WO2007021993A1 - Helical electrode geometries for electroporation - Google Patents

Abstract

Electrode geometries for electroporation. An electroporation chamber includes a non-rectangular cross section and electrodes configured to generate a substantially uniform field within the chamber, the field effecting electroporation of a sample. The electroporation chamber may be a flow electroporation chamber. Two electrodes may be positioned helically, spaced substantially equidistant from one another, and having a common axis.

Description

Description

HELICAL ELECTRODE GEOMETRIES FOR ELECTROPORATION

Background of the Invention The present invention claims priority to U.S. Provisional Patent Application

Serial No. 60/707,655, filed on August 12, 2005, which is hereby incorporated by reference.

1. Field of the Invention The present invention relates generally to electroporation. More particularly, it concerns electrode geometries for use with electroporation processes, including but not limited to flow electroporation processes.

2. Description of Related Art The ability to load various macromolecules into living cells is a key method used in modern biological and biomedical research as well as medical therapies. A number of technologies have been developed that enable macromolecules to enter living cells. Electroporation is a suitable technology for many applications including ex vivo clinical applications. Electroporation of living cells is based on the transient creation of pores in the cell membrane through which molecules, including macromolecules such as nucleic acid or protein, can enter the cytoplasm and nucleus of the cell. Electroporation is typically accomplished by using electrodes, connected to a controllable voltage source, to generate a transient electric field having a sufficient strength and duration to create pores in cells. The transient electric field should be such so that a maximal number of cells are loaded with a desired molecule via the pores, while a minimal number of cells are damaged or killed during the process. While electroporation is a common objective when one treats cells with a transient electric field, treatment of cells with an electric field may be used to differentially kill cells in a heterogeneous population or to affect the metabolism of the treated cells. The electric fields used for these purposes may be similar to those used for electroporation. It is desired that the electric field produced between the electrodes be as uniform as possible so that as high a fraction of cells as possible experience the optimal electric field. Perfect uniformity of the electric field is not necessary, as cells that experience electric fields that are not fully optimal may nevertheless become loaded with an efficiency that approaches a maximum efficiency, while not suffering undue damage or mortality. The geometry of electrodes can play a major role in the uniformity, or non-uniformity, of the electric field.

Conventional electroporation, whether carried out in a batch-wise, static manner, on in a flow-electroporation manner involves an electrode geometry to generate a substantially uniform electric field based on having two plate-like electrodes positioned parallel to one another. While this arrangement of electrodes provides for efficient electroporation, it may not be optimal for all applications, e.g., for flow electroporation.

The use of one or more pairs of plate-like electrodes typically requires an electroporation chamber having a rectangular cross section. This shape may lead to less than optimal flow of cells though the chamber and therefore less than optimal electroporation. For example, poor flow may be especially pronounced at corners where substantially flat walls meet. Cells that are located at such corners may not traverse the flow cell at a desired rate and may experience inefficient electroporation conditions. Additionally, a flow electroporation chamber using parallel plate electrodes and a rectangular cross-section typically require custom-designed sealing fixtures. Additionally, electric fields at the edges of parallel plate electrodes are typically not the same as over the bulk of a chamber, and these anomalous electric fields may be sub-optimal for electroporation and may even exhibit deleterious arcing. Additionally, fluid leakage may result from imperfections resulting from the assembly of a rectangular chamber and/or custom fittings or components for the chamber.

( Referenced shortcomings of conventional methodologies mentioned above are not intended to be exhaustive, but rather are among several that tend to impair the effectiveness of previously known techniques concerning electroporation and electrode geometry. Other noteworthy problems may also exist; however, those mentioned here are sufficient to demonstrate that methodology appearing in the art have not been altogether satisfactory and that a significant need exists for the techniques described and claimed here.

Summary of the Invention Certain shortcomings of the prior art are reduced or eliminated by the techniques disclosed here. These techniques are applicable to a vast number of applications, including but not limited to any application involving electroporation, such as applications involving flow electroporation.

