ELECTRICAL IMPEDANCE TOMOGRAPHY TO CONTROL ELECTROPORATION

FIELD OF THE INVENTION This invention relates to the field of electroporation and mass transfer across cell membranes in general and the transport of ions across a cell membrane in particular.

BACKGROUND OF THE INVENTION

Electroporation is a technique that is used for introducing chemical species into biological cells, and is performed by exposing the cells to an electric potential that traverses the cell membrane. While its mechanism is not fully understood, electroporation is believed to involve the breakdown of the cell membrane lipid bilayer leading to the formation of transient or permanent pores in the membrane that permit the chemical species to enter the cell by diffusion. The electric potential is typically 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 type and development stage of the cell. As a method of introducing chemical species into cells, electroporation offers numerous advantages: it is simple to use; it can be used to treat whole populations of cells simultaneously; it can be used to introduce essentially any macromolecule into a cell; it can be used with a wide variety of primary or established cell lines and is particularly effective with certain cell lines; and it can be used on both prokaryotic and eukaryotic cells without major modifications or adaptations to cell type and origin.

Electroporation is currently used on cells in suspension or in culture, as well as cells in tissues and organs.

Electroporation is currently 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. The pore formation, or permealization, of the membrane occurs at the cell poles, which are the sites on the cell membranes that directly face the electrodes and thus the sites at which the transmembrane potential is highest Unfortunately, the degree of permeahzauon occurring in electroporation varies with the cell type and also varies among cells in a given population Furthermore, since the procedure is performed m large populations of cells whose properties vary among the individual cells m the population, the electroporation conditions can only be selected to address the average qualities of the cell population, the procedure as currently practiced cannot be adapted to the specific characteπstics of individual cells Of particular concern is that under certain conditions, the electncal potential is too low for a cell membrane to become permeabilized, while under other conditions electroporation can induce irreversible pore formation and cell death A high electπc field, for example, may thus produce an increase in transfection efficiency in one portion of a cell population while causing cell death in another A further problem with known methods of electroporation is that the efficiency of transfection by electroporation can at times be low In the case of DNA, for example, a large amount of DNA is needed in the surrounding medium to achieve effective transformation of the cell Many of the problems identified above are a consequence of the fact that the process of electroporation in both individual cells and tissues cannot be controlled in real time There are no means at present to ascertain in real time when a cell enters a state of electroporation As a result, the outcome of an electroporation protocol can only be determined through the eventual consequences of the mass transfer process and its effect on the cell These occur long after the mass transfer under electroporation has taken place These and other deficiencies of current methods of electroporation are addressed by the present invention

Also relevant to the present invention are current techniques for the study and control of mass transfer across cell membranes Knowledge of mass transfer across cell membranes in nature, both in cells that are functioning normally and in diseased cells, is valuable in the study of certam diseases In addition, the ability to modify and control mass transfer across cell membranes is a useful tool in conductmg research and therapy in modern biotechnology and medicine The introduction or removal of chemical species such as DNA or proteins from the cell to control the function, physiology, or behavior of the cell provides valuable information regarding both normal and abnormal physiological processes of the cell The most common method of effecting and studying mass transfer across a cell membrane is to place the cell in contact with a solution that contains the compound that is to be transported across the membrane, either with or without electroporation This bulk transfer method does not permit precise control or measurement of the mass transfer across the membrane The composition of the solution at specific sites is not known and is variable In addition, when an electπc field is present, the local field intensity will vary from one point to another Furthermore, the surface of the cell that is exposed to the solution is not well defined Cell surface areas vary among cells in a given population, and this leads to significant differences among the cells in the amount of mass transfer For these reasons, the amount of mass transfer achieved by bulk transfer processes is not uniform among cells, and the actual amount transfeπed for any particular cell cannot be determined

Attempts made so far to overcome the limitations of bulk transfer techniques include techniques for treating individual cells that include either the mechanical injection (microinjection) of chemical compounds through the cell membrane or electroporation with microelectrodes In injection techniques, the membrane is penetrated with a needle to deliver a chemical agent, localizing the application of the chemical agent to a small region close to the point of injection This requires manipulation of the cell with the human hand, a technique that is difficult to perform, labor-intensive, and not readily reproducible Electroporation with microelectrodes suffers these problems as well as the lack of any means to detect the onset of electroporation in an individual cell These problems are likewise addressed by the present invention

SUMMARY OF THE INVENTION

Devices, systems and particular methods are disclosed which make it possible to precisely monitor the movement of mateπals across a cell membrane The information gained from momtoπng the movement of materials across a cell membrane may be directly applied to deduce information with respect to the cell and/or its membrane Alternatively, the information obtained from monitoring may be applied in order to control the movement of mateπals across the cell membrane such as by controlling the application of electrical current Devices and systems of the invention make it possible to move charged molecules, and m particular ionic species, across a cell membrane and precisely monitor the occuπence of such When carrying out electroporation using the devices, systems and methods of the mvention the information obtained from monitoπng the movement of the charged particles across the cell membrane is used to control the process of mass transfer across a cell membrane Specifically, the system is used to obtain measurements and changes in electrical impedance across a cell membrane while the mass transfer properties of the cell are changed by the application of electncal cuπent Thus, information obtamed on electrical impedance changes brought by the application of electncal cuπent are used, in real time, in order to control the movement of charged molecules across a cell membrane One aspect of the invention is a method compnsmg creating an electncal charge differential between a first point and a second point separated from the first point by an electπcally conductive medium comprising a biological cell A first electrical parameter between the first and second points is then measured A second electrical parameter is then adjusted based on the measuring of the first electncal parameter The first electrical parameter may be any parameter such as one selected of the group consisting of current, voltage and electncal impedance The second electncal parameter may be any parameter (the same as or different from the first electrical parameter) such as one selected from the group consisting of cuπent, voltage or a combination of cuπent and voltage In a prefeπed embodiment the method further includes placing a material m the electncally conductive medium, and adjustmg the second electncal parameter in order to move the matenal into the biological cell The material placed withm the electπcally conducted medium may be any matenal such as a pharmaceutically active compound or drug, a nucleotide sequence, a fluorescent dye, or a crystal which is specifically designed to effect the cell m a desired manner In accordance with the method vanous conditions are adjusted so that the electncal potential between the two points is sufficiently high so as to cause the cell to be permeabihzed However, the conditions between the two points are further adjusted so that electroporation is reversible and as such does not cause cell death unless that is a result specifically bemg sought In another aspect of the invention the electroporation is not carried out for the purpose of movmg matenal into or out of a cell but rather to analyze the cell or group of cells and provide information or diagnosis of the tissue or individual which contams the tissue In accordance with this method an electncal charge differential is created between a first point and a second point separated from the first point by an electncally conducted medium compnsmg a biological cell A first electncal parameter is then measured between the first and second pomts The measuring of the first electncal parameter is then analyzed in order to determine a character of the cell and m particular a characteπstic of a membrane of the cell The first electncal parameter may be any parameter and is preferably selected from the group consistmg of current, voltage and electncal impedance A second electncal parameter is preferably adjusted m a manner which effects the membrane of the cell or cells present m the medium and the second electncal parameter is any parameter but preferably selected from cuπent, voltage or a combmation of both

