WO2006112870A1 - Device and method for controlled electroporation and molecular delivery in cells and tissue - Google Patents

Abstract

An electroporative device and method for directing electrical current through biological cells is provided, the current flow substantially being required to traverse the cells, and not being able to bypass through a surrounding conductive medium. The invention provides a substantially controlled electric field imposed across the subject cell population at level that is optimal for the electroporative transfer of erogenous molecules into the cell and for survival of the cell population. Optimal levels are determined by monitoring of current flow as a function of voltage to determine the threshold voltage level, and feedback control of electrical parameters permits automation of protocols, tailored in real time to characteristics of the cells, that control the magnitude of the applied electric field. The invention thereby provides for high electroporative efficiency, a uniform level of electroporation throughout the subject cell population, and a high survival rate through the electroporation procedure even for large populations of cells processed simultaneously. The method and apparatus are suitable to a wide variety of cell types, including cultured cells as well as biopsy samples and primary cells derived therefrom, as well as tissue slices. The invention is scalable, compatible with standard cell culture materials and handling equipment, and appropriate both for small scale applications, as in diagnostic or individualized medical procedures, as well as high volume and high throughput industrial, cellular engineering processes.

Description

UNITED STATES PCT PATENT APPLICATION

DEVICE AND METHOD FOR CONTROLLED ELECTROPORATION AND MOLECULAR DELIVERY IN CELLS AND TISSUE

Inventors: YONG HUANG, a citizen of the Peoples Republic of China, residing at: 715 Towhee Court Fremont, California 94539

JIM BORNINSKI, a citizen of the United States of America, residing at:

1612 Walnut St, Apt. 2 S Berkeley, California 94709

LAURA T. MAZZOLA, a citizen of the United States of America, residing at:

2439 Whitney Court

Mountain View, California 94043

Assignee: Excellin Life Sciences, Inc.

1455 Adams Drive, Suite 2050 Menlo Park, CA 94025

TABLE OF CONTENTS

UNITED STATES PATENT APPLICATION 1

DEVICE AND METHOD FOR CONTROLLED ELECTROPORATION AND MOLECULAR DELIVERY IN

CELLS AND TISSUE 1

Table of Contents 2

DEVICE AND METHOD FOR CONTROLLED ELECTROPORATION AND MOLECULAR DELIVERY

INTO CELLS AND TISSUE 3

CROSS-REFERENCE TO RELATED APPLICATIONS 3

FIELD OF THE INVENTION 3

BACKGROUND OF THE INVENTION 3

SUMMARY OF THE INVENTION 4

BRIEF DESCRIPTION OF THE DRAWINGS 7

DETAILED DESCRIPTION OF THE INVENTION 10

1. Various Conventions and Terms 10

2. Electroporation device configuration and operation 1 1

3. Methods for blocking pores on a porous membrane with cells 14

4. Intrinsic cell membrane potential 16

5. Transepithelial/transendothelial impedance measurement 16

6. Methods for using feedback for controlled electroporation 16

7. Description of electrical pulses 18

8. Device for controlled electroporation in tissue 20

9. Configuration, fabrication, and methods of operation of a modular electroporative cartridge 20 a. Configuration and fabrication of a modular electroporative cartridge 20 b. Principles of the operation of modular electroporative cartridge 25 c. Use of the modular electroporative cartridge for preventing culture contamination 27 d. Methods for blocking pores on a porous membrane with cells 28 i. Blocking pores through cell growth 28 ii. Blocking pores with mechanical methods 28 e. Use of the modular electroporative cartridge for controlled electroporation of tissue 31 f. Processing multiple electroporative cartridges 32 g. Correcting for electric field non-uniformity 33 h. Cell layer visualization 33 i. Bubble prevention 34 j. Embodiments of the membrane 34

10. Examples 35 a. Materials 35 b. Electrical parameters study 35 c. Transfection study 36 d. Electroporation electrical measurement results 36 e. Electroporation efficiency and cell viability assessment 37 f. Gene transfection 38 g. Transfection of siRNA 39 h. Transfection of peptides and protein 39 i. Comparison of the inventive controlled electroporation method with conventional methods 39

CLAIMS 41

DEVICE AND METHOD FOR CONTROLLED ELECTROPORATION AND MOLECULAR DELIVERY IN

CELLS AND TISSUE 44

ABSTRACT 44 DEVICE AND METHOD FOR CONTROLLED ELECTROPORATION AND MOLECULAR DELIVERY INTO CELLS AND TISSUE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Patent Application No. 11/007661 filed on

December 7, 2004, which claims priority to provisional U.S. Patent Application No. 60/528147 of Huang et al. filed on December 8, 2003. The present application additionally claims priority to U.S. Provisional Patent Application No. 60/563155, filed on April 19, 2004. All of the aforementioned patent applications are incorporated in their entirety herein by this reference.

FIELD OF THE INVENTION

[0002] This invention is related to the field of cell electroporation and molecular delivery in general, which specific reference to controlling electroporation in biological and synthetic cells, tissue, and lipid vesicles.

BACKGROUND OF THE INVENTION

[0003] Exposing the cells to an applied electric potential that traverses the cell membrane has various effects on the cell membranes that promote cell-cell fusion and the permeabilization of the lipid membrane to molecules that otherwise would not be able to pass through. The membrane permeabilization, while still not fully understood in detail, is considered to involve the creation of transient pores, as indicated by the term electroporation, and such pores allow the passage of molecules that otherwise are not able to traverse the membrane. Typically, an electric potential is applied in pulses, and whether the pore formation is reversible or irreversible depends on such parameters as the amplitude, length, shape and repetition rate of the pulses, in addition to the variables associated with the types of cells, and their stage of development, overall health, and/or position in the cell cycle. Electroporation is conventionally performed by placing one or more cells, in suspension or in tissue, between two electrodes connected to a generator that emits pulses of a high-voltage electric field, for example as commonly delivered through capacitive discharge. Electroporation as a transfecting technique, whereby exogenous molecules are introduced into cells, has wide application in experimental biology, and in the more practically directed and larger scale applications of interest to the biotechnology and pharmaceutical industries.

[0004] A number of variables inherent to conventional electroporation of cells, commonly done on cells in suspension, present problems in attaining high transfection efficiency, high survival rates and general predictability in the outcome of procedures. First, the voltage differential as experienced by the cell, and consequent current flow, have a certain optimal level for success. Voltage that is too low is ineffective in promoting permeabilization; voltage that is too high damages the membrane to the point of irreversibility, significantly damaging or killing the cell. Further, the current as it travels through solution is not homogeneous throughout the solution, and thus cells within a population, while subjected broadly to the same voltage as averaged throughout the chamber volume, in fact, individually experience a wide range in the level of traversing current. Generally, electroporation procedures that are considered successful in contemporary terms, use high voltage differentials, and depend on the selection of appropriate surviving transfected cells - to obtain the desired useful cell populations. [0005] A series ot U.S. Patents to Rubinsky and Huang, including U.S. Patent No. 6,482,619, entitled

"Cell/tissue analysis via controlled electroporation", published November 19, 2002, U.S. Patent No. 6,300,108, entitled "Controlled electroporation and mass transfer across cell membranes" published October 9, 2001 , and U.S. Patent No. 6,562,604, entitled "Controlled electroporation and mass transfer across cell membranes", published May 13, 2003, have described an electroporation approach that allows the isolation of a single cell stably held with a physical support, such that the cell represents the only available path for an electroporative current. These patents are hereby incorporated into this patent application, in their entirety, by this reference. This approach of Rubinsky and Huang offers the benefit of a well controlled delivery of a voltage within a range that is high enough to permit a high degree of successful electroporation, but low enough that the electroporation is reversible and the cell survival rate is high.

[0006] Significant challenges remain, however, in the development of equipment and procedures that would permit the application of optimal voltages, specifically tunable to subject cell populations in real time, applicable to large numbers of cells in a high-throughput manner, and with uniformity in terms of the level of applied field to which individual cells within the population are exposed. Further developments in the technology along these lines would be desirable for their application to industrial scale electroporation technology.

SUMMARY OF THE INVENTION

[0007] In the present invention, a device and method for directing or "focusing" the electrical current through biological cells is provided, the current flow substantially being required to traverse the cells, and not being able to bypass through a surrounding conductive medium. One of the effects of current traversing cells is a process of electroporation, in which the normally impermeable cell membrane becomes permeable, allowing for influx of exogenous molecules into the interior of the cell and/or efflux of materials from the cell interior. Another consequence, under the appropriate conditions, of current traversing cells is the promotion of fusion of neighboring biological cells. As provided by the invention, the voltage applied to the cells is tuned to an effective threshold, a specified minimally prescribed level to achieve electroporation. Such optimal voltage, generally far lower than that used by conventional electroporation procedures, is sufficient to achieve high efficiency membrane permeabilization, but is well below levels that would make the membrane permeabilization irreversible, thereby damaging or killing the subject cell. The use of this optimal voltage level, or "tuning" of the electrical field, is permitted by the ability of the invention to detect the initiation of electroporation of cells through the electronic feedback mechanisms, and to impose a controlled or substantially controlled electric field across the subject cells in real time, throughout the process, as further described below.

[0008] The invention provides for a means of substantially limiting the electrical current to a path through biological cells. Typically a porous structure is situated in the electrical flowpath, generally a porous structure to which cells can be made to cover, thereby creating a current flow path across substantially only the biological material. Such biological material may consist of a plurality of cells from a cell culture environment, primary cells extracted directly from a living body, or cells comprising a native form as exemplified by tissue slices.

[0009] The combination of an electrical cell that inescapably includes within its electrical flowpath a portion that consists strictly or substantially of a biological cell, and the ability, as provided by the invention, to control or tune the voltage level to an optimal level, appropriate to the subject cells in real time, provides for the generation of a suDstantiauy conironeα eieciricai πeiα across ihe biological cells. For example, electrodes can be designed to ensure that substantially all of the cells experience a substantially uniform electric field, thus promoting substantially uniform electroporation across the cell layer. Alternatively, electrodes can also be designed for controlled non-uniformity of the applied electric field, as in the case where a voltage gradient across the cell layer is desired. Such control of electrical field exposure supports a uniformity of the electroporative process experienced by the constituent individual cells within a larger exposed cell population. A uniform electroporation provides for a high degree of uniformity with regard to the penetration of transfecting molecules or particles into the biological cells, thereby creating a substantially homogeneously transfected cell population.

[0010] As provided by the invention, biological cells are an exemplary electroporative target, but non-living constructs or artificial cells, or any of a variety of forms of lipid vesicles or micelles with aqueous compartments are also included within the scope of the invention. Biological cells of any kind, prokaryotic or eukaryotic are included within the scope of the invention. Biological cells may be from cell culture environments, and may include cell populations that grow in suspension or grow attached to physical substrates to varying degree. Such cells may grow in vitro as isolated single cells, or they may grow as aggregates or clumps, or as organized spheroids.

[0011] Further, biological cells may be derived from primary sources such as blood or tissue samples, such as biopsies from patients or experimental subjects, or tissue slices, or from primary cells in culture derived from such sources. The invention is particularly well suited to primary cells, as they are valuable for being rare or irreplaceable, generally delicate, individualized in nature, and uncharacterized in comparison to cell populations from well established continuously growing cell lines. The tunable feature of the present invention permits very efficient use of limited quantities of such cells, which could otherwise be ineffectively electroporated, damaged or killed, if subjected to high and inappropriate voltages, non-homogeneous electrical fields, as occurs with conventional electroporation processes.

[0012] The control of the electrical pathway allows, as provided by the invention, the ability to measure the current flow accurately and ascribe such flow to the characteristic impedance of the cell. Further, by knowing the current flow as a function of applied voltage, the invention provides a way to control the voltage, and consequently the rate of current flow through the cells. Control of the magnitude of the applied voltage is important; while cell membranes require a threshold level of voltage in order to achieve electroporation, an excessive voltage can damage and kill cells as they are not able to recover from electroporation and its consequences. Furthermore, controlled applied voltages enable a more homogeneous and reproducible process of electroporation - enabling controllably robust and high efficiency transfection while maintaining high cell viability. Thus, the present invention provides for a controlled applied voltage across the cells, and for a control over process to one that is optimal for an electroporative procedure as well as recovery from it. Thus, the invention provides for a universal approach to a wide variety of cells, and because of the efficiency, simultaneity, homogeneity, and survivability it provides, a data-density in results and quickness of their availability all converge to create time, volume, and cost efficiencies.

[0013] The invention provides for various methods to create the biological wall through which current must pass. For example, subject cells grown in vitro, may be grown to confluence such that they substantially cover the porous sπppoπ enureiy. iτ coniiuence is not attainable or desirable in the cell culture context, exposed portions of the porous support through which electrical current could pass, as provided by the invention, are covered by non- conductive particles or materials. If subject cells grow in suspension, or if they do not naturally attach well or adhere to the porous support, as provided by the invention, they may be held against the porous support by a pressure differential such as centrifugation, or by other directly applied physical force.

[0014] As provided by the invention, the material comprising the porous support upon which subject cells rest or attach is non-conducting, impermeable to electrical current. Current, of course, can pass through the pores of the support. The pores within the porous support are generally smaller than the diameter of cells growing on the support, such that the pores are covered by the cells, thereby creating a substantially effective electrically resistive seal. On the whole, thus, in a confluent cell culture, for example, where the substantial majority of the pores are covered by biological material, thus creating an effective biological barrier through which current must pass. Various approaches to achieving substantial cellular coverage of pores are described, including adhesion, affinity immobilization (the use of biological recognition sites to adhere cells to a surface, for example antibody or protein immobilization), and the application of a pressure differential. As recited above, should biological coverage of the electrically permeable pores be insufficient, as would, for example be the case in a sub-confluent culture, methods for plugging open pores are provided.

[0015] The inventive electroporative apparatus comprises an electrical chamber, in which the electroporative process occurs. Additionally, the invention comprises a power source, a computer comprising hardware and software, and electronic components that monitor a first electrical parameter, such as voltage, current, or resistance within the electrical cell, as well as the capability to regulate a second parameter, such voltage or current in response to the monitored values of the first electrical parameter. Through such real time feedback control, the invention provides for the ability to evaluate or gauge the effectiveness of the resistive seal provided by cells on the porous support, as well as to achieve a minimum effective threshold voltage required for electroporation. Thus, through the use of control algorithms and protocols that determine the amplitude as well as duration, and sequence, of voltage pulses, electroporation processes can be optimized. Electroporation processes can be automated and controlled entirely by the software, enabling "intelligent" control through an empirical process that can be controlled by the software or exerted electively by the operator. Further, artificial intelligence procedures may be implemented, whereby the software-controlled protocols incorporate experience learned in previous procedures, and make according adjustments.