In different embodiments, the techniques of this disclosure are applied to an electroporation chamber (an electroporation flow path in flow electroporation applications) that is substantially circular in cross-section. A circular-cross-section device exhibits fluidic behavior having advantages over that from a rectangular cross- section. The circular design provides for uniform and efficient electroporation of cells within the electroporation chamber, or flowing within the chamber. A cylindrical design does not need corners or exhibit areas that would lead to especially- retarded flow. Such a chamber does not easily allow for parallel plate electrodes.

According to different embodiments, an electrode design that provides an electric field of substantial uniformity but that, nevertheless, allows for an electroporation chamber having a substantially circular cross-section is desired. In addition, a substantially cylindrical electroporation chamber conveniently allows the use of stock pipes, rods, wires, threads, fittings, ports, and gaskets that are inexpensive, readily available, and made out of various materials. An electrode geometry that avoids electrical problems associated with the edges of plate-like electrodes also is desired.

In one respect, the invention involves an electroporation chamber including a non-rectangular cross section and electrodes configured to generate a substantially uniform field within the chamber, the field effecting electroporation of a sample. The electroporation chamber may be a flow electroporation chamber. The electrodes may be positioned symmetrically about a common axis. The chamber may include a circular cross section and the electrodes may be positioned helically. The chamber may define one or more non-rectangular flow electroporation channels.

In another respect, the invention involves a chamber for treating cells with an electric field including two electrodes positioned helically, spaced substantially equidistant from one another, and having a common axis. A pitch of each electrode helix may be equal to about two-times the diameter of each electrode helix. The chamber may be an electroporation chamber. The chamber may also include one or more cooling elements positioned among one or more spaces within helices formed by the electrodes.

In another respect, the invention involves a chamber for treating cells with an electric field including two non-flat electrodes having the same size and shape and being positioned symmetrically about a common axis. The chamber may also include one or more cooling elements positioned among one or more spaces of the symmetrical electrodes.

In another respect, the invention involves an electroporation system including a housing, electrodes, and a signal generator. The housing has a non-rectangular cross section, and the housing defines an electroporation chamber. The electrodes are positioned helically about the electroporation chamber, and the electrodes are configured to generate a substantially uniform field within the chamber. The signal generator is coupled to the electrodes and is configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber. The electroporation system may be a flow electroporation system, and the housing may define a flow electroporation chamber configured to electroporate a sample that flows continuously or intermittently through the chamber. The housing may have, e.g., a curved, circular, or elliptical cross section. The housing and chamber may define one or more non-rectangular flow electroporation channels. The electrodes may include, e.g., conducting wires or bands. The electrodes may be substantially equidistant from one another. A pitch of each electrode helix may be equal to about twice a diameter of each electrode helix. The electrodes may be positioned helically about a common axis. The system may include two electrodes. The system may also include one or more cooling elements positioned among one or more spaces within helices formed by the electrodes. The electrodes may be configured to generate a field within the chamber that is uniform within 20%, 10%, 5%, or 1%.

In another respect, the invention involves an electroporation system including a cylindrical housing, two or more wire electrodes, and a signal generator. The cylindrical housing defines a cylindrical electroporation chamber. The two or more wire electrodes are positioned helically about the electroporation chamber, and the electrodes are spaced substantially equidistant from one another and configured to generate a substantially uniform field within the chamber. The signal generator is coupled to the electrodes and is configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber.

hi another respect, the invention involves an electroporation system including a housing, two non-flat electrodes, and a signal generator. The housing defines an electroporation chamber. The two non-flat electrodes are positioned symmetrically about a common axis, and the electrodes have the same size and shape. The electrodes are configured to generate a substantially uniform field within the chamber. The signal generator is coupled to the electrodes and is configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber.

hi another respect, the invention involves a method for treating cells with an electric field. Cells are positioned (e.g., placed according to a conventional static protocol or flowed according to a flow protocol) between two electrodes that are positioned helically, spaced substantially equidistant from one another, and having a common axis. Treating the cells may include electroporating the cells.

hi another respect, the invention involves a method of electroporation, where a sample is introduced into an electroporation chamber having a non-rectangular cross section. Electrical energy is applied to electrodes positioned helically about the electroporation chamber. A substantially uniform field is generated within the electroporation chamber using the electrodes, and the sample is electroporated using the field. The sample may be electroporated while it flows continuously or intermittently through the chamber. The chamber may be cylindrical. The chamber may define one or more non-rectangular flow electroporation channels. The method may also include cooling the sample using one or more cooling elements positioned among one or more spaces within helices formed by the electrodes. The field within the chamber may be uniform within 20%., 10%, 5%, or 1%.

