Process and device for controlling electroporation
Abstract
sending an electric current between a first point and a second point separated from the first point by an electrically conductive means comprising tissue; create an image of the tissue in which the image is based on the electrical impedance of the tissue; and adjusting an electrical parameter based on the image to obtain a desired degree of electroporation of biological cells in the tissue; wherein the electrical parameter is selected from the group consisting of current, voltage and a combination of current and voltage.

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9 claims: 2 independent, 7 dependent
- 1ES 2 317 844 T3 REIVINDICACIONES 1. Un método, que comprende la etapas de:enviar una corriente eléctrica entre un primer punto y un segundo punto separado del primer punto por un medio eléctricamente conductor que comprende tejido;crear una imagen del tejido en el que la imagen se basa en la impedancia eléctrica del tejido;y ajustar un parámetro eléctrico basado en la imagen para obtener un grado deseado de electroporación de células biológicas en el tejido;en el que el parámetro eléctrico se selecciona del grupo que consiste en corriente, voltaje y una combinación de corriente y voltaje.
- 2El método de la reivindicación 1, que comprende adicionalmente:poner un material en el medio eléctricamente conductor y ajustar la corriente eléctrica en base a la imagen, de una manera que mueva el material dentro de las células biológicas en el tejido.
- 3El método de la reivindicación 1, en el que la imagen se crea usando tomografía de impedancia eléctrica.
- 4El método de la reivindicación 1, en el que la imagen es una imagen de impedancia creada a partir de entradas de corriente conocidas y voltaje de entrada medido usando un algoritmo de reconstrucción.
- 5El método de la reivindicación 1, en el que la imagen es una imagen de impedancia creada a partir de un voltaje de entrada conocido.
- 6El método de la reivindicación 1, en el que la imagen es una imagen de impedancia creada a partir de una entrada de corriente medida.
- 7El método de la reivindicación 1, en el que la imagen es una imagen de impedancia creada a partir de una combinación de una entrada de voltaje conocida y una entrada de corriente medida.
- 8Un dispositivo, que comprende:un medio para crear una corriente eléctrica a través de un medio eléctricamente conductor;un medio para analizar la impedancia eléctrica del medio eléctricamente conductor para crear una imagen;y un medio para ajustar un segundo parámetro eléctrico basado en la imagen para obtener un grado deseado de electroporación de células biológicas en el medio eléctricamente conductor;en el que el segundo parámetro eléctrico se selecciona del grupo que consiste en corriente, voltaje y una combinación de corriente y voltaje.
- 9El dispositivo de la reivindicación 8, en el que el medio para crear corriente eléctrica comprende electrodos.
Independent claims9
137 paragraphs in 7 sections, as filed
ES 2 317 844 T3
DESCRIPTION
Procedure and device to control electroporation.
Electrical impedance tomography to monitor electroporation.
Field of the invention
This invention relates generally to the field of electroporation and mass transfer across cell membranes and ion transport across a particular cell membrane.
Background of the invention
Electroporation is a technique used to introduce chemical species into biological cells and is done by exposing the cells to an electrical potential that crosses the cell membrane. Although its mechanism is not fully understood, electroporation is believed to involve degradation of the lipid bilayer of the cell membrane leading to the formation of transient or permanent pores in the membrane that allow chemical species to enter the cell by diffusion. Electric potential is typically applied in pulses and whether pore formation is reversible or irreversible depends on parameters such as amplitude, length, shape, and repetition rate of the pulses, as well as the type and stage of development 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 simultaneously treat entire populations of cells; 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 can be used in both prokaryotic and eukaryotic cells without fundamental 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.
Currently, electroporation is carried out 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. Pore formation, or membrane permeabilization, occurs at cell poles, which are the sites on cell membranes that are directed directly toward the electrodes and therefore, the sites where the transmembrane potential is highest. Unfortunately, the degree of permeability that occurs in electroporation varies with cell type and also varies between cells in a given population. Additionally, since the procedure is performed on large populations of cells whose properties vary among individual cells in 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 tailored to the specific characteristics of individual cells. It is of particular interest that under certain conditions the electrical potential is too low for a cell membrane to become permeable, while under other conditions electroporation can induce irreversible pore formation and cell death. For example, a high electric field can therefore cause an increase in transfection efficiency in one part of a cell population while causing cell death in another. A further problem with known electroporation methods is that the electroporation transfection efficiency can sometimes be low. For example, in the case of DNA, a large amount of DNA is needed in the surrounding environment to achieve efficient transformation of the cell.
Many of the problems identified above are a consequence of the fact that the electroporation process in both individual cells and tissues cannot be controlled in real time. At present there is no means to determine in real time when a cell enters a state of electroporation. As a result, the effect of an electroporation protocol can only be determined by the ultimate consequences of the mass transfer process and its effect on the cell. This occurs long after the electroporation mass transfer has taken place. These and other shortcomings of current electroporation methods are addressed in the present invention.
Current techniques for the study and control of mass transfer across cell membranes are also relevant to the present invention. 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 certain diseases. Furthermore, the ability to modify and control mass transfer across cell membranes is a useful tool in conducting research and therapies in biotechnology and modern 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 normal and abnormal physiological processes of the cell.
The most common method to achieve and study mass transfer across a cell membrane is to contact the cell with a solution containing the compound to be transported across the membrane, with or without electroporation. This mass transfer method does not allow for precise control or measurement of mass transfer across the membrane. The composition of the solution at specific sites is unknown and is variable. Also, when an electric field is present, the intensity of the local field will vary from point to point. Additionally, the surface of the cell that is exposed to the solution is not well defined. Cell surface areas vary between cells in a given population and this leads to significant differences between cells in the
ES 2 317 844 T3 mass transfer quantity. For these reasons, the amount of mass transfer achieved by mass transfer processes is not uniform between cells and the actual amount transferred for any particular cell cannot be determined.
Attempts so far to overcome the limitations of mass transfer techniques include techniques for treating individual cells that include mechanical injection (microinjection) of chemicals through the cell membrane or electroporation with microelectrodes. In injection techniques, the membrane is penetrated with a needle to deliver a chemical agent, locating the application of the chemical agent in a small region close to the injection point. This requires manipulation of the cell manually, a technique that is difficult to perform, requires a lot of work, and is not easily reproducible. Microelectrode electroporation experiences these problems as well as the lack of any means to detect the onset of electroporation in an individual cell.
Schmukler, RE: "Impedance Spectroscopy of Biological Cells." Proceedings of the 16th Annual International Conference of the IEEE. November 1994, Volume 1, page A47 describes performing complex impedance measurements on living cells by impregnating the cells into the pores of a filter. Porous electrodes are used and an extracellular solution is modified during impedance experiments to provide additional information. Cell membranes can be electroporated providing a way to study the impedance of living cells.
Summary of the invention
According to a first aspect of the present invention a method is provided, comprising the steps of:
sending an electrical current between a first point and a second point separated from the first point by an electrically conductive medium comprising tissue;
creating an image of the tissue in which the image is based on the electrical impedance of the tissue; and adjusting an electrical parameter based on the image to obtain a desired degree of electroporation of biological cells in the tissue;
wherein the electrical parameter is selected from the group consisting of current, voltage, and a combination of current and voltage.
According to a second aspect of the present invention a device is provided, comprising:
a means for creating an electrical current through an electrically conductive medium;
means for analyzing the electrical impedance of the electrically conductive medium to create an image; and means for adjusting a second image-based electrical parameter to obtain a desired degree of electroporation of biological cells in the electrically conductive medium;
wherein the second electrical parameter is selected from the group consisting of current, voltage, and a combination of current and voltage.