[0016] In terms of the electroporative method provided by this invention, it can be described as steps that include imposing a voltage across an electrical cell containing a porous membrane that has been populated with biological cells associated with the membrane such that the cells and membrane form a substantially resistive barrier to current flow, thus restricting the electric current flow within the electric cell to a flowpath that includes the biological cells and substantially without a route that provides a bypassing of the cells; monitoring the value of current, voltage or electrical impedance across the electrical cell; regulating the current, voltage or a combination of current and voltage in response to the monitored value; imposing a substantially controlled electric field across the biological cells; monitoring the electric current flow through the electric cell to evaluate the permeability of the biological cells at a voltage that is below the threshold for electroporation; and modifying the applied voltage to the minimal level sufficient to achieve electroporation. The method is supported by computer hardware and software, appropriate 'Algorithms ^'wϊth'irt^' thβ'Softwarer, artd a software interface. The method further provides that such steps may be initiated by a single click at the computer software interface.

[0017] Included within embodiments of the device and method is a modular electroporative cartridge that provides economic benefit and scale up efficiency. The electroporative cartridge embodiments are forms of the invention's electrical cell or electroporative chamber that are particularly adapted for cell culture processes such as a sterile, single-use consumable cartridge. As such, these embodiments may be sterilely wrapped, and of dimensions that are compatible with standard cell culture equipment formats for culturing and handling.

[0018] These and various other aspects, features and embodiments of the present invention are further described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure Ia is a schematic illustration of the electroporation apparatus design and electronics configuration.

[0020] Figure Ib is the system diagram of the feedback control electronics of the electroporation apparatus.

[0021] Figures Ic - Ie depict various configurations of loop-gain adjustment circuits of the electroporation apparatus.

[0022] Figure 2 illustrates methods for blocking pores on a porous membrane by Fig. 2a) forming a confluent cell layer by growing adherent cells on the porous membrane and Fig. 2b) forcing suspended cells/lipid vesicles to block the pores with pressure field.

[0023] Figure 3 shows a method for blocking pores that are not covered by cells with non-conductive particles using pressure Fig. 3a) before suction pressure is applied and Fig. 3b) when suction pressure is applied.

[0024] Figure 4 (collectively Figures 4a - 4d) depicts waveforms of various electroporation pulses: Fig. 4a) single step pulse, Fig. 4b) three-step pulse, Fig. 4c) four-step pulse, Fig. 4d) sinousoid pulse, and Fig. 4e) sinusoid- superpositioned step pulse.

[0025] Figure 5 is a schematic of controlling electroporation in tissue slice with a four-electrode electroporation apparatus.

[0026] Figure 6a is a cross-sectional schematic of a modular electroporative cartridge with a bottom electrode assembly attached to the chamber body. Figure 6b is the schematic of the device with the bottom electrode attached to the membrane. Figure 6c is the schematic with the top unit and bottom chamber molded around the top and bottom electrode assemblies respectively. Herein, Figures 6a, 6b, and 6c may be collectively referred to as Figure 6.

[0027] Figure 7 represents the electrode assembly of the modular cartridge realized as a flexible printed circuit. [OO.i^'8] Figure 8 is a cross^'-sectiόnarsch'ematic of the modular cartridge depicting an alternate connection method.

[0029] Figure 9a represents the electrode assembly of the modular cartridge realized as a flexible printed circuit. Figure 9b represents the flexible printed circuit to contact an electrode on the membrane. Herein, Figures 9a and 9b may be collectively referred to as Figure 9.

[0030] Figure 10 represents an alternative electrode geometry.

[0031] Figure 11 represents a rigid printed circuit board electrode.

[0032] Figure 12 depicts the measurement and control circuitry.

[0033] Figure 13 illustrates an embodiment for applying pressure difference by providing a seal between the top unit and the cell insert, and applying a positive pressure through one or more small holes in the top unit. The seals are realized through the use of O-rings.

[0034] Figure 14 illustrates a method to provide a seal between the bottom chamber and the cell insert, and a negative pressure is applied through one or more small holes in the bottom chamber. The seals are realized through the use of o-rings.

[0035] Figure 15a is a schematic representation of mechanical technique for the application of a pressure differential using a disc. Figure 15b represents the application of a pressure differential using the top electrode assembly as the disc. Herein, Figures 15a and 15b may be collectively referred to as Figure 15.

[0036] Figure 16 represents a flow cartridge for applying a pressure differential to immobilize cells.

[0037] Figure 17 represents a multiplexing method where each of the electronics connectors is connected to four multiplexers. The multiplexers simultaneously select inputs from the same device, thus connecting only that device to the measurement and control electronics.

[0038] Figure 18a depicts a side view of a realization of a multiple cell layer device that is compatible with the standard 6-well plate format for cell inserts. Figure 18b depicts the embedding of electrode assemblies in the top unit and bottom chamber, and the cell inserts have been shown as individual units. Herein, Figures 18a and 18b may be collectively referred to as Figure 18.

[0039] Figure 19 illustrates a plurality electrode assemblies embodied as flexible printed circuits.

[0040] Figure 20 illustrates the use of a plurality of concentric top electroporation electrodes along with a plurality of top probe electrodes.

[0041] Figure 21a depicts a microscopy-compatible embodiment which accommodates the use of opaque Ag/AgCl electrodes. Figure 21b depicts grid or mesh electrodes. Herein, Figures 21a and 21b may be collectively referred to as Figure 21. [0042J Figure 221^'depϊcts ^"ϊh^'e bottom electrode assembly realized as a flexible printed circuit.

[0043] Figure 23 illustrates a means for allowing bubbles to escape of their own accord by making the top electrode assembly "convex."

[0044] Figure 24 illustrates a flexible printed circuit electrode for use on a convex top electrode assembly.

[0045] Figure 25 illustrates a typical three-step electroporation pulse used to measure electrical resistance of cells before, during and after electroporation.

[0046] Figure 26 contains the electrical responses of fibroblast cells grown on a porous membrane under three- step electroporation pulses: Fig. 26a) the first pulse and Fig. 26b) second pulse applied 1 minute later.

[0047] Figure 27 shows the electrical responses of MDCK cells grown on a porous membrane under a four-step electroporation pulse: Fig. 27a) the first pulse and Fig. 27b) second pulse applied 1 minute later.

[0048] Figure 28 illustrates the electrical responses of mouse liver tissue slice under three-step electroporation pulses: Fig. 28a) fresh liver tissue, 2.0 mm thick sample, Fig. 28b) fresh liver tissue, 2.5 mm thick sample and Fig. 28c) dead liver tissue, 2.5mm thick.

[0049] Figure 29 is a fluorescent image of electroporated MDCK cells (Madin-Darby Canine Kidney cell line) electroporated with PI (Propidium Iodide) (transfection efficiency >90%).

[0050] Figure 30 is a fluorescent image of MDCK cells stained with PI dye after electroporation, showing virtually no cell death induced by the controlled electroporation (cell viability >95%).

[0051] Figure 31 is a fluorescent image of electroporated differentiated MDCK monolayer expressing GFP (Green Fluorescent Protein) reporter gene (expression efficiency >95%).

[0052] Figure 32 is a fluorescent image of electroporated satellite stem cells (human adult cardiac cells) expressing GFP reporter gene (expression efficiency >95%).

[0053] Figure 33 is a fluorescent image of electroporated primary mouse fibroblast cells expressing GFP reporter gene (expression efficiency >90%).

[0054] Figure 34 contains images of Fig. 34a) fibroblast cells before transfection and Fig. 34b) Myotube cells by transfection of fibroblast cells with MyoD gene (~40Kb).

[0055] Figure 35 demonstrates simultaneous co-transfection of CHO cells using GFP and DsRed reporter genes. Figure 35A is the fluorescent image of only the expressed GFP; Figure 35B is the fluorescent image of only the expressed DsRed. The overlaid image of Figure 35C indicates nearly total co-transfection efficiency (co- transfection efficiency >90%).

[0056] Figure 36 demonstrates the effect of siRNA transfection upon induced apoptosis in H460 cells (human lung cancer cell line). Figure 36 A is a fluorescent image of H460 cells after transfection with fluorescenated siRN'A (FTTC-siRNAj

95%) using the invented apparatus and methods described. Figure 36B is a western blot demonstrating siRNA efficiency in protein knockdown compared to a siRNA control. Figure 36C compares flow cytometry data for the H460 for this inventive apparatus compared to siRNA delivery using conventional chemical transfection (lipofection).

[0057] Figure 37 demonstrates peptide and protein delivery. Figure 37A is a fluorescent image of CHO cells (canine hamster ovary cell line) after transfection with fluorescenated anti-mouse antibody (FlTC-Ab); (transfection efficiency >85%). Figure 37B is a fluorescent image of Huh7 cells (hepatocyte-derived cell line) after transfection with fluorescenated peptide (rhodamine-peptide); (transfection efficiency >90%).

[0058] Figure 38 is a bubble chart comparison of controlled electroporation performance (upper right corner) to conventional electroporation (horizontal stripes) and lipofection (vertical stripes). Bubble diameter indicated the overall efficiency of the process (delivery x viability).

DETAILED DESCRIPTION OF THE INVENTION 1. Various Conventions and Terms

[0059] In the description of the invention herein, unless implicitly or explicitly understood or stated otherwise, it will be understood (1) that the meaning of a words appearing as either singular or plural encompass the respective counterpart, (2) that for any given component described herein, any of the possible candidates or alternatives listed for that component, may generally be used individually or in combination with one another, and (3) that any list of such candidates or alternatives, is merely illustrative, not limiting. It will be understood that the meaning of the verb "to include", in every instance, means "to include but not to limit to", or words to that effect, per the meaning in standard English. Various terms shown in quotation marks immediately below are described to facilitate an understanding of the invention, and the description of these various terms applies to linguistic or grammatical variations of these terms. It will also be understood that the invention is not limited to the specific terminology used herein, or the descriptions thereof, for the description of particular embodiments. Merely by way of example, the invention is not limited to any particular types of biological cells or non-living cell-like constructs, cell culture environments, electroporated molecules or materials, or electroporation protocols. Neither is the invention limited to any particular use to which the inventive embodiments may be directed, whether, by way of example, toward research, diagnostic, information generating, manufacturing, or therapeutic use.

[0060] The phrase "characterize cell" is intended to include the assessments including membrane integrity; the effectiveness with which a cell blocks a pore; cell health; and cell viability, cell growth and any combination thereof.

[0061] The phrase "characterize electroporation" is intended to include determinations of the onset, the extent and the duration of electroporation, as well as an assessment of the recovery of cell membranes after electroporation, and any combination thereof.

[0062] The term "charged entity" shall include any positively or negatively charged molecule or polymer, and can be of biological origin, such as a peptide, a protein or a nucleic acid, and any combination thereof. [0063] Tie^'term ''Biological^' entity" ό^'r "cell", when used in a biological context, refers to any entity with a lipid membrane, and includes any kind of biological cell, and further includes as well, artificial cells or lipid vesicles that resemble biological cells in the basics of their lipid-enclosing-an-aqueous-environment construction, and any combination thereof. Without loss of generality, the term "cell" shall refer to such a biological entity, and similarly without loss of generality, the term "cell layer" or "cell barrier" will include cases in which cells cover the membrane fairly uniformly, in a monolayer or sub-confluent monolayer, or when they preferentially congregate over pores. Other examples of cell layers include biological tissue pieces as obtained, for example, by biopsy, biological tissue slices, primary cells, spheroids, cultures of adherent and non-adherent cells, collections of cells and spheroids deposited by some mechanical means, and cells and spheroids preferentially blocking pores, and any combination thereof.

[0064] The terms "impedance" and "resistance" are used to indicate a ratio of current to voltage, both terms express an electrical circuit's opposition to current flow. The practical difference between impedance and resistance is that impedance changes as a function of frequency. In this invention, impedance is used as a more general term, except when distinguished from resistance in the text.

[0065] The term "modular" refers to aspects of articles that include being of standardized dimensions, and designed to be easily moved in and out of position within a more encompassing apparatus, and designed to enable processing of large numbers of replicates. "Modular" also refers to the variations in specifics of form, with commonality in functional connectivity to the more encompassing apparatus. Such variations in specific form may correspond, for example, to various standard cell culture formats, which themselves can be understood as being modular.

[0066] The term "pore" is throughout this application; this term can have separate meaning in two contexts, but it will be clearly understood in the context in which it occurs. In a first context, "pore" refers to the transient opening that occurs in a cellular membrane in response to an applied electrical field. In a second context, "pore" may also refer to the openings or passages in the supported porous membrane that supports or holds biological cells within the electroporative chamber.

[0067] Further by coincidence of language, "cell" is a term that occurs in the context of an "electric- or electrical cell" and a "biological cell". In cases where "cell" is not explicitly associated with "electric" or "biological", the meaning will be understood according the immediate context. In referring to biological cells, the phrase "subject cell" refers to the cell being subjected to electroporation. Generally when "cell" is used in a biological sense, it is a reference to a generic or prototypical subject cell without an intention to specifically refer to any "single" cell. Further, any biological cell referred to is generally a member of a larger cell population being subjected to electroporation.

2. Electroporation device configuration and operation

[0068] Figure Ia shows the cross-section schematic of the inventive electroporative device or chamber

(200), while Figure Ib shows the larger electronic configuration of the encompassing inventive electroporative apparatus (100), which also includes the components of the electronic control and power (300), as it is configured for monitoring and controlling electroporation of cells, including biological cells, lipid vesicles, cell cultures, cell monolayers, spneroiαs, Dioiogicai tissue ana tissue slices and any combination thereof, on porous membranes. The electroporative chamber (200) comprises three parts: The top electrode unit (1), a supported porous membrane (9), for example embodied here as a cylindrical cup with a thin, non-electrically conductive and porous membrane base; hereafter referred to as a cell insert (10), and the bottom electrode chamber (14). . The cell insert rests on feet (11) to keep the membrane (10) from touching the bottom chamber (14). Alternately, a flange along the top rim of the cell insert (9) allows the cell insert to hang from a ledge built into the bottom chamber, such that the membrane (10) is separated at a desired distance from the bottom electrode (14) A top electroporation electrode (3), embodied here of silver and silver chloride (Ag/ AgCl), is attached to the base of the top unit body (2) as shown in the Figure Ia. The surface of the top electroporation electrode (3) has roughly the same area and shape as the surface of the porous membrane (10). In practice, and as shown in Figure Ia, it may be necessary to make it slightly smaller than the membrane due to constraints imposed by cell insert (9). In a preferred embodiment, the top electroporation electrode (3) would actually be larger than the membrane. A small hole (4) is provided in the top electroporation electrode through which a probe electrode (5) is inserted. In other embodiments, the probe electrode may not intrude into the electroporation electrode surface area. Nonconductive filling (6) is used as a spacer for the probe electrode to prevent electrical connection between the two electrodes. Electrical wires (7, 8) connect the top electroporation electrode (3) and the probe electrode (5) to external electronic apparatus.

[0069] The bottom chamber comprises a body (14) and a bottom electroporation electrode (15) attached to the inside of the chamber. While it may be possible to use closely matching dimensions for both the top and bottom electroporation electrodes, in practice, the mechanical dimensions are likely to differ. For example, as shown in Figure Ia, it may be desirable to make the surface area of the bottom electroporation electrode (15) larger than that of the top electroporation electrode (3) in order to reduce fringing effects when a voltage is applied across these electrodes. As in the top unit, a probe electrode (17) is inserted in the bottom electroporation electrode (15) through a hole (16) and nonconductive filling (18) is used to insulate the electrodes. Electrical wires (19 and 20) provide electrical access to the two electrodes.