As used herein, "flow" electroporation refers to electroporation associated with a sample that flows continuously or intermittently (e.g., intermittently in cycles or recurrently) by electrodes of an electroporation chamber within an electroporation system that handles flowing samples. "Flow" electroporation is therefore distinguished from traditional, static electroporation systems that process samples statically, batch by batch, within a system that does not handle flowing samples. Different "flow" electroporation techniques are discussed in, for example, U.S.

Provisional Patent Application Serial No. 60/570,317; U.S. Patent Application Serial

No. 10/225,446; PCT Publication No. WO 03/018751; U.S. Patent No. 5,612,207;

U.S. Patent No. 5,720,921; U.S. Patent No. 6,074,605; U.S. Patent No. 6,090,617;

U.S. Patent No. 6,485,961; U.S. Patent No. 6,617,154; and/or U.S. Patent No. 6,773,669 (each of which is incorporated here by reference). Likewise a "flowing" sample refers to continuous or intermittent (e.g., intermittently in cycles or recurrently) flow of a sample.

As used herein, an "electroporation apparatus" refers to any equipment used directly or indirectly to effect electroporation. An electroporation apparatus therefore encompasses electroporation chambers, pumps, valves, reservoirs, and associated elements. An electroporation apparatus also encompasses electronics and computer equipment used directly or indirectly for the electroporation process. Likewise, a

"flow electroporation apparatus" refers to any equipment used directly or indirectly to effect flow electroporation. A flow electroporation apparatus therefore encompasses flow electroporation chambers, pumps, valves, reservoirs, and associated elements. A flow electroporation apparatus also encompasses electronics and computer equipment used directly or indirectly for the flow electroporation process. As used herein, a "chamber" refers to the area in which actual electroporation occurs. Accordingly, a "chamber" may refer to one or more channels within an electroporation apparatus.

As used herein, the term "substantially" should be given its plain meaning to those having ordinary skill in the art. When applied to measurements such as distances or electric fields "substantially" should encompass differences within 20% and preferably refers to differences within 15%, more preferably within 10%, even more preferably within 5%, and most preferably within 1 %.

The terms "a" and "an" are defined as one or more than one unless this disclosure explicitly requires otherwise.

The terms "comprise" (and any form of comprise, such as "comprises" and

"comprising"), "have" (and any form of have, such as "has" and "having"), "include" (and any form of include, such as "includes" and "including") and "contain" (and any form of contain, such as "contains" and "containing") are open-ended linking verbs. As a result, a method or device that "comprises," "has," "includes" or "contains" one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more elements. Likewise, a step of a method or an element of a device that "comprises," "has," "includes" or "contains" one or more features possesses those one or more features, but is not limited to possessing only those one or more features. Furthermore, a device or structure that is configured in a certain way is configured in at least that way, but may also be configured in ways that are not listed.

Other features and associated advantages will become apparent with reference to the following detailed description of specific, example embodiments in connection with the accompanying drawings. Brief Description of the Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The drawings do not limit the invention but simply offer examples.

FIG. 1 is a schematic diagram of an electroporation system, in accordance with embodiments of this disclosure.

FIG. 2 is a schematic diagram of an electroporation chamber including electrodes positioned helically, in accordance with embodiments of this disclosure. Electrode spacing is substantially equal and, in this embodiment, is measured parallel and perpendicular to the longitudinal axis of the chamber.

FIG. 3 is a schematic diagram of the electroporation chamber of FIG. 2, and shows that electrode spacing may be measured using parallel lines tangent to the helix.