Devices, systems, and methods that allow the movement of materials across a cell membrane to be accurately monitored are described and illustrated later in this document. Information gained from monitoring the movement of materials across a cell membrane can be applied directly to deduce information regarding the cell and / or its membrane. Alternatively, the information obtained from monitoring can be applied to control the movement of materials across the cell membrane such as by controlling the application of electrical current. Such devices and systems make it possible to move charged molecules, and in particular ionic species, through a cell membrane and precisely monitor their appearance. When electroporation is performed using these devices, systems and methods the information obtained from monitoring the movement of 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 as the mass transfer properties of the cell are changed by the application of electrical current. Therefore, the information obtained from the changes in electrical impedance produced by the application of electrical current is used, in real time, to control the movement of charged molecules through a cell membrane.
Hereinafter a method is described which comprises creating an electrical charge differential between a first point and a second point separated from the first point by an electrically conductive means comprising a biological cell. Then a first electrical parameter is measured between the first and second points. Then a second parameter is set based on the measurement of the first electrical parameter. The first electrical parameter can be any parameter such as one selected from the group consisting of current, voltage, and impedance.
ES 2 317 844 T3 electrical power. The second electrical parameter can be any parameter (equal to or different from the first electrical parameter) such as one selected from the group consisting of current, voltage, or a combination of current and voltage.
In a preferred embodiment the method further includes placing a material in the electrically conductive medium and adjusting the second electrical parameter to move the material into the biological cell. The material placed within the electrically conductive medium can be any material such as a pharmaceutically active compound or drug, a nucleotide sequence, a fluorescent dye, or a crystal that is specifically designed to affect the cell in a desired way. According to the method, various conditions are adjusted so that the electrical potential between the two points is high enough to cause permeabilization of the cell. However, the conditions between the two points are further adjusted so that the electroporation is reversible and as such does not cause cell death unless it is a specifically sought result.
Electroporation is not performed for the purpose of moving material into or out of a cell but to analyze the cell or group of cells and provide information or diagnosis of the tissue or individual containing the tissue. In this method, an electrical charge differential is created between a first point and a second point separated from the first point by an electrically conductive medium comprising a biological cell. Then a first electrical parameter is measured between the first and second points. The measurement of the first electrical parameter is then analyzed to determine the nature of the cell and, in particular, a characteristic of a cell membrane. The first electrical parameter can be any parameter and is preferably selected from the group consisting of current, voltage, and electrical impedance. A second electrical parameter is preferably set in a way that affects the membrane of the cell or cells present in the medium and the second electrical parameter is any parameter but is preferably selected from current, voltage, or a combination of both.
A device is described which preferably comprises a first electrode, a second electrode, a source of electricity that can later be connected to the electrodes but is optionally present when the device is marketed. Furthermore, the device includes a means for preventing the flow of electric current between the first and second electrodes except for the continuous flow of electric current through a defined path. Furthermore, the device includes a means for measuring an electrical parameter such as electrical current, voltage or impedance by the defined pathway and a means for adjusting the source of electricity based on the measured electrical parameter. The means for preventing the flow of electrical current preferably comprise a non-conductive material and a defined pathway comprising one or more openings, each with a diameter smaller than that of a biological cell so that the cell can fit into the defined pathway and have a current flow through but preferably not around the cell.
The device and systems can be used in the method to move a wide range of materials into or out of the biological cell to obtain a desired result. The process can be carried out in an individual cell, a group of cells, cells within a cell culture or within a living organism, for example, cells in invertebrates and vertebrates including mammals as well as in plants. When the process is performed on a plurality of cells (eg, tissue) a process can be used to image the tissue and adjust the electrical current in real time based on images. One imaging technology that can be applied is electrical impedance tomography (EIT). This technology relies on differences in bioelectrical attributes within the body or within an organism (eg, a human) to produce an image. EIT imaging can be used in the method in the same way that the measurement step is used when the process is performed on a single biological cell. In essence, EIT technology allows you to "see" the effect of the increased electrical current flow that occurs as a result of electroporation, thereby providing information that can be used to fine-tune the flow of electrical current so that cell membranes they permeate without being permanently altered.
The method of the invention comprises sending an electric current between a first point and a second point separated from the first point by an electrically conductive medium comprising tissue. After the current is delivered, an image of the tissue is created where 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 to obtain a desired degree of electroporation of biological cells in the tissue. Electroporation will change the electrical impedance and that change can be visualized in the created image. The adjusted electrical parameter is selected from the group consisting of current, voltage, or a combination of both. In a preferred embodiment, a material is placed in the electrically conductive medium such as by injecting it into the tissue and current adjustment is performed, based on the image, in a manner that moves the material into the 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 algorithm. The impedance image can be created from a known voltage input, a measured current input, or the combination of the known voltage input and the measured current input.
Another aspect of the invention is a device for carrying out this method, the device of which includes a means for creating an electric current through an electrically conductive medium. Furthermore, the device includes a means for analyzing the electrical impedance of the electrically conductive medium to create an image and a means for adjusting a second electrical parameter based on the image to obtain a desired degree of electroporation of biological cells in the electrically conductive medium. The second electrical parameter is selected from the group consisting of current, voltage, or a combination of both. The current is preferably created by a plurality of electrodes placed around an area of tissue on which the electroporation will be performed.
ES 2 317 844 T3
The present invention arises in part from the observation that the onset and extent of electroporation in a biological cell can be related to changes in electrical impedance (the term used herein to refer to the ratio of current to voltage) of the biological cell or of a conductive medium that includes the biological cell. When the cell membrane becomes permeable due to pore formation or by cell damage or other modes of pore formation in the cell membrane, an increase in the ratio of current to voltage occurs through a biological cell. Similarly, when fluid draws a biological cell to the region between the electrodes in a continuous-flow electrical cell, a decrease in the current-to-voltage ratio occurs through a flowing conductive liquid. Thus, by monitoring the impedance of the biological cell or of an electrolyte solution in which the cell is suspended, the point in time at which pore formation occurs in the cell membrane can be detected, as well as the relative degree of permeability of the cell membrane due to pore formation. This information can then be used to establish that a given cell has, in fact, undergone electroporation or to control the electroporation process by directing the selection of the electrical parameters of the process, eg, the magnitude of voltage. This observation is also useful in the simultaneous electroporation of a multitude of cells in a cell culture or in vertebrates, invertebrates or plants. Specific embodiments apply the invention to mammals including humans. The process provides a direct indication of the actual occurrence of electroporation and an indication of the average degree of electroporation of all cells undergoing the process. Similarly, the description is useful in electroporation of biological tissue (masses of biological cells with continuous membranes) for the same reasons.
The benefits of this process include a high level of control over the onset and degree of electroporation, along with a more detailed knowledge of the occurrence and degree of permeability created in particular individual cells or cell masses. When applied to individual cells or a series of individual cells, this process ensures that individual cells are in fact made permeable and are in fact 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 insufficient effect on others.
