Large volume ex vivo electroporation method

Abstract

An object of the invention is to provide an electroporation method for treating vesicles with exogenous material for insertion of the exogenous material into the vesicles which includes the steps of: a. retaining a suspension of the vesicles and the exogenous material in a treatment volume in a chamber which includes electrodes, wherein the chamber has a geometric factor (cm.sup.−1) defined by the quotient of the electrode gap squared (cm.sup.2) divided by the chamber volume (cm.sup.3), wherein the geometric factor is less than or equal to 0.1 cm.sup.−1, wherein the suspension of the vesicles and the exogenous material is in a medium which is adjusted such that the medium has conductivity in a range spanning 50 microSiemens/cm to 500 microSiemens/cm, wherein the suspension is enclosed in the chamber during treatment, and b. treating the suspension enclosed in the chamber with one or more pulsed electric fields. With the method, the treatment volume of the suspension is scalable, and the time of treatment of the vesicles in the chamber is substantially uniform.

Claims

1. An apparatus for electroporation comprising: a sterile electroporation chamber with a geometric factor defined by the quotient of the electrode gap squared (cm.sup.2) divided by the chamber volume (cm.sup.3), wherein the geometric factor is less than or equal to 0.1 cm.sup.−1; wherein the chamber is configured for sequential electroporation and the chamber has an inlet and an outlet configured for sequential passage of suspensions and fluids during electroporation; the chamber comprising: a first reservoir in fluid communication with the inlet for containing the suspensions and fluids prior to introducing into the chamber for treatment, a second reservoir in fluid communication with the inlet for containing flushing material for flushing the suspensions and fluids after treatment out from the chamber, and a third reservoir in fluid communication with the outlet for receiving the suspensions and fluids after treatment flushed out from the chamber; a vent for removing air when the chamber is filled with a fluid; parallel plate electrodes; and a source of pulsed voltages electrically connected to said parallel plate electrodes configured to provide pulse waveforms and an uniform electric field between the parallel plate electrodes, wherein the uniform electric field is greater than 100 volts/cm and less than 5,000 volts/cm and substantially uniform throughout the chamber.

2. The apparatus of claim 1, wherein the vent is a filter member in the wall of the chamber.

3. The apparatus of claim 1, wherein the vent is a vent-cell in fluid communication with the chamber.

4. The apparatus of claim 1, wherein the chamber further comprises an elastomeric seal.

5. The apparatus of claim 1, wherein the first reservoir contains a low conductivity medium for suspending vesicles for electroporation, wherein the conductivity of the medium is between 50 microSiemens/cm and 500 microSiemens/cm.

6. The apparatus of claim 1, wherein the volume of the chamber is between 2 ml and 10 ml.

7. The apparatus of claim 1, wherein the first reservoir, the second reservoir, and the third reservoir comprise flexible bags.

8. The apparatus of claim 1, wherein an inlet valve is connected between the chamber inlet and the first reservoir and the second reservoir, and an outlet valve is connected between the chamber outlet and the third reservoir.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The invention will be better understood and the above objects as well as objects other than those set forth above will become more apparent after a study of the following detailed description thereof. Such description makes reference to the annexed drawing wherein:

(2) FIG. 1A is a schematic illustration of apparatus employed with carrying out the method of the invention.

(3) FIG. 1B is a schematic illustration of the electroporation chamber with its parts of the electroporation apparatus.

(4) FIG. 2 is a graph illustrating the operating range of the method of the invention, inside the triangle, and how the operating range of the invention is outside operating ranges of prior art electroporation methods, indicated by small blocks outside the triangle.

(5) FIG. 3 is a graph illustrating the relationship between charging time (in microseconds) of biological cells and media conductivity (in microSiemens/cm) for cells having three different diameters, namely 1 micrometer, 10 micrometers, and 100 micrometers.

(6) FIG. 4 is a graph showing Time Constant versus Conductivity as it relates to the method of the invention.