Another aspect of the mvention is the device which ts preferably comprised of a first electrode, a second electrode, a source of electricity which may later be connected to the electrodes but is optionally present when the device is sold. The device further includes a means for hindering the flow of electrical current between the first and second electrodes except for electrical current flow through a defined route. Further, the device includes a means for measuring an electrical parameter such as current, voltage or electrical impedance through the defined route and a means for adjusting the source of electricity based on the measured electrical parameter. The means for hindering electrical current flow is preferably comprised of a non- conductive material and defined route comprised of one or more openings each with a diameter less than that of a biological cell so that a cell can fit within the defined route and have a current flow through but preferably not around the cell. The device and systems of the invention can be used within the method in order to move a wide range of materials into or out of the biological cell in order to obtain a desired result. The process can be carried out on an individual cell, a group of cells, cells within a cell culture or within a living organism, e.g. cells within invertebrates and vertebrates including mammals as well as in plants. When carrying out the process on a plurality of cells (e.g. a tissue) a process of imaging the tissue and adjusting electrical cuπent in real time based on images may be used. An imaging technology which may be applied is electrical impedance tomography (EIT). This technology relies on differences in bioelectrical attributes within the body or an organism (e.g. a human) to produce an image. In the method of the invention EIT images can be used in the same manner as the measuring step is used when the process is carried out on a single biological cell. In essence, the EIT technology makes it possible to "see" the effect of increased electrical cuπent flow resulting from electroporation thereby providing information which can be used to precisely adjust the flow of electrical cuπent so that cell membranes are permeabilized while not permanently disrupted.

Another aspect of the invention is a method which comprises sending an electrical cuπent between a first point and a second point separated by the first point by an electrically conductive medium comprising tissue. The tissue may be present within a living organism such as a vertebrate or invertebrate and specifically includes mammals and humans. After the cuπent is sent an image of the tissue is created wherein the image is based on an electrical parameter such as the electrical impedance of the tissue. Using the image as a guide an electrical parameter is adjusted in order to obtain a desired degree of electroporation of biological cells in the tissue.

Electroporation will change electrical impedance and that change can be visualized on the image created. The electrical parameter adjusted may be any parameter such as cuπent, voltage or a combination of both. In a preferred embodiment a material is placed in the electrically conducted medium such as being injected into the tissue and the adjustment of the cuπent is carried out, based on the image, m a manner so as to move the material mto biological cells of the tissue The image created is preferably an impedance image created from known current inputs and measured input voltage using a reconstruction algonthm The impedance image may be created from a known voltage input, a measured cuπent input, or combination of known voltage input and measured current mput

A device for carrying out this method is another aspect of the mvention which device includes a means for creating an electrical cuπent across an electrically conducted medium The device further includes a means for analyzing a first electncal parameter of the electrically conductive medium in order to create an image and a means for adjustmg a second electncal parameter based on the image to obtain a desired degree of electroporation of biological cells m the electrically conductive medium The first electncal parameter is preferably electncal impedance and the second electrical parameter is preferably selected from the group consisting of cuπent, voltage or a combination of both The current is preferably created by a plurality of electrodes positioned about an area of tissue upon which the electroporation is to be earned out The present mvention anses in part from the discovery that the onset and extent of electroporation in a biological cell can be coπelated to changes in the electrical impedance (which term is used herein to mean the ratio of cuπent to voltage) of the biological cell or of a conductive medium that mcludes the biological cell An increase in the cuπent-to-voltage ratio across a biological cell occurs when the cell membrane becomes permeable due to pore formation or because of cell damage or other modes of cell membrane poration Likewise, a decrease in the cuπent-to-voltage ratio through a flowing conductive fluid occurs when the fluid draws a biological cell mto the region between the electrodes m a flow-through electnc cell Thus, by momtonng the impedance of the biological cell or of an electrolyte solution m which the cell is suspended, one can detect the point m time in which pore formation in the cell membrane occurs, as well as the relative degree of cell membrane permeability due to the pore formation This information can then be used to establish that a given cell has m fact undergone electroporation, or to control the electroporation process by governing the selection of the electncal parameters of the process e g the voltage magnitude This discovery is also useful in the simultaneous electroporation of multitudes of cells m a cell culture or m vertebrates, invertebrates or plants Specific embodiments apply the mvention to mammals including humans The process provides a direct indication of the actual occuπence of electroporation and an indication of the degree of electroporation averaged over all the cells being subjected to the process The discovery is likewise useful in the electroporation of biological tissue (masses of biological cells with contiguous membranes) for the same reasons The benefits of this process include a high level of control over the onset and degree of electroporation, together with a more detailed knowledge of the occuπence and degree of permeability created in particular individual cells or cell masses. When applied to individual cells or to a succession of individual cells, this process assures that the individual cells are indeed rendered permeable and are indeed transformed by the introduction of chemical species. The process also offers the ability to increase the efficiency of electroporation by avoiding variations in the electrical environment that would destroy some cells while having an insufficient effect on others.

The invention can be understood by describing a simple embodiment which involves the use of an electrical device or system in which a biological cell can be placed and that contains a baπier that directs the electric cuπent flow and hence the ion flow through a flow path that passes through the biological cell while permitting substantially no electric cuπent to bypass the biological cell. In some of these embodiments, the invention involves the use of an apparatus containing two liquid-retaining chambers separated by a barrier that is substantially impermeable to an electric cuπent. The baπier contains an opening that is smaller than the biological cell such that the biological cell once lodged in the opening will plug or close the opening. To achieve electroporation, the biological cell is secured over the opening by mechanical, chemical and/or biochemical means, preferably in a reversible manner so that the biological cell can later be removed without damage to the biological cell. Once the biological cell is secured over the opening, a voltage is imposed between the two chambers and across the biological cell residing in the opening. The passage of cuπent between the chambers is thus restricted to a path passing through the opening and hence through the biological cell. By monitoring the current-voltage relation in the electric cell, the onset of electroporation is detected and the degree of pore formation is controlled, to both assure that electroporation is occurring and to prevent excessive pore formation and cell death. The user is thus afforded a highly precise knowledge and control of the condition of and the flux across the biological cell membrane.

In another series of embodiments, this invention is useful in the diffusive transport of chemical species into or out of a biological cell. In these embodiments, the cell is again divided into two chambers separated by a baπier, and the biological cell is lodged across an opening in the barrier in such a manner that the passage of liquid around the cell from one chamber to the other is substantially prevented. A liquid solution of the species to be introduced into the biological cell is placed in one or both of the chambers. The concentration of the species in the solution differs from that in the cell (either higher or lower, depending on whether one seeks to introduce or remove the species from the cell), or the concentration in one chamber differs from that m the other chamber

In prefeπed methods of applying this invention to diffusive transport, the solutions in the two chambers differ m concentration such that the driving force for the diffusive transport is between the two chambers themselves rather than between the chambers and the interior of the biological cell Knowledge and controlled monitoring of the concentrations in each of the two chambers on a penodic or continuous basis as the diffusion proceeds, together with the precise knowledge of the dimensions of the opening, enables the user to precisely observe and control the rate and amount of the species that enters the cell The diffusion time can be controlled by imposing stepwise changes in the concentrations in either or both of the chambers, thereby imposmg or removing the concentration differential An application of particular interest is the combination of this type of diffusive transport of a chemical species with controlled electroporation as described in the preceding paragraph