The invention can be understood by describing a simple embodiment that involves the use of an electrical device or system in which a biological cell can be placed and that contains a barrier that directs the flow of electrical current and therefore the flow of ions. through a flow path that passes through the biological cell while substantially not allowing electrical current to be diverted from the biological cell. In some of these embodiments, the invention involves the use of an apparatus that contains two liquid retention chambers separated by a barrier that is substantially impermeable to an electrical current. The barrier contains an opening that is smaller than the biological cell so that the biological cell plugs or closes the opening once it lodges in the opening. To achieve electroporation, the biological cell is secured in the opening by mechanical, chemical and / or biochemical means, preferably in a reversible manner so that the biological cell can be removed later without damage to the biological cell. Once the biological cell has been secured in the opening, a voltage is applied between the two chambers and across the biological cell residing in the opening. In this way, the passage of current between the chambers is limited to a path that passes through the opening and, therefore, through the biological cell. By monitoring the current-voltage relationship in the electrical cell, the onset of electroporation is detected and the degree of pore formation is monitored, both to ensure that electroporation is occurring and to avoid excessive pore formation and death. mobile. Thus, highly accurate knowledge is provided to the user and control of the state of and 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 barrier and the biological cell is introduced through an opening in the barrier in such a way as to substantially prevent the passage of liquid around the cell from one chamber to the other. other. A liquid solution of the species to be introduced into the biological cell is placed in one or both chambers. The concentration of the species in the solution differs from that of the cell (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 of the other chamber.
In preferred methods for applying this invention to diffusive transport, the solutions in the two chambers have different concentrations so that the driving force for diffusive transport is between the two chambers rather than between the chambers and the interior of the biological cell. The knowledge and controlled monitoring of the concentrations in each of the two chambers on a periodic or continuous basis as the diffusion proceeds, together with the precise knowledge of the aperture dimensions, allows the user to precisely observe and control the proportion and quantity of the species that enter the cell. Diffusion time can be controlled by applying gradual changes in concentrations in either or both chambers, thereby applying 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 previous paragraph.
In addition to being useful in connection with electroporation technology the present invention can provide valuable information regarding a cell or group of cells or tissue containing a group of cells by monitoring electrical impedance and thereby providing information regarding integrity. of a cell membrane. Specifically, measurements are made regarding the movement of charged particles across a cell membrane. These measurements are related to the amount of electrical current required to diffuse through a cell membrane. The information obtained can be analyzed directly or in comparison
ES 2 317 844 T3 with previous measurements of the same tissue or measurements made on diseased or normal tissue thereby providing an indication of the amount of change that has occurred in the tissue being measured (based on previous measurements of the same tissues) or the amount of variation between the tissue being measured and tissue with altered cell membranes (e.g. diseased cells) or a normal cell or tissue. The method is performed in a manner similar to that used to conduct electroporation. However, no material needs to be added to the medium surrounding the cells. The device is similar in that it is divided into two parts with a positive electrode on one side and a negative electrode on the other side separated by a barrier, with the cells positioned along openings in the barrier in a way that allows charged particles to pass through. through the cell and through the opening in the barrier from one electrode to another. The barrier obstructs or completely eliminates the flow of charged particles except through the openings. Measurement of the electrical impedance between the electrodes makes it possible to distinguish between cells with an intact membrane and cells with altered membranes. By making measurements more precisely it is possible to make determinations regarding the integrity of a normal cell membrane in relation to an altered (eg diseased) cell membrane.
Each of the various embodiments of this invention can be used with two or more (ie, a plurality of) biological cells simultaneously or cell masses such as in tissue that may be in 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 barrier to limit current or diffusive transport, to a flow path that passes through all cells while avoiding diversion around the cells. A further application of the concepts of this invention is the electroporation of biological cells suspended in a fluid liquid. The electrodes are placed in fixed positions in the flow channel and a voltage is applied between the electrodes while the current flow between the electrodes is monitored. Biological cells entering the region between the electrodes will decrease the current, the impedance serving as an indication of the presence of one or more cells in the region and optionally also as a signal to initiate the application of a sufficient higher voltage. to achieve electroporation.
A further application of the device, system and method of the invention is the electroporation of biological cells present in a tissue whose tissue may be present in a living organism such as a mammal. The electrodes are placed in fixed positions in the tissue and voltage is applied between the electrodes while monitoring the passage of current between the electrodes. Biological cells with intact membranes in the region between the electrodes will increase electrical impedance. Consequently, a measurement of electrical impedance provides an indication of the presence of one or more cells in the region. Electroporation will decrease the amount of impedance measured. When the process is carried out in a tissue, the electrical impedance measurement is a statistical average of the cells present between the electrodes.
The electroporation methodology of the invention can be performed on tissue in a living organism using imaging technology that allows determining when (and preferably to some extent the degree) cell membranes are transformed to allow the flow of electrical current through of their membranes. The preferred imaging technology is electrical impedance tomography (EIT) which provides a changing image created from information of differences in bioelectrical attributes of the tissue being imaged. A typical EIT image is obtained by injecting electrical currents into the body and measuring the resulting voltages across a series of electrodes. An impedance image is then produced from the known current inputs and the measured voltage data using a reconstruction algorithm. EIT is particularly suitable for the practice of the invention in tissue because it actually maps electrical impedances. Therefore, the region of tissue that will undergo electroporation and where the equivalent electrical impedance of cells will change accordingly will be imaged by EIT. The image is used to adjust electrical parameters (eg, electrical current flow) in a way that allows electroporation to occur without damaging cell membranes.
Among the advantages this invention offers over the prior art are the ability to treat cells individually and tailor treatment conditions to the needs of individual cells. In embodiments where voltage is applied, impedance monitoring provides the user with knowledge of the presence or absence of pores and shows the development of pore formation and whether irreversible pore formation has occurred that can lead to cell death.
An advantage of the barrier and aperture apparatus is the high sensitivity of the signal-to-noise ratio due to limiting the current to a current flow path through the aperture.
Yet another additional advantage is the ability of the apparatus and the method of being integrated into an automated system whereby the status of each cell is monitored by instrumentation and individual cells are housed in the aperture and then removed at times determined by the monitored conditions. .
One aspect of the invention is a method for monitoring the electroporation of biological cells in real time by adjusting an electrical parameter (for example, voltage and / or current) applied to a system based on real-time measurements of the changes detected in the current.
An element of the invention is that the general concepts can be applied to electroporation in a cell, in multiple cells, in a tissue or areas of tissues in a living animal.
ES 2 317 844 T3
An advantage of the invention is that a precise amount of electroporation can be obtained and cell damage avoided by controlling any given electrical parameter (e.g., current and / or voltage) applied based on real-time measurements of changes in current that occur. it relates to the amount of electroporation that is obtained.
Another advantage of the invention is that it can be used to transfect cells with nucleotide sequences without the need to package the sequences into a viral vector for delivery, thereby avoiding the cellular specifications of such vectors.
Still other advantages are that the processes can be performed relatively quickly with a relatively low degree of technical knowledge.
Yet another advantage is that the process can be used to transfect cells without generating an immune response.
Still another advantage is that the process is not limited by the size of the DNA (ie, the length of the DNA sequences) and the amount of DNA that is introduced into a cell can be controlled.
Another element of the invention is that imaging technologies such as EIT can be used to detect changes in impedance in a volume of cells.
Another element of the invention is that you can use EIT to map the 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., current flow and / or voltage) to obtain the desired electroporation.
These and other elements, advantages and objects of the invention will be better understood from the following description.
Brief description of the drawings
Figure 1 is a cross section of a microdiffusion device useful in the practice of the present invention to infuse a chemical species into a biological cell without the aid of an electrical current to effect electroporation.
Figure 2 is a cross section of a microelectroporation device useful in the practice of the present invention to achieve pore formation in a biological cell and optionally, to infuse a chemical species into the cell with the aid of electroporation.
Figure 3a is a longitudinal cross section of an electroporation device according to this invention, designed for a mobile suspension of biological cells. Figure 3b is a longitudinal cross section of the device shown in Figure 3a.
