of Figure 1 1 . DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In the following descriptions, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be understood by one skilled in the art, however, that the present invention may be practiced without some or all of these specific details. In other instances, well known elements have not been described in detail in order not to unnecessarily obscure the description of the present invention. The application of the present invention is focused on biological samples growing as a monolayer on a surface of a culture plate, for example, adherent cells growing in standard surfaces. The only way to accomplish this goal is applying electric field pulses directly in situ, and as cells are attached to a non-conductive material, electroporation pulses need to be applied from the top of the cell layer. When using such a small dimensions in the construction of the electrode arrays, electrode assembly is needed to be positioned closely to the cell layer so that cells are exposed to enough high intensity electric fields. In order to reduce the invasiveness of the operation and to avoid causing any physical stress to the cells, no direct contact between parts of the electrode assembly and the cells is desirable.

According to these requirements, a preferred embodiment of the electrode assembly according to the invention may be seen in Figure 1 . This figure shows a perspective view of a preferred embodiment of an electrode assembly 10 according to the invention. The electrode assembly may comprise a substrate 1 1 of a non-conductive material and an electrode array 12 placed on a first side of the substrate 1 1 , the electrode array comprising two independent closely spaced electrodes 13, 14. Further, with the aim of avoiding direct contact between parts of the electrode assembly and the cells, the electrode assembly 10 may comprise a plurality of separators 15 placed on the first side of the substrate 1 1 . Alternatively to the described separators, a system based on electrical spectroscopy bioimpedance measurements (for example, a feedback control system) is proposed to be used. Performing these measurements in a proper way and taking the advantage of the small electrode dimensions, a precise estimation of the position of the electrode assembly 10 with respect to the cell layer may be calculated with about Ι μηι precision. In any case, any of these elements (separators or a system based on electrical spectroscopy bioimpedance measurements) allows positioning the electrode assembly 10 closely parallel to the growing surface. On the other hand, this electrical spectroscopy bioimpedance measurement system may be connected to the electrode assembly 10 may perform two, three or four wire bioimpedance measurements.

The technology proposed for the construction of this preferred embodiment of the electrode assembly 10 (more specifically the electrode array 12) is any of the ones used in large scale standard thick layer production so that costs are reduced. Double Layer Printed Circuit Board (PCB) technology is presented as a preferred option, this solution being a compromise solution between the technical needs of the present invention and the reduction in costs. In large scale production this technology permits achieving such a low cost per unit, comparing to Thin Film Technologies (TFT), that disposable electrode assemblies may be produced. This way, the substrate 1 1 may be made of a dielectric material (for example, any dielectric material used in large volume standard thick layer fabrication technology), for example FR-2, FR-3 or FR4 in PCB. The geometry of the substrate 1 1 may be in accordance to the geometry of the receptacle (not shown) where the biological sample is growing and may cover the whole surface of this receptacle. The thickness of the substrate may be for example in the range of 500

Mm to 2000 Mm.

On the other hand, the independent electrodes 13, 14 may be conductive lines that may be made of a conductive material used in large volume standard thick layer fabrication technology, for example copper, platinum, gold, stainless steel, titanium, aluminium or alloys thereof. In PCB fabrication, usually copper is the preferred material. Thickness of the conductive lines 13, 14 achieved using this fabrication technology is thick if compared with chemical vapour deposition (e.g. sputtering and other TFT) and it is usually in the range of tens of microns (e.g. between 10 and 30 μιτι). Also, minimum line width achieved in this technology is in the range of several tens of microns. This way, as preferred dimensions, substrate 1 1 may be 1000 μηι thick and conductive lines 13, 14 may be 20 μηι thick. Lines width and spacing between them varies from 50 μηι to 200 μηι, preferably spacing is double the lines width.

Further, although electrodes are not supposed to be in direct contact with the biological material, it is necessary to use non-cytotoxic materials in their fabrication. In case these electrodes 13, 14 are made of any cytotoxic substance, a final non-cytotoxic conductive layer (e.g. gold or platinum) covering the conductive lines 13,14 may be added. As an example, if cooper is the conductive material used for the fabrication of the conductive lines 13, 14 a thin film of gold may be deposited with standard electrolytic processes.

