WO2007021994A2

WO2007021994A2 - Electrode shielding for electroporation

Info

Publication number: WO2007021994A2
Application number: PCT/US2006/031478
Authority: WO
Inventors: Linhong Li; Cornell Allen; Linda Liu; Sergey M. Dzekunov; Nicholas J. Chopas
Original assignee: Maxcyte, Inc.
Priority date: 2005-08-12
Filing date: 2006-08-11
Publication date: 2007-02-22
Also published as: WO2007021994A3

Abstract

Electrode shielding for electroporation. An apparatus includes (a) an electroporation chamber, (b) an electrode (402) in operative relation with the chamber, and (c) a conductive and water permeable barrier (404) in operative relation with the electrode, the barrier being configured to substantially prevent cells in a sample being electroporated within the chamber from coming into contact with the electrode during use.

Description

Description Electrode Shielding for Electroporation

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

Serial No. 60/707,636, 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 techniques to protect samples being electroporated and to improve the electroporation efficiency.

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. Different cell types are known to be differentially sensitive to such fields. 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. The electrodes are a key component of any electroporation apparatus. However, the interfacial areas between electrodes and aqueous space can trigger at least two kinds of problems. A first problem involves generation of toxic electrochemical products at the electrode surfaces, including both the generation and release of metal ions from electrodes and the formation of toxic gas, and their introduction into the aqueous space occupied by the cells being electroporated. A second problem involves the formation of complexes between cells and the toxic gases from electrochemical reaction products, which loosely adhere to electrodes but may leave the electrodes and mix with healthy cells (especially during sample collection), and reduce the yield of electroporated cells. Such complexes generate byproducts derived from damaged or lysed cells on or near electrode surfaces, thereby reducing electroporation efficiency or leading to a less efficacious post- electroporation cell suspension. Generation of these products can become an especially serious problem if electrodes are repeatedly employed for processing large volumes of cells, such as in the case of large volume flow electroporation. If one desires to reuse electrodes it is further desirable that the electrodes have not contacted cells from a prior electroporation.

Materials that are substantially electrochemically inert may be used in electroporation electrodes to minimize generation and release of metal ions. For example, gold electrodes may be used. However, such materials are generally more costly than less inert materials, and the ability to use less inert, and less costly, electrode materials without substantial degradation of electroporation performance is desired. Moreover, even use of inert electrodes does not effectively address problems associated with toxic gas formation generated during electroporation and the formation of complexes of cells and the gases that generate cell debris (e.g., proteins and genomic DNA from dead cells).

The shortcomings mentioned above are not intended to be exhaustive, but rather are among many that tend to impair the effectiveness of previously known techniques concerning electrodes for electroporation; however, those mentioned here are sufficient to demonstrate that the methodologies appearing in the art have not been altogether satisfactory and that a significant need exists for the techniques described and claimed in this disclosure.

Summary of the Invention

Certain shortcomings of the prior art may be 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 effectively shield electrodes from samples to be electroporated. Different materials may be used to form a barrier about all, or part, of electrodes. The barrier is preferably selectively permeable. For example, the barrier may be permeable to small ions but impermeable to macromolecules and cells, or cell fragments. The shielding may reduce or even eliminate harmful byproducts that are produced during electroporation and may provide a barrier spatially separating cells being electroporated from electrode surfaces and potentially toxic or interfering materials produced at or near those electrode surfaces.

In one respect, the invention involves an apparatus for electrically treating cells, including a chamber, an electrode, and a conductive and water permeable barrier. The electrode is in operative relation with the chamber. The conductive and water permeable barrier is in operative relation with the electrode. The barrier is configured to substantially prevent cells in a sample being treated within the chamber from coming into contact with the electrode during use. The barrier may be configured to prevent over 90% of the sample from coming into contact with the electrode. Treating cells may involve electroporation, and the chamber may therefore be an electroporation chamber. The electroporation chamber may be a flow electroporation chamber. The flow electroporation chamber may include an input and output. The apparatus may include two electrodes and two barriers. The apparatus may include two electrodes and one barrier, and the one barrier may be in operative relation with a positive side of an electrode. The electrode may include iron or brass. The electrode may be part of a helical arrangement. The barrier may include a dialysis membrane. The barrier may surround a portion of the electrode. The barrier may be spaced from the electrode. The barrier may be spaced from the electrode by one or more gaskets.