MODES FOR CARRYING OUT THE INVENTION

(7) As previously described a significant problem is the conductivity of the media used in electroporation. In the process of the invention, a low conductivity medium is employed to keep the total resistance of the suspension greater than one ohm, wherein heating in the chamber is limited to low levels. Not just any medium conductivity can be used. As the ionic content of the medium is reduced, the number of free ions that are available to build charge (voltage) across the cell member is decreased. The effect is to increase the amount of time it takes to charge the membrane. This process is described by the equation in Electroporation and Electrofusion in Cell Biology, edited by Eberhard Neumann, Arthur Sowers, and Carol Jordan, Plenum Press, 1989, on page 71. Assuming a typical cell diameter of 10 microns, the charging time is 20 microseconds at 80 μS/cm. Below 80 μS/cm the charging time become too long and the pathways in cell membrane stop forming. The TABLE 6 below illustrates the resistance of the media as a function of electrode chamber volume and conductivity.

(8) TABLE-US-00006 TABLE 6 Chamber Suspension Resistance - ohms Volume ml 17,000 μS/cm 200 μS/cm 80 μS/cm 0.5 19.2 160 0   4000 1 9.6 800 2000 5 1.92 160 400 10 0.96  80 200 50 0.19  16 40

(9) Ex vivo electroporation has been demonstrated in numerous published research projects. At this point commercial applications, such as clinical transfection to produce a vaccine for the patient, requires large electrodes or chambers to process millions of cells at one time. The static parallel plate chamber provides the most uniform amplitude and most uniform electric field direction of any configuration available. This uniformity is required to insure uniform treatment of the target cells. It is also important not to use very high-density cell concentration such as 30 million cells/ml to insure local uniform electric fields about the cells. This invention applies to chambers in a range spanning 2 to 10 milliliters.

(10) Using larger chambers results in high current flow when voltage is applied. The equations for chamber resistance vs. conductivity of the cell and media mixture and the chamber dimensions are as follows:

(11) $\mathrm{Volume}\mathrm{of}\mathrm{material}=l\times A$$\mathrm{Resistance}\mathrm{of}\mathrm{Material}=\rho \frac{l}{A}=\frac{1}{\sigma }\frac{l}{A}=\frac{1}{\sigma }\frac{l^2}{\upsilon }=\frac{GF}{\sigma }\mathrm{ohms}$ ρ=resistivity in ohm-cm σ=1/ρ in Siemens/cm υ=volume of material being treated l=gap between electrodes (cm) A=area of electrode (cm.sup.2)

(12) There is a Geometric Factor (GF), which is a constant for any chamber dimension. As the volume of the chamber gets larger the resistance of the material in the chamber gets smaller thus increasing current flow.

(13) The present invention uses an electrode with large capacity in combination with an electroporation buffer of defined low conductivity. This process exposes all cells to the same treatment conditions, provides control over the amperage required and can process large numbers of cells. Since the cell suspension statically remains in the chamber during application of pulsed electric fields, complex waveforms can be used.

(14) Another aspect of the invention further increases capacity by alternately filling and emptying the gap between the electrodes. In this manner, all desired properties are met during a specific treatment and the electrodes can be re-used for subsequent treatments in a sequential batch process.

(15) This present invention specifies a range of medium and suspension conductivities, which can be used versus the chamber dimensions, the larger the volume the smaller the conductivity. This invention specifies an operating area for use with the larger volume chambers. This is illustrated in FIG. 2. Operating points of prior art published results are also presented in FIG. 2 as squares. For chambers with a Geometric Factor less than 0.1 there are two limiting factors, which are related. The first is the absolute value of the chamber resistance. In this invention the chamber resistance is one ohm or greater. Operating below one ohm is viewed as impractical. The other constraint is the conductivity of the medium and suspension in the chamber. As the conductivity decreases the charging time of the cell membrane increases because there are fewer ions external to the cell membrane.