Furthermore, according to the PCB technology, the separators 15 described above are easy to fabricate as a standard layer of any photosensible material, usually epoxy resins. Thickness of these separators 15 may be in the range of several microns, for example between 10 μηι and 20 μηι according to preferred embodiments.

According to Figure 2, the electrode assembly 12 may also comprise a plug 20 that comprise an electrical connector (not shown) for each conductive line 13; 14. The plug 20 is placed on the other side of the substrate 1 1 and each connector is connected to the associated conductive line 13; 14 through the substrate 1 1 , for example, by means of metalized microvias 21 . In the preferred standard fabrication technology proposed (i.e. PCB technology) is typical to use this type of connections in double layer circuits. The easy way to connect the electrode assembly 10 using a standard plug 20 as shown in Figure 2 facilitates its use by research personnel and the replacement of the electrode assembly 10 in disposable embodiments of the invention. The utilization of disposable electrodes improves sterility in laboratory conditions and reduces the cost and the time spent in sterilization processed in prior known systems.

A simplification of another embodiment of the electrode array 12 is shown in Figure 3 where an array of circular interdigitated electrodes comprising two independent conductive lines 13a, 14a is hold in the circular substrate 1 1 . The circular shape of the substrate is designed specifically to fit in a standard multi-well plate and to cover all the surface of each well. In Figure 4 another embodiment of the electrode array 12 is shown. In this case the interdigitated conductive lines 13b, 14b are parallel between them (i.e. two independent straight conductive lines) and also cover the whole surface of the substrate 1 1 (as in the case of Figure 3). On the other hand, Figure 5 depicts a different embodiment of the electrode array 12 comprising six independent spiral lines 13c, 14c,50a,51 a,52a,53a placed on the circular non-conductive substrate 1 1 covering its whole surface.

Finally, Figure 6 is given as an example of the electrode array 12 comprising parallel lines, as in Figure 4, but with six independent conductive lines 13d, 14d, 50b, 51 b, 52b, 53b placed on the same side of the circular non-conductive substrate 1 1 . This number of conductive lines configures three active areas 54,55,56 shown in the figure with different tones. In all of the electrode arrays 12 described above other number of independent conductive lines is possible, always being an even number, so that it is possible to form active pairs between them as shown in Figure 6.

Further, in all the embodiments, when electroporation is performed, each adjacent independent conductive line is connected to the cathode V- and to the anode V+ respectively as shown in Figure 4 (that is, line 13b to the anode V+ and line 14b to the cathode V-). In the case of Figure 6, one independent line in each active area is connected to the anode V+ and the other three corresponding lines are connected to the cathode V-.

It is important to highlight that the use of different active areas is useful to apply different electric field pulses in different areas of the same growing well plate such that it can be used as a test probe to find the optimal parameters to perform reversible electroporation. In the case of Figure 5, different effects in different areas of the same well plate can also be achieved with long spiral lines. If each spiral line is long enough, there is a resistance gradient between the beginning and the end of this line that can be traduced in a voltage gradient between the external and the central part of the substrate. Due to these differences in voltage applied, the electric field is higher between external adjacent lines and decreases in central direction. One of the main objects of the invention is to reduce the electric current needed to cause electroporation of biological sample membranes and to increase the uniformity in the effect performed in the surface where biological sample is attached. Figure 7 depicts the use of non-conductive materials 70 in the space between adjacent lines 13e, 14e placed on the substrate 1 1 . The effect in the current density distribution if compared with the absence of any material is an increase in such current density in the region below the conductive lines 13e, 14e, vertically expanding the area where said current density is high enough to create an electric field with the proper intensity to perform electroporation. Taking into account that the electrode array 12 is positioned relatively close but not in contact, this increment in vertical direction optimizes the current passing through the biological sample. Additionally, if borders of the conductive lines 13e, 14e are also covered, the undesirable current density accumulation caused by border effect is reduced, improving subsequently the uniformity of the electric field applied. More variable conductivity materials may be added as a film coating in the remaining electrode surface to force electric current to flow even more apart from the electrode surface concentrating said electric current in the area where the biological sample resides. In general, the filling variable conductivity materials may be deposited with standard thick layer fabrication techniques an may be preferably standard materials as epoxy photoresins.