Figure 4 is a plot of current versus voltage in a series of electroporation experiments conducted using a microelectroporation device similar in structure to that of Figure 2.
Figures 5a, 5b, 5c and 5d are plots of current versus voltage in a series of additional electroporation experiments conducted using a microelectroporation device of similar structure to that of Figure 2.
Figure 6a shows current flow around cells before electroporation and Figure 6b shows electric current flow through cells after (during) electroporation.
Figure 7 shows a typical electrical impedance tomography (EIT) system for use with the invention.
Figure 8a is an image of current flow through cells with irreversible electroporation and Figure 8b is an image of current flow through cells with reversible electroporation.
Figure 9 is a graphical schematic view of a finite element mesh showing a circular region of tissue surrounded by electrodes (dark dots) - the domain has two different impedances.
Figure 10 schematically shows a typical electrode configuration, measured electrical variables and equipotential lines in a circular domain having an inclusion with a different electrical impedance.
Figure 11 shows a real image in the upper left while impedance mapping is shown in the lower right showing differential impedance mapping.
Description of the invention and specific embodiments
When a range of values is provided, it is understood that each intermediate value is also specifically described, up to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limits of that range . The invention spans every smallest interval
ES 2 317 844 T3 between any indicated value or intermediate value in a indicated range and any other indicated or intermediate value in the indicated range. The upper and lower limits of these smaller ranges may be independently included or excluded in the range and the invention also encompasses every range in which either, neither, or both limits are included in the smaller ranges, subject to any specifically excluded limits in the indicated interval. When the stated range includes one or both limits, ranges that exclude either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to whom this invention pertains. Although any of the methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned in this document are incorporated herein by reference to set forth and describe the methods and / or materials in relation to those cited in the publications.
It is to be noted that as used herein and in the appended claims, the singular forms "a", "and" and "the" include plural referents unless the context clearly indicates 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 of skill in the art and so on.
The publications discussed in this document are provided solely for description prior to the filing date of this application. Nothing in this document should be construed as an acknowledgment that the present invention does not entitle such a publication to be back dated due to a prior invention. Also, the publication dates provided may be different from the actual publication dates which may independently require your confirmation.
Definitions
The term "electrode" is intended to refer to any conductive material, preferably a metal, most preferably a non-corrosive metal that is used to establish the flow of electrical current from that electrode to another electrode. "Electrically conductive" means to transmit electrical current that can be referred to in any way, for example, current or voltage. The electrodes are made of a variety of different electrically conductive materials and can be pure metals or alloys such as copper, gold, platinum, steel, silver, silver chloride, and alloys thereof. Furthermore, the electrode may comprise a non-metal that is electrically conductive such as a silicon-based material used in connection with microcircuits. Typical electrodes used in tissue electroporation are preferably rod-shaped, flat plate-shaped or hollow needle-shaped structures. The electrodes can be used to continuously supply electrical current or to supply pulses. Electrodes can be very application specific and comprise of parallel stainless steel plates, implanted leads, pairs of needles, and series of needles. Those skilled in the art will design specific electrodes that are particularly useful with respect to the desired results to obtain electroporation in accordance with the present invention.
The term "tissue" will mean a plurality of cells. Cells can be of the same type or of several different types. These cells are preferably organized to perform a specific function. Tissue includes tissue present in a living organism as well as removed tissue and can refer to in vivo or in vitro situations. Furthermore, the tissue can be from any organism including plants and animals or a tissue developed using genetic engineering and therefore be from an artificial source. In one embodiment the tissue is a plurality of cells present in a defined area of a human.
The term "device" and the term "electroporation device" are used interchangeably to describe any device as set forth and described throughout this document. The device preferably includes a first electrode and a second electrode where the first and second electrodes are connected to a source of electricity in a manner to provide the electrodes with positive and negative charges respectively. The device also preferably includes a means to impede the flow of electricity between the two electrodes except through one or more specific openings. For example, the means for impeding flow can be a non-conductive material having one or more openings therein wherein the openings are designed to specifically hold a biological cell or group of biological cells. Thus, the electric current has to flow through the aperture and through the cells to the other electrode. The device preferably also includes a means for measuring the flow of electrical current between the electrodes. The measurement means can include a voltmeter, an ammeter, or any device known to those skilled in the art that is capable of measuring the flow of electrical current in any way. Furthermore, the device preferably includes means for adjusting the amount of electric current flow between the electrodes. Thus the voltage, current, or other desired electrical current flow parameter can be specifically adjusted based on the measured flow to obtain optimal 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 to move a compound such as a drug or DNA sequence into a cell.
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The terms "power source", "source of electricity" and the like are used interchangeably herein to describe any means for providing electrical energy, current or voltage thereby creating a flow of electrical current between the electrodes. Preferably the device is capable of providing a controlled amplitude and mode and can provide constant DC current or AC current and provide pulsed voltage or continuous voltage. Preferred devices are capable of causing an exponential drop in voltage, ramp voltage, ramp current, or any other combination. For example, an electrical supply can be used in combination with a chip of the type used in connection with microprocessors and provide high speed power amplification relative to a conventional wall circuit by providing alternating current at 110 volts. The pulse shape can be generated by a microprocessor device such as a Toshiba laptop running a Lab View program supplying the output power to a power amplifier. A wide range of commercially available electrical supplies can provide the desired function. The electrical potential supplied for electroporation is usually indicated in terms of the voltage gradients that develop in the affected region which are defined in units of v / cm developed in the tissue. The 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 specific for amplification and can be extended outside the range for any desired application. Electrical pulses generally vary from microseconds to milliseconds. However, other pulse ranges can be used depending on the desired results.
Invention in general
Although this invention encompasses a variety of structures, methods, and applications, this part of the specification will illustrate certain specific structures and methods in detail, from which the concepts of the invention will become apparent in their entirety.
A wide range of different devices and systems can be used to perform the method of the invention. The device has to comprise a first electrode having a first voltage and a second electrode having a second voltage. Furthermore, the device will comprise a means for detecting the flow of charged particles between the electrodes and a means for varying the electrical current between the electrodes based on data obtained by detecting changes in the flow of charged particles between the electrodes. The device further comprises means for analyzing the electrical impedance of the electrically conductive medium to create an image. Preferably the device further comprises a component that prevents or substantially reduces the flow of charged particles between the electrodes except for the flow that occurs through one or more biological cells positioned between the first and second electrodes.
Any desired material can be added to the medium to move that material into a cell that is present in the medium. Furthermore, the invention does not necessarily include a process step of inclusion of a material in the medium that has to be brought into a cell. The process can be done simply to determine the changes that occur in a cell membrane based on the applied electrical current. This information can be valuable to determine characteristics about the cell or group of cells present in the medium and, specifically, it can be used to compare with information from normal and diseased cells or to determine the differences between cells previously tested and those that have been tested. they are currently rehearsing.
The first structure to be discussed is an electroporation cell with an internal support to support a single biological cell and an internal barrier that limits the flow of electrical current in the electrical cell to a flow path that passes through the biological cell. When no voltage is applied, the framework can be used for diffusive transport only, without the aid of voltage-induced pore formation.