(16) The relationship between the Transmembrane Voltage (TMV) and conductivity and cell diameter is as follows, taken from Newman et al stated below:
Transmembrane Voltage=TMV
TMV=−1.5Er|cos δ|f(λ) where: E=electric filed in volts/cm r=cell radius in cm δ=angle from electric field line in degrees

(17) $f\left(\lambda \right)=\mathrm{composite}\mathrm{conductivity}$$f\left(\lambda \right)=\frac{{\lambda }_0{\lambda }_1(2\frac{d}{r}\left(\right)}{\left(2{\lambda }_o+{\lambda }_i\right){\lambda }_m+\left(2\frac{d}{r}\right)\left({\lambda }_o-{\lambda }_m\right)\left({\lambda }_i-{\lambda }_m\right)}$ where: λ.sub.o=conductivity of media external to cell milliSiemens/cm λ.sub.i=conductivity of cytoplasm λ.sub.m=conductivity of cell membrane d=thickness of cell membrane Reference: Electroporation and Electrofusion in Cell Biology Edited by Eberhard Neumann, Arthur Sowers, and Carol Jordon Plenum Press, 1989

(18) Below 1 microSiemens/cm there are so few ions that the time to change the cell membrane is unrealistically large.

(19) The preferred operating region of the present invention is then: Cell diameter>1 micrometer Chamber volume 2 to 10 milliliters Conductivity of Material to be treated 50 microSiemens/cm to 500 microSiemens/cm Total resistance of material to be treated in chamber>1 ohm Geometric Factor of Chamber<0.1 cm.sup.−1

(20) The invention uses a static chamber with large volume to insure that all cells in suspension are subject to the same electric field intensity and direction and the density of the cells and treating material are uniform. With this invention any waveform may be used. This invention includes a voltage waveform generator connected to electrodes in the form of parallel plates, and, which has a low conductivity medium and a suspension in the static chamber, having a cell density of 20 million cells or less.

(21) A component of the invention is the use of low conductivity medium within a defined range to limit amperage and heat while simultaneously providing enough ions to effectively electroporate cells. Typically the medium used will have a conductivity in a range spanning 50 microSiemens/cm to 500 microSiemens/cm.

(22) The invention may be used in clinical applications and with a closed sterile chamber into which the cells and large molecules are inserted and removed.

(23) One aspect of the invention further increases capacity by alternately filling and emptying the chamber. In this manner, all desired properties are met during a specific treatment and the chamber can be re-used for subsequent treatments in a sequential batch process.

(24) The conductivity of the medium used in electroporation is an important aspect of this invention. In this process, a low conductivity medium is employed to keep the total resistance of the medium small and virtually eliminate heating. There is a limit to the lower conductivity medium that can be used. As the ionic content of the medium is reduced the number of free ions that are available to build charge (voltage) across the cell membrane is decreased. The effect is to increase the amount of time it takes to charge the membrane. This process is described by the equation in Neumann, p 71. Assuming a typical cell diameter of 10 microns, the charging time is 20 microseconds at a conductivity of 80 microSiemens/cm. For a typical cell diameter of 10 microns, below 80 microSiemens/cm, the charging time becomes too long and the pathways in cell membranes stop forming.

(25) Using an electrode with a 4 mm gap, TABLE 6 illustrates the resistance of the medium as a function of electrode chamber volume and conductivity.

(26) In one aspect of the invention, a chamber with two electrodes is used as shown in FIG. 1. An example of electrode dimensions that can be used is a gap of 0.4 cm, electrode height of 2 cm and electrode length of 10 cm. The chamber can be used with a commercial electroporator such as the Cyto Pulse Sciences, Inc. PA-4000 electroporator.

(27) An example of a medium that can be used with the chamber is one with the following formula: Sorbitol 280 millimoles Calcium Acetate, 0.1 millimoles Magnesium Acetate, 0.5 millimoles

(28) FIG. 3 is a graph illustrating the relationship between charging time (in microseconds) of biological cells and media conductivity (in microSiemens/cm) for cells having three different diameters, namely 1 micrometer, 10 micrometers, and 100 micrometers. From FIG. 3 it is clear that for media conductivity below 1 microSiemen/cm, the charging time would be so large that electroporation would not work.