According to another preferred embodiment of the invention, the electrode assembly 10 may comprise a complementary electrode 80 placed relatively spaced from the electrode array 12 (for example, the complementary electrode may be placed in the substrate 1 1 but in the opposite side of the electrode array 12). In Figure 8 an electrode assembly 10 comprising the complementary electrode 80 is shown. In general, it can be understood that the electrode array 12 and the complementary electrode 80 may be of the same composition to avoid generation of electric fields and/or currents that may occur when dissimilar electrode material is used. The geometry of such complementary electrode 80 is designed with the same shape than the substrate 1 1 of the electrode assembly 10, covering the entire surface of the well plate and positioned on the opposite side where the electrode array is placed. The distance between the complementary electrode 80 and the electrode array 12 is about one order of magnitude bigger is compared with the inter-electrode distance of the electrode array 12. Such distance is long enough to contain, between the complementary electrode 80 and the bottom of the well plate, all the volume of buffer solution (see Figure 9, reference 93) where molecular species are dissolved.

As described above, the use of such electrode assembly 10 according to the invention is conceived, for example, when charge molecules as DNA are expected to be introduced into cells growing as a monolayer on a surface of a culture plate (electroporation). This way, Figure 9, part B illustrates an embodiment of the method according to the invention of performing electroporation to the cells by means of the electrode assembly described above, the method comprising:

o Positioning the electrode assembly 10 on the top of the cells, avoiding direct contact between the electrode assembly 10 and the cells; o Connecting at least one of the independent electrodes (for example the electrode 13b shown un Figure 4) of the electrode array 12 to a positive polarity V+;

o Connecting at least one other of the independent electrodes (for example the electrode 14b shown in Figure 4) of the electrode array

12 to a negative polarity V-;

o Applying short duration and high amplitude pulses (see Figure 10, reference 100) between the independent electrode 13b connected to the positive polarity V+ and the independent electrode 14b connected to the negative polarity V- in order to generate electric field pulses on the cells and thus to introduce molecules (for example

DNA) into the cells.

On the other hand, when such type of molecules are the object of the electroporation treatment described above, the electrophoretic movement of these molecules may be used to create a method where both electrophoresis and electroporation are combined to enhance transfection rate. It is important to highlight that this combination improves considerably the electroporation treatment.

Thus, Figure 9, part A shows a first electrophoretic treatment performed before the electroporation treatment described above having the object of increasing the charged molecule concentration nearby the cell layer. In this first electrophoretic treatment, as can be seen in the figure, all the independent conductive lines of the electrode array 12 are short-circuited and connected to positive polarity of the electric field pulses generator (not shown) and the complementary electrode 80 is connected to negative polarity of the generator and a single attracting electrophoretic pulse (see Figure 10, reference 101 - a long duration and low amplitude pulse) is applied between them. This way, the charged molecules 90 move and concentrate in the space between the cell layer and the electrode array

12. This increase in concentration around the cell membranes contributes to improve the entrance of such molecules 90 in the cells. As long as only a high concentration of such molecules 90 is relevant in the closely volume near the cell layer, the initial global concentration of molecules added can be reduced if compared with other known systems. This reduction in concentration is traduced also in a cost reduction since the most expensive biochemical agent is always the charged active molecules to be introduced.

Next, the complementary electrode 80 is unconnected and all conductive lines of the electrode array 12 are connected following the electroporation configuration described above (see Figure 9, part B). A train of electroporation pulses (see Figure 10, reference 100 - a short duration and high amplitude pulses) is then generated.