In another respect, the invention involves a method for electrically treating a sample by electroporating the sample with an apparatus described in the above paragraph. The sample may include cells, which may be human cells.

In another respect, the invention involves a method, in which cells are loaded into a chamber. The cells are subjected to an electric field sufficient to treat the cells using an electrode that is in operative relation with the chamber. The cells being treated are substantially prevented from coming into contact with the electrode during use with a barrier that is in operative relation with the electrode. Treating the cells may include electroporating the cells. The chamber may be a flow electroporation chamber, and loading the sample may involve flowing the sample through the flow electroporation chamber. The electrode may include iron or brass. The barrier may include a dialysis membrane.

In another respect, the invention involves a method for reducing electroporation byproducts in an electroporation chamber. A conductive and water permeable barrier is formed around an area of an electrode within the chamber, the barrier being sufficient to substantially prevent electroporation byproduct material from leaking into an aqueous space within the chamber for processing samples. The byproduct material may include electrode material or cell debris. The cell debris may include DNA.

In another respect, the invention involves a method for electroporation in which cycles of cells are subjected to an electric field sufficient to effect electroporation. A conductive and water permeable barrier is formed around areas of electrodes such that the electrodes are capable of repeatedly processing more than 30 cycles without a substantial lessening of cell viability or transfection efficiency. The cells may include Jurkat cells. The electrodes may be capable of repeatedly processing more than 50 cycles without a substantial lessening of cell viability or transfection efficiency. The electrodes may be capable of repeatedly processing more than 75 cycles without a substantial lessening of cell viability or transfection efficiency. The electrodes may be capable of repeatedly processing more than 100 cycles without a substantial lessening of cell viability or transfection efficiency. The electrodes may be capable of repeatedly processing at least 120 cycles without a substantial lessening of cell viability or transfection efficiency.

In another respect, the invention involves a method for electroporation in which flowing cells are subjected to an electric field sufficient to effect electroporation. A conductive and water permeable barrier is formed around areas of electrodes such that the electrodes are capable of processing at least 420 mL of cells without a substantial lessening of cell viability or transfection efficiency. The cells may include K562 cells.

As used herein, "treating" a sample refers to any procedure affecting the sample (e.g., affecting structure, behavior, or functionality) and, in a preferred embodiment, can refer to treatment by electroporation. In other embodiments, treating a sample involves, e.g., differentially killing cells of the sample or affecting the metabolism of cells of the sample. Those having ordinary skill in the art will recognize many other types of treatments can be achieved electrically using techniques of this disclosure.

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.

The terms "a" and "an" are defined as one or more unless this disclosure explicitly requires otherwise. The term "approximately" and its variations are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The term "substantially" and its variations are defined as being largely but not necessarily wholly what is specified as understood by one of ordinary skill in the art, and in one-non and in one non-limiting embodiment the substantially refers to ranges within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5% of what is specified. If a barrier is said to "substantially" prevent a sample being electroporated from coming into contact with the electrode, then a large percentage of that sample will be prevented from coming into contact with the electrode during use. In one embodiment, more than 90% of the sample is prevented contact, more preferably more than 95%, more preferably more than 99%, and most preferably more than 99.5%.

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.

The term "coupled," as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.

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, and encompasses equipment such as that used with, e.g., static electroporation systems. An electroporation apparatus 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. 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.

The term "permeable" should be interpreted according to its plain and ordinary meaning as understood by those having ordinary skill in the art. The term should generally encompass meanings such as penetrable. The term should generally encompass concepts such as having pores or openings that permit, e.g., liquids or gases to pass through. In the context of embodiments of this disclosure, a barrier that is water permeable allows water molecules and small inorganic ions to diffuse through it to a degree such that an electric field used for electroporation is either not substantially altered by the barrier or altered in a defined way so that it can be compensated-for electrically. In the context of certain embodiments of this disclosure, a barrier may prevent the passage of intact cells through the barrier. Such a barrier may or may not prevent or impede the diffusion of proteins or other macromolecules.

The term "conductive" should be interpreted according to its plain and ordinary meaning as understood by those having ordinary skill in the art. A material that is conductive should be interpreted as allowing an electric current to pass through it with relatively little resistance relative to other components in the current path, hi the context of embodiments of this disclosure, a barrier is conductive if it allows an electric field to pass such that the electric field is either not substantially altered or altered in a defined way that can be compensated electrically so as not to effect electroporation of cells.