The configuration of the barrier and the two chambers in embodiments that include two chambers is not critical to the invention and can vary widely while continuing to serve the purposes and advantages of the invention. However, since biological cells are microscopic in size, the preferred type of apparatus for the practice of this invention in each of its various forms is one in which the entire structure and / or its chambers are the size of chips. electronic, manufactured by microfabrication techniques such as those used in the manufacture of electronic chips. Furthermore, it is preferred that the chambers are constructed as continuous flow chambers to allow the passage of liquids in continuous flow, intermittent flow or flow in the direction of the user and to allow changes in concentrations, pressure and other conditions as necessary to achieve strict control over the passage of species through the biological cell membrane. Accordingly, a preferred structure and method of manufacturing the apparatus are those which involve forming the apparatus in layers or platelets with appropriate openings that form flow passages when the layers or platelets are bonded together.
Flow-through chambers offer the advantage of allowing single cells to enter and remove consecutively so that a large number of cells can be treated in series. Continuous flow chambers also allow the sourcing of solutions poor in solute so that concentration gradients can be continuously maintained when desired. An additional function that continuous flow chambers can offer is increasing and decreasing pressure, a function that is useful for various purposes as described later.
The support of the biological cell in this structure can be any structure that secures the biological cell in a fixed position and that allows the passage of electrical current. The most convenient support is an opening in the barrier. Securing a biological cell over the opening serves to close, seal, or plug the opening, thereby directing
ES 2 317 844 T3 mode the passage of electrical current, diffusive transport, or both, through the cell and eliminating or minimizing leakage around the cell. A convenient mechanical means to achieve this is to apply a pressure differential across the aperture in a direction that will press the cell against the aperture. The diameter of the opening will be smaller than that of the cell and the cell will pass into one of the two chambers after entering the apparatus. By increasing the pressure in the chamber in which the cell resides or by decreasing the pressure in the other chamber, the cell will be forced against the opening, closing it. Once the procedure is complete, the cell is easily released from the opening by equalizing the pressures in the two chambers or reversing the differential so that the higher pressure is in the chamber other than the chamber in which the cell was inserted. Then the flow of liquid into the chamber in which the cell was introduced will remove the cell from the opening, exposing the opening for another cell.
An alternative method of sealing the opening with the cell is by using a coating on the surface of the barrier, or on the edge of the opening, of a substance that binds to the cell membrane. Since biological cell membranes are negatively charged, the coating can be a substance that carries a positive charge, such as polylysine, polyarginine, or polyhistidine. The biological cell can be directed into the opening by a pressure differential across the opening and held in place by the coating. Once the procedure is complete, the cell can be released from the coating by momentarily increasing the flow rate of the liquid in the chamber on the side of the opening that the cell is on or by applying a reverse pressure differential across the opening to propel the cell away from the opening.
The size of the aperture is not critical to the invention as long as the aperture exposes sufficient surface area on the cell membrane to achieve the desired degree of mass transfer, the passage of an electrical current, or both, within a period of time. controllable and economically reasonable. Therefore, the optimal size will vary with the particular cells being treated or studied. In general, the aperture is preferably circular or roughly circular in shape and, depending on the size of the cell, preferably ranges in 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. Preferably the barrier in which the hole is formed and which separates the two chambers is made of a rigid dielectric material which is impermeable to both water and solutes and which will withstand a sufficient pressure differential to secure a cell against the opening. For devices manufactured by microfabrication techniques, silicon nitride is a suitable material for the barrier. Other materials that will serve equally well will be readily apparent to those of skill in the art.
An additional element of preferred embodiments of this invention is the use of apparatus made of transparent materials. This allows the user to observe the interior of the cells and the microdiffusion and microelectroporation processes through a microscope as they occur.
Electroporation used in in vivo therapy
The electroporation techniques of the present invention are useful in connection with the treatment, analysis or diagnosis of an organism including mammals and humans in need of treatment. In general, treatment can be performed by continuously injecting a material or in a rapid bolus into an area of tissue to be treated. The electrodes are placed adjacent to the tissue and current or voltage is applied and continuously measured to determine when the desired level of electroporation is obtained thereby allowing the injected material to be moved towards the cells of the tissue being treated.
The pharmaceutically active compound that is injected can be a conventional drug commonly referred to as a small molecule or be a protein or nucleotide sequence encoding a protein. Furthermore, the composition injected into the tissue can be administered before, during or even after the application of electrical pulses from the electroporation device. The overall goal of the process is to provide pore opening through electroporation and thereby introduce the compounds into cells whose cell membranes would not normally penetrate the compounds. For example, it is possible to introduce bleomycin or various gene and / or plasmid constructs into cells of the tissue being treated. This is achieved by generating electrical potentials and currents through cells in the tissue to be treated where electrical potentials are generated as electrical impulses. It is preferable to use a plurality of electrodes as opposed to a single electrode to generate the pulses.
An example of a useful electrode design is one comprising two flat steel strips 10mm wide and 0.6mm thick. The electrodes are spaced at a fixed distance of approximately 6 to 7 m from each other. A second electrode design comprises two, up to a maximum of 8, 20mm flat steel squares. The electrodes are connected to a PS15 electro-pusher. The pulses can be delivered by placing the electrodes on the skin with the flat side on the skin or by placing the electrodes around skin tumors. Contact with the skin can be achieved through the use of materials conventionally used in connection with the performance of electrocardiograms such as gels or electro-conductive saline solutions. To perform the procedure a patient may receive one or a plurality of pulses and preferably receives a plurality of pulses. Different configurations can be designed to electroporate tissue within an organism such as within a human body, that is, without applying electrodes outside of the skin. Such configurations may comprise arrays of needles comprising a plurality of needle electrodes. As an example, the positive and negative electrodes may each comprise six or more needles of 0.5mm diameter and comprising stainless steel,
ES 2 317 844 T3 cm in length connected to a BTX 820 pulse generator. Electrodes can be inserted in parallel into the tissue around the cells to be influenced by electroporation. The electrodes can be placed in circles of various diameters ranging from 5mm to 1cm. An electrode voltage ratio in the range of about 1300 v / cm can be used. Although any number of pulses can be delivered it is preferable to start the process by supplying approximately six pulses at one second intervals with a pulse width of 100 microseconds. The present invention is particularly desirable in relation to electroporation of tissue in that the method can determine whether electroporation is occurring without the use of dyes and labels to track material being introduced into the cell.
As shown in Figures 6a and 6b electrical current can flow around cells (Figure 6a) or through cells (Figure 6b) after electroporation has taken place. The process of the invention makes it possible to determine the point at which the transition occurs between what is shown in Figure 6a and what occurs in Figure 6b and, in addition, it makes it possible to avoid the appearance of irreversible effects on cell membranes. As shown in Figure 8a electroporation can be performed to such a great extent that cell membranes are damaged thereby resulting in irreversible effects on cells. In general, this is undesirable. However, it is possible to obtain electroporation without significant damage to cell membranes by modulating the amount of electric current, thereby obtaining a reversible situation as shown in Figure 8b.
As shown in Figures 6a and 6b cells create electrical impedance and the present invention is concerned with accurately determining the degree of that electrical impedance and adjusting the current 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 to measure other effects of current in creating electroporation in a plurality of cells in the tissue. Electrical impedance is a measurement of how electricity travels through a given material. Each material has a different electrical impedance determined by its electrical composition. Some materials have high electrical impedance and others have low electrical impedance. Malignant (cancerous) breast tissue has a much lower electrical impedance (conducts electricity much better) than normal tissue or a benign (non-cancerous) tumor.