In addition to being useful in connection with electroporation technology the present invention can provide valuable information relating to a cell or group of cells or tissue containing a group of cells by monitoring electncal impedance and thereby providing information regarding the integrity of a cell membrane Specifically, measurements are earned out regarding the movement of charged particles across a cell membrane These measurements are related to the amount of electncal cuπent needed to cany out the diffusion across a cell membrane The mformation obtamed can be analyzed directly or compared to previous measurements of a same tissue or measurements earned out on diseased or normal tissue thereby providmg an indication of the amount of change which has occuπed m the tissue being measured (based on an earlier measurement of the same tissues) or the amount of vaπance between the tissue bemg measured and tissue with impaired cell membranes (e g diseased cells) or a normal cell or tissue The method is earned out in a manner similar to that used for conducting electroporation However, no matenal needs to be added to the medium surroundmg the cells The device is similar in that it is divided mto two portions with a positive electrode on one side and a negative electrode on another side separated by a baπier with the cells bemg positioned along openings on the baπier m a manner which allows for the passage of charged particles through the cell and through the openmg m the barner from one electrode to another The baπier hinders or completely ehmmates the flow of charged particles except through the openmgs The measurement of electncal impedance between the electrodes make it possible to distinguish between cells with an mtact membrane and cells with impaired membranes By more precisely carrying out the measurements it is possible to make determinations with respect to the integnty of a normal cell membrane relative to an impaired (e g diseased) cell membrane

Each of the vanous embodiments of this invention may be used with two or more (1 e a plurality of) biological cells simultaneously, or cell masses such as m tissue which may be m an animal or plant during the process The apparatus described above can be adapted for use with two or more biological cells by arranging the baπier such that the cuπent or diffusive transport will be restricted to a flow path that passes through all of the cells while preventmg bypass around the cells A further application of the concepts of this invention is the electroporation of biological cells suspended in a flowing liquid Electrodes are placed in fixed positions in the flow channel, and a voltage is imposed between the electrodes while current passmg between the electrodes is monitored Biological cells entenng the region between the electrodes will lower the current, the impedance serving as an indication of the presence of one or more cells m the region, and optionally also as a signal to initiate the application of a higher voltage sufficient to achieve electroporation A further application of the device, system and method of the mvention is the electroporation of biological cells present within a tissue which tissue may be present withm a living organism such as a mammal Electrodes are placed m fixed positions withm the tissue, and voltage is applied between the electrodes while cuπent passing between the electrodes is monitored Biological cells with mtact membranes m the region between the electrodes will increase the electncal impedance Accordingly, a measurement of the electncal impedance provides an indication of the presence of one or more cells in the region Electroporation will decrease the measured amount of impedance When the process is carried out on a tissue then the measurement of electncal impedance is a statistical average of the cells present between the electrodes Electroporation methodology of the invention can be carried out on tissue m a living organism usmg an imaging technology which makes it possible to determine when (and preferably to some extent the degree) cell membranes are transformed so as to allow the flow of electncal cuπent through their membranes The prefeπed imaging technology is electncal impedance tomography (EIT) which provides a changing image created from information on differences m bio-electncal attributes of the tissue bemg imaged A typical EIT image is acquired by injecting electrical cuπents into the body and measuring the resulting voltages through an electrode aπay An impedance image is then produced from the known cuπent inputs and the measured voltage data usmg a reconstruction algorithm EIT is particularly appropnate for the implementation of the invention in tissue because it actually maps electncal impedances Therefore, the region of tissue that will undergo electroporation and m which, consequently, the equivalent electrical impedance of the cells will change will be imaged by EIT The image is used to adjust the electncal parameters (e g flow of electrical current) m a manner which allows electroporation to occur without damaging cell membranes Among the advantages that this invention offers relative to the pπor art are the ability to treat cells individually and to adapt the treatment conditions to the needs of individual cells In embodiments where voltage is applied, the monitoring of the impedance affords the user knowledge of the presence or absence of pores and shows the progress of the pore formation and whether irreversible pore formation that might lead to cell death has occuπed An advantage of the barπer-and-openmg apparatus is the high sensitivity of the signal to noise ratio by virtue of its restnction of the current to a current flow path passmg through the openmg

A still further advantage is the ability of the apparatus and method to be integrated mto an automated system whereby the condition of each cell is monitored by instrumentation and mdividual cells are lodged in the openmg and then removed at times governed by the monitored conditions

An aspect of the invention is a method of controlling electroporation of biological cells in real time by adjusting an electrical parameter (e g voltage and/or cuπent) applied to a system based on real time measurements of changes in current detected A feature of the mvention is that the general concepts can be applied to cany out electroporation on a cell, multiple cells, a tissue or areas of tissues in a living animal

An advantage of the mvention is that a precise amount of electroporation can be obtamed and cell damage avoided by controlling any given electncal parameter (e g cuπent and/or voltage) applied based on real time measurements of changes in cuπent which relates to the amount of electroporation bemg obtamed

Another advantage of the invention is that it can be used to transfect cells with nucleotide sequences without the need for packaging the sequences m a viral vector for delivery, thereby avoiding the cellular specificities of such vectors

Still other advantages are that the process can be earned out relatively quickly with a relatively low degree of technical expertise

Yet another advantage is that the process can be used to transfect cells without generatmg an immune response Still another advantage is that the process is not limited by the size of the DNA (i.e. the length of the DNA sequences) and the amount of DNA brought into a cell can be controlled.

Another feature of the invention is that imaging technologies such as EIT can be used to detect changes in impedance in a volume of cells.

Another feature of the invention is that it can use EIT in order to map impedance of an area of tissue and thereby detect changes in cell impedance in a volume of cells to adjust any given electrical parameter (e.g. cuπent flow and/or voltage) to obtain desired electroporation.

These and further features, advantages and objects of the invention will be better understood from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross section of a microdiffusion device useful in the practice of the present invention for infusing a biological cell with a chemical species without the assistance of an electrical cuπent to effect electroporation. FIG. 2 is a cross section of a microelectroporation device useful in the practice of the present invention for achieving pore formation in a biological cell, and optionally for infusing the cell with a chemical species with the assistance of electroporation.

FIG. 3a is a longitudinal cross section of an electroporation device in accordance with this invention, designed for a mobile suspension of biological cells. FIG. 3b is a transverse cross section of the device shown in FIG. 3a.

FIG. 4 is a plot of current vs. voltage in a series of electroporation experiments conducted using a microelectroporation device of the structure similar to that of FIG. 2.

FIGS. 5a, 5b, 5c, and 5d are plots of cuπent vs. voltage in a further series of electroporation experiments conducted using a microelectroporation device similar to that of FIG. 2.

FIG. 6a shows current flow around cells prior to electroporation and FIG. 6b shows electrical current flow through cells after (during) electroporation.

FIG. 7 shows a typical electrical impedance tomography (EIT) system for use with the invention. FIG. 8a is an image of current flow through cells with iπeversible electroporation and FIG. 8b is an image of cuπent flow through cells with reversible electroporation. FIG 9 is a graphic schematic view of a fimte element mesh showing a circular region of tissue bounded by electrodes (dark dots) — the domain has two different impedances

FIG 10 schematically shows typical electrode configuration, measured electncal vanables and equipotential lines in a circular domain having an inclusion with a different electncal impedance

FIG 11 shows an actual image m the top left whereas the impedance mappmg is shown in the bottom nght which shows differential impedance mapping

DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS Before the present devices and methods including methods for carrying out electroporation are descπbed, it is to be understood that this invention is not limited to particular methods and devices described, as such may, of course, vary It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims

Where a range of values is provided, it is understood that each mtervemng value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed Each smaller range between any stated value or mtervemng value in a stated range and any other stated or mtervemng value in that stated range is encompassed withm the invention The upper and lower limits of these smaller ranges may mdependently be included or excluded m the range, and each range where either, neither or both limits are included m the smaller ranges is also encompassed withm the mvention, subject to any specifically excluded limit m the stated range Where the stated range includes one or both of the limits, ranges excluding either or both of those mcluded limits are also mcluded in the invention