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 shielded electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure.

FIG. 3 is a schematic diagram of shielded electrodes, particularly well suited for flow electroporation, in accordance with embodiments of this disclosure. FIG. 4 is a schematic diagram of shielded electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure.

FIGS. 5-6 are schematic diagrams of shielded helical electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure.

FIG. 7 is a flow chart showing example steps of a method for electroporation, in accordance with embodiments of this disclosure.

FIGS. 8-11 present data associated with embodiments of this disclosure. The data and underlying experiments are discussed in the Examples section of this disclosure.

Description of Illustrative Embodiments

The description below is directed to specific embodiments, which serve as examples only. Description of these particular examples should not be imported into the claims as extra limitations because the claims themselves define the legal scope of the invention. With the benefit of the present disclosure, those having ordinary skill in the art will comprehend that techniques claimed and described here may be modified and applied to a number of additional, different applications, achieving the same or a similar result. The attached claims cover all such modifications that fall within the scope and spirit of this disclosure.

The techniques of this disclosure can be applied to many different types of systems, including any application involving electroporation, such as but not limited to flow electroporation. For example, shielding techniques of this disclosure can be applied to an electroporation apparatus, including a flow electroporation apparatus.

Electrodes create electrochemical products (metal ion release and gas formation) at the electrode surfaces. Some of these electrochemical products can be deleterious to cells, especially at elevated concentrations. As these electrochemical products are produced at the electrode surface they are most concentrated near the electrode surface, and cells that may be located near the electrodes will experience higher concentrations of these products. Electrochemical products do not only affect cells close to electrodes. They may also affect all cells in a chamber — especially if a sample collection procedure facilitates an even distribution of those products in an entire sample. Techniques of this disclosure address these and similar problems.

In addition, damage to cells near the electrode surface can result in the accumulation of cell debris at the electrode surface, which can negatively affect the performance of electroporation. In a flow electroporation system designed to load large volumes of cells, accumulation of debris can be an especially substantial problem. Formed debris occupies volume in a chamber, which reduces the chamber's effective volume for cells. The effective chamber volume can be an important factor for flow electroporation. Minimizing the effect of debris, as taught in this disclosure, can improve the efficiency of electroporation in terms of loading of cells and post electroporation viability regardless of the electrode material and may permit the use of less expensive electrode materials that would otherwise be unacceptable because of generation of damaging electrochemical products at the electrode surface.

Costs associated with electrode fabrication can represent a significant expense in the manufacture of electroporation chambers. Electrodes made of certain inexpensive materials, such as brass or iron, may provide for suitable electric fields and be inexpensive to make but may produce excessive levels of electrochemical products that damage or kill cells, thereby reducing the overall performance of the electroporation process. Even though certain materials such as gold, which produce less damaging electrochemical products (metal ion release), can be used to reduce the generation of damaging' electrochemical products at the electrode surface. However, first, such materials can add substantially to the cost of fabrication. Furthermore, such materials do not reduce the gas formation originated from electrochemical reaction and will generate cell debris especially when used repeatedly. Electrodes that can provide the benefits of electrode surfaces such as gold at lower cost and reduce the formation of cell debris are desired and can broaden the commercial application of electroporation. An electrode improvement that provides equivalent performance at lower cost or improved performance at the same cost is desired. Techniques of this disclosure may provide such improvements, among other things.

Electrodes preferably should provide low resistance to minimize generation of heat within the electrodes and to allow most of the voltage provided by an electrical power source to exist between the electrodes where the cells to be electroporated reside. To accomplish this, the material through which current passes should be of high conductivity and of a shape to reduce the overall resistance within the electrode.

When electroporation on cells is carried out, the individual cells in the sample being electroporated are located at different distances from the electrodes. Most electroporation apparatuses are designed to provide an approximately uniform electric field between the electrodes to provide the same electric field strength to all cells in suspension. Designs of this type provide an electric field such that substantially all cells experience a similar electrical environment. However, some cells will be closer to an electrode surface than others, and these cells will be exposed to higher concentrations of electrochemical products created at electrode surfaces than cells located farther from an electrode. A design that minimizes the exposure of cells to these deleterious electrochemical products can improve the overall performance of electroporation.

