WO2010064991A1

WO2010064991A1 - Electroporation apparatus and method

Info

Publication number: WO2010064991A1
Application number: PCT/SG2008/000458
Authority: WO
Inventors: Mark Richards
Original assignee: Nanyang Polytechnic
Priority date: 2008-12-01
Filing date: 2008-12-01
Publication date: 2010-06-10

Abstract

An electroporation apparatus comprising a main body dimensioned to be moved relative to a colony of cells; and an electrode disposed on the main body and being configured to discharge an electrical field for inducing poration in cells within a focal area of said colony of cells.

Description

ELECTROPORATION APPARATUS AND METHOD

Technical Field

The present invention generally relates to an electroporation apparatus and method for discharging an electrical field for inducing poration in cells with a focal area.

Background Electropermeabilization or electroporation results in a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. Typically, this method is used in molecular biology as a way of introducing substances into a cell, such as molecular probes, drugs or pieces of foreign DNA. However, known methods of electroporation have several drawbacks in relation to the electroporation of certain cell lines, such as Immortal and pluripotent human embryonic stem cells (hESCs) . hESCs are stem cells that are capable of differentiating into somatic cells of all three primordial germ layers. hESCs can also be maintained in a non-transformed, undifferentiated state indefinitely in vitro without any apparent loss of self-renewal ability or differentiation potential. In particular, diploid hESC lines have the capacity to differentiate into the complete range of about 210 different types of highly specialized cells that form the human.

Hence, it can be seen that hESC lines are useful in vitro systems that allow the study of the genetic and embryological events and the production of tissues for transplantation therapy in the human. The unique differentiation potential of hESCs may also be harnessed for drug and pharmacological testing. hESC-based in vitro drug and toxicity screens can also provide a platform to analyse product safety and behaviour in drug development with the added advantage of high-throughput compatibility. hESC-based in vitro systems are a reliable and standardized system that also have the advantage of being completely animal-free and therefore, this technology has the potential of reducing and possibly eliminating the reliance on some forms of animal testing.

The ability to genetically modify hECSs is critical for the widespread use of hECSs as a platform for pharmaceutical discovery. One problem affecting progress in this area is the availability of reporter and genetically marked hESC lines. A major hurdle has been the resistance of hESCs to established electroporation, infection and chemical transfection methodologies to introduce ectopic genes. In one known method of electroporation, an electric field is applied throughout the cell culture dish to electroporate the cells contained within. Because hESCs are social cells which form colonies, they often require feeder support when in culture. Hence, when this method of electroporation is used to electroporate hESCs, the electric field that has been generally applied to the entire culture dish causes an indiscriminate electroporation of the hESCs and the feeder cells. As a result, when vectors, construct are subsequently introduced into the cell culture, there may be unwanted transfection of feeder cells with the vector constructs. This may undesirably alter the feeder cells ability to support the hESCs in culture. Moreover, a large portion of these vector constructs may be wasted in inadvertently transecting these feeder cells, affecting the overall transfection efficiency of the hESCs. Because of the general electric field that is applied throughout the culture dish, localized variation of the electric field

(to vary the level of permeability) for each hESC colony would not be possible. In view of the above, other attempts have been made to isolate hESCs into single cell suspension for single- cell electroporation (for example the patch clamp technique) . To isolate these hESCs from their cell colonies, caustic chemicals are sometimes used. Such harmful chemicals may inevitably increase the mortality rate of the hESCs. Furthermore, beacause hESCs are social cells which form colonies, dissociating hESC colonies into single-cell suspensions or into ^• small clumps of cells (ie about 10 to about 20 cells per clump) for the purpose of transfection often results in cell death and low plating efficiencies. hESCs are also very sensitive to perturbations in the culture environment and chemical transfection methodologies often have a toxic effect on these cells . There is a need to provide an electroporation apparatus and a method of electroporating cells, such as hESC cell lines, that overcome or at least ameliorate one or more of the disadvantages described above. Summary

According to a first aspect, there is provided an electroporation apparatus comprising a main body dimensioned to be moved relative to a colony of cells; and an electrode disposed on the main body and being configured to discharge an electrical field for inducing poration in cells within a focal area of said colony of cells.

Advantageously the disclosed electroporation apparatus allows localized electroporation of cell types which are colony-forming.