Unless defined otherwise, all technical and scientific terms used herem have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs Although any methods and materials similar or equivalent to those descnbed herem can be used in the practice or testmg of the present invention, the prefeπed methods and mateπals are now descnbed All publications mentioned herein are incorporated herein by reference to disclose and descnbe the methods and/or materials in connection with which the publications are cited It must be noted that as used herem and in the appended claims, the smgular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise Thus, for example, reference to "a biological cell" includes a plurality of such biological cells and reference to "an electrode" includes reference to one or more electrodes and equivalents thereof known to those skilled in the art, and so forth

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed

DEFINITIONS

The term "electrode" is intended to mean any conductive material, preferably a metal, most preferably a non-corrosive metal that is used to establish the flow of electncal cuπent from that electrode to another electrode "Electrically conductive" means for transmitting electncal cuπent that can be refeπed to in any manner, e g cuπent or voltage Electrodes are made of a vaπety of different electπcally conductive mateπals and may be alloys or pure metals such as copper, gold, platinum, steel, silver, silver chloride, and alloys thereof Further, the electrode may be compnsed of a non-metal that is electrically conductive such as a silicon-based matenal used m connection with microcircuits Typical electrodes used in tissue electroporation are preferably rod-shaped, flat plate-shaped or hollow needle-shaped structures Electrodes may be used to deliver electncal cuπent continuously or to deliver pulses The electrodes may be very application-specific and be comprised of parallel stainless steel plates, implanted wires, needle pairs and needle aπays Those skilled in the art will design specific electrodes that are particularly useful m connection with the desired results of obtaining electroporation in accordance with the present invention

The term "tissue" shall mean a plurality of cells The cells may be of the same or of a number of different types These cells are preferably organized to carry out a specific function Tissue mcludes tissue present withm a living organism as well as removed tissue and may refer to in vivo or in vitro situations Further, the tissue may be from any orgamsm mcludmg plants and animals or a tissue developed using genetic engmeeπng and thus be from an artificial source In one embodiment the tissue is a plurality of cells present withm a distinct area of a human The terms "device" and "electroporation device" are used interchangeably here for describing any a device as disclosed and described throughout The device preferably includes a first electrode and a second electrode wherein the first and second electrodes are connected to a source of electricity in a manner so as to provide the electrodes with positive and negative charges respectively The device also preferably includes a means for hmdenng the flow of electncity between the two electrodes except through one or more specific openmgs For example the means for hmdenng flow can be a non-conductive matenal which has one or more opemngs therein wherein the openings are designed so as to specifically hold a biological cell or group of biological cells Thereby the electncal current must flow through the openmg and through the cells to the other electrode The device also preferably includes a means for measunng the flow of electncal cuπent between the electrodes The means for measunng can include a volt meter, amp meter or any device known to those skilled in the art which is capable of measunng the flow of electncal current in any manner Further, the device preferably includes a means for adjusting the amount of electncal cuπent flow between the electrodes Thereby the voltage, cuπent or other desired parameter of electncal cuπent flow can be specifically adjusted based on the measured flow so as to obtain optimum electroporation of the cell or cells positioned between the electrodes When the term "electroporation" is used it does not necessarily mean that the device is being used m order to move a compound such as a drug or DNA sequence mto a cell The terms "power source", "source of electncity" and the like, are used interchangeably herem to describe any means for providmg elqctπcal power, cuπent or voltage thereby creatmg a flow of electncal cuπent between the electrodes The device preferably is capable of providmg for a controlled mode and amplitude and may provide constant DC current or AC cuπent, provide pulse voltage or continuous voltage Preferred devices are capable of exponentially decaymg voltage, ramp voltage, ramped cuπent, or any other combination For example, a power supply may be used in combmation with a chip of the type used m connection with microprocessors and provide for high-speed power amplification in connection with a conventional wall circuit providing alternating current at 110 volts The pulse shape may be generated by a microprocessor device such as a Toshiba laptop running on a Lab View program with output fed mto a power amplifier A wide range of different commercially-available power supplies can provide the desired function The electrical potential delivered for electroporation is usually quoted in terms of the voltage gradients that develop in the affected region that is defined in units of V/cm developed in the tissue Ranges include a range of 10 V/cm to 100,000 V/cm or more preferably 100 V/cm to 10,000 V/cm However, the range is amplification-specific and can be extended outside the range for any desired application Electncal pulses range from microseconds to milliseconds m general However, other ranges of pulsing may be utilized depending on the desired results

INVENTION IN GENERAL While this invention extends to a vanety of structures, methods, and applications, this portion of the specification will illustrate certain specific structures and methods m detail, from which the concepts of the mvention as a whole will become apparent

A wide range of different devices and system can be used to cany out the method of the invention The device must be comprised of a first electrode havmg a first voltage and a second electrode havmg a second voltage Further, the device will compnse a means for detectmg charged particle flow between electrodes and a means for varying the electncal cuπent between electrodes based on data obtained by detecting changes in charged particle flow between electrodes Preferably the device is further comprised of a component that prevents or substantially reduces charged particle flow between electrodes except for flow occurring through one or more biological cells positioned between the first and second electrodes

Any desired matenal can be added to the medium in order to move that matenal mto a cell which is present in the medium Further, the invention does not necessanly mclude a process step of mcludmg a material mto the medium which is to be brought mto a cell The process can be earned out merely to determme changes which occur in a cell membrane based on the electncal cuπent applied That information can be valuable to determine charactenstics about the cell or group of cells present m the medium and, specifically, can be used to compare with information on normal and diseased cells or to determine the differences between previously tested cells and those currently being tested

The first structure that will be discussed is an electroporation cell with an internal support to hold a single biological cell and an internal baπier that restncts the electπc current flow in the electnc cell to a flow path that passes through the biological cell When no voltage is applied, the structure can be used for diffusive transport alone, unassisted by voltage-mduced pore formation

The configuration of the baπier, and the two chambers m embodiments that include two chambers, is not cntical to the invention, and can vary widely while still serving the purposes and advantages of the invention Since biological cells are microscopic m size, however, the preferred type of apparatus for the practice of this invention m each of its vanous forms is one m which the structure as a whole and/or its chambers are the size of electronic chips, fabπcated by microfabncation techniques such as those used in electronic chip manufacture It is further prefeπed that the chambers are constructed as flow-through chambers to allow the passage of the liquids m continuous flow, intermittent flow, or flow at the direction of the user, and to allow changes m the concentrations, pressure, and other conditions as needed to achieve close control over the passage of species across the biological cell membrane Accordingly, a prefeπed structure and method of manufacture of the apparatus are those that involve the formation of the apparatus m layers or platelets with appropnate openings that form flow passages when the layers or platelets are bonded together

Flow-through chambers offer the advantage of pemutting the successive entry and removal of mdividual cells so that large numbers of cells can be treated m succession Flow- through chambers also permit replenishment of solute-depleted solutions so that concentration gradients can be continuously maintained when desired A further function that can be served by flow-through chambers is the increase and decrease of pressure, a function that is useful for vanous purposes as described below