Where electroporation is carried out involving repeated use of the same electrodes with successive samples of cells, accumulation of undesirable products at the electrode surface can be especially problematic. A design that minimizes exposure of cells to these products is desired and is taught in this disclosure. As discussed below, embodiments of this disclosure block samples (e.g., cells) from electrodes during electroporation.

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 shielding 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, hi another embodiment, connection 106 is wireless, hi 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. In 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. In 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. In 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 shielded electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure. Electrode configuration 200 includes electrodes 202, gaskets 204, and barriers 206. In one embodiment, gaskets 204 may be 3 mm thick so that electrodes are separated from cells by a 3 mm gap, but in other embodiments, a different gap (>0 mm) may be used, if gaskets or other separators are used in this or a different configuration. Electrode configuration 200 may be in operative relation with any electroporation chamber within any electroporation apparatus, such as that shown in FIG. 1.

Electrodes 202 may be made from metallic or non-metallic conductors. In different embodiments, electrodes 202 may include iron, brass, gold, platinum, tantalum, or carbon (graphite, diamond). Those having ordinary skill in the art will recognize that other materials may also be used.

In one embodiment, barriers 206 may be porous. They may be conductive and water permeable. They may be made from a dialysis membrane although other materials may be used as will be appreciated by those having ordinary skill in the art with the benefit of this disclosure. For example, any material that acts to block samples from unwanted byproducts associated with electroporation electrodes while, at the same time, allowing sufficient electrical fields to be formed within the chamber to effect electroporation, may be used. Placement of barriers 206 is such that they substantially prevent a sample being electroporated from coming into contact with the electrode. In one embodiment, barriers 206 allow fluid flow through their bodies, while substantially not allowing pass-through of sample or byproducts from electrodes.

The size, number, and placement of barriers 206 may vary according to need. In the illustrated embodiment, barriers 206 are spaced apart from electrodes 202 by gaskets 204. In other embodiments, barriers may, e.g., encompass all or a portion of electrodes 202. Different barriers may be spaced differently than another or made from different materials. Porosity and electrical field permeability may vary, but in preferred embodiments, such variables should not be allowed to vary to a degree so as to detract from the electroporation process.

Barriers may be shaped differently according to need. In one embodiment, a barrier may be substantially circular when viewed in cross-section so that cells flow or reside within a substantially circular channel that resides within an electric field that is substantially uniform. Dialysis membranes may be used as a barrier, and they are commonly manufactured as cylindrical tubes, making them attractive from a manufacturing standpoint. More than one substantially cylindrical tube of barrier membrane may be placed in a single electric field between electrodes, and each may contain the same or a different cell type or compound to be loaded by electroporation. FIG. 3 is a schematic diagram of shielded electrodes, particularly well suited for flow electroporation, in accordance with embodiments of this disclosure. Electrode configuration 300 includes electrodes 302, gaskets 304, barriers 306, and sample holding gasket 308. Electrodes 302 have output ports 316 and 320. They have input ports 318 and 322. In one embodiment, the input/output ports may be in gasket 304, instead of in the electrodes. Sample holding gasket 308 includes air in/out port 310, sample in port 312, and sample out port 314. In one embodiment, gaskets 304 may be 1 mm thick, but in other embodiments, different thickness may be used, if gaskets are used in this or a different configuration. Electrode configuration 300 may be in operative relation with any electroporation chamber within any electroporation apparatus, such as that shown in FIG. 1.

Configuration 300 shares the same or similar barrier considerations as does configuration 200 shown in FIG. 2, so description of that system will not repeated. By way of the electrode- and sample-holding-gasket ports, configuration 300 of FIG. 3 is particularly well suited for any flow electroporation apparatus, such as continuous or intermittent flow systems. Such an apparatus may greatly benefit from having sample flow paths effectively insulated from potentially-harmful byproducts. For example, sample flow paths may be kept clean of, for significant amounts of time, any toxic byproducts from electrodes themselves or any debris that may accumulate near electrodes.

FIG. 4 is a schematic diagram of shielded electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure. FIG. 4 is a more generalized diagram, illustrating a configuration 400 including electrodes 402 and barriers 404. Arrow 404 represents flow of a sample in a flow electroporation apparatus or the sample loading direction in a static electroporation apparatus. FIG. 4 illustrates that the use of gaskets or spacers may not be necessary and that the size, positioning, and material of barriers may vary.