In one embodiment, the main body of the electroporation apparatus is generally longitudinal with the electrode being disposed on one end of said main body. Advantageously, the longitudinal main body of the electroporation apparatus provides a mechanical advantage for the manipulation of the apparatus. More advantageously, the longitudinal main body is dimensioned to be manipulated relative to said colony of cells. In one embodiment, the electrode of the electroporation apparatus comprises a generally curvilinear-shaped part. Advantageously, the curvilinear- shaped part provides a substantially enclosed area around the cell colony to be electroporated. The electrode may also comprise a tip-shaped part disposed within said curvilinear-shaped part. Advantageously, this allows an electrical field to be generated in a radial direction from the tip-shaped part.

In another embodiment, the end of said main body adjacent said electrode is generally convergent along the longitudinal axis of the main body in the direction towards said electrode. Advantageously, this ensures that the focal area of the electric field created to be independent of the dimensions of the main body and allows electroporation of higher locality. The main body of the apparatus may also be generally circular-shape in cross-section extending along the longitudinal axis. This allows for better portability and manipulation by hand or by an automated robotic device that is programmed to move the main body relative to the colony of cells.

The electrode of the apparatus may also be electrically coupled to a power source and the apparatus may further comprise a switch configured to control the supply of power from said power source to said electrode. This beneficially allows the user to easily control when the electric field is to be discharged. The power source may be configured to vary the magnitude of the electrical field being discharged by the electrode.

In one embodiment, the apparatus further comprises an outlet conduit adjacent to the electrode for allowing transmission of liquid containing biological material to the cells while they undergo poration for transfection of said biological material into said cells. Advantageously, the apparatus may have the further function of functioning as a dispensing device which can immediately dispense a desired amount of biological material to cells as soon as they are being electroporated. More advantageously, this also allows localized dispensation of the biological material. The apparatus may also further comprise a reservoir disposed within the main body for holding the biological liquid for transmission to said outlet liquid conduit adjacent to said electrode. The reservoir serves as a holding unit for the biological material to hold a predetermined amount of biological material therein so that the apparatus does not have to be refilled after each dispensation operation. Advantageously, because there exist a supply of biological material within the apparatus, the apparatus can be used as a standalone, electro-transfection device without the need for a separate dispensing apparatus.

The apparatus may also further comprise an inlet conduit for receiving the biological material and being disposed on said main body in fluid communication with said reservoir. This provides access to the reservoir during a refilling operation.

In a second aspect, there is provided a method of electroporating cells comprising the steps of selectively applying an electrical field to a focal area of a cell colony in a solid culture to thereby induce poration of said cells within said focal area.

The method may also comprise the step of providing said electrode on an electroporation apparatus having a main body dimensioned to be moved relative to said cell colonies. The electrode provides the source of electric field required to electroporate the cells. The method may comprise the step of contacting said porated cells with a liquid containing biological material during said selectively applying step. This step serves to transfect the electroporated cells with a target biological material. In one embodiment, the disclosed method further comprises the steps of transfecting a first biological material into a first cell colony while inducing poration of said first cell colony; and transfecting a second biological material into a second cell colony while inducing poration of said second cell colony; wherein the constituents of the first and second biological materials are similar or different.

Definitions

The following words and terms used herein shall have the meaning indicated:

As used herein, the term "biological material" includes molecules and macromolecules within a wide range of molecular weight, from the size of ions and small molecules up to proteins, antibodies, and large enzymes. Hence, the molecules include, but are not limited to, lipoproteins, nucleic acids or nucleic acid molecules, including RNA, DNAl ribozymes, siRNA, and other nucleic acids, proteins, polypeptides, amino-acids, including modified proteins or polypeptides, antibodies, antibody fragments, receptors, and other proteins or polypeptides, as well as other organic molecules suitable for introduction into cells by the present method. The term "generally cylindrical housing" as used herein is not meant to limit a cross-section of the housing in a plane perpendicular to the longitudinal axis of the housing being strictly circular or elliptical and may include other shapes in a plane perpendicular to the longitudinal axis of the housing such as stepped or conical configurations. The term "cell colony" or "colony of cells" herein indicates a cell aggregate formed as a result of cell growth. These terms are used irrespective of the number of cells or cell layers constituting the colony. For example, the term "human embryonic stem cell colony" or "hES cell colony" indicates a colony containing human embryonic stem cells, and the term is used irrespective of the number of the human embryonic stem cells constituting each colony or the proportion of the human embryonic stem cells in the colony.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated ^'value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value .