The support for the biological cell in this structure can be any structure that secures the biological cell m a fixed position and that allows the passage of electnc cuπent The most convement support is an opening m the baπier Securement of a biological cell over the openmg serves to close, seal or plug the openmg, thereby directmg the passage of electric current, diffusive transport, or both, through the cell and eliminating or minimizing leakage around the cell A convenient mechanical means of achievmg this is to impose a pressure differential across the openmg m a direction that will press the cell agamst the opening The diameter of the openmg will be smaller than that of the cell, and the cell upon entenng the apparatus will pass into one of the two chambers By increasing the pressure in the chamber in which the cell resides, or lowering the pressure m the other chamber, the cell will be forced agamst the openmg, closmg it off Once the procedure is completed, the cell is readily released from the openmg by equalizing the pressures in the two chambers or by reversing the differential such that the higher pressure is in the chamber other than the chamber in which the cell was introduced The flow of liquid in the chamber m which the cell was introduced will then remove the cell from the openmg, exposmg the openmg for another cell

An alternative method of sealmg the openmg with the cell is by the use of a coating on the baπier surface, or over the nm of the openmg, of a substance that binds to the cell membrane Smce biological cell membranes are negatively charged, the coatmg may be a substance that bears a positive charge, such as polylysme, polyargimne, or polymstidine The biological cell can be directed to the openmg by a pressure differential across the openmg, and held m place by the coating Once the procedure is completed, the cell can be released from the coating by momentarily increasing the flow rate of the liquid m the chamber on the cell side of the opemng, or by imposing a reverse pressure differential across the openmg to urge the cell away from the openmg

The size of the opening is not critical to the invention provided that the openmg exposes sufficient surface area on the cell membrane to achieve the desired degree of either mass transfer, the passage of an electric cuπent. or both, withm a controllable and economically reasonable period of time The optimal size will thus vary with the particular cells being treated or studied In general, the opening is preferably circular or approximately circular m shape, and dependmg on the cell size, preferably ranges m diameter from about 1 micron to about 100 microns, more preferably from about 1 micron to about 50 microns, and most preferably from about 2 microns to about 20 microns The baπier in which the hole is formed and which separates the two chambers is preferably of a rigid dielectric matenal that is impermeable to both water and solutes and that will hold a pressure differential sufficient to secure a cell agamst the openmg For devices that are manufactured by microfabncation techniques, a convenient material for the barrier is silicon mtπde Other matenals that will serve equally well will be readily apparent to those skilled m the art

A further feature of prefeπed embodiments of this invention is the use of apparatus made of transparent materials This enables the user to observe cell mteπors and the processes of micro diffusion and microelectroporation through a microscope as they occur

ELECTROPORATION USED IN IN VIVO THERAPY The electroporation techniques of the present mvention are useful in connection with treating, analyzing or diagnosing an organism mcludmg mammals and humans in need of treatment In general, treatment may be carried out by injecting a matenal contmuously or m a rapid bolus mto an area of tissue to be treated Electrodes are placed adjacent to the tissue and current or voltage are applied and measured contmuously m order to determine when the desired level of electroporation is obtamed thereby making it possible to move the mjected material mto the cells of the tissue bemg treated

The pharmaceutically active compound that is injected may be a conventional drug normally refened to as a small molecule or be a protein or nucleotide sequence that encodes a protein Further, the composition injected into the tissue may be administered before, dunng or even after the application of electncal pulses from the electroporation device The overall goal of the process is to provide for the openmg of pores via electroporation and thereby introduce the compounds mto the cells which compounds would not normally penetrate the cell membrane For example, it is possible to mtroduce bleomicyn or various gene constructs and/or plasmids into cells of the tissue being treated This is accomplished by generating electrical potentials and currents across the cells withm the tissue to treated wherein the electrical potentials are generated as electncal pulses It is preferable to utilize a plurality of electrodes as opposed to a single electrode in order to generate the pulses

An example of a useful electrode design is one that is comprised of two flat steel strips 10 mm in width and 0 6 mm m thickness The electrodes are spaced at a fixed distance of approximately 6 to 7 mm from each other A second electrode design is compnsed of two to as much as eight flat steel squares of 20 mm The electrodes are connected to a PS 15 electropulsator Pulses can be delivered by placing the electrodes on the skm with the flat side on the skm or by placing the electrodes around skm tumors Skm contact can be achieved by the use of matenals conventionally used m connection with performing electrocardiographs such as electro-conductive gels or salme In order to carry out the procedure a patient can receive one or a plurality of pulses and preferably receives a plurality of pulses Different configurations can be designed in order to carry out electroporation of tissue mside an organism such as mside a human body, l e without applymg electrodes outside the skm Such configurations can be compnsed of needle arrays that compnse a plurality of needle electrodes As an example the positive and negative electrodes can each be compnsed of six or more needles that are 0 5 mm in diameter and compnsed of stamless steel, 1 cm in length connected to a BTX 820 pulse generator The electrodes can be inserted in parallel mto the tissue around the cells to be affected by electroporation The electrodes can be positioned in circles of various diameters ranging from 5 mm to 1 cm Voltage electrode ratio m the range of approximately 1300 V/cm can be used Although any number of pulses can be delivered it is preferable to begm the process by delivering approximately six pulses m one second intervals with a pulse width of 100 microseconds The present invention is particularly desirable m connection with electroporation of tissue m that the method can determine whether electroporation is occurring without the use of dyes and tags in order to track the matenal being brought mside the cell

As shown in FIGS 6a and 6b the electrical cuπent can flow around the cells (FIG 6a) or through the cells (FIG 6b) after electroporation has taken place The process of the mvention makes it possible to determine the point when the transition is occurring between what is shown m FIG 6a and what is occumng m FIG 6b and further makes it possible to prevent the occuπence of irreversible effects on the cell membranes As shown in FIG 8a the electioporation can be earned out to such a great extent that cell membranes are damaged thereby resultmg in irreversible effects on the cells In general, this is undesirable However, by modulating the amount of electncal cuπent it is possible to obtain electroporation without significant damage to the cell membranes thereby obtaining a reversible situation as shown in FIG 8b

As shown in FIGS 6a and 6b cells create electrical impedance and the present invention relates to precisely determining the degree of that electrical impedance and adjustmg cuπent so as to obtain desired results with respect to electroporation However, when large numbers of cells are involved such as in a tissue it may be desirable to use other mechanisms for measunng other effects of the current in creating electroporation on a plurality of cells m the tissue Electncal impedance is a measurement of how electncity travels through a given matenal Every material has a different electncal impedance determined by it's electncal composition Some mateπals have high electncal impedance and others have low electncal impedance Breast tissue which is malignant (cancerous) has much lower electncal impedance (conducts electncity much better) than normal tissue or a benign (non-cancerous) tumor

Impedance is a measurement of the degree to which an electncal circuit resists electiical-current flow when voltage is impressed across its termmals Impedance expressed m OHMS, is the ratio of the voltage impressed across a pair of termmals to the cuπent flow between those terminals In direct-cuπent (DC) circuits, impedance coπesponds to resistance In alternating cuπent (AC) circuits, impedance is a function of resistance, inductance, and capacitance Inductors and capacitors build up voltages that oppose the flow of cuπent This opposition is refened to as reactance, and must be combined with resistance to define the impedance The resistance produced by inductance is proportional to the frequency of the alternating cuπent, whereas the reactance produced by capacitance is inversely proportioned to the frequency