FIGS. 5-6 are schematic diagrams of shielded helical electrodes, well suited for static or flow electroporation, in accordance with embodiments of this disclosure. FIG. 5 is a schematic diagram of an electroporation chamber 500 including wire electrodes 502 and 504 positioned helically (in this embodiment as a double helix). Electrode spacing is represented by distances A and B and, in one embodiment, may be substantially equal. Barrier 506 is placed within the cylinder formed by the electrodes 502 and 504 to effectively insulate any sample within from potentially harmful byproducts, hi one embodiment, the barrier 506 may include dialysis membrane components, hi other embodiments, any material that does not disrupt electrical fields to a degree to prevent electroporation may be used. Any material may be used to also provide a desirable filtering or flow-through of particular components — e.g., a material that blocks byproducts while allowing, e.g., water to flow through.

FIG. 6 is a schematic diagram of an electroporation chamber 600 including electrodes positioned helically. As drawn the electrodes are positioned on the surface of the cylindrical chamber. The electrodes need not be at this surface and can be located away from the inside surface of the chamber, thereby allowing material to reside between the electrodes and the inside of the chamber. Cylindrical tubing may form the input and output of the chamber. Advantageously, this tubing 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 secure the helically positioned electrodes and may be conductors so as to convey an electrical signal, shown generically as a square pulse. Similar to barrier 506 described above, barrier 602 of FIG. 6 is placed within the helix and is configured to substantially block the sample flowing therein from coming into contact with the electrodes.

In the embodiments of FIGS. 5-6, or other embodiments, the barrier may be shaped as a cylindrical tube within which or through which cells may reside or flow. The cylindrical tube may be positioned between the electrodes, and desirably may be positioned so that it allows sample to occupy where the electric field is most uniform and optimal for electroporation or other treatment of the cells. Fluid may flow between the barrier and electrode, and such a fluid flow may help remove heat associated with electroporation or remove toxic materials.

FIG. 7 is a flow chart showing example steps of a method 700 for electroporation, in accordance with embodiments of this disclosure. In step 702, a sample is loaded into an electroporation chamber. The chamber may be part of a static or flow electroporation apparatus. In step 704, the sample is subjected to an electric field sufficient to effect electroporation. One or more electrodes are involved in the generation of the electric field, and those electrodes are typically placed within the electrode chamber (e.g., about opposite ends of the chamber) although different arrangements (e.g., helical electrodes) may be used. In step 706, the sample being electroporated is substantially prevented from coming into contact with the electrodes. In a preferred embodiment, this is done by using a barrier that is in operative relation with the electrode. For example, barriers such as those described in FIGS. 2-6 may be used.

Gel and Paper Embodiments

In alternative embodiments, gels and/or paper-like materials may be used as an effective barrier. For example, one may coat one or both electrodes with a gel containing an electroporation buffer. The gel may contain, but is not limited to, materials such as Agarose or polyacrylamide. Current flows through such a gel with about the same resistance as it does in the same un-gelled buffer, but cells are not able to penetrate the gel and therefore will get no closer to the electrode than the gel-fluid boundary. Since electrochemical byproducts would diffuse slowly through the gel, the gel protects the cells from those byproducts. In another alternative embodiment, one may use a material such as paper to overlay electrodes.

Mathematical Considerations

The analysis below addresses whether the addition of a permeable (e.g., micropore) membrane to an electrode surface would impede normal current flow and cause formation of a voltage drop across the membrane, thus decreasing the electric held applied to cells. As snown below, the electrical resistance of a porous membrane can be low enough, and therefore need not be a concern.

Consider two metal electrodes, each having the surface area A, separated by the distance d and the space between them is filled with saline with resistivity p. The resistance R_s of this saline will be

A porous membrane can be characterized by its thickness λ, average area (or diameter) of the pores and the number of pores per unit area of the membrane. Equivalently, the last two parameters can be combined into the fractional area F of the membrane surface that is occupied by pores. If the membrane is of the same size as an electrode, and its pores are filled with the same saline, its resistance R_1n is

In order not to cause a decrease in electric field between the electrodes, the membrane resistance should be much lower than the resistance of saline. If we require

that R '_n", = —

lo —o , then the above eq ^Huations y^Jield the relationship ^f: — _F = ₁₀₀ ,^> or

F = that should be satisfied. d

With realistic values of d = 1 cm and λ= 10 μm the membrane should have the fractional pore area of 0.1 (i.e. 10 percent of its surface is covered by pores), which is readily achievable. It is believed that such a membrane may provide at least a 10-fold decrease in the flux of chemical species from the electrode surface into the bulk of saline. A thicker membrane with the same fractional pore area will have higher electrical resistance while giving the same protective effect. Therefore, in preferred embodiments one may use thinner membranes with lower pore size and density, although the invention is not so-limited. The following examples are included to demonstrate aspects of specific experiments related to this disclosure. FIGS. 8-11 present data associated with embodiments of this disclosure and are explained in the following examples. Subject matter presented as an example may be encompassed by the present claims or added to the claims to define protected subject matter.