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges ' as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Detailed Disclosure of Embodiments Exemplary, non-limiting embodiments of an electroporation apparatus, will now be disclosed.

There is provided an electroporation apparatus that comprises a main body dimensioned to be moved relative to a colony of cells; and an electrode disposed on the main body and being configured to discharge an electrical field for inducing poration in cells within a focal area of said colony of cells.

The main body may be generally longitudinal with said electrode being disposed on one end of said main body. In one embodiment, the main body has a generally circular shape in cross section extending along the longitudinal axis. The longitudinal main body may be dimensioned to be manipulated relative to said colony of cells. Such manipulation may be manual or automatic. In one embodiment, the manipulation is carried out by hand. In another embodiment, the manipulation is carried out by a robotic arm. The longitudinal main body may also be a generally cylindrical housing. In one embodiment, the longitudinal main body may be coated with a synthetic or natural elastomer. The main body may be coated with an elastomer selected from the group consisting of polychloroprene, natural rubber, styrene butadiene rubber, nitrile rubber, silicone rubber. In one embodiment, the main body is made of polyoxymethylene and is available under the trade name Delrin™ made by Du Pont Corporation. It should be appreciated that any polymer could be used that that can function as an electrical insulator and which has sufficient mechanical strength such that it can be handled. Advantageously, this improves the grip on the apparatus when the apparatus is manipulated by hand. Advantageously, the main body is thermally and electrically resistant, which protects the user during use.

In one embodiment, the electrode of the apparatus comprises a generally curvilinear shaped part. The curvilinear -shaped part may be in the form of a circle, oval or ellipsoid. In another embodiment, the electrode of the apparatus comprises a polygonal shaped part. The polygonal shaped part may be in the form of a square, rectangle or rhombus. Advantageously, when in use, the curvilinear shaped part or the polygonal shaped part forms a substantial enclosure that localizes the cells to be electroporated. In one embodiment, the curvilinear shaped part or the polygonal shaped part is adjustable to vary the area that substantially encloses the cells to be electroporated. In one embodiment, when the curvilinear shaped part is circular, the diameter of the circular shaped part can be varied to fit the specific size of the cell colony. Advantageously, this ensures that localization of the electroporation is still possible for differing sizes of cell colonies. In one embodiment, the electrode comprises a tip- shaped part disposed within said curvilinear shaped part. In another embodiment, the electrode comprises a tip- shaped part disposed within said polygonal shaped part. The tip shaped part may be capable of causing an electric field to radiate from it. In one embodiment, the tip shaped part has a diameter ranging from about 0.5 mm to about 4.0 mm. The tip shaped part may be disposed in a substantially central position within the curvilinear shaped part or the polygonal shaped part. Alternatively, the tip shaped part may also be disposed at a position that is offset from the centre of the curvilinear shaped part or the polygonal shaped part.

The tip shaped part, curvilinear shaped part and/or the polygonal shaped part of the electrode may be a metal. The metal may be selected from the group consisting of Group IB, Group VIB, Group VIIIB, Group IVA, Group IVB, Group HB and Group HIA of the Periodic Table of Elements, as well as their alloys and combinations thereof.

In one embodiment, the metal may be selected from the group consisting of aluminum, cobalt, copper, gold, indium, molybdenum, nickel, palladium, platinum, silver, tin, titanium, tungsten, zinc and combinations thereof. In one embodiment, the metal is gold. It will be appreciated that in other embodiments, the metal may be any ferrous or non-ferrous material that can function as an electrical conductor.

In one embodiment, the tip shaped part and either the curvilinear shaped part or the polygonal shaped part of the electrode are of different polarities. For example, the tip shaped part may be a negative electrode while the curvilinear shaped part or the polygonal shaped part may be a positive electrode. Alternatively, the tip shaped part may be a positive electrode while the curvilinear shaped part or the polygonal shaped part may be a negative electrode.

The end of the main body adjacent to the electrode may also be generally convergent along the longitudinal axis of the main body in the direction towards said electrode. In one embodiment, the main body adjacent to the electrode comprises an adjustable portion that is capable varying the size of the curvilinear shaped part or the polygonal shaped part such that the area that substantially encloses the cells to be electroporated is also varied. For example, curvilinear shaped part or the polygonal shaped part may be comprised a plural electrode sub-parts that are configured to be moved towards a central axial point or away from a central axial point so that the electrode sub-parts are capable of moving toward and away from each other to thereby adjust the size of the area of the electrode and thereby adjust the electrode for the particular size of cell colony being electroporated.