The basic concepts described above are utilized in the basic aspects of the present mvention and are also applicable to descπbmg electrical impedance imaging also refened to as electncal impedance tomography (EIT) It should be noted that a number of different terminologies may be used to describe the same technique and those mclude applied potential tomography (APT) These imagmg technologies make it possible to produce images based on the spatial vanation of the electrical properties of the biological tissue Techniques such as APT and EIT could be utilized to carry out the invention m connection with tissue The applied potential tomography (APT) relies for its physical basis on the measurement of a potential distnbution on a surface of a biological material, when an electrical cuπent is applied between two points of its surface Other researchers have utilized the techmque and refened to it as electncal impedance imagmg, conductivity imagmg, electrical impedance tomography, etc Herein, the technology is generally refened to as EIT or electrical impedance tomography Accordingly, within the remamder of the disclosure the technology is refened to only as EIT technology and an example of such is shown within Example 3 below

Those skilled in the art will contemplate different means of determining changes in electncal cuπent upon reading this disclosure A prefeπed method for determining such when carrying out the invention on tissue is to use imaging technology and specifically electncal impedance tomography (EIT) which monitors and analyzes differences m bio-electncal attributes of the sample being monitored m order to produce an image The EIT technology can be used in connection with the present invention by creating an EIT image and using that image to adjust cuπent flow to obtain desired results Specifically, the EIT image is created by injecting electncal cuπents mto the tissue and measunng the resultmg voltages through an electrode array This makes it possible to produce an impedance image from the known cuπent inputs and the measured input voltage data usmg a reconstruction algonthm The use of EIT technology is particularly desirable m connection with the present invention as applied to tissue in that EIT imagmg provides a map of electncal impedances The map of electrical impedances essentially allows the user to visualize when electroporation is beginning When electroporation begins the user can stabilize the amount of cuπent bemg applied and thereby avoid applymg so much cuπent as to result in lπeversible damage to cells as shown m FIG 8a The EIT technology makes it possible for the region of tissue undergoing electroporation to be visualized based on changes m equivalent electrical impedance of the cells withm tissue being monitored FIG 7 shows a conceptual view of an EIT system bemg used to cany out a process of the present invention on tissue 71 A current source 72 is controlled by a signal generator 73 and is used to drive an electncal cuπent into the tissue sample 71 through a pair of computer controlled multiplexers 74 and 75 which lead to a differential amplifier 76 and demodulator 77 The measured signals are compared to the original in order to record amplitude and phase data for later image construction The controllmg computer 78 typically chooses which pair of electrodes will mject cunent while readmg the remaining electrode voltages There are a number of different hardware configurations which can be utilized m connection with the present invention

The EIT system as shown in FIG 7 is generally refened to as a senal system because of it's single cuπent source and measurement amplifier Varying degrees of parallelism (multiple cuπent sources and voltage measunng amplifiers) have been utilized in other systems thereby increasmg the flexibility and speed of the current injection system

Reconstruction algorithms are used in order to take the voltage measured on an outer surface of a region of interest m the body (the injected cuπent data) and information relatmg to the electrode geometry, and produce an image which represents spatial tissue impedance tissue distnbution inside the region of the tissue 71 There are a number of methods which can be used to create an impedance image Static imaging is the production of an absolute impedance distnbution Cook, R D et al ACT3 a high speed, high precision electrical impedance tomography IEEE, Trans Biomed Eng 41. 713-22 (1994) Differential imagmg methods produced distributions based on differences between two data sets Barber, D C m Advances in Biomed Eng (ed Benek m, W , Thevenm, V ) 165-173 (IOS Press. Amsterdam, 1995) This type of technique provides an image of how the impedance distribution has changed from one baseline measurement Multi frequency impedance imagmg takes advantage of the frequency dependence of tissue impedance Groffiths, H The importance of phase measurement in electncal impedance tomography Physics in Medicine and Biology 32, 1435-44 (1987) Quasi-static images can be produced usmg the above differential techmque with a low frequency image used as the baselme Accordmgly the system makes it possible to produce a type of static imaging without the difficulties of true static imagmg

In order to provide for reconstruction and thus and image , a mathematical model of how the cuπent behaves in the tissue is used In general a model governing current flow in EIT is provided by the well-known Poisson equation The type of mathematical analysis that is needed in EIT image reconstruction as well as many other medical imaging technologies, belongs to a general class known as boundary value problems There are a number of different methods of solving boundary value problems However, these problems can all be classified mto either analytical or numeπcal iterative techniques and those skilled in the art can apply such in order to cany out the present invention

The vast majonty of reconstruction algonthms cuπently m use employ iterative numencal solutions to the Poisson equation Most iterative numerical approaches attempt to solve the boundary value problem by guessing an impedance distribution in the tissue and repeatedly solving the forward problem (finding the voltage and cuπent densities given an impedance distnbution) and adjusting the impedance guesses correspondmgly, until the voltage and currents measured fit those calculated The forward problem must be solved numencally and is usually done so using finite element or finite difference schemes The FEM is a very powerful and popular method of forward problem solution, and because of this, tends to dommate engineering solutions across many interdisciplinary fields

An example of a micro diffusion apparatus m accordance with this invention for a single biological cell, for transporting matenals across the cell membrane without the application of an electric field, is shown in FIG 1 This components of this apparatus, from the bottom up, are an acrylic base 11, an intermediate silicon layer 12 (1 micron in thickness) with a portion 13 carved out to define the lateral boundaries of the lower of the two liquid chambers, a silicon nitπde layer 14 serving as the barrier between the two chambers, a silicon washer 15 defining the lateral boundaries of the upper liquid chamber 16, and a glass cover plate 17 A hole 18 in the silicon nitnde baπier serves as the opening, and a cell or contiguous cell mass such as tissue 19 is shown coveπng the hole Channels extend through the acrylic base to serve as mlet and outlet channels for the liquids that pass through the upper and lower chambers, as shown by the arrows in the Figure

When the pressure m the upper chamber 16 is higher than that m the lower chamber 13, the cell will be retained m position over the hole, serving as a plug separating the liquids m the two chambers from each other When the composition of the solutions in the two chambers differs from that of the cell interior, mass transfer occurs across the cell membrane between the chambers and the cell When the composition of the solution m one chamber differs from that in the other, mass transfer occurs through the cell from one chamber to the other By precisely controlling the compositions of the solutions m the two chambers, one can precisely control the mass transfer rate and direction withm the cell Since the diameter of the openmg 18 is known, one can precisely determine the mass transfer that occurs through the opening

The numerous applications of this micro diffusion device will be readily apparent For example, the device can be used to infuse a cell with a cryopreservative such as glycerol by filling the upper chamber 16 with physiological saline and the lower chamber 13 with glycerol When usmg a cell 19 for which the mass transfer coefficient of glycerol across the cell membrane is known, one can readily calculate the amount of glycerol that will enter the cell and adjust the concentrations and exposure times to mfuse the cell with the amount that is known to be required for cryopreservation