Examples Inventors have performed experiments associated with embodiments of the present disclosure, which are discussed here. Experimental Procedures:

Inventors assembled more than one chamber as illustrated in FIG. 2 with gold-coated electrodes and naked-iron electrodes, hi the experiment, each of the chambers was used as a static chamber. Three stacked silicon gaskets being 3 mm- thick each (about total 9 mm in electrode gap after assembly) were used.

The electroporation area was divided into three parts by using two water-and- inorganic-ion porous membranes (dialysis bag membrane, 3.5k MW cut off): (1) the area close to positive electrode side, (X) the area close to negative electrode side, and (3) the area away from both electrodes. These areas may be referred to as positive or +, negative or -, and middle areas, respectively. Cell samples (Jurkat cells) from the same cell/DNA suspension were loaded into an individual area before electroporation and collected separately after electroporation.

For flow electroporation, inventors assembled a chamber as illustrated in FIG. 3. K562 cells, separated from electrodes with the same dialysis bag membranes mentioned above by about 1 mm distance, were processed. Transfected cells were collected for each two cycles. During process, total cell volume of 45 mL cells (4e7 cells/mL) was divided into 3 parts, 15 mL each. The first 15 mL was processed and collected for each fraction. The second 15 mL of cells was processed and all fractions were collected together. Immediately after the finishing of the total 15 ml sample processing, the 15 ml sample was processed again. In this way, this 15 ml sample was processed for a total of 4 times (4 x 15 mL = 60 mL was processed in total by the chamber for this 15 ml sample) and the cell fraction was not analyzed because of over processing. The final 15 mL of cells was processed and collected for each fraction, the same as the one for the first 15 mL of cells. There was little delay of time between the process of the first and the second and the third 15 mL of cells. Taken together, the total processed cell volume by the chamber was 90 mL. The maximal chamber volume capacity was approximately 1 - 1.2 mL, and at each cycle 0.75 mL was processed. During the whole processing period, electroporation buffer continuously flowing through input/output ports (see FIG. 3, elements 320 and 322) to make the electric contact. Cells were processed using standard variable flow protocol. Briefly, cells were processed 0.5 s after being inside the chamber and collected 0.5 s post processing with 1 ml/s flow rate and about 1 kV/cm, 400 us pulse width, 4 pulses with 0.5s pulse interval.

The cells were transfected with pTM2 at 100 μg/mL (DO 106) for static mode and 40 μg /mL for flow mode. Cells were processed at lkV/cm, 400 μs, 4 pulses. The transfected cells were seeded at 3e5 cells into 24 well plates with 1 mL/well, and were stained with propidium iodide prior to FACS analysis at 24 hr post transfection to distinguish live cells from dead cells and to enumerate each. Student's T test with paired 2 tailed distribution was used for the statistic analysis.

Results:

FIG. 8 shows the effect of electroporation ("EP") area on transfection efficiency of Jurkat cells. Jurkat cells were transfected in the chamber described in FIG. 2. EP areas were created by using porous membranes. Iron electrodes or gold- coated electrodes were used. After the standard transfection, the cells were analyzed at 24h post transfection. For the chamber with iron electrodes, negative (-) and middle areas resulted in high cell viability (70-80%) and transfection efficiency (60-70%), without significant difference. However, the positive area gave rise to significantly lower cell viability (10%) and transfection efficiency (2%) (pO.OOl). For the chamber with gold-coated electrodes, all three areas resulted in similar high cell viability (70-80%) and transfection efficiency (60-70%). Toxic effects of iron electrodes appear to mostly, or only, affect the cells located between the barrier and the positive electrode. The porous membrane successfully shielded the cells in the area between the barriers and between the barrier and the negative electrode. These results were in triplicate experiments.