In one embodiment,- the electrode is electrically coupled to a power source. In one embodiment, the power source is a direct current (DC) power source. In another embodiment, the power source is an alternating current

(AC) power source. The power source may also be located within the apparatus (ie such as a battery power source) or may be an independent power source located outside the apparatus. In one embodiment, the power source is in the form of electrical cells. The power source may also operate from a voltage of about 0 volts to a voltage of about 5,000 volts.

The apparatus may further comprise a switch configured to control the supply of power from the power source to said electrode. Preferably, the switch is disposed at a position that can be easily accessed by the user.

In one embodiment, the apparatus further comprises an outlet conduit adjacent to the electrode for allowing transmission of liquid containing biological material to the cells while they undergo poration for transfection of said biological material into cells. In one embodiment, the diameter of the outlet conduit is from about 0.5 mm to about 5 mm. In one embodiment, the dispensation of biological fluid from the apparatus is actuated by a pressure differential. In one embodiment, the pressure differential is created by a pump that is selected from the group consisting of pneumatic and electro-mechanical devices.

In one embodiment, the apparatus further comprises a reservoir disposed within the main body for holding the biological liquid for transmission to said outlet liquid conduit adjacent said electrode. In one embodiment the reservoir is a container that is made of bio-compatible polymeric material. Said bio-compatible polymer materials may be selected from a group consisting of a polyvinyl resin, a vinyl acetate-ethylene copolymer, a vinyl polymer, an acrylic resin, a cellulose derivative and a polyolefin. Advantageously, said bio-compatible polymer material may be selected from the group consisting of poly (ethyl vinyl acetate), polyethylene, polypropylene, polystyrene and polyamide . The reservoir may also be made up of a material that will not undesirably change in its properties when in contact with the biological fluid contained therein. Advantageously, the container is made up of a material that is non-toxic. The container may also be sterilized at temperatures of more than about 110⁰C and pressures at more than about 15 psi (-104 KPa), without an appreciable change in its desired form and properties.

.There is also provided a method of electroporating cells comprising the steps of selectively applying an electrical field to a focal area of a cell colony in a solid culture to thereby induce poration of said cells with said focal area.

It should be appreciated that any cell colony may be electroporated, such as embryonic stem cell colonies, keratinocytes colonies and ^• any other colony-forming epitheloid, neuronal or fibroblast-like cell type such as human embryonic stem cell (hESC) colonies. In another embodiment, the disclosed method and apparatus may also be suitable for electroporating bacteria colonies.

The disclosed method may also comprise the step of providing the electrode on an electroporation apparatus having a main body dimensioned to be moved relative to said ^' cell colonies. In one embodiment, the method also comprises the step of contacting said porated cells with a liquid containing biological material during said selective applying step. In another embodiment, the method further comprises the step of providing plural cell colonies in said solid culture. In yet another embodiment , the method further comprises the steps of transfecting a first biological material into a first cell colony while inducing poration of said first cell colony; and transfecting a second biological material into a second cell colony while inducing poration of the second cell colony. The first biological material and the second biological material may be comprised of the same material or may be comprised of different material. Similarly, the first cell colony and the second colony may be comprised of the same cells or may be comprised of different cells .

Brief Description Of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of

^• illustration only, and not as a definition of the limits of the invention. Fig. 1 shows a perspective view of the electroporation apparatus in accordance with one embodiment disclosed herein.

Fig. 2 shows a perspective view from the electroporation tip-shaped part and an enlarged view of the tip-shaped part of the electroporation apparatus in accordance with one embodiment disclosed herein.

Fig. 3 shows a cross-sectional view of the electroporation apparatus as shown in Fig. 1. Fig. 4 shows a perspective view of the electroporation apparatus in accordance with another embodiment disclosed herein.

Fig. 5 shows a cross-section view of the electroporation apparatus shown in FIG 4.

Detailed Description of the drawings

The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.

Now referring to Fig. 1, there is provided a perspective view of the electroporation apparatus in accordance with one embodiment disclosed herein. As shown in Fig. 1, the in electroporation apparatus 1 comprises a main body in the form of a probe holder 10 with one proximal end 11 and one distal end 12 and an electrode assembly20 being integrally attached onto the distal end 12. The proximal end 11 is electronically coupled with a power supply providing electrical power to- the electrode assembly 20 during electroporation. The probe holder 10 preferably has a cylindrical shape with the distal end 12 being taped off. It is to be noted that the shape and configuration of the probe holder 10 is not so limited; any design or configuration may be suitable as long as it does not impede the performance of the electroporation apparatus 1.