FIG. 4 is a schematic diagram of the electroporation chamber of FIG. 2, which shows electric field lines in two directions.

FIG. 5 is a schematic diagram of the electroporation chamber of FIG. 2, which shows electric field lines in around a fixed point.

FIG. 6 is a schematic diagram of an electroporation chamber including electrodes positioned helically, in accordance with embodiments of this disclosure. Cylindrical tubing forms the input and output of the chamber. A signal is shown being applied to the helically positioned electrodes.

FIG. 7 is a top-view schematic diagram of an electroporation chamber including electrodes positioned helically, in accordance with embodiments of this disclosure. FIGS. 8 and 9 are schematic diagrams of electroporation chambers including band electrodes positioned helically, in accordance with embodiments of this disclosure.

FIG. 10 is a flow chart showing example steps of a method for electroporation, in accordance with embodiments of this disclosure.

FIGS. HA and HB are graphs modeling helically configured electrodes, in accordance with embodiments of this disclosure.

Description of Illustrative Embodiments

FIG. 1 is a schematic diagram of an electroporation system 100, in accordance with embodiments of this disclosure. FIG. 1 shows an electroporation apparatus 104 coupled to computer 102 via connection 106.

Electroporation apparatus 104 is meant to indicate, in general, any electroporation apparatus in which the electrode geometry techniques of this disclosure may be applied. In a preferred embodiment, electroporation apparatus 104 is a flow electroporation apparatus, although in other embodiments it may be a conventional, static device. In flow electroporation embodiments, the electroporation apparatus 104 may include any flow electroporation equipment described in at least U.S. Provisional Patent Application Serial No. 60/570,317; U.S. Patent Application Serial No. 10/225,446; PCT Publication No. WO 03/018751; U.S. Patent No. 5,612,207; U.S. Patent No. 5,720,921; U.S. Patent No. 6,074,605; U.S. Patent No. 6,090,617; U.S. Patent No. 6,485,961; U.S. Patent No. 6,617,154; and/or U.S. Patent No. 6,773,669 (each of which has been incorporated by reference). Electroporation apparatus 104 may also encompass any other flow electroporation apparatus known or available in the art. Those having ordinary skill in the art will appreciate that such flow electroporation apparatuses may be modified and/or or optimized and still be suitable for implementing the techniques of this disclosure.

Connection 106 is meant to indicate any connection suitable for allowing computer 102 to communicate with electroporation apparatus 104. In one embodiment, connection 106 is a wired connection. In another embodiment, connection 106 is wireless. In a wireless embodiment, connection 106 may be a network connection over a network such as the Internet. Such an embodiment allows for remote control of electroporation apparatus 104 from virtually any computer in the world connected to the Internet. hi one embodiment, computer 102 and electroporation apparatus 104 are integral, hi such an embodiment, connection 106 may be an internal connection.

Computer 102 is meant to indicate any computing device capable of executing instructions for controlling one or more aspects of electroporation apparatus 104. hi one embodiment, computer 102 is a personal computer (e.g., a typical desktop or laptop computer operated by a user of the flow electroporation apparatus 104). Such a computer may include one or more appropriate boards for interfacing with electroporation apparatus 104. hi another embodiment, computer 102 may be a personal digital assistant (PDA) or other handheld computing device, hi another embodiment, computer 102 and electroporation apparatus 104 may be integral, and in such embodiment, computer 102 may simply constitute one or more boards (e.g., a motherboard including a processor) among other electronic boards and equipment, hi one embodiment, a computer may not be used or used minimally, where electroporation is accomplished in a more manual manner.

