U.S. patent application number 10/265133 was filed with the patent office on 2003-04-17 for method and apparatus for treating materials with electrical fields having varying orientations.
Invention is credited to King, Alan D., Walters, Derin C., Walters, Richard E..
Application Number | 20030070939 10/265133 |
Document ID | / |
Family ID | 22913747 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030070939 |
Kind Code |
A1 |
Walters, Richard E. ; et
al. |
April 17, 2003 |
Method and apparatus for treating materials with electrical fields
having varying orientations
Abstract
The object of the invention is to provide a method and apparatus
for treating membrane-containing material with electrical fields,
in vitro, and with an added treating substance. With the method, a
plurality of electrodes are arrayed around the material to be
treated and are connected to outputs of an electrode selection
apparatus. Inputs of the electrode selection apparatus are
connected to outputs of a pulse sequence generator. A treating
substance is added to the membrane-containing material. Electrical
pulses are applied to the electrode selection apparatus and are
routed through the electrode selection apparatus in a
predetermined, computer-controlled sequence to selected electrodes
in the array of electrodes, whereby the membrane-containing
material is treated with the added treating substance and with
electrical fields. The routing of applied pulses through the
electrode selection apparatus to selected electrodes can be done in
an enormous number of ways.
Inventors: |
Walters, Richard E.;
(Columbia, MD) ; King, Alan D.; (Takoma Park,
MD) ; Walters, Derin C.; (Austin, TX) |
Correspondence
Address: |
Marvin S. Towsend
Patent Attorney
8 Grovepoint Court
Rockville
MD
20854
US
|
Family ID: |
22913747 |
Appl. No.: |
10/265133 |
Filed: |
October 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10265133 |
Oct 7, 2002 |
|
|
|
09637380 |
Aug 15, 2000 |
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Current U.S.
Class: |
205/688 ;
204/229.4; 204/229.5; 204/230.2; 205/701 |
Current CPC
Class: |
C12M 35/02 20130101;
C12N 13/00 20130101 |
Class at
Publication: |
205/688 ;
205/701; 204/229.4; 204/229.5; 204/230.2 |
International
Class: |
C25C 003/16 |
Claims
1. A method of treating material with electrical fields and an
added treating substance, comprising the steps of: arranging a
plurality of electrodes in an array of locations around the
material to be treated, connecting the electrodes to outputs of an
electrode selection apparatus, connecting inputs of the electrode
selection apparatus to outputs of an electrical pulser apparatus,
contacting the material with the added treating substance, applying
electrical pulses to the electrode selection apparatus, wherein
electrical pulses applied to the electrode selection apparatus are
in a sequence of at least three non-sinusoidal electrical pulses,
having field strengths equal to or greater than 100 V/cm, to the
material, wherein the sequence of at least three non-sinusoidal
electrical pulses has one, two, or three of the following
characteristics: (1) at least two of the at least three pulses
differ from each other in pulse amplitude; (2) at least two of the
at least three pulses differ from each other in pulse width; and
(3) a first pulse interval for a first set of two of the at least
three pulses is different from a second pulse interval for a second
set of two of the at least three pulses, and routing applied pulses
through the electrode selection apparatus in a predetermined
sequence to selected electrodes in the array of electrodes, whereby
the material is treated with the added treating substance and with
electrical fields of sequentially varying directions.
2. The method of claim 1 wherein at least one of the electrodes is
a pulse-receiving electrode and wherein at least one of the
electrodes is a pulse-returning electrode.
3. The method of claim 2 wherein any electrode, in any combination
of electrodes, functions as either a pulse-receiving electrode, a
pulse-returning electrode, or an non-pulse-conveying electrode.
4. The method of claim 1 wherein the sequence of at least three
non-sinusoidal electrical pulses is an agile pulse sequence.
5. The method of claim 4 wherein the electrical pulses have field
strengths equal to or greater than 200 V/cm.
6. The method of claim 1 wherein a selected number of electrodes
receive applied pulses simultaneously.
7. The method of claim 1 wherein N applied pulses are routed to N
groups of electrodes in sequence.
8. The method of claim 7 wherein successive groups of electrodes in
the N groups of electrodes are comprised of different individual
electrodes.
9. The method of claim 7 wherein successive groups of electrodes in
the N groups of electrodes are comprised of the same individual
electrodes.
10. The method of claim 1 wherein N pulse patterns can be routed to
N selected groups of electrodes in sequence.
11. A method of treating membrane-containing material in vitro with
electrical fields, comprising the steps of: arranging the in vitro
material in an array of locations, arranging a plurality of
electrodes in an array of locations corresponding to the array of
in vitro material locations, connecting the electrodes to outputs
of an electrode selection apparatus, connecting inputs of the
electrode selection apparatus to outputs of an electrical pulser
apparatus, applying electrical pulses to the electrode selection
apparatus, routing applied pulses through the electrode selection
apparatus in a predetermined sequence to selected electrodes in the
array of electrodes, whereby the in vitro material in the array of
locations is treated with electrical fields sequentially in the
array of locations.