Now referring to FIG 2, there is provided a perspective view from the electrode assembly 20 and an enlarged view of the electrode assembly 20 of the electroporation apparatus in accordance with one embodiment disclosed herein. As shown in Fig. 2, the electroporation tip assembly 20 has a frusto-conical configuration. It should also be noted that other configuration can also be used as long as it is suitable for performing the designated function electroporation apparatus. The electrode assembly 20 comprises an external layer 21, a curvilinear shaped part in the form of a positive electrode 22 and a tip shaped part in the form of a negative electrode 23, whereby the electrical field formed between the positive electrode 22 and negative electrode 23 is used for electroporation. The positive electrode 22 has a circular configuration, and the negative electrode 23 being positioned in the central point of the circular positive electrode has a point configuration; thus an electrical field having a circular configuration is formed, making it suitable for electroporating a focal area such as colonies of cells and bacteria. The positive and negative electrodes 22, 23 are made of copper. Now referring to Fig. 3, there is provided a cross- sectional view of the electroporation apparatus as shown in Fig 1. The probe holder 10 has been so configured to allow the electrode assembly 20 be connected to an external power supply. The connection can be made possible by providing a central channel or throughhole. The probe holder is preferably made of Delrin™ for having electrical and thermal resistances. It is to be noted that other available electrical and thermal resistant materials can also be used. The positive and negative electrodes 22, 23 have tip protrudes for enabling the electrodes to penetrate tissues and solidified culture media such as agarose.

Now referring to Fig. 4, there is provided a perspective view of the electroporation apparatus in accordance with another embodiment disclosed herein. As shown in Fig. 4, the electroporation apparatus 100 is similar to ^'the electroporation apparatus 1 shown in Fig. 1 with the exception of the presence of an additional solution holding reservoir 130. The additional solution holding reservoir 130 is capable of storing the solution containing for example expression vectors before the solution being released into the electrode assembly 120 for being electroporated into cells.

Now referring to Fig. 5, there is provided a cross- section view of the electroporation apparatus shown in Fig. 4. The solution holding reservoir 130 comprises a solution inlet/outlet port 131 for receiving to-be- electroported solution, a storage body -132 for storing the received to-be-electroporated solution, and a releasing mechanism 133 for releasing the to-be- electroporated solution into the electrode assembly 120 after the electrode assembly has been positioned at the locale for electroporation. Therefore, the electroporation apparatus in this configuration has the capacity of delivering any expression vectors into the localized cells without disturbing the adjacent cells or exposing the expression vectors to the cells not being included for electroporation; this is especially important when many colonies are present in one culturing plate. Now there is provided a brief description of electroporation method employing a localized electrical field being generated by the electroporation apparatus as described above. After a culture plate with one or more colonies of cells is over-layed with the solution containing any desired expression vectors, the electroporation apparatus is positioned above one designated colony, then power supply is switched on, resulting in an electrical field being generated around the colony. The power supply is then turned off after an appropriate amount of time has lapsed and the electroporation apparatus is removed. When multiple expression vectors are required to be electrotransfected into different colonies, one method is to overlay one solution at a time, and then washing away the existing solution after the electrotransfection of the first solution has been completed. After which, another solution over-layed in the ^• culture dish. The electroporation apparatus with the solution holding reservoir can also be used conveniently to deliver different expression vectors into different colonies without the use of overlaying method sdescribed above.

While the present invention has been described with reference to particular embodiments, it will be understood that the embodiments are illustrative and that the invention scope is not so limited. Alternative embodiments of the present invention will become apparent to those having ordinary skill in the art to which the present invention pertains. Such alternate embodiments are considered to be encompassed within the spirit and scope of the present invention. Accordingly, the scope of the present invention is described by the appended claims and is supported by the foregoing description.

App1ications The electroporation apparatus disclosed is suitable for delivering substances such as expression vectors into cells such as hECSs. The apparatus is capable of localised electroporation which in turn avoids the need for enzymatic dissociation of hESC colonies into single cells, thereby eliminating the problem of low plating efficiencies commonly observed with hESCs following conventional electroporation protocols. Such localised electroporation also avoids interruption of the cell cycle and intercellular communication by eliminating the need to detach adherent cells for the purpose of electroporation.