FIG. 9 shows the effect of EP area on transfection efficiency of Jurkat cells. The mean fluorescence intensity of the transfected Jurkat cells from the experiment described in FIG. 8 is plotted in FIG. 9. When iron electrodes are used, the cells close to the anode electrode give rise to lower transgene expression.

FIG. 10 shows the effect of the chamber of FIG. 3 on large volume variable flow transfection. K562 cells were transfected using standard variable flow protocol. 1 - 1.2 mL cell volume holding gasket was used. This cell hold gasket is separated from electrodes by two, 1 mm thick gaskets sandwiched with two porous membranes, as shown in FIG. 3. AU cells were processed as described above. The first 15 mL was processed once and collected every two cycles together for analysis. The second 15 mL was processed 4 times and the collections were not analyzed because of over processing. The last 15 mL fresh cells were processed once again and collected every two cycles together for analysis. Therefore, the chamber processed a total of 90 ml cell volume for this experiment, and performed 120 cycles of electroporation. The static electroporation was conducted in duplicates before the flow electroporation experiment and in triplicates after the flow process to examine incubation time effect on cell/DNA mixture. No significant variability on cell viability and transfection efficiency was observed for the whole process, up to 120 cycle process.

The percentage of the GFP positive cells in variable flow EP was approximately 10% lower than that in static EP. Such slightly lower loading is commonly but not always observed when carrying out flow EP compared to static EP. Without being bound by theory, the 10% lower percentage may be because (a) a not- exactly-matched electric field was used for static and variable flow, or (b) a slightly lower electric field caused by the porous membranes (while not low enough to prevent electroporation). Either reason, however, can be compensated readily and easily, as understood by those of ordinary skill in the art having the benefit of this disclosure.

FIG. 11 shows the effect of the chamber of FIG. 3 on large volume variable flow transfection. Comparison of transfection efficiency of the transfected K562 cells from large volume EP (pooled together, flow) and static EP (Static). No significant difference was observed from these two samples.

Discussion:

In these examples, inventors demonstrate that electroporation chambers can block mammalian cells from electrodes via barrier dialysis membranes (3500 MW cut off). This, among other things, can reduce or substantially prevent the toxic effect of electrochemical products generated from iron electrodes during electroporating Jurkat cells in a static mode. This can also lead to the processing of K562 cells repeatedly using the same electrodes for at least 120 cycles without compromising cell viability and transfection efficiency in a variable flow electroporation mode.

Testing electroporation chambers for large volume transfection may be time, labor, and cost intensive. In the experiments of these examples, inventors used a strategy to process 1/3 of the cell volume {e.g. 15 mL) multiple times in the middle of the whole process, which provided an equivalent, much-larger processed cell volume.

By doing this, one can cut down the required cell numbers but achieve large volume results. This method is considered being more stringent than directly processing exact, large volumes because electroporating the same cells multiple times (4 times for this experiment) should lead to more dead cells resulting in more detrimental materials from dead cells. Therefore, it is fair to conclude that the system works for a large volume when it passes this process.

The advantage of blocking cells' direct contact with electrodes is significant.

The toxicity of, e.g., an anode electrode can be substantially prevented resulting high transfection efficiency while maintaining high cell viability. By this configuration, high cost electrodes made of gold or other expensive materials can be replaced by low cost electrodes without substantially reducing transfection efficiency or cell viability.

Previously, it was shown processing K562 cells by a CL-2 chamber, the maximum electroporation cycles were 28 before the transfection efficiency declined.

Here, inventors demonstrate no significant changes up to 120 cycles. The chamber fill in volume for this experiment was 0.75 mL. If the fill in volume for each cycle is

3.5 mL as it is shown for CL-2 chamber or maybe higher (a CL-2 chamber holds 4.5 mL, but because of the accumulated cell debris during process, the fill up volume is set at 3.5mL), chambers designed using the techniques of this invention can process upwards of 420 mL of cells.

* * *

With the benefit of the present disclosure, those having ordinary skill in the art will recognize 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, barriers may be used in virtually any size and/or shape including but not limited to cylindrical or rectangular shapes of a size suitable to sufficiently block an electrode. Additionally, barriers may placed at different locations relative to a chamber to achieve different flow patterns. One example flow pattern may involve different flow paths with liquid flowing at different rates or even different directions. For example, a barrier may, alone or with other components, define an inner flow path and an outer flow path having different flow characteristics.