[0070] As practiced by this invention, during an electroporative procedure, biological entities block substantially all of the pores (13) on the porous membrane (10), in some cases forming a continuous layer of cells across the membrane. The middle supported porous membrane, here embodied by a cell insert (9) is placed in the bottom chamber (14). The proper amount of electroporation buffer. Electroporation buffer consists of a conductive electrolyte solution, including but not limited to, PBS (phosphate buffered saline) or cell culture medium; more preferably medium that does not contain serum. In some cases the conductive capacity of the electroporation buffer is preferably modified, for example a low-conductivity buffer, through modification of the electrolytes, salt concentrations or ionic capacity and concentrations of the molecules within the buffer. Entities for molecular transfer or delivery can be placed in either the upper or lower electroporation reservoir or both-. (The two reservoirs may also contain different entities and/or a plurality of entities at different concentrations.) The top unit is inserted in the cell insert. By design, the electroporation electrodes on bottom chamber (15) and the top unit (3) maintain a fixed distance to the cell and the porous membrane and the intervening space is filled with a electroporation buffer. The top electroporation electrode (3) is connected to the output of a power amplifier (21) via the wire (7). The bottom electroporation electrode is connected to a transimpedance amplifier (22). The top probe electrode (5) and bottom probe electrode (17) are connected to the two inputs of a high input-impedance differential voltage amplifier (23) through electrical wires (8) and (20) respectively. [007Ϊ] " During ah e'[eciτbpό"ratϊve"proOedure, electrical pulses are applied to the cells through the two electroporation electrodes (3 and 15). When the cells are electroporated, electrical current flows through the cell membrane(s) via electrical field-induced pore formation, a process that can be understood as permeabilization of an otherwise electrically impermeable volume. Once having traversed the cell membrane, current continues to traverse through the interior of the cell, creating a situation in which, the biological cell is an electrically conductive medium. It should be noted that "pore formation" is a phrase is somewhat theoretical in that the nature of the effect of survivable electrical current on cells is both transient and difficult to observe. Nevertheless the terms "electroporation" and "pore formation" are well understood by practitioners of the technology, and the present invention is not bound by any particular theory. Various functional consequences of electroporation are known. During this process, for example, molecular or particulate entities can be delivered into the cell by transport mechanisms including passive transport (diffusion), electrophoretic force, electrokinetic or electroosmotic flow, or any combination thereof. Similarly, by various mechanisms both polar and non-polar molecules can escape from the cell into the surrounding environment. The described invention enables the user to select the polarity of the applied field to achieve directional molecular transport of charged materials. Another possible consequence of electrically-induced poration is biological cell fusion, wherein the disrupted membranes of adjacent cells fuse, thereby uniting two parent cells into a new cell. This form of manipulation of biological cells also has utility in a variety of biotechnological procedures. Whether the consequence of electrical manipulation is cell fusion or transport of molecular entities, the magnitude of this electrical current flow through the cells is dependent on the degree of cellular electroporation.

[0072] This electroporation-induced electrical current can be measured with the transimpedance amplifier

(22) and can be used to monitor the process of cell electroporation. In addition to the current measurement, the two probe electrodes are used to determine the applied voltage imposed across the cell layer throughout the electroporation process. Because of the voltage drop at the electrode-electrolyte interfaces, the voltage applied to the two electroporation electrodes (3 and 15) is not the same as the voltage across the biological cell layer. The two probe electrodes (5 and 17) are connected to the high input-impedance amplifier (23). Thus, since substantially no current flows through these electrodes (5 and 17), there is no appreciable voltage drop at the electrode-electrolyte interfaces, and the differential voltage between them more accurately reflects the voltage across the biological cell layer. The electrical impedance of the cell layer can thus be approximately calculated, for example, by a computer; the relative impedance measurement indicates the magnitude of applied voltage required to achieve electroporation of the biological cell layer. This biological cell impedance can be used as feedback to fine-tune electroporation variables (for example the magnitude and duration of the applied field), in order to achieve highly controlled electroporation of the cells, as well as for monitoring the permeable state during electroporation and recovery process of the cell membranes.

[0073] This process of electroporation can be repeated within the scope of a single experiment, for example by performing additional sequential cycles or "pulses" of the previously described process to the cell layer. Sequential cycles may be desirable to increase the efficiency of electroporation and mass transfer into the cell population. A particular benefit of this invention is the ability to monitor the recovery process of the cell membranes, enabling the researcher to time the cycles to membrane recovery, thus ensuring minimal damage or exhaustion of the cellular membranes throughout the process. ό. Methods ior DiocKing pores on a porous membrane with cells

[0074] Effective blocking of the pores on the porous membrane (for example, with cells or other pore- blocking matter) is highly preferred, in order to achieve highly controlled electroporation using the device described above. Pores that are not blocked by cells, as would be the case in a sub-confluent cell layer, allow parasitic currents pathways when electroporation pulses are applied, which reduces accuracy in trans-membrane current measurement and also deteriorates the "focusing" effect of this configuration, resulting in less effective electroporation of the cells. Moreover, unblocked pores which are distributed non-uniformly across the membrane can result in electric field asymmetries across the membrane surface. It is important to note that while incomplete coverage of pores may result in these adverse effects, the inventive electroporative apparatus can still achieve electroporation at applied voltages much lower than those used for conventional electroporation. Thus, blocking pores of the porous support to which cells are attached, supported by, or adhered to is desired, and such methods and materials that block pores are described in this section.

[0075] The significance of membrane pore blockage is that such a configuration causes the biological material, as a whole, to become a resistive or impeding bottleneck through which substantially all current transiting across the electroporative chamber must flow. Inasmuch as the individual cells of the cell population in the cell chamber are homogeneous with regard to the electrical resistance or impedance they exert in the face of a voltage differential, the biological portion of the electrical circuit will be substantially uniform across its area. This aspect of the electrical flowpath, coupled with the various approaches to exerting control over the voltage and current as described herein, allow for the creation of a substantially controlled electric field to be placed across the biological material, as a whole, and substantially uniform with respect to the electrical field exerted on each cell within the population of cells being subjected to the electroporative procedure.

[0076] For the purpose of forming a substantially controlled electric field, thus, this invention provides several ways to effectively block pores such that electrical currents are forced to flow completely or substantially through cells during electroporation process. "Blocking" pores, as used in describing this invention, means blocking or substantially blocking the flow of electrical current between two points except through the biological material being electroporated. In such a case, it can be understood that electrically conductive medium is biological material such as a plurality of cells. Figure 2a illustrates the first method of blocking pores, which is the formation of a confluent layer of adherent cells to cover a porous membrane (10). In this process, adherent cells are cultured on a porous membrane that is constructed of materials such as polycarbonate, poly(ethylene terephthalate) (PET) or polytetrafluoroethylene (PTFE), and is tissue culture treated and/or coated with cell growth permissive coatings (including, by way of example, collagen, fibronectin, or polylysine or any combination thereof). When cells grow into an interconnected monolayer that covers the entire porous membrane, they effectively electrically block all pores. In this scenario, there is insignificant current flow between the two electroporation electrodes (3 and 15) if the applied electrical voltage is not of the threshold value required to induce electroporation in the cells. In the absence of electroporation, the lipid membrane of the cells typically exhibit very high electrical impedance. Upon electroporation, a continuous electrical flowpath is created by the continuity of electrolyte-containing aqueous solutions on either side of the cell, and within it. As such, this path represents the primary electrical path between the two electrodes. In practice, a small leakage current may develop due to imperfect sealing of the cells to the pores, as well as uncovered pores. In many cases, it is difficult or undesirable to grow cells into a 100% confluent layer, inis may resun in many uncovereα pores, which as discussed previously, may reduce device performance. To solve this problem, non-conductive substances, including but not limited to glass or polymer micro- or nano- sized particles, may be added to block the uncovered pores through various mechanisms. As illustrated in Figure 3, one method to achieve this is through generating a pressure difference between the two sides of the porous membrane to pull the substances toward the uncovered pores and block/clog them.

[0077] Suspension cells normally do not attach to surfaces and form an adherent layer. For these kinds of cells, a mechanical means is preferred for sealing of pores. One such mechanical means is generating a pressure difference between two sides of the porous membrane such that the suspended cells are pulled toward pores; thus the cells can effectively block the pores as illustrated in Figure 2b. Because an excessive pressure difference can cause mechanical damage to cell membrane, the pressure must be properly regulated so as to produce a good seal between cells and pores, but avoid damaging the cells. This pressure difference may be generated by: providing a seal between the top unit (1) and the supported porous membrane (9), and applying a positive pressure through a small hole in the top unit; providing a seal between the bottom chamber (14) and the supported porous membrane (9), and a negative pressure is applied through a small hole in the bottom chamber; or some external device prior to insertion of the cup (9). In the former two cases, the pressure may be applied throughout an experimental procedure. Another mechanical method of moving the cells into a position whereby they block the porous membrane is by hydrodynamic flow, which can be achieved by the flow of liquid through (perpendicular flow) or across (transverse flow) the porous membrane, or any combination thereof. Embodiments of hydrodynamic flow include but are not limited to gravity-induced hydrodynamic flow; it can also be achieved by centrifugation. The entire supported porous membrane (9) or cartridge, along with cells or lipid vesicles in a suspension of liquid, is placed in a centrifuge such that the cells are carried by the liquid to block the pores under the action of the centrifuge. These mechanical techniques, pressure differential or hydrodynamic flow can be used together. They can also be used in conjunction with an adherent cell layer resulting from cell growth, as described above; for example, it may be necessary to block the pores in areas not covered by the adherent cell layer. Either of these mechanical techniques, pressure differential or hydrodynamic flow, can be combined with the use of non- conductive substances, such as glass or polymer particles, to block any uncovered pores, as described above. For example, in the case where it is desired to electroporate a population of cells that is lower in number than pores on the porous membrane (10), an effective procedure, for example, may include adding the cells in liquid suspension to the supported porous membrane (9); applying a pressure differential to move the cells to block pores; adding sufficient micro- or nano-sized inert particles to cover the remaining pores; and applying a pressure differential to move the particles to plug the remaining pores. The cells and particles may also be combined in one step for convenience.

[0078] The percentage of pores that are effectively blocked can be evaluated simply by measuring the overall impedance of the cell-covered porous membrane. Data of this kind is diagnostic of the level of pore blockage, because when a pore is blocked by a cell whose membrane impedance is very large, the effective impedance of this cell-pore unit is far larger than that of an uncovered pore. Therefore, the more pores that are effectively covered by cells or blocked by non-conductive substances, the larger the overall impedance of the cell-membrane complex will be. The correlation between pore coverage and overall impedance can be readily established; by applying a low voltage to electroporation electrodes (3 and 15) that does not induce electroporation in cells, and by measuring the corresponding impedance, the effectiveness of pore-coverage can be evaluated. This impedance measurement can De very πcipiui in iaicr ucierminaiion oi ine optimal electroporation voltages.

4. Intrinsic cell membrane potential

[0079] Due to charge accumulation within biological cells and on cell membrane surfaces, a cell membrane can be described as having a built-in or intrinsic potential. During an electroporation procedure, this potential contributes to, or subtracts from, the externally supplied voltage; thus, highly controlled electroporation requires knowledge of the membrane built-in potential. The present invention allows measurement of this intrinsic cellular potential prior to electroporation. For such a measurement, the top and bottom electroporation electrodes (3 and 15) must be electrically disconnected from the power amplifier (21) and the transimpedance amplifier (22) respectively. These electrodes (3 and 15) may be allowed to float. Alternatively, the top electroporation electrode (3) may be connected to the top probe electrode (5) and the bottom electroporation electrode (15) may be connected to the bottom probe electrode (17). In the case that the differential amplifier (23) common mode rejection is inadequate, it may be necessary to connect either the top (5) or the bottom (17) probe electrode to a defined potential, such as the reference ground of the differential amplifier (23).

5. Transepithelial/transendothelial impedance measurement

[0080] Studies of the barrier and transport functions of epithelia and endothelia commonly rely on measurements of the electrical impedance of monolayers of such cells. This property is termed the transepithelial or transendothelial impedance. As described above, the present invention is capable of performing such impedance measurements. Thus, the present invention is uniquely qualified to assess barrier and transport function changes as a result of electroporation, as well as barrier and transport function changes due to the transfer of any exogenous or foreign substance into or through the cells during electroporation.

[0081] Furthermore, there is a unique aspect of cell orientation that can be exploited by using the above- described method and device for controlled electroporation. The porous membrane provides a natural support for tissue-derived cell growth, and thus allows a more natural state of the cell or cell layer for in situ electroporation. The device also provides a means for controlling orientation of the cell and/or cell layer. By way of example, cells like the MDCK (Madin-Darby Canine Kidney) epithelial cells are known to differentiate as a function of development and cell density - they naturally develop a polarized aspect (cell polarity) with apical and basal lateral membranes that differ in lipid and protein composition. The inventors have observed a vector-dependence to the electroporation performance with MDCK cells, meaning that direction (polarity) of the applied electric field may depend on the differentiated membrane orientation, i.e., apical-to-basal lateral vs. basal lateral-to-apical applied fields. Given that most tissues and tissue-derived cells have defined growth vectors (motility) and orientation preferences, the device has novel use in determining and optimizing cell engineering for adherent cells and tissue.

6. Methods for using feedback for controlled electroporation

[0082] As described above, the voltage applied by the power amplifier (21) to the electroporation electrodes

(3 and 15) is not the same as that seen by the cells. However, the voltage measured by the differential amplifier (23) through the probe electrodes (5 and 17) is a more accurate representation of the voltage that drops over the cells. Thus, the operator of the present invention can use the voltage produced by the differential amplifier (23) as guidance, or 'feedback,' when attempting to apply a desired voltage to the cells; specifically, the operator may inticase me vuiiage appneα oy ine power ampniier (21) until the voltage measured by the differential amplifier (23) reaches the desired value. Alternately, the operator can use the current measured by the transimpedance amplifier (22) as feedback; as described above, the magnitude of the electrical current is dependent on the degree of electroporation of the cells. Thus, the operator may increase the voltage applied by the power amplifier (21) until the current measured by the transimpedance amplifier (22) reaches the desired value. Finally, the operator can use the impedance measurement of the cells as feedback. As described above, the electrical impedance of the cell layer can be estimated or calculated with cross-cell voltage measurement from differential amplifier (23) and crosscurrent measurement from transimpedance amplifier (22). The impedance measurement reveals the degree of electroporation of the cell layer since cell membrane impedance is dependent on the extent of membrane electroporation. Thus, the operator may increase the voltage applied by the power amplifier (21) until the calculated cell layer impedance decreases to the desired value. The "desired value", in this case, maybe considered one that delivers a threshold- or minimally prescribed electric field to the cell, one that is large enough to be highly effective with regard to inducing cellular electroporation, but sufficiently low that it does not irreversibly damage the cells.