An example of a microelectroporation apparatus in accordance with this invention for a single biological cell, is shown in FIG 2 The apparatus is similar m construction to the micro diffusion apparatus of FIG 1. Its structural components, from the bottom up, are an acrylic base 21, a lower silicon layer 22 with a portion carved out to define the lateral boundanes of the lower liquid chamber 23, a silicon nitnde layer 24 (1 micron m thickness) serving as the banner between the two chambers, an upper silicon layer 25 defining the lateral boundanes of the upper liquid chamber 26, and a cover consistmg of an n+ poly-silicon layer (5,000 A m thickness) 27 and a silicon nitnde layer (1 micron m thickness) 28 A hole 29 m the silicon nitnde baπier 24 serves as the openmg, and a cell 30 (or cell mass) covers the hole Channels extend through the acrylic base to serve as inlets and outlets for the liquids that pass through the upper and lower chambers, as shown by the arrows in the Figure A further layer of n+ poly-silicon (5,000 A) 31 resides above the acrylic base 21, and this layer, together with n+ poly-silicon layer 27 above the upper chamber 26 serve as the two electrodes Each electrode is joined by electπc leads to a pnnted circuit board 32 which controls the voltage applied between the electrodes and measures the cuπent passing between them The microelectroporation apparatus shown in FIG 2 can be fabricated by conventional microfabncation techniques, typically involving chemical vapor deposition, maskmg, etchmg and sputtenng The operation of the apparatus will be analogous to the operation of the microdiffusion apparatus of FIG 1 The movement of biological cells through the apparatus is achieved by suspending the cells m the liquid used to fill the upper chamber, and cells are drawn to the openmg, one at a time, by imposing a pressure differential between the chambers, which also holds a cell in place once the cell has been drawn to the openmg A convenient method of imposing such a pressure differential is to maintain atmosphenc pressure m the upper chamber while lowering the pressure in the lower chamber below atmosphenc by attaching a syringe to the lower chamber and pulling on the syringe plunger Care should be taken to limit the pressure differential to one that will not damage the cell

FIGS 3a and 3b illustrate to a different apparatus and method within the scope of this mvention This apparatus and method involve a fluid suspension of biological cells flowing through a conduit or flow channel, in which the cells pass through a region between a pair of electrodes The longitudinal cross section of FIG 3a shows the walls 41 of the channel, and a biological cell 42 passmg downward through the lumen of the channel (in the direction of the arrow) The transverse cross section of FIG 3b shows that the channel is rectangular in cross section, although other cross-sectional geometries may be used Electrodes 43, 44 are formed as coatings on two opposmg walls of the channel The electrodes are connected through leads to a pnnted circuit board 45 which measures the impedance and controls the voltage applied to the electrodes The biological cell 42 is shown passmg through the region between the two electrodes

The area of the cross section of the channel is large enough to permit the cell to pass through essentially unimpeded by the channel walls, and yet small enough that only one cell can pass through the mter-electrode region at a time In addition, each electrode 43, 44 is either approximately equal in length or slightly larger in length than the diameter of the biological cell, so that the cell upon entermg the region causes a significant or measurable decrease in the cuπent passmg through the region due to the voltage applied across electrodes The spacing of the electrodes, i e , the distance between them, is likewise subject to the same considerations The biological cells are suspended m a liquid solution of the species to be introduced mto the cells, and the suspension is passed through the channel A voltage is applied between the electrodes as suspension flows through the channel, and the current between the electrodes (or the impedance) is monitored A significant drop in the current indicates the presence of a biological cell in the mter-electrode region Once the cell is detected in this manner, an electroporation pulse can be applied to the electrodes while the cell is still in the inter-electrode region, and impedance can be observed further to detect the onset of electroporation The species dissolved m the liquid solution will enter the cell as a result of the electroporation

Variations on these structures and methods will be readily apparent to those skilled in the art For example, the barriers described above can be minimized or avoided by using microelectrodes that are the same size as or smaller than the biological cells Examples of such microelectrodes are carbon fiber microelectrodes (such as ProCFE, Axon Instruments, Foster City, California, USA) used in conjunction with high-graduation micromampulators (such as those available from Naπshige MWH-3, Tokyo. Japan) Microelectrodes can be used m place of the electrodes shown m FIG 2 or in place of those shown in FIGS 3a and 3b

EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and descnption of how to make and use the present invention, and are not mtended to limit the scope of what the inventors regard as their mvention nor are they intended to represent that the expeπments below are all or the only expenments performed Efforts have been made to ensure accuracy with respect to numbers used (e g amounts, temperature, etc ) but some expeπmental errors and deviations should be accounted for Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is m degrees Centigrade, and pressure is at or near atmosphenc

EXAMPLE 1

A senes of experiments was performed using a microelectroporation system consisting of the microelectroporation device descπbed above and shown m FIG 2, combmed with flow and pressure control units and pressure gauges for the liquids to be circulated through the upper and lower chambers, a variable DC power supply, a pulse generator and power amplifier for imposing voltage pulses across the device, a digital oscilloscope for monitoring the pulses, a fluorescent microscope, a CCD (charge coupled device) camera, and a computer with image processing and waveform processing software Both chambers of the device were filled with physiological saline and cells were introduced into the upper chamber Liquid motion in the top and bottom chambers was controlled by The pressure in the upper chamber was atmospheric while the pressure in the lower chamber was reduced below atmospheric by pulling on the baπel of the syπnge connected to that chamber The voltage was applied m single square pulses ranging from zero to 120V m magnitude and from 2 microseconds to 100 milliseconds in duration The distance between the electrodes m the upper and lower chambers was 900 microns

The tests in this example were performed using ND-1 human prostate adenocarcmoma cells with a typical diameter of 20 microns The opening in the microelectroporation device was 5 microns in diameter A rectangular voltage pulse was applied with a duration of 60 milliseconds, and the pulse was applied at various amplitudes rangmg from 10V to 60V in mcrements of 5 volts With each pulse, the electric cunent passmg through the openmg was measured Experiments were performed with the cells and were repeated both with the openmg stopped by a glass bead and with no obstruction at all m the opemng The results m each case were expressed as microamperes of cuπent vs volts of pulse amplitude and are plotted m FIG 4, in which the upper curve (data pomts represented by x's) represents the unobstructed openmg, the lower curve (data pomts represented by astensks) represents the data taken with the glass bead residmg m the opening, and the three middle curves (open squares, open upnght tnangles, and open inverted triangles) represent data taken with three different ND-1 cells residmg m the openmg The upper curve shows that the current increases m a substantially steady manner as the voltage increases when there is no baπier to the passage of current through the opemng The lower curve also shows a substantially steady rise as the voltage mcreases, although at a much lower level The cuπent values shown in the lower curve represent stray cuπents through the device The curves of data taken with the ND-1 cells across the openmg show that at low voltages the cuπent is close m value to that obtamed when the openmg is closed by the glass bead while at high voltages the cunent nses to the levels obtamed with an unobstructed opening The transition is a sharp mcrease which is indicative of the formation of pores in the cell membrane through which an electnc current can pass, i e , the onset of electroporation In all three cells, the transition occurred at voltages between 30V and 40V In two of the three cells (open squares and open upnght triangles), the onset of electroporation occuπed essentially at the same voltage, while in the third (inverted tnangles). the onset occuπed at a voltage that was lower than the other two by about 5V This illustrates the value of controlling the process for individual cells to achieve optimal results After the data shown in FIG 4 was generated, the pulses were reapphed in descending order of amplitude values, and the resultmg curves displayed hysteresis, i e , the curves obtained with descending amplitudes were higher in voltage than those obtamed with ascending amplitudes This indicated that the electroporation in these expeπments was irreversible