Computer 102 can be a networked device and may constitute a terminal running software from a remote server, wired or wirelessly. Input from a user may be gathered through one or more known techniques such as a keyboard and/or mouse. Output, if necessary, can be achieved through one or more known techniques such as an output file, printer, facsimile, e-mail, web-posting, or the like. Storage can be achieved internally and/or externally and may include, for example, a hard drive, CD drive, DVD drive, tape drive, floppy drive, network drive, flash, or the like. Computer 102 may use any type of monitor or screen known in the art. For example, a cathode ray tube (CRT) or liquid crystal display (LCD) can be used. One or more display panels may also constitute a display. In other embodiments, a traditional display may not be required, and computer 102 may operate through appropriate voice and/or button commands. FIG. 2 is a schematic diagram of an electroporation chamber 200 including wire electrodes 206 and 208 positioned helically (in this embodiment as a double helix), in accordance with one embodiment of this disclosure. Electrode spacing is substantially equal and, in this embodiment, is measured parallel and perpendicular to the longitudinal axis of the chamber 200. Specifically, the distances A and B shown in FIG. 2 are substantially equal (and, here, equal to a helical pitch). The distance A is measured parallel to the axis of the chamber 200, and the distance B is measured perpendicular to the axis of the chamber 200. Electrodes 206 and 208 in this embodiment have the same size and shape and are positioned symmetrically about a common axis (a longitudinal axis running up-and-down through the center of the double helix).

The electric field generated by electrodes 206 and 208 is substantially uniform within the electroporation chamber 200 when electrodes 206 and 208 are substantially equidistant. Additional electrode arrangements may be used to create a substantially uniform field, and commercially-available electric field modeling software may be used to this end.

While electrodes 206 and 208 are positioned helically about a common axis in the embodiment of FIG. 2, the electrodes in other embodiments may not share an axis.

Electroporation chamber 200 is defined by housing 204. In the illustrated embodiment, housing 204 is cylindrical, having a circular cross section. In other embodiments, the cross section of housing 204 may vary. For example, it may be any non-rectangular cross section. It may have a curved cross section or an elliptical cross section.

Although shown as wires, electrodes 206 and 208 may take a different form.

For example, electrodes 206 and 208 may include bands of material (discussed below in relation to FIGS. 8 and 9). Electrodes 206 and 208 may be constructed out of any material or materials known in the art. Electrodes 206 and 208 may be made from metallic or non-metallic conductors. In different embodiments, electrodes 206 and 208 may include gold, platinum, tantalum, or carbon (graphite, diamond).

Although shown with two helical electrodes 206 and 208, electroporation chamber 200 may include more than two electrodes.

Base 210 of FIG. 2 is representative of any input or output to electroporation chamber 200 and, in practice, may entail tubing, seals, pumping equipment, and the like, as is known in the art.

Electroporation chamber 200 of FIG. 2 may include cooling elements 209 positioned among spaces within the helices formed by electrodes 206 and 208. Cooling elements 209 may be piezoelectric or other cooling elements known in the art. Positioning cooling elements 209 among electrode surfaces does not cause significant non-uniformity of electric fields as it would in conventional designs. In particular, the design of the chamber 200 affords the option of putting cooling elements in the space between helical electrodes, thereby making the cooling elements almost, but not actually, part of the electrodes themselves, hi other embodiments, however, the cooling elements may be made physically integral with electrodes themselves. Cooling elements 209 may be used to cool a sample being electroporated, which may lead to more effective treatment of, e.g., cells.

The non-rectangular cross section of electroporation chamber 200 is advantageous because the lack of corners avoids areas of retarded flow. Moreover, the cylindrical electroporation chamber 200 allows one to use standard pipes, rods, wires, threads, fittings, ports, and gaskets that are inexpensive, readily available, and made out of various materials. Still further, the electrode geometry of FIG. 2 avoids electrical problems associated with the edges of plate-like electrodes — e.g., arcing problems are reduced or eliminated.

FIG. 3 is a schematic diagram of the electroporation chamber of FIG. 2, and shows that electrode spacing may be measured using parallel lines tangent to the helix, hi FIG. 3, the two electrodes are also substantially equidistant, but the illustrated distance A is measured slightly differently than in FIG. 2. In FIG. 3, the distance A is measured not along the longitudinal axis, but instead with reference to parallel lines tangent to the electrodes. Electrodes are substantially equidistant if the spacing of the electrodes is about equal, using either measurement scheme (measured along a longitudinal axis or with reference to parallel tangent lines).