Finally, Figure 9, part C shows a second electrophoretic treatment performed after the electroporation treatment described above having the object of pushing charged molecules 90 against cell membranes. The conductive lines of the electrode array 12 and the complementary electrode 80 are connected in the same configuration according to the first described electrophoretic treatment but with inverse polarity. A repulsing electrophoretic pulse (see Figure 10, reference 102 - a medium duration and low amplitude pulse) is then applied to move the charged molecules 90 in direction to the cell layer. When applying electroporation pulses, cell membranes remain opened for a variable period of time so that this second electrophoretic treatment exploits this state of the cell membranes and pushes the charged molecules 90 through the remaining pores. Basically, the meanings of the features of the described pulses may be the following:

• Low amplitude: in the range of 50 to 500 V/cm;

• High amplitude: in the range of 1000 to 3000 V/cm;

• Long duration: in the range of 200 to 1000 ms;

· Medium duration: in the range of 50 to 100 ms;

• Short duration: in the range of 1 με to 10 ms. Figure 10 shows a possible sequence of the pulses applied during the described method according to the invention. Firstly, a single low amplitude and long duration pulse 101 is applied; next, a train of high amplitude and short duration pulses 100 is applied; and finally a low amplitude and medium duration pulse 102 with inverse polarity with respect to the first pulse 101 is applied. The real voltage or current magnitude applied by a pulse generator are not indicated because such magnitudes depend on the final realization of the electrode assembly 10 and their dimensions and also in the characteristics of the generator used. Nevertheless, said magnitudes must be appropriate to create electric fields between 50 to 500 V/cm when low amplitude is needed and fields between 1000 to 3000 V/cm in the case of high amplitudes. The preferred waveform comprises unipolar square pulses in the steps of electrophoresis whereas the preferred waveform comprises bipolar square pulses in the steps of electroporation. On the other hand, a computer system suitable for carrying out the described method is required. This way, this computer system may comprise:

o Computer means for positioning the electrode assembly 10 on the top of the biological sample (e.g. cells) growing as a monolayer on the surface of the culture plate, avoiding direct contact between the electrode assembly and the biological sample;

o Computer means for connecting at least one of the independent electrodes (e.g. see Figure 4, reference 13b) of the electrode array 12 to the positive polarity V+;

o Computer means for connecting at least one other of the independent electrodes (e.g. see Figure 4, reference 14b) of the electrode array 12 to the negative polarity V-;

o Computer means for applying short duration and high amplitude pulses between the independent electrode connected to the positive polarity V+ and the independent electrode connected to the negative polarity V- in order to generate electric field pulses on the biological sample. In case of the first electrophoretic treatment is required, the computer system may also comprise:

o Computer means for short-circuiting all the independent electrodes of the electrode array 12;

o Computer means for connecting the short-circuited electrodes to a positive polarity V+;

o Computer means for connecting the complementary electrode to a negative polarity V-;

o Computer means for applying at least one long duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to increase the negatively charged molecule concentration nearby the biological sample.

Finally, in case of the second electrophoretic treatment is required, the computer system may comprise:

According to preferred embodiments of the invention, the computer system may be implemented by software (e.g. a computer program product), by hardware (e.g. logic gates) or by a combination of software and hardware. Thus, for example, the computer system may be implemented by a memory and a processor, embodying instructions stored in the memory and executable by the processor, the instructions comprising functionality to perform the steps of the method previously described.