Impedance is a measurement of the degree to which an electrical circuit resists the flow of electrical current when voltage is impressed across its terminals. Impedance, expressed in OHMS, is the ratio of the voltage that is printed across a pair of terminals to the current flow between those terminals. In direct current (DC) circuits, impedance equals resistance. In alternating current (AC) circuits, impedance is a function of resistance, inductance, and capacitance. Inductors and capacitors create voltages that oppose the flow of current. This opposition is called reactance and has to be combined with resistance to define impedance. The resistance produced by the inductance is proportional to the frequency of the alternating current, while the reactance produced by the capacitance is inversely proportional to the frequency.
The basic concepts described above are used in the basic aspects of the present invention and are also applicable to describe electrical impedance imaging also called electrical impedance tomography (EIT). It should be noted that several different terminologies can be used to describe the same technique and these include applied potential tomography (APT). These imaging technologies make it possible to produce images based on the spatial variation of the electrical properties of biological tissue. Techniques such as APT and EIT could be used to carry out the invention in relation to tissue. Applied Potential Tomography (APT) depends for its physical basis on the measurement of a potential distribution on a surface of a biological material, when an electrical current is applied between two points on the surface. Other researchers have used the technique and refer to it as electrical impedance imaging, conductivity imaging, electrical impedance tomography, etc. In this document, the technology is generally referred to as EIT or Electrical Impedance Tomography. Accordingly, in the remainder of the description the technology is referred to as EIT technology only and an example thereof is shown below in Example 3.
Those skilled in the art will consider different means of determining changes in electrical current after reading this description. A preferred method of determining such changes when performing the invention on tissue is to use imaging technology and specifically electrical impedance tomography (EIT) that monitors and analyzes differences in the bioelectrical attributes of the sample being monitored to produce. an image. EIT technology can be used in connection with the present invention by creating an EIT image and using that image to adjust current flow to obtain desired results. Specifically, the EIT image is created by injecting electrical currents into the tissue and measuring the resulting voltages across a series of electrodes. This allows an impedance image to be produced from known current inputs and measured input voltage data using a reconstruction algorithm. The use of EIT technology is particularly desirable in relation to the present invention when applied to tissue where EIT imaging provides a map of electrical impedances. The electrical impedance map essentially allows the user to visualize the start of electroporation. When electroporation begins the user can stabilize the amount of current being applied and thereby avoid applying so much current that it results in irreversible cell damage as shown in Figure 8a. EIT technology enables the region of tissue undergoing electroporation to be visualized based on changes in the equivalent electrical impedance of cells in the tissue being monitored.
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Figure 7 shows a conceptual view of an EIT system used to perform a process of the present invention on tissue (71). A current source (72) is controlled by a signal generator (73) and is used to conduct an electrical current in the tissue sample (71) through a pair of computer controlled multiplexers (74) and (75). leading to a differential amplifier (76) and a demodulator (77). The measured signals are compared to the original to record amplitude and stage data for later image construction. The control computer (78) typically chooses which pair of electrodes to inject current while reading the voltages of the remaining electrodes. There are several different hardware configurations that can be used in connection with the present invention.
The EIT system as shown in Figure 7 is generally called a series system due to its single current source and measurement amplifier. In other systems, varying degrees of parallelism (multiple current sources and voltage measurement amplifiers) have been used, thereby increasing the flexibility and speed of the current injection system.
Reconstruction algorithms are used to take the voltage measured on an outer surface of a region of interest in the body (the injected current data) and information related to the geometry of the electrode and produce an image representing the tissue spatial distribution of impedance. tissue within tissue region (71). There are several methods that can be used to create an impedance image. Static imaging is the production of an absolute impedance distribution. Cook, RD et al. ACT3: a high speed, high precision electrical impedance tomography. IEEE, Trans. Biomed. Eng. 41, 713-22 (1994). Differential imaging methods produced distributions based on the differences between two data sets. Barber, DC in Advances in Biomed. Eng. (Ed. Benek en, W., Thevenin, V.) 165-173 (IOS Press, Amsterdam, 1995). This type of technique provides a picture of how the impedance distribution has changed from an initial measurement. Multi-frequency impedance imaging takes advantage of the dependence of tissue impedance on frequency. Groffiths, H. The importance of phase measurement in electrical impedance tomography. Physics in Medicine and Biology 32, 1435-44 (1987). Quasi-static images can be produced using the above differential technique using a low-frequency image as the initial measure. Accordingly, the system enables one type of static imaging to be produced without the difficulties of true static imaging.
A mathematical model of how the current behaves in the tissue is used to provide the reconstruction and thus the image. In general, a model is provided for directing current flow in the EIT using 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 several different methods for solving boundary value problems. However, all of these problems can be classified into iterative analytical or numerical techniques and can be applied by those skilled in the art to carry out the present invention.
The vast majority of reconstruction algorithms in use today employ iterative numerical solutions to the Poisson equation. Most iterative numerical study procedures attempt to solve the boundary value problem by estimating an impedance distribution in the tissue and repeatedly solving the direct problem (finding the voltage and current densities with a given impedance distribution) and adjusting the rough estimates of impedance proportionally, until the measured voltages and currents correspond to those calculated. The direct problem has to be solved numerically and is usually done using finite element or finite difference schemes. The Finite Element Method is a very powerful and popular method of direct problem solving and because of this it tends to dominate engineering solutions across many interdisciplinary fields.
An example of a microdiffusion apparatus in accordance with this invention is shown in Figure 1 for a single biological cell, for transporting materials across the cell membrane without the application of an electric field. These components of this apparatus, from bottom to top, are an acrylic base (11), an intermediate silicon layer (12) (1 micrometer thick) with a part (13) carved to define the lateral limits of the lowest of the two liquid chambers, a layer of silicon nitride (14) that serves as the barrier between the two chambers, a silicon washer (15) that defines the lateral limits of the upper liquid chamber (16) and a glass cover ( 17). A hole (18) in the silicon nitride barrier serves as the opening and a contiguous cell or cell mass such as tissue (19) is shown covering the hole. Channels extend through the acrylic base to serve as inlet and outlet channels for liquids passing through the upper and lower chambers, as shown by the arrows in the Figure.
When the pressure in the upper chamber (16) is higher than in the lower chamber (13), the cell will remain in position over the hole, serving as a plug that separates the liquids in 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 in one chamber differs from that of the other, mass transfer occurs through the cell from one chamber to the other. By precisely controlling the compositions of the solutions in the two chambers, the amount and direction of mass transfer within the cell can be precisely controlled. Since the diameter of the aperture (18) is known, the mass transfer that occurs through the aperture can be accurately determined.
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The many applications of this microdiffusion device will be readily apparent. For example, the device can be used to infuse a cryopreservative such as glycerol into a cell by filling the upper chamber (16) with physiological saline and the lower chamber (13) with glycerol. When using a cell (19) for which the mass transfer coefficient of glycerol across the cell membrane is known, the amount of glycerol that will enter the cell can be easily calculated and the concentrations and exposure times adjusted to infusing the cell with an amount known to be required for cryopreservation.
An example of a microelectroporation apparatus according to this invention for a single biological cell is shown in Figure 2. The apparatus is similar in construction to the microdiffusion apparatus of Figure 1. Its structural components, from bottom to top, are an acrylic base (21), a lower silicon layer (22) with a carved part to define the lateral limits of the lower liquid chamber (23), a silicon nitride layer ( 24) (1 micrometer thick) that serves as the barrier between the two chambers, an upper silicon layer (25) that defines the lateral limits of the upper liquid chamber (26) and a cover consisting of a layer of poly silicon n + (5,000 A thick) (27) and a layer of silicon nitride ( 1 micrometer thick) (28). A hole (29) in the silicon nitride barrier (24) serves as the opening 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. Above the acrylic base (21) resides an additional layer of poly silicon n + (5,000 A) (31) and this layer, together with the layer of poly silicon n + (27) above the upper chamber (26) serve as the two electrodes. Each electrode is connected by electrical cables to a printed circuit board (32) that controls the voltage applied between the electrodes and measures the current that passes between them.