FIG. 4 is a schematic diagram of the electroporation chamber of FIG. 2, which shows electric field lines in two directions. The field distribution is the superposition of all such field vectors. The field distribution, if desired, may be acquired using commercially available software that models electric fields for various configurations of conductors. The embodiment of FIG. 4 is intended to convey that, using helical electrodes, one may arrive at a substantially uniform electric field throughout the interior of a non-rectangular (here, cylindrical) electroporation chamber.

FIG. 5 is a schematic diagram of the electroporation chamber of FIG. 2, which shows electric field lines in around a fixed point. As with FIG. 4, the field distribution would be the superposition of all such points.

FIG. 6 is a schematic diagram of an electroporation chamber including electrodes positioned helically, in accordance with embodiments of this disclosure. Cylindrical tubing 606 forms the input and output of the chamber. Advantageously, tubing 606 may be stock tubing, widely available commercially. Gaskets and other sealing mechanisms may likewise be standard, which may reduce costs and improve maintenance of electroporation equipment. Caps 608 and 610 secure the electrodes and may be conductors so as to convey electrical signal 612.

A generic signal 612 is shown being applied to the helically positioned electrodes through caps 608 and 610. In practice signal 612 may be any signal sufficient to effect electroporation, static or flow. For example, signal 612 may be a signal of a particular strength and duration for causing the formation of pores in a particular sample type. Those having ordinary skill in the art will understand what types of signals should be applied to various samples. Signal 612 may be generated from any signal generator known in the art and capable of producing electroporation signals.

Advantageously, as long as the ends of the electrodes in FIG. 6 are secured (in the illustrated embodiment by caps 608 and 610), the electrodes are self-aligned by tension. Also advantageously, the electroporation chamber of FIG. 6 can be assembled with very few parts, and several if not all of those parts may be stock equipment readily available and not needing customization. Also advantageously, the electroporation chamber may be made without machining, drilling, grinding, lapping, polishing or extruding. Also advantageously, the system is simple and compatible with a large array of stand fittings, connectors, and adaptors. All of these example advantages may lead to a lower cost system, even for exotic electrode materials such as platinum or gold.

FIG. 7 is a top-view schematic diagram of an electroporation chamber including electrodes 206 and 208 positioned helically, in accordance with embodiments of this disclosure. FIG. 7 more clearly illustrates the ability to form a circular cross section electroporation chamber, which provides advantages such as those mentioned above. FIGS. 8 and 9 are schematic diagrams of electroporation chambers including metallic, band electrodes positioned helically, in accordance with embodiments of this disclosure, hi these embodiments, the bands are used instead of the wire electrodes 206 and 208 of FIG. 2. In other embodiments, differently shaped conductors may be used for the electrodes. In particular, any shape may be used that may interact with an electroporation chamber to create a field suitable for electroporation. Additionally, conductors of various materials may be used.

Electrodes may be placed symmetrically in embodiments of this disclosure. For example, in one embodiment, a chamber including two or more non-flat electrodes positioned symmetrically about a common axis may be utilized. "Non- flat" electrodes simply refers to electrodes that are not the typical, rectangular flat conducting elements used in conventional electroporation apparatuses. The symmetry of the electrodes aids in forming a substantially uniform field. One example of a symmetrical chamber may involve helical electrodes, while those having ordinary skill in the art will appreciate that other symmetrical shapes may be used equally as well.

FIG. 10 is a flow chart showing example steps of a method for electroporation, in accordance with embodiments of this disclosure. In operation, an electroporation system using techniques of this disclosure may be used to (1) introduce a sample into an electroporation chamber having a non-rectangular cross section as shown in step 1002, (2) apply electrical energy to electrodes positioned helically about the chamber as shown in step 1004, (3) generate a substantially uniform electric field within the chamber as shown in step 1006, and (4) electroporate the sample as shown in step 1008.

This method (along with the other techniques of this disclosure) may be applied for conventional, static electroporation or flow electroporation.

* * *

The following examples are included to demonstrate aspects of specific experiments related to this disclosure.

Example !

A prototype cuvette was built from a serological pipette and tin-plated copper wire. The coils serving as electrodes were hand-wound. The coil spacing was adjusted by eye to approximately match the coil core diameter.