[0083] The preceding paragraph describes the manual use of measured data by the operator of the present invention as feedback for achieving desired results. Specifically, since the voltage applied to the cells is not the same as that applied to the electroporation electrodes (3 and 15), the operator is required to adjust the voltage applied to the electroporation electrodes (3 and 16) until the voltage applied to the cells, as measured by the differential amplifier (23) reaches the desired value. In this case, the electronic circuit is configured in an open-loop fashion, as shown in Figure Ia. An alternate technique is to design the electronic circuitry in a closed-loop configuration in order to force the voltage between the probe electrodes (5 and 17) to a desired value. Figure Ib depicts one such configuration. Negative feedback is accomplished through the use of an operational amplifier (24). A switch (25) allows the circuit to be configured as closed-loop or open-loop. The position of the switch (25) shown in Figure Ib is the position required for closed-loop operation. The closed-loop circuit may become unstable due to poles contributed by: amplifiers 21, 23 and 24; the electrodes 3, 5 and 17; and the cells or cell layer. Three optional compensation elements (26, 27 and 28) can be used to ensure the stability of the closed-loop circuit. Elements 26 and 27 may be configured as shown in Figures Ic and Id respectively, in which case they would both serve as phase lead elements, in addition to allowing adjustment of loop gain. An example configuration of 28, shown in Figure Ie, is used for adjusting loop gain. In the exemplary configuration presented above, where A_dllT is the gain of the differential amplifier (23), V_wavcgen is the output of the waveform generator (29) and V_cen is the voltage imposed across the cell layer, the voltage imposed across the cells is directly controlled by the waveform generator (29) output according to the following algorithm or relationship:

1 R_S] + R_S2

V c,ell ' V wavegen X — J ώff R 82

[0084] In one embodiment of the invention, the waveform generator (29) is controlled by a computer (30).

The output of the differential amplifier (23), which represents the voltage imposed across the cells, is converted to digital form by the analog-to-digital converter 31, while the output of the transimpedance amplifier (22), which represents the current flowing through the cells, is converted to digital form by the analog-to-digital converter 32. Analog to digital converters 31 and 32, both, in turn, pass on the digital information to the computer (30). As described ^'above, the electrical impedance can thus be calculated using a computer. This impedance measurement can in turn be used by computer software to change the output of the waveform generator (29). Thus, the voltage applied to the cells can be adjusted to achieve a desired cell impedance; for example, if the calculated impedance is higher than the desired impedance, the computer (30) can increase the magnitude of the output of the waveform generator (29), thus increasing the voltage applied to the cells. The computer will continue to increase the voltage applied to the cells until the degree of electroporation of the cells results in the impedance decreasing to the desired value.

7. Description of electrical pulses

[0085] As described above, the unique configuration of the present invention allows electroporation voltage pulses more than two orders of magnitude smaller than those used for electroporation of cells in suspension; this in turn allows generation of arbitrary pulse shape and duration without adding complexity to the power amplifier (21). The polarity of a pulse is defined as follows: a positive pulse is one in which the potential of the top electroporation electrode (3) is positive with respect to the potential of the bottom electroporation electrode (15); a negative pulse reverses this electrode polarity.

[0086] The simplest such pulse is a step pulse, that is, a step from ground potential to some constant voltage, which is maintained for some period of time, followed by a step from this constant potential back down to ground potential. Such a pulse is shown in Figure 4a. In order to initiate reversible electroporation, the potential drop across the cell layer, as measured by the differential amplifier (23) through the probe electrodes (5 and 17), should be roughly greater than about 20OmV and less than about 300OmV, depending on cell type and charge. (Due to intrinsic cell charge, the absolute value of the threshold may be different for opposite polarity pulses.) To achieve this, the voltage that must be generated by the power amplifier (21) is typically less than 20V. The width of this pulse should be greater than approximately 100msec and less than approximately 10000msec, as longer pulses may cause irreversible electroporation or other damage to the cells. It may be desirable to both immediately precede and follow this step pulse by contiguous step pulses of lower amplitude, as shown in Figure 4b, where this amplitude is sufficiently low (20-5OmV) such that it does not cause electroporation of the cells. The low amplitude pulse (33) preceding the electroporation pulse (34) allows measurement of the impedance of non-electroporated cells. This measurement serves as comparison for the impedance measured during the electroporation pulse (34); a decrease in impedance during the electroporation pulse (34) as compared to that measured during the pre-electroporation pulse (33) indicates that electroporation has taken place. The low amplitude pulse following electroporation (35) allows assessment of the recovery of cells from electroporation; an increase in impedance during the post-electroporation pulse (35) as compared to that measured during the electroporation pulse (34) indicates that the cells have begun to recover from electroporation.

[0087] As described above, the electroporation pulse (34) should be limited to ensure cell viability. However, it may be desirable to extend the time during which mass transfer can take place, and to help drive mass transfer through field-induced mobility such as electrophoretic force, electrokinetic or electroosmotic flow, or any combination thereof. This may be particularly valuable, given the direct current (DC) nature of the pulses described. The experience of the inventors is that once cells are electroporated, the potential required to maintain a given degree of electroporation is in the range of 10OmV to 50OmV, and as such is lower than the threshold value required for initiation of electroporation. When set in this potential range, a pulse may be several seconds long. l neretore, it may oe aαvaniageous to αiviαe tne electroporation pulse (34) from Figure 4b into two segments, as shown in Figure 4c. The first part of the electroporation pulse (36), is intended to initiate electroporation. The second part of the electroporation pulse (37) has a lower amplitude than the first part (36), and is intended to maintain electroporation. The two portions of the electroporation pulse need not be the same polarity; for example, if the intrinsic charge of the cell membrane is positive, it may be desirable to make the first part of the electroporation pulse (36) negative. However, if the molecule to be transferred is, for example, positively charged, it may be advantageous to make the second portion of the electroporation pulse (37) positive in order to assist in field-induced mobility as described above.

[0088] Under certain circumstances, a sinusoidal pulse, defined as a finite number of periods of a sinusoid with a constant amplitude and frequency, is preferred over the step pulses described above. For example, a sinusoidal pulse prevents deterioration of the electroporation electrodes (3 and 15). Moreover, the step pulses described above may result in polarization of the electrodes, which in turn could lead to measurement errors. Finally, a sinusoidal pulse may result in more efficient transfer of molecules or in increased cell survival for certain cell types. The cell or lipid vesicle layer can be modeled as a resistor in parallel with a capacitance, and thus the impedance of the layer will have a low pass filter response. During electroporation, the resistance of the cell layer will decrease while the capacitance will remain largely unchanged. Thus, the cutoff frequency of the filter, f-3dB = 1 /(2πR_<;_eiiC_ceiι), will actually increase during electroporation. Given the typical small values of C_cen, measuring f- 3dB shift as a means of detecting electroporation may even may improve system sensitivity, particularly for cell layers with a low equivalent resistance. Estimation of R^n or f-3dB requires information at a number of distinct frequencies. Therefore, a sum of the sinusoidal pulses described above, where the frequency of the sinusoid used to generate each individual pulse is unique, can be used. The frequencies may be chosen such that an integer number of periods of each sinusoid is completed in the duration of pulse; for example, the frequencies may be separated by a factor of two. The amplitude of the resultant pulse is defined as the magnitude of the maximum excursion of the summation. For the sake of clarity, references to such summations of sinusoidal pulses will be henceforward referred to as simply sinusoidal pulses and figures referring to summations of sinusoidal pulses will depict a single frequency.

[0089] As described above for step pulses, contiguous sinusoidal pulses of varying amplitudes can be useful

(Figure 4d). The low amplitude pulse (38) preceding the electroporation pulse (39 and 40) allows measurement of the impedance of non-electroporated cells. This measurement serves as comparison for the impedance measured during the electroporation pulse (39 and 40); a decrease in impedance during the electroporation pulse as (39 and 40) compared to that measured during the pre-electroporation pulse (38) indicates that electroporation has taken place. The first part of the electroporation pulse (39), is intended to initiate electroporation. The second part of the electroporation pulse (40) has a lower amplitude than the first part (39), and is intended to maintain electroporation. The low amplitude pulse following electroporation (41) allows assessment of the recovery of cells from electroporation; an increase in impedance during the post-electroporation pulse (41) as compared to that measured during the electroporation pulse (39 and 40) indicates that the cells have begun to recover from electroporation.

[0090] The step pulse technique can be combined with a sinusoidal component. This may be desirable in the case where the step pulses offer the most efficient electroporation for a given cell type, but where the sinusoid, for the reasons described above, provides a superior impedance measurement. Such a pulse can be realized through the summation ot a low amplitude (20 - 5UmVj sinusoid with the electroporation step pulses (39 and 40) shown in Figure 4c. The resultant pulse is shown in Figure 4e. Low amplitude sinusoidal pulses (42 and 45) are used for measurement before and after electroporation. Step pulses with a superimposed sinusoid (43 and 44) accomplish electroporation. The first part of the electroporation pulse (43), is intended to initiate electroporation. The second part of the electroporation pulse (44) has a lower amplitude than the first part (43), and is intended to maintain electroporation.

8. Device for controlled electroporation in tissue

[0091] The device described above can also be adjusted to control electroporation in tissue, as shown in

Figure 5. In a typical electroporation procedure for tissue, the tissue sample is placed on the cell insert membrane (10), and the cell insert is placed between the two electroporation electrodes (3 and 15) as shown in Figure 5, for in vitro electroporation. The tissue sample should be sized such that it covers the majority of the membrane (10). Alternately, or additionally, the tissue sample may be allowed to culture on the membrane (10) such that it attaches and spreads to cover the membrane (10) fully. An electrolyte is introduced to generate good contact between the tissue and the electrodes. Then, electrical pulses are applied to the tissue through the two electroporation electrodes (3 and 15), which are connected to the power amplifier (21) and the transimpedance amplifier (22). Measuring the electrical current through this electrical circuit is dependent on the overall and average degree of electroporation that the cells in the tissue sample between the electrodes experience.

[0092] Once the cells are electroporated, there is increased electrical current flow through the cells and the magnitude of the electrical current becomes dependent on the degree of electroporation of the cells. This cross-cell electrical current can be measured with the transimpedance amplifier (22) and can be used to monitor the process of electroporation of the cell membranes. In addition to the current measurement, the two inserted probe electrodes (5 and 17) are used to precisely measure the voltage drop across the tissue during the electroporation process. The electrodes (5 and 17) are connected to the high input-impedance amplifier (23). Thus, since no current flows through these electrodes (5 and 17), there is no voltage drop over the electrode-electrolyte interfaces, and the differential voltage between them provides an accurate measurement of the voltage across the tissue. Precise electrical impedance of the tissue is thus calculated from cross-tissue voltage measurement with the probe electrodes (5 and 17) and crosscurrent measurement with the circuit attached to the electroporation electrodes (3 and 15). The impedance measurement reveals the degree of electroporation of the cells in tissue since cell membrane impedance is directly dependent on the extent of membrane electroporation. In addition to monitoring the electroporation, the electrical current measurement, as well as membrane impedance measurement, can be used as feedback for fine-tuning of electroporation pulses that allow the generation of a minimally prescribed electric field, specific for cell type and other particulars of the cell and/or the environment within the electrical cell, to achieve highly controlled electroporation of the cells in tissue.

9. Configuration, fabrication, and methods of operation of a modular electroporative cartridge a. Configuration and fabrication of a modular electroporative cartridge

[0093] Embodiments of the present invention can vary in terms of physical dimensions of (linear measurements, surface area, volume) and aspect ratio of the electroporation chamber described above (Figure Ia), without differing in any of the basic aspects of the electronic hardware or operation of the apparatus (Figure Ib). 5>ome emDoαimenis or ine invention, merely oy way of example, may include a single bottom chamber with relatively large and robust base, with square dimensions of 5 inches by 5 inches. Such an embodiment has utility as a bench top device, and can have utility as a long-lived, durable research tool. Other embodiments of the present invention have the form and dimensions of modular and consumable articles, very much in the same sense that cell culture articles are available in standard formats, are scalable, and are considered consumable inasmuch as they are generally single use. Associated with the single-use aspect of cell culture articles is the fact that they are commonly sterile wrapped, and disposed not for wear or damage incurred during use, but the maintaining sterility or the biological isolation of cells being handled. Embodiments described in this present section, including the embodiments depicted in Figures 6 — 24, are representative of this modular, consumable, scalable, and cell culture- compatible class of articles. Further, the association of these embodiments with cell culture includes compatibility with various cell culture material holders and robotic devices that handle the movement of cells and liquids during high volume or high throughput cell culture operations. These cell culture associated embodiments do not differ the embodiments broadly described above in sections 2 - 8 in terms of fundamentals of their architecture or operation, but the cell culture-related embodiments are described in this present section separately in order that their particular features may be detailed. These cell culture-friendly embodiments will generally be referred to as electroporative "cartridges" 220, but it will be understood that a "cartridge" is but a form of an electrical cell, as is the basic electroporation chamber 200 of Figure Ia.

[0094] In its most basic form the cartridge (220) depicted in Figure 6a is an electric cell capable of holding within it a biological cell or plurality of cells in a substantially stable position, attached or affixed to solid but porous membrane or substrate. The substrate is positioned in such a way that that it represents a potential impediment to an electrical flowpath from an electrode on one side of the support to an electrode on the other side. The cartridge is modular in that it is of standardized dimensions, easily moved in and out of position within a larger electroporation instrument, designed to enable processing of large numbers of replicates, and consumable in a manner common to many cell culture materials. Figures 6a - 6c show the cross-sectional schematic of a modular electroporative cartridge, a device for monitoring and controlling electroporation of cells (biological cells, lipid vesicles, cell cultures, cell monolayers, spheroids, biological tissue and tissue slices) on porous membranes. The electroporative cartridge comprises three parts: a top unit (A), a middle supported porous membrane, here embodied as a cell insert (B) and a bottom chamber (C). The middle cylindrical cell insert has a thin, non- electrically conductive and porous membrane (Bl), on which cells form a layer. Biological tissue, spheroids, and tissue slices may also be placed and/or cultured on the membrane. A flange along the top rim of the cell insert (B2) allows the cell insert to hang from a ledge built into the bottom chamber, such that the porous membrane (Bl) is separated at a desired distance from the bottom chamber (C). Alternately, the cell insert rests on built-in feet (not shown) to keep the porous membrane (Bl) from touching the bottom chamber (C). In other embodiments, the porous membrane may be integrated, either temporarily or permanently, into the cartridge housing for support. The top unit body (A) is composed of a low-cost, biocompatible material, such as polystyrene, and can be formed by injection molding, stereolithography or machining.

[0095] A variation of the electroporative cartridge (220) that exemplifies modularity in another way is an embodiment (not shown) that comprises the top unit (A) and the bottom chamber (C), but does not include the supported porous membrane, here embodied as a cell insert (B). This embodiment is, however, configured to receive such a middle cell insert (B) or equivalent, and upon insertion of such an insert of equivalent, the assembled cartridge oecomes pπysicaiiy ana runciionaiiy equivalent to the complete cartridge (220). Further embodiments of the porous membrane portion (Bl) of the insert (B) are described below in section (J). Similarly, the middle cell insert (B) portion of electroporative cartridge (220), alone, is an embodiment of this invention, such insert being configured to be compatible with the embodiment comprising (A) and (C), above. Thus any subset of the combination of a top unit (A), a supported porous membrane (B), and a bottom chamber (C) represents an embodiment of this invention, provided each subset is compatible with an integration into the full combination embodied as the above described cartridge (220).