Advantageously, the electroporation apparatus and the- electroporation method disclosed herein allow selective colony transfection and avoids t^'ransfection of feeder cells. As a result, there would also be high target cell transfection efficiency. -Likewise, different colonies on the same tissue culture dish can be transfected with different vector constructs. This enables the study of transient gene expression on cell- cell/colony-colony interaction by virtue of having different colonies in the same dish expressing different vector constructs.

The electroporation apparatus disclosed herein also allows electroporation of all cell types which grow in colonies including keratinocytes and many other epithelioid cell types . Advantageously, the electroporation apparatus also allows immediate analysis/observation of fast-acting small molecules, proteins, nucleic acid molecules as there is no waiting time for cells to adhere to tissue- culture surface and resume cell cycle since the cells that are electroporated by the electroporation apparatus in situ are ^' already adhered to the tissue-culture surface .

More advantageously the electroporation apparatus disclosed herein can also be used to electroporate multicellular structures/tissues grown „ in petri dishes (eg. embryoid bodies and 3-D tissue engineering cell/scaffold constructs) .

While reasonable efforts have been employed to describe equivalent embodiments of the present invention, it will be apparent to the person skilled in the art after reading the foregoing disclosure, that various other modifications and adaptations of the invention may be made therein without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims .

Claims

1. An electroporation apparatus comprising: a main body dimensioned to be moved relative to a colony of cells; and an electrode disposed on the main body and being configured to discharge an electrical field for inducing poration in cells within a focal area of said colony of cells.

2. An apparatus as claimed in claim 1, wherein said main body is generally longitudinal with said electrode being disposed on one end of said main body.

3. An apparatus as claimed in claim 2, wherein said longitudinal main body is dimensioned to be manipulated by human or robotic hand relative to said colony of cells.

4. An apparatus as claimed in claim 1 or claim 2, wherein the electrode comprises a generally curvilinear-shaped part.

5. An apparatus as claimed in claim 4, wherein the electrode comprises a tip-shaped part disposed within said curvilinear-shaped part.

6. An apparatus as claimed in claim 3, wherein the circular-shaped part is a positive electrode.

7. An apparatus as claimed in claim 5, wherein the tip- shaped part is a negative electrode.

8. An apparatus as claimed in claim 2, wherein the end of said main body adjacent said electrode is generally convergent along the longitudinal axis of the main body in the direction towards said electrode .

9. An apparatus as claimed in any one of claims 2 to 8, wherein the main body has a generally circular-shape in cross-section extending along the longitudinal axis .

10. An apparatus as claimed in any one of the preceding claims, wherein the electrode is electrically coupled to a power source.

11. An apparatus as claimed in claim 9, further comprising a switch configured to control the supply of power from said power source to said electrode.

12. An apparatus as claimed in any one of the preceding claims, further comprising an outlet conduit adjacent to the electrode for ^"allowing transmission of liquid containing biological material to the cells while they undergo poration for transfection of said biological material into said cells.

13. An apparatus as^' claimed in claim 12, further comprising a reservoir disposed within the main body for holding the biological liquid for transmission to said outlet liquid conduit adjacent said electrode .

14. An apparatus as claimed in claim 12, further comprising an inlet conduit for receiving the biological material and being disposed on said main body in fluid communication with said reservoir.

15. A method of electroporating cells comprising the steps of selectively applying an electrical field to a focal area of a cell colony in a solid culture to thereby induce poration of said cells within said focal area.

16. A method as claimed in claim 15, comprising the step of providing said electrode on an electroporation apparatus having a main body dimensioned to be moved relative to said cell colonies .

17. A method as claimed in claim 15 or claim 16, comprising the -step of contacting said porated cells with a liquid containing biological material during said selectively applying step.

18. A method as claimed in any one of claims 15 to 17, comprising the step of providing plural cell colonies in said solid culture.

19. A method as claimed in claim 18 when dependent on claim 17, comprising the steps of: transfecting a first biological material into a first cell colony while inducing poration of said first cell colony; and transfecting a second biological material into a second cell colony while inducing poration of said second cell colony; wherein the constituents of the first and second biological materials are the same or different .

20. A method as claimed in any one of claims 15 to

19, comprising the step of providing said electrode having a generally curvilinear -shaped positive part and a tip-shaped negative part encompassed within said generally circular-shaped positive part.