Results of loading FITC-conjugated dextran (MW ~300,000) loading into Jurkat cells are shown below. In the data that follows, the relative amount of "positive" cells corresponds to the viable cells that accumulated FITC, whereas some cells remained viable without getting permeabilized by electrical field ("negative" cells). Therefore, the positive, the negative, and the dead cells in any sample add up to 100 percent. (a) Lower field strength (~1 kV/cm)

87.7% positive 98.3% viable

(b) Higher field strength (~1.25 kV/cm) 87.3% positive

91.9% viable

The same experiment was repeated 10 days later (FITC-conjugated dextran):

(a) Lower field strength (~1 kV/cm)

79.1% positive 97.9% viable

(b) Higher field strength (-1.25 kV/cm) 81.0% positive

95.1% viable

(c) Standard MaxCyte cuvette (1.0 kV/cm)

88.1% positive 99.5% viable

Results for the same experiment, but this time for DNA transfection using ~2 kb DNA plasmid encoding green fluorescent protein (eGFP) at concentration 50 ug/mL:

(a) Lower field strength (~1 kV/cm) . 29.4% positive 67.9% viable

(b) Higher field strength (~1.25 kV/cm)

10.6% positive 25.9% viable (c) Standard MaxCyte cuvette (1.0 kV/cm) 71.6% positive 90.5% viable

Example 2

Modeling was performed to analyze certain aspects of embodiments of this disclosure.

Au electrical field around a line of charge decreases with the distance from the line, and an electrical field between two lines of charge (of opposite signs) is constant. With this and other fundamental physics bases, one can model electrical field properties about helical electrodes.

Without being bound by theory, if electrode lines are not straight but the distance between them is constant, the electrical field between the electrodes is substantially constant if the lines' curvature is small. If, however, the curvature is not small, the field distribution may become distorted, but the field will still be governed primarily by the distance (and its variation) between the lines.

FIG. HA shows the variation of the distance between a fixed point on one helical coil and the nearest turn of another one (shown by arrows on FIG. 11 B). In FIG. HA, in the top trace, pitch is increased 40% and the variation is minimal in the range +/- 1.5 radians (half turn). In the middle trace, pitch is increased 15% and variation does not exceed 10% in the range +/- 2.5 radians. In the lower trace, coil pitch is 2x the diameter.

With the benefit of the present disclosure, those having ordinary skill in the art will comprehend that techniques claimed here and described above may be modified and applied to a number of additional, different applications, achieving the same or a similar result. For example, while description is directed to electroporation embodiments, the techniques of this disclosure may be applied to any application in which a sample is electrically treated (e.g., any procedure affecting the sample, its structure, behavior, or functionality). For example, treatments to kill cells of a sample or treatments to affect metabolism of cells of a sample are contemplated. The attached claims cover all modifications that fall within the scope and spirit of this disclosure.

References

Each of the following references is hereby incorporated by reference in its entirety:

U.S. Provisional Patent Application Serial No. 60/570,317

U.S. Patent Application Serial No. 10/225,446

PCT Publication No. WO 03/018751

U.S. Patent No. 5,612,207

U.S. Patent No. 5,720,921 U.S. Patent No. 6,074,605

U.S. Patent No. 6,090,617

U.S. Patent No. 6,485,961

U.S. Patent No. 6,617,154

U.S. Patent No. 6,773,669

Claims

Claims

1. An electroporation chamber comprising a non-rectangular cross section and electrodes configured to generate a substantially uniform field within the chamber, the field effecting electroporation of a sample.

2. The chamber of claim 1, the electroporation chamber being a flow electroporation chamber.

3. The chamber of claim 1, where the electrodes are positioned symmetrically about a common axis.

4. The chamber of claim 1, where the chamber comprises a circular cross section and the electrodes are positioned helically.

5. The chamber of claim 1, where the chamber defines one or more non-rectangular flow electroporation channels.

6. A chamber for treating cells with an electric field comprising two electrodes positioned helically, spaced substantially equidistant from one another, and having a common axis.