12. The method of claim 11 wherein at least one of the selected
electrodes serves as a pulse-receiving electrode and wherein at
least one of the selected electrodes serves as a pulse-returning
electrode
13. The method of claim 12 wherein any electrode, in any
combination of electrodes, must be either a pulse-receiving
electrode, a pulse-returning electrode, or an non-pulse-conveying
electrode.
14. The method of claim 11 wherein N applied pulses are routed to N
groups of electrodes in sequence.
15. The method of claim 14 wherein successive groups of electrodes
in the N groups of electrodes are comprised of different individual
electrodes.
16. The method of claim 14 wherein successive groups of electrodes
in the N groups of electrodes are comprised of the same individual
electrodes.
17. The method of claim 11 wherein at least two successive pulses
applied to a selected electrode are reversed in electric field
direction.
18. The method of claim 11 wherein the electrodes are arrayed as M
pairs of electrodes associated with M wells in a testing plate.
19. The method of claim 11 wherein the electrodes are arrayed as a
pair of electrodes associated with a cuvette.
20. The method of claim 11 wherein the electrodes are arrayed as P
electrodes associated with a cuvette, wherein P is greater than
two.
21. A method of treating tissues in vivo with an added treating
substance and with electrical fields, comprising the steps of:
arranging a plurality of electrodes in an array of locations around
the in vivo tissues to be treated, connecting the electrodes to
outputs of an electrode selection apparatus, connecting inputs of
the electrode selection apparatus to outputs of an electrical
pulser apparatus, adding a treating substance to the in vivo
tissues to be treating, applying electrical pulses to the electrode
selection apparatus, wherein the electrical pulses are in a
sequence of at least three non-sinusoidal electrical pulses, having
field strengths equal to or greater than 100 V/cm, to the material,
wherein the sequence of at least three non-sinusoidal electrical
pulses has one, two, or three of the following characteristics: (1)
at least two of the at least three pulses differ from each other in
pulse amplitude; (2) at least two of the at least three pulses
differ from each other in pulse width; and (3) a first pulse
interval for a first set of two of the at least three pulses is
different from a second pulse interval for a second set of two of
the at least three pulses, and routing the applied pulses through
the electrode selection apparatus in a predetermined sequence to
selected electrodes in the array of electrodes by programmable
computer-controlled switches in the electrode selection apparatus,
whereby the in vivo tissues are treated with the added treating
substance and with electrical fields of sequentially varying
directions.
22. An apparatus for treating materials with electrical pulses,
comprising: an agile pulse sequence generator, an electrode
selection apparatus electrically connected to said agile pulse
sequence generator, wherein said electrode selection apparatus
includes a set of input connectors electrically connected to said
agile pulse sequence generator, a set of at least two output
connectors, and an array of selectable switches connected between
said input connectors and said output connectors, and a set of
electrodes electrically connected to at least two of said output
connectors of said electrode selection apparatus.
23. The apparatus of claim 22 wherein said selectable switches are
manually operated switches.
24. The apparatus of claim 22 wherein said selectable switches are
computer controlled switches.
25. The apparatus of claim 22 wherein said selectable switches are
programmable computer controlled switches.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of applying a
defined pattern of pulsed electrical fields to materials,
especially living cells. More specifically, the present invention
is especially concerned with the fields of electroporation,
electrofusion, and electromanipulation.
BACKGROUND ART
[0002] Electroporation and electrofusion are related phenomena with
a variety of uses in manipulation of prokaryotic and eukaryotic
cells. Electroporation is the destabalization of cell membranes by
application of a brief electric potential (pulse) across the cell
membrane. Properly administered, the destabalization results in a
temporary pore in the membrane through which macromolecules can
pass while the pore exists. Therefore, in electroporation,
membranes of membrane-containing material open to admit treating
substances. Electrofusion is the fusion of two or more cells by
application of a brief electric potential across a cell membrane.
In electrofusion, membranes of membrane-containing material open to
merge with membranes of other membrane-containing material. In this
respect, one membrane-containing material may be regarded as a
treating substance for another membrane-containing material. The
physical and biological parameters of electrofusion are similar to
those of electroporation.
[0003] The potential applied to cell membranes is applied using
instruments delivering various pulse shapes. The two most common
pulse shapes are exponential decay and rectangular wave. The
exponential decay pulse is generated with capacitance discharge
pulse generators. It is the least expensive pulse generator and
gives the operator the least control over pulse parameters. The
rectangular wave pulse generator is more expensive, gives more
control over pulse parameters and generates a pulse that is less
lethal to cells. With both pulse shapes, the energy needed to
generate resealable pores in cells is related to cell size, shape,
and composition.
[0004] With electrofusion, cells must be in contact at the time of
membrane destabalization. This is accomplished by physical means
such as centrifugation, biochemical means such as antibody
bridging, or electrical means through dielectrophoresis.
Dielectrophoresis is the creation of a dipole within a cell by
application of a low voltage potential across a cell membrane in an
uneven electrical field. The dipole can be created in DC or AC
fields. Since DC fields tend to generate unacceptable heat, radio
frequency AC is often used for dielectrophoresis.