Figure 1 1 shows a block diagram of an embodiment of an apparatus for performing electroporation. This apparatus may comprise a control and a user interface unit 1 10 whereby the user of the system can act with the different parts of the system according to the invention (for example, the computer system described above may be implemented in this unit); an electroporation power supply 1 1 1 (that is, the pulse generator), which provides the electric field pulses required for the electroporation treatment (as can be seen in the figure, the power supply is controlled by the control unit, establishing the type of pulse to provide, that is, pulses with low/high amplitude and short/medium/long duration); an electrical bioimpedance measurements block 1 12, which determines in real time the distance between the electrode assembly 10 and the cells for avoiding direct contact between the electrode assembly and the cells; a connection switching block 1 13 for interconnecting all blocks of the system, which is controlled by the control and a user interface unit 1 10 (this way, it is possible, for example, to connect the conductive lines of the electrode array and/or the complementary electrode to the positive polarity or to the negative polarity or to short-circuit the conductive lines); and a positioning system 1 14 for positioning the electrode assembly 10 on the top of the cells to be electroporated according to data provided by the electrical bioimpedance measurements block 1 12 with the object to avoid direct contact between the electrode assembly and the cells.

Figure 12 depicts a possible commercial realization of the system for performing electroporation to cells by means of the electrode assembly previously described, this system comprising:

- The pulse generator 1 1 1 , which provides the electric field pulses required for the electroporation treatment;

- The electrode assembly 10;

- An arm 121 comprising a complementary plug to the plug of the electrode assembly 10 for attaching and electrically interconnecting the electrode assembly with the pulse generator 1 1 1 ; - The computer system described above;

- The housing comprising the said computer system inside.

The positioning system 1 14 controls the arm 121 so that the electrode assembly 10 is placed on the top of the cells growing as a monolayer on a surface of a standard multi-well plate 122, based on the information received from electrical bioimpedance measurements block 1 12. Once the electrode assembly is placed on the top of the cells, the computer system controls the power supply 1 1 1 for obtaining the required type of pulses according to the treatment to be applied and the connection switching block 1 13 for connecting the conductive lines of the electrode array 12 and the complementary electrode 80 if required according to said treatment, that is, according each step of the method to be performed.

Although this invention has been disclosed in the context of certain preferred embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. Thus, it is intended that the scope of the present invention herein disclosed should not be limited by the particular disclosed embodiments described before, but should be determined only by a fair reading of the claims that follow.

Further, although the embodiments of the invention described with reference to the drawings comprise computer apparatus and processes performed in computer apparatus, the invention also extends to computer programs, particularly computer programs on or in a carrier, adapted for putting the invention into practice. The program may be in the form of source code, object code, a code intermediate source and object code such as in partially compiled form, or in any other form suitable for use in the implementation of the processes according to the invention. The carrier may be any entity or device capable of carrying the program.

For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means.

When the program is embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means.

Alternatively, the carrier may be an integrated circuit in which the program is embedded, the integrated circuit being adapted for performing, or for use in the performance of, the relevant processes.

Claims

1 . An electrode assembly for generating electric field pulses to perform electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, the electrode assembly being adapted to be positioned on the top of the biological sample avoiding direct contact between the electrode assembly and the biological sample, the electrode assembly comprising:

- A substrate of a non-conductive material;

- An electrode array placed on a first side of the substrate, the electrode array comprising at least two independent closely spaced electrodes;

- One electrical connector associated to each electrode, the connectors being placed on a second side of the substrate and being connected to the electrodes through the substrate.

2. The electrode assembly according to claim 1 , wherein the non-conductive material of the substrate is a dielectric material, preferably FR-2, FR-3 or FR-4.

3. The electrode assembly according to any of claims 1 or 2, wherein the at least two independent closely spaced electrodes are made from a conductive material, preferably copper, platinum, gold, stainless steel, titanium, aluminium or alloys thereof.

4. The electrode assembly according to any of claims 1 to 3, wherein the at least two independent closely spaced electrodes are covered with one or more non- cytotoxic materials, preferably gold or platinum.

5. The electrode assembly according to any of claims 1 to 4, wherein the spacing between adjacent electrodes of the electrode array is in the range of 50 to 150 μηι.

6. The electrode assembly according to any of claims 1 to 5, wherein the spacing between adjacent electrodes is filled with a non-conductive material, preferably non-cytotoxic epoxy resins.

7. The electrode assembly according to any of claims 1 to 6, wherein the width of each independent electrode of the electrode array is in the range of 50 to 150 μηι.