7. The chamber of claim 6, where a pitch of each electrode helix is equal to about two-times a diameter of each electrode helix.

8. The chamber of claim 6, where the chamber comprises an electroporation chamber for electroporating cells.

9. The chamber of claim 6, further comprising one or more cooling elements positioned among one or more spaces within helices formed by the electrodes.

10. A chamber for treating cells with an electric field comprising two non-flat electrodes having the same size and shape and being positioned symmetrically about a common axis.

11. The chamber of claim 10, further comprising one or more cooling elements positioned among one or more spaces of the symmetrical electrodes.

12. An electroporation system comprising: a housing having a non-rectangular cross section, the housing defining an electroporation chamber; electrodes positioned helically about the electroporation chamber, the electrodes being configured to generate a substantially uniform field within the chamber; and a signal generator coupled to the electrodes and configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber.

13. The system of claim 12, where the electroporation system is a flow electroporation system and where the housing defines a flow electroporation chamber configured to electroporate a sample that flows continuously or intermittently through the chamber.

14. The system of claim 12, where the housing has a curved cross section.

15. The system of claim 12, where the housing has a circular cross section.

16. The system of claim 12, where the housing has an elliptical cross section.

17. The system of claim 12, where the housing and chamber define one or more non- rectangular flow electroporation channels.

18. The system of claim 12, where the electrodes comprise conducting wires.

19. The system of claim 12, where the electrodes comprise conducting bands.

20. The system of claim 12, where the electrodes are substantially equidistant from one another.

21. The system of claim 20, where a pitch of each electrode helix is equal to about two-times a diameter of each electrode helix.

22. The system of claim 12, where the electrodes are positioned helically about a common axis.

23. The system of claim 12, comprising two electrodes.

24. The system of claim 12, further comprising one or more cooling elements positioned among one or more spaces within helices formed by the electrodes.

25. The system of claim 12, where the electrodes are configured to generate a field within the chamber that is uniform within 20%.

26. The system of claim 12, where the electrodes are configured to generate a field within the chamber that is uniform within 10%.

27. The system of claim 12, where the electrodes are configured to generate a field within the chamber that is uniform within 5%.

28. The system of claim 12, where the electrodes are configured to generate a field within the chamber that is uniform within 1%.

29. An electroporation system comprising: a cylindrical housing defining a cylindrical electroporation chamber; two or more wire electrodes positioned helically about the electroporation chamber, the electrodes being spaced substantially equidistant from one another and configured to generate a substantially uniform field within the chamber; and a signal generator coupled to the electrodes and configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber.

30. An electroporation system comprising: a housing defining an electroporation chamber; two non-flat electrodes positioned symmetrically about a common axis, the electrodes having the same size and shape and being configured to generate a substantially uniform field within the chamber; and a signal generator coupled to the electrodes and configured to supply electrical energy to the electrodes sufficient to generate the substantially uniform field and effect electroporation within the chamber.

31. A method for treating cells with an electric field comprising positioning the cells between two electrodes positioned helically, spaced substantially equidistant from one another, and having a common axis.

32. The method of claim 31, where treating the cells comprises electroporating the cells.

33. A method of electroporation, comprising: introducing a sample into an electroporation chamber having a non- rectangular cross section; applying electrical energy to electrodes positioned helically about the electroporation chamber; generating a substantially uniform field within the electroporation chamber using the electrodes; and electroporating the sample using the field.

34. The method of claim 33, where the sample is electroporated while it flows continuously or intermittently through the chamber.

35. The method of claim 33, where the chamber is cylindrical.

36. The method of claim 33, where the chamber defines one or more non-rectangular flow electroporation channels.

37. The method of claim 33, further comprising cooling the sample using one or more cooling elements positioned among one or more spaces within helices formed by the electrodes.

38. The method of claim 33, where the field within the chamber is uniform within 20%.

39. The method of claim 33, where the field within the chamber is uniform within 10%.

40. The method of claim 33, where the field within the chamber is uniform within 5%.

41. The method of claim 33, where the field within the chamber is uniform within 1%.