The microelectroporation apparatus shown in Figure 2 can be manufactured by conventional microfabrication techniques, typically involving chemical vapor deposition, masking, etching, and sputtering. The operation of the apparatus will be analogous to the operation of the microdiffusion apparatus of Figure
1. The movement of biological cells through the apparatus is achieved by suspending the cells in the liquid used to fill the upper chamber and the cells are drawn into the opening, one by one, applying a pressure differential between the chambers, which also maintains a cell in place once the cell has been drawn into the opening. A convenient method of applying such a pressure differential is to maintain atmospheric pressure in the upper chamber while lowering the pressure in the lower chamber below atmospheric by attaching a syringe to the lower chamber and pulling the plunger of the syringe. Care must be taken to limit the pressure differential so that it does not damage the cell.
Figures 3a and 3b illustrate a different apparatus and method within the scope of this invention. This apparatus and method involves 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 Figure 3a shows the channel walls (41) and a biological cell (42) passing down through the channel lumen (in the direction of the arrow). The cross section of Figure 3b shows that the channel is rectangular in cross section, although other cross section geometries can be used. The electrodes (43, 44) are formed as coatings on two opposite walls of the channel. The electrodes are connected by wires to a printed circuit board (45) that measures impedance and controls the voltage applied to the electrodes. The biological cell (42) is shown passing through the region between the two electrodes.
The cross-sectional area of the channel is large enough to allow the cell to pass essentially unimpeded through the channel walls and, at the same time, small enough that only one cell can pass through the inter-electrode region at a time. Additionally, each electrode (43, 44) is of length approximately equal to or slightly greater than the diameter of the biological cell, so that after entering the region the cell causes a significant or appreciable decrease in the current that passes through the region. due to the voltage applied across the electrodes. The spacing of the electrodes, that is, the distance between them, is likewise subjected to the same considerations. The biological cells are suspended in a liquid solution of the species to be introduced into the cells and the suspension is passed through the channel. A voltage is applied between the electrodes as the suspension flows through the channel and the current between the electrodes (or impedance) is monitored. A significant drop in current indicates the presence of a biological cell in the inter-electrode region. Once the cell is detected in this way, an electroporation pulse can be applied while the cell is still in the inter-electrode region and further impedance can be observed to detect the onset of electroporation. Species dissolved in the liquid solution will enter the cell as a result of electroporation.
Variations in these structures and methods will be readily apparent to those of skill in the art. For example, the barriers described above can be minimized or avoided by using microelectrodes that are the same size or smaller than biological cells. Examples of such microelectrodes are carbon fiber microelectrodes (such as ProCFE, Axon Instruments, Foster City, California, USA) used in conjunction with high grade micromanipulators (such as those available from Narishige MWH-3, Tokyo, Japan). Microelectrodes can be used in place of the electrodes shown in Figure 2 or in place of those shown in Figures 3a and 3b.
Examples
The following examples are set forth to provide those skilled in the art with a full explanation and description of how to prepare and use the present invention and are not intended to limit the scope of what the inventors consider their invention nor are they intended to represent that the following experiments are all experiments or
ES 2 317 844 T3 only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperature, etc.) but some experimental errors and deviations must be assumed. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
A series of experiments were performed using an electroporation system consisting of the microelectroporation device described above and shown in Figure 2, combined with flow and pressure control units and pressure indicators for the liquids that have to circulate through the upper and lower chambers, a variable DC power supply, a pulse generator and current amplifier to apply voltage pulses through the device, a digital oscilloscope to monitor the pulses, a fluorescence microscope, a CCD (charge coupling device) camera, and a computer with signal processing software imaging and waveform processing. Both chambers of the device were filled with physiological saline and the cells were introduced into the upper chamber. The movement of liquid in the upper and lower chambers was controlled by syringes. The pressure in the upper chamber was atmospheric while the pressure in the lower chamber was reduced below atmospheric by pulling the barrel of the syringe connected to that chamber. The voltage was applied in single square pulses ranging from zero to 120 v in magnitude and from 2 microseconds to 100 milliseconds in duration. The distance between the electrodes in the upper and lower chambers was 900 microns.
The assays in this example were performed using ND-1 human prostate adenocarcinoma cells with a typical diameter of 20 microns. The opening in the microelectroporation device was 5 microns in diameter. A rectangular voltage pulse with a duration of 60 milliseconds was applied and the pulse was applied at various amplitudes ranging from 10v to 60v in 5-volt increments. With each pulse, the electrical current passing through the aperture was measured. Cell experiments were performed and repeated both with the opening covered with a glass microsphere and without any obstruction in the opening. In each case the results were expressed as current microamps versus pulse width volts and are plotted in Figure 4, where the upper curve (data points represented by x) represents the unobstructed opening, the lower curve ( data points represented by asterisks) represents the data obtained with the glass microsphere residing in the aperture and the three intermediate curves (open squares, open vertical triangles and open inverted triangles) represent data obtained with three different ND-1 cells residing in the aperture.
The upper curve shows that the current increases in a substantially continuous manner as the voltage increases when there is no barrier to the passage of current through the aperture. The lower curve also shows a substantially continuous increase as the voltage increases, albeit at a much lower level. The current values shown in the lower curve represent leakage currents through the device. The data curves obtained with the ND-1 cells through the aperture show that at low voltages the current has a value close to that obtained when the aperture is closed by the glass microsphere, while at high voltages the current increases to levels obtained with a clear opening. The transition is a sharp increase that is indicative of the formation of pores in the cell membrane through which an electrical current can pass, that is, the beginning of electroporation. In all three cells, the transition occurred at voltages between 30 v and 40 v. In two of the three cells (open squares and open vertical triangles), the start of electroporation occurred at essentially the same voltage, while in the third (inverted triangles), the start occurred at a voltage that was lower than the other two at about 5v. This illustrates the value of controlling the process so that individual cells achieve optimal results.
After the data shown in Figure 4 were generated, the pulses were reapplied in descending order of amplitude values and the resulting curves presented hysteresis, that is, the curves obtained with descending amplitudes were of higher voltage than those obtained with amplitudes. ascending. This indicated that the electroporation in these experiments was irreversible.
Example 2
Using the same electroporation system used in Example 1, a series of assays were performed on rat hepatocytes (American Culture Collection Type No. CRL-1439), whose typical cell diameter was 20 microns and the microelectroporation apparatus had a 4 micrometer diameter opening. Rectangular voltage pulses having a duration of 60 milliseconds were also used here, varying in amplitude from 10v to 37.5v with increases of 5V in the 10v part to 30v and with 2.5v increases in the part of 30 v to 37.5 v. In some cases the experiments were performed only by increasing the amplitudes and in others by first increasing and then decreasing the amplitudes to assess reversibility. The results are represented in the graphs shown in Figures 5a, 5b, 5c and 5d. In each case, the upper curve (data points represented by circles) are the data obtained without a cell or a glass microsphere residing in the aperture, the lower curve (data points represented by squares) are the data obtained with a glass microsphere in the aperture and the intermediate curve (data points represented by triangles) are the data obtained with a hepatocyte in the aperture, using different hepatocytes for each of the four Figures.