[0096] Returning to details of the configuration of electrodes of the electroporative cartridge, top electrode assembly (D) is attached to the top unit body (A) as shown in the Figure 6. Possible modes of attachment between these two components include the use of a biocompatible pressure-sensitive adhesive. The top electrode assembly, one configuration of which is shown in Figure 7, can be realized as a flexible printed circuit (FPC). The materials used to construct this assembly are preferably biocompatible and more preferably cell-culture compatible. In one embodiment, the substrate of the electrode assembly (Dl) is formed from a polyester-based laminate.

[0097] The top electroporation electrode (D2) and the top probe electrode (D3) are deposited on the substrate; screen printing a conductive ink is a preferred technique. The electrodes can consist of a number of biocompatible metallic compounds; a mixture of silver and silver chloride is preferred for applications where minimal electrode polarization is desired, such as when the electoroporation pulses contain DC components. The surface of the top electroporation electrode (D2) has approximately the same area and shape as the surface of the porous membrane (Bl). In practice, it may be necessary to make it slightly smaller than the porous membrane. The top probe electrode (D3) need only be as large as is necessary to provide reliable connection with the electroporation buffer (see below). By design, when the top electrode assembly (D) is attached to the top unit (A), and the top unit is, in turn, fully inserted into the cell insert (B), the electroporation electrode on the top unit (D2) maintains a fixed distance to the porous membrane (Bl) of 0.1mm to 10mm.

[0098] Electrical traces (D4 and D5) provide electrical access to the electrodes (D2 and D3, respectively).

While they must form electrical connections with the electrodes, the traces need not be composed of the same material as the electrodes. Applicable processing techniques include both screen printing and a combination of lithographic pattern definition with chemical etching. Both traces are covered by an electrically insulating material (D6). This insulating material is likely to cover the entire surface of the assembly (D), excepting the electrodes (D2 and D3) and the terminals (see below). In addition to preventing electrical connection between the traces and an electroporation buffer (described below), the insulating material prevents the top probe electrode trace (D5) from contacting the top electroporation electrode (D2).

[0099] The electrical traces (D4 and D5) are terminated in such a way as to ensure convenient and inexpensive electrical connection. The terminals (D7 and D8) shown in Figure 2 are extensions of the traces that are not covered by the insulating material (D6). It may be desirable to plate the terminals with an additional metal, such as tin, or a metallic compound, in order to improve connectivity. These terminals are particularly suitable for mating with a low insertion force (LIF) connector. Alternately, these terminals can mate with spring-loaded contacts or other spring-based contact systems. To facilitate connection, the terminals may also be dimpled. Options for mating with such terminals include other dimpled terminals, spring-loaded contacts or other spring- oaseα contacts, ine iup eiectroαe assemoiy (u) may extend beyond the top unit (A), as shown in Figure 8, such that the portion of the top electrode assembly with the terminals (D7 and D8) does not adhere to the top unit. Moreover, in the case that the top electrode assembly is realized as a FPC, it may be stiffened in the vicinity of the terminals in order to improve the reliability of the electrical connection.

(00100] A bottom electrode assembly (E) is attached to the chamber body (C) as shown in Figure 6a. As with the top unit and top electrode assembly, modes of attachment include the use of a biocompatible pressure-sensitive adhesive. The bottom electrode assembly, one configuration of which is shown in Figure 9a, can be realized as a flexible printed circuit (FPC). Similar to the top electrode assembly, it comprises a substrate (El), a bottom electroporation electrode (E2), a bottom probe electrode (E3), electrical traces (E4 and E5), insulating material (E6) and terminals (E7 and E8). The fabrication and composition of the bottom electrode assembly (E) and its constituents is substantially the same as the top electrode assembly (D). While it may be possible to use an identical design for both the top and bottom electrode assemblies, in practice, the mechanical dimensions are likely to differ. For example, it may be desirable to make the surface area of the bottom electroporation electrode (E2) larger than that of the top electroporation electrode (D2) in order to reduce fringing effects when a voltage is applied across these electrodes. By design, when the bottom electrode assembly (E) is attached to the bottom chamber (C), and the supported porous membrane (B) is fully inserted in the bottom chamber (C), the electroporation electrode on the bottom unit (E2) maintains a fixed distance to the porous membrane (Bl) of 0.1mm to 10mm. The bottom electrode assembly (E) may extend beyond the bottom chamber (C), as shown in Figure 8, such that the portion of the top electrode assembly with the terminals (E7 and E8) does not adhere to the top unit. Moreover, in the case that the bottom electrode assembly is realized as a FPC, it may be stiffened in the vicinity of the terminals in order to improve the reliability of the electrical connection.

[00101] In an alternate embodiment, the bottom electroporation electrode (E2) and the bottom probe electrode (E3) adhere directly to the bottom surface of the porous membrane (Bl). This could be accomplished by screen printing a conductive ink on the bottom surface of the membrane. A conductive adhesive is used to connect the electrodes (E2 and E3) to uninsulated pads on the traces (E4a and E5a) on a flexible printed circuit (E). In this embodiment of the invention, the bottom flexible printed circuit (E) would adhere to the outside of the cell insert (B) rather than the inside of the chamber (C). Figures Ib and 4b show the modular cartridge device and bottom flexible printed circuit respectively.

[00102] The electrode assemblies (D and E) need not involve FPCs. For example, in the cases of a multi-well configuration, as described in section (f), and/or high volume production, assembly time of the top unit and bottom chamber with FPCs may become prohibitively long. Embedding the electrode assemblies in the top unit and bottom chamber eases assembly issues. In one embodiment, the top unit and bottom chamber (A and C) are molded around the top and bottom electrode assemblies (D and E) respectively (Figure 6c). The molding compound chosen, along with economic considerations (e.g., capital costs, volume, throughput), will dictate the choice of molding technique; transfer molding and injection molding are among the likely candidates. This embodiment is recognizable to practitioners of the art manufacturing of plastic encapsulated integrated circuits (ICs); the manufacturing techniques outlined below are well known to the practitioner. By way of further demonstrating the compatibility of the required manufacturing with existing IC packaging technologies, the top unit and bottom chamber shown in Figure 6c are recognizable to those skilled in the art as similar to a "pre-molded" plastic IC pacKage. /vs in a pre-moiαeα - package, wnere the die pad surface is not encapsulated by plastic (allowing for incorporation of the die post-molding), the electrode surfaces of the top unit and bottom chamber are left uncovered by plastic.

[00103] The electrode assemblies are constructed in a manner similar to that used for IC leadframes. A sheet of metal, usually a copper alloy, is stamped or chemically etched to define leads and electrodes. Before molding, the electrodes and leads are selectively plated. The electrodes are preferably plated with silver, which is frequently used for plating IC leadframes; the silver plating is later chloridized (as described below), resulting in the preferred silver chloride electrode surface. Finally, the electrodes and leads are pre-formed. The leadframes for ICs are typically processed as "strips" of a plurality of individual leadframes; the entire strip is placed in a mold, which in turn defines the outline of the package for each individual leadframe. Only after molding are the IC packages singulated. Similarly, the electrodes and leads for a plurality of top or bottom electrode assemblies form part of a single strip. After molding, the separate top or bottom electrode assemblies are separated from the strip, thus singulating the individual top units or bottom chambers.

[00104] Chloridization of the electrodes can occur before or after singulation; alternately, it can take place after the units are partially singulated, such that electrodes from many devices are still electrically connected together, while others are disconnected. For example, it may be desirable to chloridize all the probe electrodes in a strip together, but separately from the electroporation electrodes. It is evident from Figure 6c that if an electrolyte is added to the bottom chamber (C), the only portions of the bottom electrode assembly (E) in contact with the fluid are the electrodes (E2 and E3). Similarly, if the top unit (A) is dipped (as opposed to submerged) in an electrolyte, the only portions of the top electrode assembly (D) are the electrodes (D2 and D3). Thus, the molding process has provided a way in which to selectively chloridize only the electrode portions of the electrode assemblies. Suitable parameters for chloridization, including voltages and concentration and type of electrolytic solution, are well covered in the literature, and well known to practitioners of electrochemistry.

[00105] The placement of a probe electrode in the center of an electroporation electrode may require special consideration. To extend the comparison with IC leadframes, the electrodes are similar to die pads, in that they will be (partially) encapsulated, and are located central to the final device; such a comparison will be evident to those skilled in the art, and is not meant as anything more than a way to demonstrate the applicability of existing processing techniques. A typical IC has only one die pad, while the device described above has two electrodes, moreover, the location of the probe electrode requires that its lead runs below (or above) the electroporation electrode. This electrode configuration actually resembles the "over/under" leadframe configuration employed in optocouplers (also known as opto isolators and solid state relays); in such devices, a photoemitting diode is placed directly above a photodetector, with both components on separate die pads. It will be apparent to practitioners of the art manufacturing of optocouplers that the same techniques used to achieve an over/under configuration can be applied to create the electrode configuration required.

[00106] The probe electrode is not restricted to a center location. As shown in Figure 10, the probe electrode can be located at the outer edge of the electroporation electrode. The centered probe electrode provides the advantage of matching the symmetry of the overall electrode geometry. The off-center probe electrode provides the advantage of minimizing the area of membrane covered by the probe. Furthermore, the probe electrode position is not limited to the same plane as the'electroporation electrode, for example the probe electrode can be positioned above or below the plane of the electroporation electrode.

[00107] In addition to the use of flexible printed circuits for the electrode assemblies, rigid printed circuits may also be implemented. Figure 11 depicts a cross sectional view of a bottom electrode assembly. The advantage provided by the rigid printed circuit is that existing printed circuit board fabrication technologies allow the use of vias in the electrode assembly. In traditional printed circuit board fabrication, a via provides a feed-through to bring electrical contact or trace from one face of the board to the other. As illustrated in Figure 11, the terminals (E7, E8) can extend and be a conductive underlayer to the electroporation and probe electrodes (E2 and E3, respectively). The vias bring the electrode contact to the backside of the rigid circuit board (El), allowing a much simpler connection to the terminals.

[00108] The concept of the via illustrated in Figure 11 may also be implemented with flexible printed circuits. The via holes are placed in a flexible substrate (El), and a conductive coating is applied to both sides of the substrate (E7, E8). The electrode material (E2, E3) is applied to the conductive coating. The conductive coating becomes the terminals (E7, E8) on the backside of the flexible circuit, simplifying electrical connection to the terminals. In the case of rigid or flexible printed circuits, the open vias represented in Figure 11 could be filled with conductive compounds, or sealed by application of various coatings. These techniques are familiar to those skilled in the art of printed circuit fabrication. These techniques all share the advantage of enabling backside electrical contact.

b. Principles of the operation of modular electroporative cartridge

[00109J During the operation of the inventive electroporative cartridge device, biological entities block substantially all of the pores on the porous membrane (Bl), in some cases forming a continuous layer across the membrane. The term "biological entity" refers to any entity with a lipid membrane, and includes biological cells, artificial cells and lipid vesicles. Without loss of generality, the term "cell" shall refer to such a biological entity. Biological cells may cover the membrane fairly uniformly, or they may congregate over the membrane pores. Cells, in plural or aggregate forms, may include: biological tissue; biological tissue slices; spheroids; primary cells; blood cells; cultures of adherent and non-adherent cells; adherent cell monolayers; collections of cells, tissue and spheroids deposited by some mechanical means, as discussed in section (d)(ii); and cells and spheroids preferentially blocking pores, as discussed in section (d)(ii). Again, without loss of generality, the term "cell layer" may include a layer of cells of any one of these forms that multiple cells may assume or any combination of the preceding list. The proper amount of electroporation buffer is delivered to both the bottom chamber (C) and the supported porous membrane, here embodied as a cell insert (B). Molecular entities (including, for example, DNA, RNA, peptides, proteins, small organic and inorganic molecules, nanoparticles, etc.) for molecular transfer can be placed in either the upper or lower electroporation reservoir or both simultaneously, depending on the desired polarity of the applied electric field, and the polarity of the cell and entities. (The two reservoirs may also contain different entities and/or a plurality of entities at different concentrations.) The supported porous membrane (B) is placed in the bottom chamber (C) and the top unit (A) is inserted in the cell insert (B). The intervening spaces between the top electroporation electrode (D2) and the cell layer, and between and the bottom electroporation electrode (E2) and the porous membrane (Bl), are filled with the electroporation buffer. With this configuration in place, specifically, with a cell layer or population of biological cells associated with the supported porous membrane, the impedance measured atter imposing a voltage imposed across the described electrical cell represents the combined impedance of the membrane and the associated biological cells.

[00110] The modular cartridge device is connected to the measurement and control circuitry shown in Figure 12. The top electroporation electrode (D2) is connected to the output of a power amplifier (Fl) through an electronics connector (F2a), via the trace D4 and terminal D7. The bottom electroporation electrode (E2) is connected to a transimpedance amplifier (F3) through the electronics connector (F2b), via the trace E4 and terminal E7. The top probe electrode (D3) and bottom probe electrode (E3) are connected to the two inputs of a high input-impedance electronics connector (F4) through the electronics connector (F2), via traces D5 and E5, respectively, and terminals D8 and E8 respectively. As shown in Figure 12, the electronics connector (F2) may be composed of two separate connectors (F2a and F2b).

[00111] During an electroporation procedure, electrical pulses are applied to the cell layer through the two electroporation electrodes (D2 and E2). As cells are electroporated, electrical current flows through the cell membrane(s) through electrical field-induced pore formation. During this process, molecular entities can be delivered into the cells by various transport mechanisms, including passive transport (diffusion), electrophoretic force, electrokinetic or electroosmotic flow, or some combination of transport mechanisms. The magnitude of this electrical current is dependent on the degree of electroporation of the cells, i.e., the greater the amount of poration of the cells, the greater the flow of electrical current. This electroporation-induced electrical current can be measured with the transimpedance amplifier (F3) and can be used to monitor the process of cell electroporation. In addition to the current measurement, the two probe electrodes are used to precisely measure the voltage drop across the cell layer during the electroporation process. Because of the voltage drops at the electrode-electrolyte interfaces, the voltage applied to the two electroporation electrodes (D2 and E2) is not the same as the voltage imposed across the cell layer. The two probe electrodes (D3 and E3) are connected to the high input-impedance amplifier (F4). Thus, since substantially no current flows through these electrodes (D3 and E3), there is no appreciable voltage drop over the electrode-electrolyte interfaces, and the differential voltage between them, as measured by the high input impedance amplifier (F3), more accurately reflects an the voltage across the biological cell layer. The electrical resistance of the cell layer can thus be approximately calculated, for example, by a computer, with cross-cell layer voltage measurement and cross-current measurement; the relative resistance measurement indicates the degree of electroporation of the cell layer. In the case that the signal from the power amplifier (Fl) contains AC components, the electrical impedance of the cell layer can be computed, and thus any reference to resistance in this disclosure can be replaced by true impedance. The electrical membrane resistance or impedance can be used as feedback to fine-tune electroporation pulses, in order to achieve highly controlled electroporation of the cells. Further, the resistance can be used for monitoring the recovery process of the cell membranes after electroporation.