EXAMPLE 2

Usmg the same microelectroporation system used in Example 1, a senes of tests were performed on rat hepatocytes (ATCC #CRL-1439). whose typical cell diameter was 20 microns, the microelectroporation apparatus having an openmg that was 4 microns in diameter Here as well, rectangular voltage pulses that were 60 milliseconds in duration were used, ranging m amplitude from 10V to 37 5V in increments of 5V in the portion from 10V to 30V and m increments of 2 5V in the portion from 30V to 37 5V The expenments were performed in some cases only by increasing the amplitudes and in others by first increasing, then decreasing the amplitudes to evaluate reversibility The results are plotted in the graphs shown in FIGS 5a, 5b, 5c, and 5d In each case, the upper curve (data pomts represented by circles) is the data taken with neither a cell nor a glass bead residing m the openmg, the lower curve (data pomts represented by squares) is the data taken with a glass bead in the opemng, and the middle curve (data pomts represented by tnangles) is the data taken with a hepatocyte in the openmg, usmg different hepatocytes for each of the four Figures In FIG 5a, the amplitude was increased and not decreased, displaying an electroporation threshold voltage of between 25 V and 30V In FIGS 5b and 5c, the amplitude was first mcreased and then decreased to produce the two middle curves Although the ascendmg and descendmg curves are not differentiated, they are substantially identical m each Figure, indicating that the cell membrane in each of these two cases resealed after each voltage pulse and thus that the pore formation was reversible In the test represented by FIG 5d, the cell dismtegrated once the applied voltage exceeded 37 5V, although this is not shown in the Figure It is significant to note that despite the fact that the same cell types were used m each of FIGS 5a, 5b, 5c, and 5d, the electroporation threshold voltage differed among the individual cells, although all were withm the range of 20V to 35V Adaptation of the procedure to individual cells is readily achieved by momtormg the current in this manner to note when the electroporation threshold occurs Selection of the optimal exposure time, voltage, composition changes in the surrounding liquids, and other parameters of the system can then be made to achieve the desired treatment of the cell without destruction of the cell.

The methods described herein are useful tools in the laboratory for conducting fundamental research in the electroporation properties of biological cells, and useful tools in industry for processing large quantities of cells in a flow-through manner. By enabling one to observe and record the current flowing through individual cells, one can control the amplitude and duration of the voltage pulse to achieve optimal results. In addition, the devices described and shown herein for use in practicing the invention can be constructed with transparent parts and of a size suitable for mounting on a microscope stage. This will permit one to correlate the electrical cuπent measurements to visual observations and fluorescence measurements inside the cell. The device can be used to electrically detect, through the measurement of cuπents, the point in time when a cell becomes lodged in the opening as well as the point in time when pore formation is achieved in the cell membrane. For larger scale and industrial applications, large numbers of microelectroporation devices of the type described herein can be arranged in parallel. For each cell, electrical information indicating the trapping of a cell in the opening (such as a sharp drop in the current) can be used to generate a signal that will initiate an electroporation sequence, and further electrical information indicating the completion of electroporation (such as a sharp rise in cuπent) will generate a signal that will release the cell (for example by eliminating or reversing the pressure differential) and permit the next cell to flow toward the opening. In addition to using the device and system of the invention to move a material into or out of the cell the system and device can be used in a diagnostic or analytic mode. This is carried out by measuring electrical impedance of a cell or cells placed in a medium and using the measured electrical impedance information. It is possible to deduce information relating to the integrity of cell membranes and thus provide for analysis. It is also possible to compare the information to information previously obtained on normal or diseased cells of the same type and thereby obtain diagnostic information. For example, the electrical impedance of a cell with an intact membrane will be much high than the impedance of the same cell with impaired membrane. Thus, analytically the process can provide information with respect to the structural integrity of the cell membrane. Diagnostically the method can provide information with respect to the relative structural integrity of cell membranes. EXAMPLE 3 ELECTRICAL IMPEDANCE MAPPING OF ELECTROP ORATED DOMAINS.

In order to illustrate the ability of EIT to monitor electroporation in tissue we have solved a mathematical simulation of the problem. To provide the necessary data for electroporation imaging simulation, a simulated tissue phantom was created first using a 2-D fine-mesh FEM model (-1600 nodes, -3100 elements). The phantom, shown in FIG. 9, consisted of a circular imaging domain (20 mm radius, resistivity 500 ohm-cm for muscle with a variable number of point source electrodes equally spaced around the periphery. Within this imagmg region, a single arbitrarily shaped electroporated region was defined with a different resistivity. An opposite electrode current injection pattern was used, providing N(N-l)/2 independent voltage measurements where N is the number of electrodes. The model was solved using the adaptive mesh generation and FEM solution algorithms available in MATLAB's Partial Differential Equation Toolbox (The Mathworks Inc.). . An example mesh for the given geometry is shown in FIG. 9. The information that the phantom module makes available to the reconstruction algorithms represents data that would have been available during the electroporation part of an experiment, i.e current and voltage at the different electrodes around the tissue. From this data we attempted to reconstruct the original image of the tissue that was input in the model. (It should be noted that a DC injection current was used in place of the AC current typical to EIT in order to simplify the problem. The AC derivation and implementation is a straightforward extension of that presented here.) A typical example for the voltage and cuπent distribution in the phantom during a simulated data acquisition step for an 8-electrode EIT system is illustrated in FIG. 10.

The data obtained from the phantom was input into two EIT imaging algorithms, one using the finite element method and the second the boundary element method to generate the impedance image. The algorithms use a standard Newton Raphson technique to produce the image. FIG. 11 compares the image of a circular domain with two different electrical impedances in comparison to the image of the original phantom as recreated with the finite element technique and with the boundary element technique.

Electrical impedance tomography can be used to image the electroporated region in tissue because EIT produces an image of the tissue from a map of the electrical impedance of the tissue and electroporation produces changes in impedance. The electrodes for tissue electroporation imaging may be different than those used for the electroporation process itself or may be the same. EXAMPLE 4 ELECTRICAL DETECTION OF CHANGE IN MEMBRANE PERMEABILITY.

As part of our research on cell electroporation we have studied the electrical characteristics of cells during reversible and irreversible electroporation. In reversible electroporation the cell is not damaged by the electroporation process and the membrane reseals. In irreversible electroporation the cell membrane is damaged and does not reseal. In a set of experiments in which we have used ND-1 cells to measure cuπents through cells in the microelectroporation chip we have obtained results illustrated by FIGS. 8a and 8b. The results were obtained by exposing cells to triangular shaped electrical pulses (top curve) in 8a and 8b. The electrical cuπents flowing through the cells are shown in the bottom curve in 8a and 8b. FIG. 8a is for a cell that was iπeversible electroporated and FIG. 8b for a cell that was reversible electroporated. It can be easily noted that when the voltage was reduced in the reversible electroporated cell it retained the same values as during the voltage increase stage. However, in the iπeversible case the current through the cell with the damaged membrane had a higher cuπent than in the intact cell. This leads to the conclusion that electrical cuπents flowing through cells can provide indication on changes in membrane permeability in general and a measure of the integrity of the cell membrane in particular under a variety of situations and not only during electroporation. For instance, cell viability is often measured with trypan blue or fluorescence dyes that penetrate through damaged membranes. These results show that an alternative method for detecting cells with damaged membranes would be to measure the electrical cuπent-voltage relation across the cell. Similarly, there are compounds that induce pores in the cell membrane, such as ionophors. Measuring the cuπent-voltage (impedance relation across a cell membrane could also detect if the membrane was impaired by these chemicals). Electrical measurements would have advantage over chemical means for detecting cell membrane damage because they would produce immediate information. A possible method for detecting changes in cell membrane permeability and in particular damaged cell membranes is to use the electroporation chip as described for the process of electroporation. The measure of damage would be the difference between an intact cell impedance and a damaged cell impedance as illustrated in FIGS. 8a and 8b . In tissue it would be possible to detect cells with damaged membranes in a similar way to the methods for detection of electroporation described here.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spmt and scope of the invention In addition, many modifications may be made to adapt a particular situation, matenal, composition of matter, process, process step or steps, to the objective, spint and scope of the present invention All such modifications are intended to be within the scope of the claims appended hereto