In Figure 5a, the amplitude was increased and not decreased, presenting an electroporation threshold voltage of between 25 v and 30 v. In Figures 5b and 5c, the amplitude was first increased and then decreased to produce the
ES 2 317 844 T3 two intermediate curves. Although the ascending and descending curves do not differ, they are substantially identical in each Figure, indicating that the cell membrane in each of these two cases closed again after each voltage impulse and, therefore, that the pore formation was reversible. In the test represented by Figure 5d, the cell disintegrated once the applied voltage exceeded 37.5 v, although this is not shown in the Figure. It is significant to note that despite the fact that the same cell types were used in each of Figures 5a, 5b, 5c, and 5d, the electroporation threshold voltage was different between individual cells, although all were in the range of 20 to 35 v. Adaptation of the procedure to individual cells is easily achieved by monitoring the current in this way to observe when the electroporation threshold occurs. Selection of the optimal exposure time, voltage, composition changes in environmental fluids, and other system parameters can then be made to achieve the desired cell treatment without cell destruction.
The methods described in this document are useful tools in the laboratory for conducting fundamental research into the electroporation properties of biological cells and useful tools in industry for processing large numbers of cells in a continuous flow manner. By allowing the observation and recording of the current flowing through individual cells, the amplitude and duration of the voltage pulse can be controlled to achieve optimal results. Additionally, the devices described and shown in this document for use in the practice of the invention can be constructed with transparent parts and of a size suitable for mounting on a microscope stage. This will allow for a correlation between electrical current measurements, visual observations, and fluorescence measurements within the cell. The device can be used to electrically detect, through current measurement, the point in time at which a cell lodges in the aperture as well as the point in time at which pore formation is achieved in the opening. cellular membrane. For larger scale and industrial applications, a large number of microelectroporation devices of the type described in this document can be arranged in parallel. For each cell, electrical information indicating immobilization of a cell in the aperture (such as a sharp drop in current) can be used to generate a signal that will initiate an electroporation sequence and additional electrical information indicating completion of the electroporation. Electroporation (such as a sudden increase in current) will generate a signal that will release the cell (for example, eliminating or reversing the pressure differential) and will allow the next cell to flow into the opening.
In addition to using the device and system of the invention to move material into or out of the cell the system and device can be used in a diagnostic or analytical mode. This is done by measuring the electrical impedance of a cell or cells placed in a medium and using the measured electrical impedance information. It is possible to deduce information related to the integrity of the cell membranes and thus to anticipate the analysis. It is also possible to compare the information with information previously obtained from normal or diseased cells of the same type and thus obtain diagnostic information. For example, the electrical impedance of a cell with an intact membrane will be much higher than the impedance of the same cell with an altered membrane. Thus, the process can analytically provide information regarding the structural integrity of the cell membrane. Diagnostically the method can provide information regarding the relative structural integrity of cell membranes.
Example 3
Mapping of electrical impedance of domains subjected to electroporation
To illustrate the ability of EIT to monitor electroporation in tissue, a mathematical simulation of the problem has been solved.
To provide the data necessary for the electroporation imaging simulation, a simulated tissue surrogate was created first using a 2-D fine mesh FEM model (~ 1600 nodes, ~ 3100 elements). The surrogate, shown in Figure 9, consisted of a circular imaging domain (20 mm radius, 500 ohm-cm resistivity for muscle with a variable number of point source electrodes spaced the same distance around the periphery . Within this imaging region, a single electroporation region was defined that was arbitrarily shaped with a different resistivity. An opposing electrode current injection pattern was used, providing independent voltage measurements N (N-1) / 2, where N is the number of electrodes. The model was solved using adaptive mesh generation and FEM solution algorithms available from MATLAB's Partial Differential Equation Toolbox (The Mathworks Inc.). An example mesh for the given geometry is shown in Figure 9. The information that the surrogate module makes available to the reconstruction algorithms represents the data that would have been available during the electroporation portion of an experiment, that is, current and voltage across the different electrodes around the tissue. From these data, an attempt was made to reconstruct the original image of the tissue that was introduced into the model. (It should be noted that a DC injection current was used instead of the typical AC current in EIT to simplify the problem. The AC bypass and run is a simple extension to what is presented in this document). A typical example of the voltage and current distribution in the surrogate during a simulated data acquisition stage for an 8-electrode EIT system is illustrated in Figure 10.
The data obtained from the surrogate was fed into two EIT imaging algorithms, the first using the finite element method and the second using the boundary element method to generate the impedance image. The algorithms use a conventional Newton Raphson technique to produce the image. Figure 11
ES 2 317 844 T3 compares the image of a circular domain with two different electrical impedances compared to the image of the original surrogate as recreated with the finite element technique and with the boundary element technique.
Electrical impedance tomography can be used to image the electroporation 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. Electrodes for tissue electroporation imaging may be different from those used for the electroporation process itself or they may be the same.
Example 4
Electrical detection of changes in membrane permeability
As part of the research on cell electroporation, the electrical characteristics of cells during reversible and irreversible electroporation have been studied. In reversible electroporation the cell is not damaged by the electroporation process and the membrane closes again. In irreversible electroporation, the cell membrane is damaged and does not close again. In a set of experiments in which ND1 cells have been used to measure the currents through the cells on the microelectroporation chip, results illustrated in Figures 8a and 8b have been obtained. The results were obtained by exposing the cells to electrical impulses in a triangular shape (upper curve) in 8a and 8b. The electric currents flowing through the cells are shown in the lower curve at 8a and 8b. Figure 8a is of a cell that has been irreversibly electroporated and Figure 8b of a cell that has been reversibly electroporated. It can be easily seen that when the voltage was lowered in the cell that underwent reversible electroporation, it retained the same values as during the step of increasing the voltage. However, in the irreversible case the current through the cell with the damaged membrane had a higher current than in the intact cell. This leads to the conclusion that electrical currents flowing through cells can provide indications of changes in membrane permeability in general and a measure of cell membrane integrity in particular in a variety of situations and not just during electroporation. For example, cell viability is often measured with trypan blue or fluorescent dyes that penetrate damaged membranes. These results show that an alternative method of detecting cells with damaged membranes would be to measure the ratio of electrical current to voltage through the cell. Similarly, there are compounds that induce pores in the cell membrane, such as ionophores. Measurement of current-voltage (impedance ratio across a cell membrane) could also detect whether the membrane has been altered by these chemicals. Electrical measurements would have an advantage over chemical means of detecting cell membrane damage because they would produce immediate information. One possible method to detect changes in the permeability of the cell membrane and in particular damaged cell membranes is to use the electroporation chip as described for the electroporation process. The measure of damage would be the difference between the impedance of an intact cell and the impedance of a damaged cell as illustrated in Figures 8a and 8b. It would be possible to detect cells with damaged cell membranes in tissue in a similar manner to the electroporation detection methods described herein.
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Numbers
- Publication
- 2317844
- Publication, DOCDB
- 2317844
- Publication, EPODOC
- ES2317844T
- Application
- 950560
- Application, DOCDB
- 00950560
- Application, EPODOC
- ES20000950560T
Titles2
- Spanish
- PROCEDIMIENTO Y DISPOSITIVO PARA CONTROLAR LA ELECTROPORACION.
- English
- PROCEDURE AND DEVICE TO CONTROL THE ELECTROPORATION.
Classification
- CPC, 3
- G01N33/48728
- A61B5/0536
- C12M35/02
- IPC, 11
- C12N13 00
- G01N27 02
- A61B5 053
- A61N1 30
- C12M1 00
- C12M1 42
- C12M3 00
- C12N5 07
- C12N5 071
- C12N15 09
- G01N33 487