[00112] Most conventional electroporation devices electroporate cells in suspension, i.e., cells dispersed in an aqueous medium, unattached to a surface. One problem associated with electroporation of cells in suspension is that it is neither possible to measure the voltage drop over any individual cell, nor to exert any control over the voltage. Moreover, due to inhomogeneities in the suspension, individual cells will see a broad range of voltages. Cell death is thus a common during electroporation of cells in suspension, as electroporation at high voltage can irreversibly damage the cell membrane. In addition, there will be some population of cells in solution that do not experience a suiricieni voltage ana remain uπauected by the process. In the present invention, cells are attached or intimately associated with a porous surface, and assume the form of a contiguous layer on that surface. Accordingly, the electrical current must flow through the cells, making it possible to accurately measure, and thus precisely control, the voltage over the cell layer using the differential amplifier (F4) through the probe electrodes (D3 and E3). If adherent cells are cultured on the supported porous membrane (B) for sufficient time, the resulting layer of cells will typically form a monolayer; that is, the thickness of the cell layer will approximate the width of a single cell. It is important to note that other techniques, as will be described below, may also result in a cell layer with the thickness of a single cell. As a result, the voltage detected by the differential amplifier (F4) through the probe electrodes (D3 and E3) typically represents the voltage over a single cell. Since the present invention allows both measurement and precise control of the voltage applied to a single cell, the invention provides a means of ensuring a priori that cells are not irreversibly damaged or killed during electroporation. In cases where the cell layer is deeper than the width of a single cell (e.g., biological tissue, spheroids and slices and adherent cells that are not contact-inhibited), the determination of the appropriate voltage must take into account not only the voltage imposed across a single cell required to initiate electroporation, but also thickness of the cell layer and the impedance of the extracellular pathways.

[00113] An additional advantage of this configuration is that since the cell layer presents a large electrical impedance, the bulk electrolyte (electroporation buffer) impedance becomes negligible, and most of the voltage applied to the electroporation electrodes (D2 and E2) drops over the cell. This "focusing" of the electric field permits application of electroporation voltages to the electroporation electrodes (D2 and E2) that is very close to the actual cross-cell layer voltage required to initiate electroporation (approximately between 0.3V and 1.0V for the case of a cell layer whose thickness is the width of a single cell). Thus, voltages applied to the electroporation electrodes (D2 and E2) in the present invention can be more than three orders of magnitude lower than those used in electroporation systems where the cells are in suspension. Lower voltages, in turn, reduce the complexity, size and cost of the power amplifier (Fl), and also allow electroporation pulses of arbitrary shape and duration without adding complexity to the power amplifier (Fl). It is important to note that simply crowding cells together closely increases electrical impedance; as the spaces between cells get smaller, the impedance of the gaps increases. Thus, provided that current is not allowed to flow around regions of densely crowded cells, a certain amount of electric field 'focusing' will occur. This phenomenon will be particularly evident in the cases that the cell layer is thicker than the width of a single cell (e.g., biological tissue, spheroids and slices and adherent cells that are not contact inhibited).

c. Use of the modular electroporative cartridge for preventing culture contamination

[00114] The modular electroporative cartridge provides a sealed environment that protects the cells on the supported porous membrane (B) from contamination by any local microbial life forms that commonly infect cultured cells. Thus, the cartridge can be transported outside of a cell culture hood, obviating the necessity of bringing electroporation equipment inside the hood. Moreover, cells can be cultured while inside the cartridge; that is, the entire cartridge, including cell insert and cells, can be placed in an incubator. Features such as these exemplify the cell-culture friendly aspect of these embodiments of the inventive cartridge, as described above. d. Methods tor Clocking pores on a porous membrane with cells

[00115] Effective blocking of the pores on the porous membrane (with cells or other material) is highly preferred, in order to achieve highly controlled electroporation using the electroporative cartridge. Pores that are not blocked by cells, as would be the case, for example, in a sub-confluent cell layer, produce parasitic current pathways when electroporation pulses are applied. Such leakage of current reduces the accuracy in trans-membrane current measurement and also deteriorates the "focusing" effect of this configuration, resulting in less effective electroporation of the cells.

[00116] The percentage of pores that are effectively blocked can be evaluated by simply measuring the overall impedance of the cell-covered porous membrane. This is because the effective impedance of a biological cell is far larger than that of an uncovered pore. Therefore, the more pores that are effectively covered by cells or blocked by non-conductive substances, the larger the overall impedance of the cell-membrane complex will be. The correlation between pore coverage and overall impedance can be readily established; by applying a low voltage to electroporation electrodes (D2 and E2) that does not induce electroporation in cells, and by measuring the corresponding impedance, the effectiveness of pore-coverage can be evaluated. This impedance measurement can be very helpful in later determination of the optimal electroporation voltages.

i. Blocking pores through cell growth

[00117] There are several ways to effectively block the supported porous membrane such that electrical currents are forced to flow through cells during electroporation process. Figure 6 illustrates the first method, which is the formation of a confluent layer of adherent cells to cover a porous membrane (Bl). In this process, adherent cells are cultured on a porous membrane which is constructed of materials such as polycarbonate, poly(ethylene terephthalate) (PET) or polytetrafluoroethylene (PTFE), and has been treated by various processes, such as electrical plasma-treatment, or being coated with biological or organic molecules, such as collagen, fibronectin, or polylysine, that encourage cell attachment and growth. When cells grow into an interconnected (confluent) monolayer that covers the entire porous membrane, they effectively electrically block all pores. In this scenario, an insignificant amount of current flows between the two electroporation electrodes (D2 and E2) if the applied electrical voltage is not large enough to induce electroporation in the cells; as the cell membranes have very high electrical impedance, current cannot flow through them or through the porous membrane, which in turn provide the only current pathways between the two electrodes. In many cases, it is difficult or not desirable to grow cells into a 100% confluent layer. This may result in many uncovered pores, which as discussed previously can be detrimental to the performance of the modular cartridge device. To solve this problem, non-conductive substances, such as glass or polymer particles or additional cells, can be added to block the uncovered pores through various mechanisms. As illustrated in Figure 7, one method to achieve this is through generating a pressure difference between the two sides of the porous membrane to pull the substances toward the uncovered pores and block/clog them.

ii. Blocking pores with mechanical methods

[00118] In some cases, cell growth, as described above, will not sufficiently block the pores. Illustrative examples include: Suspension cells, which normally do not attach to substrates and form an adherent layer; spheroids, which are typically in suspension; weakly adherent cells, which may not attach sufficiently to the membrane surface; aggregates of cells, such as slices of biological tissue, that may not be attached to the membrane, and thus will not seal the pores; and cases where it may be desirable to suspend cells, even if they can be adhered or attached to the membrane. A mechanical approach is required in such situations for sealing of the pores. For the remainder of this section, the term "cells" refers to any such entity where a mechanical method is required to move it to the membrane in order to block the pores.

[00119] One such mechanical method involves generating a pressure differential between two sides of the porous membrane such that the cells are pulled toward pores; thus the deformed cells can effectively block the pores as illustrated in Figure 8. Because excessive pressure difference can cause mechanical damage to the cell membrane, the pressure must be properly regulated so as to produce a good seal between cells and pores, but avoid damaging the cells. This pressure difference may be generated by: providing a seal (G) between the top unit (A) and the supported porous membrane (B), and applying a positive pressure through one or more small holes (Al) in the top unit (Figure 13); providing a seal (H) between the bottom chamber (C) and supported porous membrane (B), and a negative pressure is applied through one or more small holes (Cl) in the bottom chamber (Figure 14); or some external device prior to insertion of the supported porous membrane (B). In the former two cases, the pressure may be applied throughout an electroporating procedure. Additionally, the pressure differential may be reversed following electroporation to assist in recovering the electroporated cells from the cartridge. Figures 13 and 14 illustrate the realization of the seals (G and H) through the use of O-rings. The holes Al and Cl are shown with fittings (A2 and C2) for mating with tubes or other fittings. A simple valve may also be incorporated so as to prevent leakage of electroporation buffer and entities for molecular transfer.

[00120] Another mechanical method of moving the cells into a position whereby they block the pores is to simply allow them to settle to the membrane under the influence of gravity. In cases where this is too time consuming, or does not achieve the desired result, more force may be required. Centrifugation can provide this force; the entire supported porous membrane, here embodied as a cell insert (B) along with cells in a suspension of liquid is placed in a centrifuge such that the cells are forced onto the membrane under the centrifugal force. The entire modular cartridge device, that is, the supported porous membrane (B), assembled with the top unit (A) and the bottom chamber (C), may be placed in the centrifuge.

[00121] A third mechanical technique for moving the cells to the membrane involves the use of a disc (I), of a surface area equal to, or slightly smaller than, the membrane (Bl). This disc is inserted in the cell insert (B) after the introduction of cells in a suspension of liquid, such that the surface of the disc and the membrane are parallel to each other. Through some mechanical means, the disc (I) is moved toward the porous membrane (Bl). One means of such movement is settling under the force of gravity. If more force is required, centrifugation is the preferred technique. In the embodiment shown in Figure 15a, a handle or plunger (H) is attached to the disc (I) to control the position of the disc relative to the porous membrane (Bl). In one embodiment, the disc (I) is itself a microporous membrane, but of significantly higher porosity than the supported porous membrane (Bl). The cells are thus pushed by the disc (I) toward the cell insert membrane. The disc (I) is pressed downward to a desired separation between the two porous membranes. It may be preferred that a mechanical stop (12) is designed to define sufficient clearance between the disc (I) and the membrane (Bl) to prevent crushing the cells. Note that in the case that gravity or centrifugation is used to move the disc (I) downward, the mechanical stop could be built into the disc (I) itself. In a preferred embodiment, the material density and dimensions of the disc (I) are carefully chosen, and moreover the force applied to the disc is limited, such that the cells themselves can act as the mechanical stop without being damaged. It may be desirable to include sufficient cells in the suspension such that when the cells reach the top surface of the membrane, they actually form more than one layer; as described above, densely crowed cells can themselves produce a 'field focusing' effect. It may also be necessary to include a flexible lip (13) to prevent cells and liquid from escaping around the disc (I) as it is pressed downward. Ideally, the thickness and porosity of the disc (1) are chosen such that when permeated with the electroporation buffer, the electrical resistance of the disc (I) becomes negligible; thus, the disc (I) may be left in the cell insert without significantly perturbing the electroporation procedure described above.

[00122] Alternately, the porosity of the disc (I) may be the same as, or comparable to, that of the supported porous membrane (Bl). A downward action generates a pressure differential that pulls the cells into the micro pores of both the porous membrane (Bl) and the disc (I). The volume and cell density of the cell suspension should be chosen such that when the plunger is fully depressed, substantially all of the pores in the membrane (Bl) and the disc (I) are blocked. Depending on the final distance between the disc (I) and the membrane (Bl), this realization may result in multiple cell layers in series, the number of layers controllable by the total number of cells delivered into the cartridge. Thus, the chance of a parasitic current pathway existing are reduced; for instance, even if a pore on the membrane (Bl) is not blocked, the pore may be electrically in series with a blocked pore on the disc (I).

[00123] Finally, the disc may be non-porous. This is exactly as described for the case in which the disc porosity is comparable to the membrane, with the exception that in this case the cells are pulled only to the pores in the membrane (Bl). In this embodiment, the number of cells must be sufficient such that when the disc reaches its final position, in one embodiment dictated by the mechanical stop (13), the pores are substantially blocked. As the disc is nonporous, it must be removed before the top unit (A) is put in place. Alternately, with a minor modification to the top unit (A), the top electrode assembly can also perform the same function provided by the disc; it is only necessary to add the lip (13) to the top unit (Figure 15b). The flange B2 should be designed to facilitate pressure relief; this is straightforward, as need not run along the entire rim of the insert. It is important to note that this embodiment operates on the same principles described above for a pressure differential. However, instead of applying a positive pressure through a small hole (Al), the positive pressure is applied by the downward movement of the top unit (A) itself. The lip (13) serves the same function as the seal (G). Through this example, it is clear that two mechanical techniques for moving cells to block the pores, in this case pressure differential and a disc can be combined.

[00124] By extension, any of these mechanical techniques, pressure differential, hydrodynamic follow, settling, centrifugation, or a disc (I), can be used in any combination. They can also be used in conjunction with an adherent cell layer resulting from cell growth, as described in section (d)(i); for example, it may be necessary to block the pores in areas not covered by the adherent cell layer. Additionally, these mechanical techniques can be combined with the use of non-conductive substances, such as glass or polymer particles, to block any uncovered pores, as described above. For example, in the case where it is desired to electroporate cells which are fewer in number than pores on the porous membrane (Bl), an effective protocol may include adding the cells in liquid suspension to the cell insert (B); applying a pressure differential to move the cells to block pores; adding sufficient particles to cover the remaining pores; and applying a pressure differential to move the particles to plug the remaining pores. Additionally, to promote immobilization or adhesion of the cells to the porous membrane (Bl), it snouiα De tissue cuirure ireaieα anα/or coaieu with cell adhesion promoting factors (such as polylysine).

[00125] A schematic for another embodiment of the present invention, a flow-enabled cartridge, is depicted in Figure 16. The flow cartridge is a closed system that allows the application of a positive or negative pressure differential across the porous membrane (Bl). This embodiment operates on the same principles and processes outlined above for a pressure differential. The membrane is contained between a top (A) and bottom (C) part. The top and bottom parts contain the electrodes (D and E). This flow cell is compatible with all of the electrode concepts presented above. The cartridge features at least two ports above (Al) the membrane, and at least two ports below (Cl) the supported porous membrane (B). In addition to liquid flow. through the membrane (perpendicular flow), the multiple ports enable flow parallel to the membrane (transverse flow), which aids the introduction of new reagents and also aids the purging of bubbles and previous reagents from the cartridge. Additionally, transverse flow may be sufficient to immobilize the cells to the membrane in some embodiments. The shape of the flow cavity and the choice of materials can be optimized to enhance the exchange of fluid and the removal of bubbles. The flow cartridge is a highly scalable embodiment, accommodating a single pore and growing to accommodate as many cells as desired. The design of the flow cartridge is amenable to high-volume production, making this cartridge a potential consumable product.

e. Use of the modular electroporative cartridge for controlled electro po ration of tissue

[00126] The inventive cartridge can also be used to control electroporation in tissue and tissue slices. In a typical electroporation procedure for tissue, the tissue sample is placed on supported porous membrane (Bl), which is then placed between the two electroporation electrodes (D2 and E2), for in vitro electroporation. The tissue sample should be sized such that it covers the majority of the membrane (Bl). Alternately, or additionally, the tissue sample may be allowed to culture on the membrane (Bl) such that it attaches and spreads to cover the membrane (Bl) fully. For some tissues, the structural support of the membrane is unnecessary, and the assembly of cells within the tissue provide adequate resistance to current flow to allow controlled electroporation without a membrane. An example tissue that meets these criteria is skin. An electrolyte is introduced to generate good contact between the tissue and the electrodes. Then, electrical pulses are applied to the tissue through the two electroporation electrodes (D2 and E2) that are connected to the power amplifier (Fl) and the transimpedance amplifier (F3). Measuring the electrical current through this electrical circuit is dependent on the overall and average degree of electroporation that the cells in the tissue sample between the electrodes experience. Once the cells comprising the tissue are electroporated, electrical current flow through the cells will increase; the magnitude of the electrical current becomes dependent on the degree of electroporation of the cells in tissue. This cross-tissue electrical current can be measured with the transimpedance amplifier (F3) and can be used to monitor the process of electroporation of the cell membranes. In addition to the current measurement, the two inserted probe electrodes (D3 and E3) are used to measure the voltage drop across the tissue during the electroporation process. The electrodes (D3 and E3) are connected to the high input-impedance amplifier (F4). Thus, while substantially no current flows through these electrodes (D3 and E3), there is no appreciable voltage drop over the electrode- electrolyte interfaces, and the differential voltage between them provides an accurate measurement of the voltage across the tissue. The electrical impedance of the tissue is thus determined from cross-tissue voltage measurement with the probe electrodes (D3 and E3) and cross-current measurement with the circuit attached to the electroporation electrodes (D2 and E2). The impedance measurement reveals the degree of electroporation of the ceiis in tissue as me ceiiuiar impedance is αirecny dependent on the extent of cellular electroporation. In addition to monitoring the electroporation, the electrical current measurement as well as cellular impedance can be used as feedback for fine-tuning of electroporation pulses to achieve highly controlled electroporation of the cells in tissue. Any feedback scheme must account for the fact that the electrical impedance of tissue depends not only by the impedance of single cells, but also the resistance of the extracellular pathways and the thickness of the tissue.