8. The electrode assembly according to any of claims 1 to 7, wherein the thickness of each independent electrode of the electrode array is in the range of 10 to 30 m.

9. The electrode assembly according to any of claims 1 to 8, wherein the at least two independent closely spaced electrodes comprise parallel, concentric or uniform ly-spaced-along lines.

10. The electrode assembly according to any of claims 1 to 9, further comprising a complementary electrode placed relatively spaced from the electrode array.

1 1 . The electrode assembly according to claim 10, wherein the complementary electrode is placed in a third side of the substrate.

12. The electrode assembly according to any of claims 10 or 1 1 , wherein the complementary electrode is made from a conductive material, preferably copper, platinum, gold, stainless steel, titanium, aluminium or alloys thereof.

13. The electrode assembly according to any of claims 1 to 12, further comprising a plug comprising the electrical connectors associated to each independent electrode.

14. The electrode assembly according to any of claims 1 to 13, further comprising a plurality of separators placed on the first side of the substrate.

15. A method of performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly according to any of claims 1 to 14, the method comprising: o Positioning the electrode assembly on the top of the biological sample growing as a monolayer on the surface of the culture plate, avoiding direct contact between the electrode assembly and the biological sample;

o Applying short duration and high amplitude pulses between the independent electrode connected to the positive polarity and the independent electrode connected to the negative polarity in order to generate electric field pulses on the biological sample.

16. The method according to claim 15, further comprising:

o Connecting the complementary electrode to a negative polarity;

17. The method according to any of claims 15 or 16, further comprising:

o Short-circuiting all the independent electrodes of the electrode array; o Connecting the short-circuited electrodes of the electrode array to a negative polarity;

o Connecting the complementary electrode to a positive polarity;

o Applying at least one medium duration and low amplitude pulse between the short-circuited electrodes and the complementary electrode in order to push negatively charged molecules against biological sample membranes.

18. The method according to any of claims 16 or 17, wherein low amplitudes are in the range of 50 to 500 V/cm.

19. The method according to any of claims 15 to 18, wherein high amplitudes are in the range of 1000 to 3000 V/cm .

20. The method according to any of claims 16 to 19, wherein long durations are in the range of 100 to 1000 ms.

21 . The method according to any of claims 17 to 20, wherein medium durations are in the range of 50 to 100 ms.

22. The method according to any of claims 15 to 21 , wherein short durations are in the range of 1 με to 50 ms.

23. A computer system for performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly according to any of claims 1 to 14, the computer system comprising:

24. The computer system according to claim 23, further comprising:

o Computer means for connecting the short-circuited electrodes to a positive polarity;

o Computer means for connecting the complementary electrode to a negative polarity;

25. The computer system according to any of claims 23 or 24, further comprising:

26. An apparatus for performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate, the apparatus comprising:

- A pulse generator;

- An electrode assembly according to any of claims 1 to 14;

- An arm comprising a complementary plug to the plug of the electrode assembly for attaching and electrically interconnecting the electrode assembly with the pulse generator; - An arm positioning system for positioning the electrode assembly in the correct position according to the biological sample growing as a monolayer on the surface of the culture plate;

- A computer system according to any of claims 23 to 25.

27. The apparatus according to claim 26, further comprising a system based on electrical spectroscopy bioimpedance measurements.

28. Computer program product comprising program instructions for causing a computer to perform a method of performing electroporation to at least one biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly according to any of claims 1 to 14, said method according to any of claims 15 to 22.

29. Computer program product according to claim 28, embodied on a storage medium.

30. Computer program product according to claim 28, carried on a carrier signal.

31 . A method of performing electrofusion to cells of a biological sample growing as a monolayer on a surface of a culture plate by means of an electrode assembly according to any of claims 1 to 14, cells of the biological sample being in physical contact, the method comprising:

- Executing the method of performing electroporation according to any of claims 15 to 22.

32. The method according to claim 31 , further comprising:

- Providing cells of the biological sample in physical contact.