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:

1. U.S. Provisional Patent Application Serial No. 60/707,655 entitled, "Electrode Geometries for Electroporation" with inventors Sergey M. Dzekunov and Nicolas J. Chopas, filed August 12, 2005.

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

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

4. PCT Publication No. WO 03/018751

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

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

7. U.S. Patent No. 6,074,605

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

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

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

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

Claims

1. An apparatus for electrically treating cells comprising: a chamber; an electrode in operative relation with the chamber; and a conductive and water permeable barrier in operative relation with the electrode, the barrier configured to substantially prevent cells in a sample being treated within the chamber from coming into contact with the electrode during use.

2. The apparatus of claim 1, the barrier configured to prevent over 90% of the sample from coming into contact with the electrode.

3. The apparatus of claim 1, where treating comprises electroporating and where the chamber is an electroporation chamber.

4. The apparatus of claim 3, the electroporation chamber comprising a flow electroporation chamber.

5. The apparatus of claim 4, the flow electroporation chamber comprising an input and output.

6. The apparatus of claim 1, comprising two electrodes and two barriers.

7. The apparatus of claim 1, comprising two electrodes and one barrier.

8. The apparatus of claim 7, the one barrier being in operative relation with a positive side of an electrode.

9. The apparatus of claim 1, the electrode comprising iron or brass.

10. The apparatus of claim 1, the electrode being part of a helical arrangement.

11. The apparatus of claim 1, the barrier comprising a dialysis membrane.

12. The apparatus of claim 1, the barrier surrounding a portion of the electrode.

13. The apparatus of claim 1, the barrier being spaced from the electrode.

14. The apparatus of claim 1, the barrier being spaced from the electrode by one or more gaskets.

15. A method for electrically treating a sample, the method comprising electroporating the sample with the apparatus of claim 1.

16. The method of claim 15., the sample comprising cells.

17. The method of claim 16, the cells comprising human cells.

18. A method comprising: loading cells into a chamber; subjecting the cells to an electric field sufficient to treat the cells using an electrode that is in operative relation with the chamber; and substantially preventing the cells being treated from coming into contact with the electrode during use with a barrier that is in operative relation with the electrode.

19. The method of claim 18, where treating the cells comprises electroporating the cells.

20. The method of claim 19, where the chamber comprises a flow electroporation chamber and where loading the cells comprises flowing the cells through the flow electroporation chamber.

21. The method of claim 18, the electrode comprising iron or brass.

22. The method of claim 18, the barrier comprising a dialysis membrane.

23. A method for reducing electroporation byproducts in an electroporation chamber, the method comprising forming a conductive and water permeable barrier around an area of an electrode within the chamber, the barrier being sufficient to substantially prevent electroporation byproduct material from leaking into an aqueous space within the chamber for processing samples.

24. The method of claim 23, the byproduct material comprising electrode material or cell debris.

25. The method of claim 24, the cell debris comprising DNA.

26. A method for electroporation comprising: subjecting cycles of cells to an electric field sufficient to effect electroporation; and forming a conductive and water permeable barrier around areas of electrodes such that the electrodes are capable of repeatedly processing more than

30 cycles without a substantial lessening of cell viability or transfection efficiency.

27. The method of claim 26, where the cells comprise Jurkat cells.

28. The method of claim 26, where the electrodes are capable of repeatedly processing more than 50 cycles without a substantial lessening of cell viability or transfection efficiency.

29. The method of claim 26, where the electrodes are capable of repeatedly processing more than 75 cycles without a substantial lessening of cell viability or transfection efficiency.

30. The method of claim 26, where the electrodes are capable of repeatedly processing more than 100 cycles without a substantial lessening of cell viability or transfection efficiency.

31. The method of claim 26, where the electrodes are capable of repeatedly processing at least 120 cycles without a substantial lessening of cell viability or transfection efficiency.

32. A method for electroporation comprising: subjecting flowing cells to an electric field sufficient to effect electroporation; and forming a conductive and water permeable barrier around areas of electrodes such that the electrodes are capable of processing at least 420 mL of cells without a substantial lessening of cell viability or transfection efficiency.

33. The method of claim 32, where the cells comprise K562 cells.

34. A method for electroporation comprising steps for isolating an electrode from a sample during electroporation to substantially prevent electroporation byproduct material from leaking into an aqueous space within a chamber for processing samples.