[00127] The advantages of this invention also extend to supported cells with a thickness greater than the width of a single cell, as in, for example, the cases of biological tissue slices and adherent cells that are not contact inhibited. First of all, as described above, the impedance measurement can be used to detect the initiation of electroporation, and thus can be used to fine-tune the applied imposed voltages to achieve electroporation. Further, the voltage required to electroporate a cell layer with a thickness of multiple cells can be predicted. This prediction must take into account not only the magnitude of voltage imposed across a single cell that is required to initiate electroporation, but also the thickness of the cell layer and the impedance of the extracellular pathways. Using the present invention, this prescribed voltage may be applied to the cell layer. Thus, the present invention provides a means of ensuring a priori that the cells comprising a tissue sample are not killed during electroporation. Finally, since the tissue presents a large electrical resistance, the bulk electrolyte (electroporation buffer) impedance becomes negligible, and most of the voltage applied to the electroporation electrodes (D2 and E2) drops over the tissue. This effect reduces the demands on the power amplifier (Fl).

f. Processing multiple electro porative cartridges

[00128] The modular electroporative cartridges are designed for processing pluralities of cells within a single electroporative procedure, and further, multiple cartridges can be handled simultaneously, thereby creating a mechanism for scaling up of standardized, high efficiency, and high throughput procedures. Processing more than one cell layer at once is easily accomplished using the present invention by including an electronics connector (F2) for each of a plurality of cartridges. The cartridges contain independent cell layers that require processing. As shown in Figure 17, each of the electronics connectors is connected to four multiplexers (F5, F6, F7 and F8). The multiplexers simultaneously select inputs from the same cartridge, thus connecting only that cartridge to the measurement and control electronics (Fl, F3 and F4). Equivalently, the four N-to-1 multiplexers can be combined into a single N-to-4 multiplexer, where N is the number of cartridges. Efficiency can also be increased by providing measurement and control electronics for each modular cartridge device; thus, all of the cell layers are processed simultaneously. This "parallel" technique can clearly be combined with the multiplexing, sequential technique. The optimal solution will depend on the number of cell layers to be processed, the required processing time and constraints on electronics cost and complexity.

[00129] For ease of handling and accommodation of workflow, the design of the cartridge itself may also be modified to include more than one cell layer. It is now convenient to use the term "well" to describe the parts of the top unit and bottom chamber, as well as the electrodes, associated with a single cell layer. Figure 18a depicts a realization of a multiple cell layer cartridge that is compatible with the standard 6-well plate format for cell inserts. Note that Figure 18a is a side view, and thus only shows three wells. Also, the cell inserts may be individual units as shown in Figure 6a, and need not be connected together as shown in Figure 18a. The top unit (J) and bottom chamber (K) accommodate 6 separate cell inserts, in two rows of three. The electrode assemblies (L and M) can be embodied as flexible printed circuits (Figure 14), as discussed in detail for the single cell layer modular cartridge αevice. i πe πexiDie printed circuits (.rrus; ior trie top and bottom electrode assemblies are similar, and differ primarily in dimensions; this, too, was described for a single cell layer. The multiple cell layer concept can be extended arbitrarily, for example including but not limited to the standard 12-, 24- and 96-well plate formats.

[00130] Embedding the electrode assemblies in the top unit and bottom chamber, as described above, may be preferable for larger arrays, where the time and complexity of assembling with a FPC may become prohibitive. This concept is demonstrated in Figure 18b; the cell inserts have been shown as individual units, but may be connected together as in Figure 18a. The manufacturing technique is much as described above for plastic encapsulated electrode assemblies, with the major exception that the singulation step only disconnects the separate wells electrically; the mold is defined such that the individual wells of the top unit are mechanically connected together, as are the individual wells of the bottom chamber. Again, this concept can be scaled up arbitrarily. It is worth noting that the realization of the electrical connector (F2) as a "bed-of-nails" is particularly suitable for this configuration.

g. Correcting for electric field non-uniformity

[00131] Near the edges of the cell insert membrane (Bl), field fringing will disrupt the uniformity of the electric field imposed on the cell layer, particularly as the size of the insert membrane decreases. Non-uniformity of the cell layer itself may also distort the applied electric field. These effects can be counteracted through the use of a plurality of concentric top electroporation electrodes (D2) along with a plurality of top probe electrodes (D3). Figure 20 illustrates such a configuration. Each probe electrode (D3a, D3b, and D3c) is connected to independent high-input impedance differential amplifiers, each of which employs the bottom probe electrode (E3) for the other side of the differential voltage measurement. Alternately, the three top probe electrodes are multiplexed to the same input of a single amplifier. Due to circular symmetry, each probe electrode senses the voltage at a specific distance from the center of the electrode assembly. The voltage detected at each probe electrode is used to control the voltage applied to the corresponding electroporation electrode; that is, the voltages at D3a, D3b, and D3c are used to control D2a, D2b, and D2c respectively. Ideally, the control of the electroporation electrodes will also involve corrections based on detailed simulations of the electric fields for any given electrode configuration. Such correction factors may even eliminate the need for the additional probe electrodes D3b and D3c; the voltages at D3b and D3c can be predicted based on the simulations and the voltage at D3a.

h. Cell layer visualization

[00132] Visualization of the cell layer is important in many applications and workflow protocols. For example, the molecular entities transported into the cells, or the byproducts of these entities, may be tagged such that fluorescent microscopy can be used to confirm successful transport. Additionally, visualization allows detection of bubbles between the electrodes and the cell layer or the membrane that will interfere with proper modular cartridge device operation. In the embodiments described above, one or both of the electrode assemblies must be transparent or translucent to permit optical observation of the cell layer. One solution is to use ITO (indium tin oxide) as the electrode material. However, in many cases, particularly when the signal from the power amplifier (Fl) contains a DC component, Ag/AgCl is the preferred electrode material because of its superior electrochemical properties. [00133] ^! -^f Ohe^rrticroscop^cdmpaitible^{^}eiribOdiment of the invention that accommodates the use of opaque Ag/AgCl electrodes is depicted in cross section in Figure 21a. Rather than covering the bottom surface of the bottom chamber, the bottom electrode assembly now takes the form of a band around the bottom of the bottom chamber sidewall. The bottom electrode assembly, realized as a flexible printed circuit, is shown in Figure 22. Use of such a bottom electrode assembly with the top electrode assembly (D) shown in Figure 7 would impose a nonuniform electric field across the cell layer. In order to ensure a more homogeneous electroporation process, the top electrode assembly may be configured and controlled as described in section (g). Construction of the bottom structure with a transparent material, for example, polystyrene, is necessary for visual access.

[00134] Another approach is to replace the opaque electrodes with grid or mesh electrodes, an example portion of which is shown in Figure 21b. When carefully engineered, the distortion in electrical field due to the electrode geometry can be confined within one hundred micrometers of the electrode surface. Beyond that, the electrical field generated by a grid/mesh electrode is very similar with that of a continuous sheet electrode; therefore, the grid/mesh electrodes can be approximated as a sheet electrode as depicted in the previous embodiments. The optical characteristics of the grid/mesh electrodes depend on the width of the line electrodes, VV, and the spacing between them, S, as defined in Figure 21b. In general, small width and large space result in better optical characteristics, but also induce more local distortion in electrical field near the electrode surface. Moreover, very narrow line electrodes result in higher current density, which is not desirable as it leads to deterioration of the electrodes. Taking into account of all the factors, the width of the line electrodes is in the range of about 20μm to about 2mm and the space between them is in the range of about 20μm to about 10mm, for the embodiments previously described.

i. Bubble prevention

[00135] Bubbles trapped between the top electrodes (D2 and D3) and the cell layer will interfere with modular cartridge device operation. Moreover, these bubbles may be difficult, or impossible, to see. An approach for allowing such bubbles to escape of their own accord is to make the associated surfaces, for example the top electrode assembly, "convex". The embodiment shown in Figure 23 is conical. The top electrode assembly shown in Figure 24 would conform to such a structure. Specifically, the portion of the assembly defining the electrodes, a sector with a radius equal to the slant height, and a length equal to the circumference of the base of the cone, is the development of the conical surface. Use of such a contoured top unit with a single top electroporation electrode (D2) would impose a non-uniform electric field across the cell layer. In order to provide a more uniform electroporation process, the top electrode assembly may be configured with multiple top electroporation electrodes as discussed in section (g).

j. Embodiments of the membrane

[00136] The porous membrane (Bl) can take many forms, but have in common the features of comprising a non-conductive material and a plurality of pores, the pores being smaller than the diameter of biological cells. Several embodiments are commercially available, and pre-assembled as a cell insert (B). In one preferred embodiment, the pores of the membrane take the form of densely packed "torturous paths." A typical off-the-shelf torturous path membrane is constructed of PTFE and is about 50 μm thick. The pore diameter typically falls within the range of about 0.1 μm - 1.0 μm. The relative pore density is such that the overall porosity of the membrane (as aeiinecroy me ratio oi me sum or me areab υi an the pores to the total area defined by the outline of the membrane) is approximately 50% to 80%. Another embodiment, the track-etched membrane, is typically constructed of PET or polycarbonate and is usually around 10 μm thick. Pore diameters from about 0.4 μm up to about 8.0 μm are commercially available. Pore densities range from about 1.OxIO⁵ to 1.0 x 10⁸ pore/cm². A third type of commercially available membrane is composed of alumina, with pore diameter ranging from 0.02 μm to 0.2 μm and porosity ranging from 25% to 50%. Such small pores are advantageous for use with very small cells, such as platelets.

[00137] The present invention is not limited to the use of commercially available membranes. In some cases, custom variations of the membrane types described above may be preferred. For example, when working with spheroids, it may be desirable to use a membrane with pores of diameter smaller than, but on the scale of, the spheroids themselves, such that a spheroid can "wedge" into a pore. As spheroids can be large (greater than 100 μm in diameter in some cases) the pores may need to be larger than the commercially available 8.0 μm. In other cases, such as when: a uniform array of pores is required; the required pores are larger than what can be created using the above-mentioned techniques; or a very small number of pores is desired, micromachined membranes may be preferred. Suitable materials include silicon dioxide and silicon nitride. The micromachining would begin with a substrate wafer, usually silicon, and would consist of several processing steps, including material deposition and/or growth, photolithographic pattern definition and chemical and/or reactive ion beam etching. Suitable techniques for micromachining are well known to those skilled in the art, and are well documented; an in-depth treatise is not within the scope of this disclosure.

10. Examples a. Materials

[00138] Studies with cells: Various types of cells were examined, including epithelial cells (such as MDCK cell line), fibroblast cells (such as NIH 3T3 cell line), lymphocytes (such as BCBL-I cell line) and primary cells (such as skeletal satellite cells). Cell layers with desirable confluence were formed on various porous cell inserts from Millipore, Corning or BD Biosciences either by 1) growing cells on the porous inserts for various length of time (from a few hours to several days, depending on the cell type), or 2) by applying a pressure differential to the cells, as described previously.

[00139] Studies with tissue: Tissue samples were obtained by slicing fresh mouse liver to a thickness ranging from lmm to 4mm. Then a disk of liver was obtained by pressing a sharp circular tube onto the sample to trim the excess tissue. The resulting sample was then placed in the device for measurement. Livers that were kept prior to resection in a refrigerator at 4 C for three days were used for negative controls.

b. Electrical parameters study

[00140] Studies with cells: Inserts with adherent layers of cells were placed into the configuration shown in Figure Ia, medium was collected, cells were washed with PBS (phosphate buffered solution) and electroporation buffer was introduced to ensure good contact between the electrodes and both sides of the confluent cell layer. The electrical impedance of the sample was measured, after which a series of electroporation pulses were applied and the electrical data recorded. [00i^!4^'iJ' Stu'dies witn tissue'TTfte fissile layer was placed between the electroporation electrodes of the device shown in Figure 5. Electroporation buffer was added to ensure good contact between the electrodes and the tissue.

c. Transfection study

[00142] To assess the efficacy of the controlled electroporation and its ability to introduce various substances into cells, cells were transfected with a variety of molecules, including fluorescent dyes (YOYO-I and PI dyes), nucleotides, such as small and large DNA (e.g., GFP and MyoD genes), and small interfering RNA (siRNA), and proteins, such as antibodies, none of which are permeable to cell membranes under normal conditions. In various experiments, the transfecting reagent was mixed with electroporation buffer at desirable concentrations, and then introduced to the cell culture inserts where cell layer was formed. Delivery of those reagent molecules was enabled by electroporating the cell layer using the methods described above. Transfection expression was evaluated at various time points following electroporation, depending on how long it took for the expression to occur (immediate results are obtained using fluorescent dyes, one to two days are required for gene expression)

d. Electroporation electrical measurement results

[00143] Figure 25 shows a typical three-step electrical pulse, as depicted in Figure 4b, used to study the process of electroporation in cell layers and tissue samples. It includes three contiguous step pulses. The amplitude of the first step pulse is significantly below what is required to produce electroporation; it is used to probe the electrical impedance of the cells or tissue prior to electroporation. The second step pulse was varied in amplitude until a change in the electrical impedance of the cells was detected, indicating occurrence of electroporation. According to the invention, the occurrence of electroporation should result in a decrease in the electrical impedance of the cells, while electrical pulses that do not produce electroporation will not affect the electrical impedance of the cells. It should be noted that the polarity of the pulse was chosen such that the top electroporation electrode was at a lower potential than the bottom electrode, in order to facilitate the insertion of negatively charged molecules (such as DNA plasmids) into the cells through electrophoresis. The third electrical pulse has the same amplitude as the first. The impedance measured during the third pulse was used to determine if the electroporation was reversible or not. In various experiments, the effect of several sets of three contiguous step pulses, separated by various intervals of time were studied.