(29) As to the manner of usage and operation of the instant invention, the same is apparent from the above disclosure, and accordingly, no further discussion relative to the manner of usage and operation need be provided.

(30) It is apparent from the above that the present invention accomplishes all of the objects set forth by providing a large volume ex vivo electroporation method which may advantageously be used for clinical and therapeutic purposes wherein all cells, ex vivo or in vitro, are subject to substantially the same process conditions. With the invention, a large volume ex vivo electroporation method is provided which is scalable from 2 to 10 milliliters so that substantially large volumes of ex vivo or in vitro cells can be processed in a relatively short period of time. With the invention, a large volume ex vivo electroporation method is provided which achieves increased biological cell capacity without increasing the size of a chamber resulting in excessively large amperage requirements. With the invention, a large volume ex vivo electroporation method is provided which limits heating within the chamber to low levels. With the invention, a large volume ex vivo electroporation method is provided which exposes substantially all ex vivo or in vitro cells to the same electric field intensity and direction. With the invention, a large volume ex vivo electroporation method provides that the density of the material to be inserted into the treatment chamber can be held constant. With the invention, a large volume ex vivo electroporation method is provided which permits variable rectangular pulse waveforms such as disclosed in U.S. Pat. No. 6,010,613 can be employed. With the invention, a large volume ex vivo electroporation method is provided which avoids problems in flow through treatment cells that are due to laminar and turbulent flow conditions. With the invention, a large volume ex vivo electroporation method is provided which permits the use of medium with lower conductivity to achieve the movement of macromolecules into mammalian cells and to allow the use of larger capacity chambers. With the invention, a large volume ex vivo electroporation method is provided which is easily scalable to large capacity without using a flow through treatment chamber for cells to be treated.

(31) The electroporation chamber with its parts 1 as shown by FIG. 1B includes the electroporation chamber 13 with a geometric factor defined by the quotient of the electrode gap squared (cm.sup.2) divided by the chamber volume (cm.sup.3), wherein the geometric factor is less than or equal to 0.1 cm.sup.−1. The chamber 13 has an inlet 2 and an outlet 3 configured for sequential passage of suspensions and fluids during electroporation. The chamber 13 includes a first reservoir 4 in fluid communication with the inlet 2 for containing the suspensions and fluids prior to introducing into the chamber 13 for treatment, a second reservoir 5 in fluid communication with the inlet 2 for containing flushing material for flushing the suspensions and fluids after treatment out from the chamber 13, and a third reservoir 6 in fluid communication with the outlet 3 for receiving the suspensions and fluids flushed after treatment out from the chamber 13. The inlet valve 10 is connected between the inlet 2 and the first reservoir 4 and the inlet valve 11 is connected between the second reservoir 5 and inlet 2, and the outlet valve 12 is connected between the outlet 9 and the third reservoir 6. The chamber 13 includes the vent 7 for removing air when the chamber is filled with a fluid. The chamber includes sealing means 9 for providing a sealed chamber. The chamber 13 includes parallel plate electrodes 8. Not shown by FIG. 1B, the chamber 13 includes a source of pulsed voltages electrically connected to said parallel plate electrodes 8 configured to provide pulse waveforms and an uniform electric field between the parallel plate electrodes 8, wherein the uniform electric field is greater than 100 volts/cm and less than 5,000 volts/cm and substantially uniform throughout the chamber 13.

(32) With respect to the above description, it should be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, form function and manner of operation, assembly and use, are deemed readily apparent and obvious to those skilled in the art, and therefore, all relationships equivalent to those illustrated in the drawings and described in the specification are intended to be encompassed only by the scope of appended claims.

(33) While the present invention has been shown in the drawings and fully described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that many modifications thereof may be made without departing from the principles and concepts set forth herein. Hence, the proper scope of the present invention should be determined only by the broadest interpretation of the appended claims so as to encompass all such modifications and equivalents.