[00144] Figures 26a and 26b illustrate a sequence of electroporation pulses applied to satellite cells. The top graph in each figure shows the voltage imposed across the cell layer in response to the three-step electroporation pulse described above; the first voltage step corresponds to the pre-electroporation impedance measurement pulse (50mV/500ms), followed by the electroporation step (300m V/l 00ms) and finally the post-electroporation impedance measurement pulse (50mV/500ms). The middle graph shows the current through the cell layer. Again, it should be noted that the current is negative and that the current during the middle electroporation pulse is larger than the current before and after the electroporation pulse. The bottom graph is the most important and illustrates the impedance of the cell layer. It should be noted that in all the figures, the cell layer impedance during the pre- electroporation measurement pulse is constant. In various experiments it was found that the impedance measured remains the same for pulses with increasing amplitude until a threshold is reached.

[00145] However, when the amplitude of the pulse reached a threshold value, a significant drop was observed in tne-,eiecιπcai impeαaπce, similar to ine αrop snown in Figures 26a and 26b during the second, higher-amplitude electroporation step pulse. It is very interesting to note that the impedance decreases gradually throughout the electroporation portion of the pulse, which is consistent with the theory of electroporation. Figure 26b, which depicts an electroporation pulse applied at one minute after the first, indicates that the cell membrane essentially seals and returns to its original impedance within the one-minute interval.

[00146] Figures 27 shows electroporation of cells using a 4-step electroporation pulse as depicted in Figure 4c. It can be seen that the electroporation portion of the 4-step pulse includes a 800mV/lsec main electroporation pulse, which was used to initiate electroporation, and a 300mV/2sec "maintaining" pulse, which was used to keep the high permeability state of the electroporated cells and to facilitate cross-membrane transfer of charged molecules via electrophoresis. As can be seen in the impedance plot, the impedance of the cell monolayer dropped significantly when the 80OmV pulse was imposed (from 22 ohms to 3.6 ohms) due to electroporation. When the pulse amplitude reduced to 30OmV (the maintaining pulse), the impedance of the cell layer still remain low at approximately 3.6 ohms, indicating the cells were kept at a highly permeable state by the low post-electroporation pulse. Figure 27b shows the data from an identical pulse applied a minute later. It clearly shows that the cell recovered during this one minute interval as the initial impedance obtained with the second pulse went back to about 17.7 ohms, which was significantly higher than the impedance of the cells in electroporated state. Figure 27b also illustrated the effect of the maintaining pulse, which kept the impedance of cells low after the cells were first electroporated by the 80OmV pulse.

[00147] Figures 28a and 28b illustrate the typical behavior of fresh liver tissue during electroporation. It is evident that in response to the three-step pulse electroporation protocol, the tissue exhibits the same behavior as the layer of cells. Obviously the impedance of the layer of tissue is higher than that of the layer of cells. However, it also shows no change in impedance during the first portion of the pulse, which does not induce electroporation. Then, during the second pulse, which induces electroporation, the impedance drops. During the third pulse, impedance returns to its initial value. Figure 28c shows the typical behavior of dead tissue. It can be seen that the impedance of the tissue slice is significantly lower than that of fresh tissue because dead cells have lower impedance than living cells, as their membranes are already impaired. It can also be clearly seen that the impedance of the dead tissue remained fairly constant during the entire pulse, indicating there was no further permeabilization in the impaired dead cell membranes even when high electrical pulses are applied. Thus, the change in impedance with electroporation is the hallmark of live cells and is what makes it possible to control the process of electroporation in live tissue, as claimed in this invention.

e. Electroporation efficiency and cell viability assessment

[00148] Extensive experiments were performed to evaluate electroporation efficiency using the apparatus described above. Cell viability analysis was also carried out to assess the degree of damage to cells due to electroporation using our methods.

[00149] Figure 29 shows illustrates the introduction of propidium iodide (PI), a fluorescent DNA stain that can not penetrate the membranes of normal cells, using our apparatus and method. Madin Darby Canine Kidney (MDCK) cells were grown on a porous cell insert (Corning) for three days to form a confluent cell monolayer. 5μL PI was added in PBS electroporation buffer, then three three-step pulses (Figure 6) with 600mv/300ms eieciropσrauoπ puises were appneα ai iiiiiiiuic iiiterval to electroporate the cells in order to introduce the membrane impermeant PI into the cells. Figure 10 was taken with a scanning fluorescent microscope under 2OX objective. From the image, more than 90% cells in the monolayer were stained (red cells) indicating that more than 90% cells were effectively electroporated. In fact, a high electroporation efficiency (from 70% to nearly 100%) was consistently achieved with this method on a variety of cells. The electroporation efficiency depends not only on electroporation pulses, but also on the confluence of the cell layer, which was explained in our previous sections.

[00150] Cell viability after electroporation was assessed by adding membrane impermeant fluorescent dyes (such as PI, EthD-2 and YOYO-I) to cell buffer after electroporation pulses. The dyes are commonly used to mark dead cells because dead cells cannot exclude the dye molecules due to their impaired membranes. Figure 30 shows MDCK cells stained with PI after the typical procedures used to obtain electroporation. The nearly completely dark image indicated that there were virtually no dead cells (dead cells should appear in red color) after electroporation, meaning the electroporation didn't induce any noticeable membrane damages due to irreversible electroporation, which is commonly associated with traditional electroporation apparatuses. In addition to MDCK cells, such viability analysis was performed on other cells, and cell viability of more than 95% was consistently achieved under our typical electroporation conditions.

f. Gene transfection

[00151] To evaluate the efficiency of gene transfection using our methods, two types of genes, GFP reporter gene and MyoD gene, were introduced into various cell types. Typically, 5μg DNA plasmids were mixed with electroporation buffer, and both three-step and four-step pulses (Figures 4b and 4c) were applied to electroporate cells for gene transfer. The polarity of applied pulses was set to be negative in order to facilitate insertion of negatively charged DNA into cells through electrophoresis. Expression of the genes was typically evaluated at 24- 72 hours after electroporation. GFP expression was observed under green filter fluorescence microscopy. MDCK cells were viewed on the same porous membrane on which they were cultured. Treated fibroblasts and satellite cells were trypsinized and centrifuged at 1800 rpm for 10 minutes at RT. Pellet was suspended in cold PBS with glucose (2.5gr/L), and centrifuged at 500 rpm for 15 minutes on glass microscope slides.

[00152] Figure 31 shows transfection of GFP reporter gene in a differentiated MDCK monolayer. From the image, more than 95% MDCK cells expressed the reporter gene (cells in green fluorescence), comparing to at most 16% transfection rate reported using other methods, such as chemical transfection (lipofection).

[00153] Figure 32 shows transfection of GFP gene in primary satellite stem cells. More than 95% of cells were positively transfected. It was also found that in every experiment in which the impedance measurements indicated electroporation gene expression occurred, and there was no expression (0%) in the negative controls where there was no electroporation.

[00154] Figure 33 shows transfection of GFP gene in mouse skin fibroblast cells (NIH 3T3 cell line), which indicates a transfection efficiency of more than 90%.

[00155] Figure 34 shows the transfection of large MyoD genes (~40Kb), which converts fibroblast cells into myotube muscle cells, using our apparatus. Through serum deprivation, the MyoD treated fibroblasts differentiated and fused into multinucleated nascent myotuoes that were stained positive for sarcomeric actin/ myosin. These morphologic and myogenic changes were observed in all impedance-monitored electroporation and absent in control fibroblasts. Figures 15a and 15b illustrate the normal fibroblast cells and the converted myotubes that were induced by transfection through electroporation of fibroblasts.

[00156] Figure 35 demonstrates simultaneous co-transfection of CHO cells using GFP and DsRed reporter genes. Figure 37 A is the fluorescent image of only the expressed GFP; Figure 37B is the fluorescent image of only the expressed DsRed. The overlaid image of Figure 37C indicates nearly total transfection efficiency, with virtually all cells expression GFP and DsRed (co-transfection efficiency >90%).

g. Transfection of siRNA

[00157] Figure 36 demonstrates our apparatus's capability of delivering siRNA (small interfering RNA) into cells. In the experiment, fluorescenated siRNA was added in electroporation buffer, and then H460 cells (human lung cancer cell line) were electroporated using the method and conditions previously described. After electroporation, H460 cells were detached from cell inserts by trypsinization, re-suspended and loaded onto a glass slide for fluorescence microscopy. Cells that were uploaded with fluorescenated siRNA molecules appeared in green under fluorescent microscope in Figure 36A. By visual inspection, the efficiency of siRNA introduction was consistently more than 90%. Figure 36B is a western blot demonstrating siRNA efficiency in protein knockdown compared to a siRNA control, indicating the specificity of the siRNA compared to a nonsense sequence control. Cells that received the active siRNA appeared to lose protein as demonstrated by a fainter protein line indicator in the Western blot image. Figure 36C compares flow cytometry data for the H460 for this inventive apparatus compared to siRNA delivery using conventional chemical transfection (lipofection). The flow cytometry data demonstrates the critical effect of the transfection efficiency upon the "observed" efficiency of apoptosis.

h. Transfection of peptides and protein

[00158] Figure 37 demonstrates the ability of the inventive apparatus to transfect cells with peptides and proteins, experiments were performed to introduce a fluorescenated protein (FITC-antimouse Ab) into CHO cells (Chinese hamster ovary cell line). Experiment protocol was similar with the one for siRNA transfection experiment. Figure 37 A shows the fluorescent image of the transfected cells. Cells that were successfully delivered with FITC-Ab appeared in green fluorescence in the image. The image showed that the efficiency of antibody transfection reached nearly 90% with the apparatus. Figure 37B is a fluorescent image of Huh7 cells (hepatocyte- derived cell line) after transfection with fluorescenated peptide (rhodamine-peptide). Cells that were successfully delivered with Rho-peptide appear red in the image. This image shows that the efficiency of peptide delivery reached nearly 100% with the apparatus.

i. Comparison of the inventive controlled electroporation method with conventional methods

[00159] In the case of traditional electroporation (Figure 37, EPl - EP4 indicate typical results), typically less than 50% of the cells survive the process and the transport efficiency is less than 50% for the cells that do survive. At the other extreme, a fraction of the overall cell population experiences no effective electroporation due to insufficient magnitude of the local electric field to which they are exposed. An overall efficiency of electroporation can be understood to be the product of the fraction of cells that are effectively electroporated multiplied by the traction ot cells that survive tne procedure. With such factors at work, traditional electroporation generally produces a highly inhomogeneous result, with less than 25% overall efficiency.

[00160] Most conventional electroporation devices electroporate cells while they are in solution (suspension), a condition in which it is not possible to measure, much less control, the voltage drop over any individual cell. Moreover, due to inhomogenieties in the suspension and on the electrodes, individual cells thus are exposed to a broad range of voltage, a result of the cell population being subjected to significant inhomogeneities in the localized electric field, resulting in significant differences in observed electroporation from cell to cell. Cells exposed to the optimal voltage electroporate optimally, and survive the procedure. On the other hand, cells exposed to a suboptima! voltage survive but do not electroporate well, and those exposed to too high a voltage electroporate irreversibly and die.

Claims

1. An electroporative apparatus for the manipulation of at least one biological cell, the apparatus comprising: an electric cell containing a support capable of holding the biological cell, the support positioned to restrict electric current flow in the electric cell to a flowpath through the supported cell; means for imposing a voltage across the electric cell and for monitoring the value of current, voltage, and electrical impedance and using the value to regulate the current, voltage or a combination of current and voltage; and means for imposing a substantially controlled electric field across the biological cell.

2. The apparatus of claim 1 , in which the electrical cell is a consumable, cell culture-compatible cartridge.

3. The apparatus of claim 1, further comprising means for a minimally prescribed electric field across the biological cell.

4. The apparatus of claim 1, wherein the apparatus imposes a substantially uniform electric field across the biological cell.

5. The apparatus of claim 1, wherein the apparatus is used to permeabilize the biological cell.

6. The apparatus of claim 5, wherein the apparatus enables molecular delivery into the biological cell.

7. The apparatus of claim 5, wherein the apparatus enables biological cell fusion.

8. The apparatus of claim 1, wherein the electric cell comprises at least one electrode, the at least one electrode comprising silver and silver chloride.

9. The apparatus of claim 1, wherein the support divides the interior of the electric cell into first and second electrode chambers.

10. The apparatus of claim 1, wherein the support comprises a material substantially impermeable to electric current, the support further comprising a plurality of pores smaller in width than the width of a biological cell.

1 1. lhe apparatus or claim lυ, lurtner comprising means for immobilizing the biological cell through adhesion or affinity immobilization to form an effective electrically resistive seal over the opening.

12. The apparatus of claim 10, further comprising means for immobilizing the biological cell through pressure differential to form an effective resistive seal over the opening.

13. The apparatus of claim 12, wherein the pressure differential is induced by hydrodynamic flow.

14. The apparatus of claim 10, wherein the assembled combination of electric cell and support provides a means for imposing a substantially controlled electric field across the biological cell.

15. An electroporative apparatus for the manipulation of at least one biological cell, the apparatus comprising: an electric cell; and means for imposing a voltage across the electric cell and for monitoring the value of at least one of the group including current, voltage, and electrical impedance; and means for using the value to regulate the current, voltage or a combination of current and voltage; wherein the apparatus is configured to receive a support capable of holding at least one biological cell, the support positioned to restrict electric current flow in the electric cell to a flowpath through the supported cell.

16. A electroporative method comprising: imposing a voltage across an electrical cell containing a porous membrane, a population of biological cells associated with the membrane; restricting substantially the electric current flow in the electric cell to a flowpath through the supported biological cells; monitoring the value of at least one of the group including current, voltage, and electrical impedance; regulating the current, voltage or a combination of current and voltage, in response to the value; and imposing a substantially controlled electric field across the biological cells.

17. The method of claim 16, further comprising monitoring the electric current flow through the electric cell.

18. 1 he method ot claim 17, further comprising evaluating the impedance of the membrane and the associated biological cells.

19. The method of claim 18, further comprising evaluating the permeability of the biological cell.

20. The method of claim 19, further comprising evaluating the permeability at a voltage that is below the threshold for electroporation.

21. The method of claim 20, further comprising estimating the minimum effective threshold voltage required for electroporation.

22. The method of claim 20, further comprising modifying the applied voltage to a minimal effective threshold voltage sufficient to achieve electroporation.

23. An electro porative method, the method comprising controlling with a computer software program the automation of the following steps: imposing a voltage across an electrical cell containing a porous membrane, a population of biological cells associated with the membrane; restricting substantially the electric current flow in the electric cell to a flowpath through the biological cells; monitoring the value of current, voltage or electrical impedance; regulating the current, voltage or a combination of current and voltage in response to the value; and imposing a substantially controlled electric field across the biological cells; monitoring the electric current flow through the electric cell to evaluate the permeability of the biological cells at a voltage that is below the threshold for electroporation; and modifying the applied voltage to the minimal effective threshold voltage sufficient to achieve electroporation.

24. The method of claim 23, further comprising controlling the computer software program with a via a software interface.

25. The method of claim 24, further comprising initiating the method by a single click at a computer software interface.