[0005] The uses of electroporation and electrofusion are many. A
partial list follows: (1) transient introduction of DNA or RNA into
both eukaryotic and prokaryotic cells; (2) permanent transfection
of DNA into both eukaryotic and prokaryotic cells; (3) permanent
and temporary transfection of DNA into human and animal cells for
gene therapy; (4) introduction of antibodies, other proteins, or
drugs into cells; (5) production of antibody producing hybridomas;
(6) pollen electrotransformation in plants; (7) electroinsertion;
(8) manipulation of animal embryos; (9) electrofusion of adherent
cells; (10) production of plant somatic hybrids; (11) DNA
vaccination; and (12) cancer therapy.
[0006] One of the ways that electroporation or electrofusion works
is to induce the formation of holes or pores in the cell membrane.
There is some controversy about the exact nature of the cell pore
induced by the application of an electrical pulse to a cell, but
the practical effect is an induced cell permeability and a tendency
to fuse with other similarly affected cells that are in close
contact. There is a DC voltage threshold for the induction of pores
in or for the fusion of cell membranes. Voltages below the
threshold will not bring about substantial cell membrane
disturbance. The threshold potential for many cells is
approximately one volt across the cell membrane. The total DC
voltage applied per centimeter between electrodes to achieve one
volt potential across the cell membrane is therefore proportional
to the diameter of a cell. Small cells such as bacteria, require
high DC voltages while larger cells, such as many mammalian cells,
require somewhat lower voltages. There are other cell specific
variables such as the structure of the cellular cytoskeleton that
affect the voltage required for that cell.
[0007] When using DC electrical pulses which are powerful enough to
bring about electroporation or electrofusion of cells, the main
problem is that the process is often lethal to an unacceptable
percentage of the cells. The lethality rate may be as high as 50%
or higher. There are a number of reasons why such high lethality
rates to cells are not desirable. When cells are treated for
further use in ex vivo gene therapy, lethality to the cells will
prevent an adequate number of cells from uptaking therapeutic
genetic material. When in vivo gene therapy is employed in a
patient, lethality to cells may not only result in less effective
treatment, but may also result in causing injury to the
patient.
[0008] A number of methods have been used to reduce cell killing in
electroporation and electrofusion. The most commonly used method is
to apply a rectangular shaped DC pulse to cells instead of an
exponential decay pulse. This method reduces the total energy
applied to the cell while applying enough DC voltage to overcome
the threshold. While the rectangular shaped pulse is an
improvement, there is still substantial cell killing during an
effective application of electrical energy to the cells.
[0009] Rectangular wave pulsers currently marketed for
electroporation and electrofusion have a number of adjustable
parameters (voltage, pulse width, total number of pulses, and pulse
frequency). These parameters, once set, are fixed for each pulse in
each pulse session. For example if a voltage of 1,000 volts per
centimeter, pulse width of 20 microseconds, pulse number equal to
10, and a pulse frequency of 1 Hz is chosen, then each of the 10
pulses will be fixed at 1000 volts per centimeter and 20
microseconds for the pulse session.
[0010] However, even when using rectangular wave pulsers that
employ fixed pulse parameters, an undesirably high lethality rate
of the cells may still occur. In this respect, it would be
desirable if wave pulses could be controlled in such a way that the
lethality rate of cells would be significantly reduced.
[0011] In an article by Sukharev et al entitled "Electroporation
and electrophoretic DNA transfer into cells" in Biophys. J., Volume
63, November 1992, pages 1320-1327, there is a disclosure that
three generators are employed to generate DC pulses. A time delay
generator controls a first pulse generator to generate a first DC
pulse to be imposed on biological Cos-1 cells. The first pulse has
an amplitude sufficient to induce pore formation in the cells. The
time delay generator causes a time delay and then controls a second
pulse generator to generate a second DC pulse which is imposed on
the cells. The second DC pulse is insufficient to sustain the
induced pores formed from the first pulse. However, the second
pulse is sufficient to bring about electrophoresis of DNA material
into the previously pulsed cells. Several key points are noted with
respect to the disclosures in the Sukharev et al article. First,
the induced pores that are formed in the cells as a result of the
first pulse begin to contract after the first pulse is over without
any additional pulse being imposed on the cells sufficient to
sustain the induced pores. Second, the Sukharev et al article does
not address the issue of cell viability after the
induced-pore-forming pulse. Third, there are only two pulses
provided with Sukharev et al. Therefore, the time period that the
DNA material can enter the cells is constrained by the effects of
only two brief pulses. In this respect, it would be desirable if a
pulse protocol were provided that sustains induced pores formed in
electroporation. Moreover, it would be desirable if a pulse
protocol were provided which is directed towards improving cell
viability in cells undergoing electroporation. Furthermore, it
would be desirable if a pulse protocol were provided which provides
three or more pulses to allow more time for materials to enter
cells undergoing electroporation.
[0012] In an article by Ohno-Shosaku et al entitled "Somatic
Hybridization between Human and Mouse Lymphoblast Cells Produced by
an Electric Pulse-Induced Fusion Technique" in Cell Structure and
Function, Vol. 9, (1984), pages 193-196, there is a disclosure if
the use of an alternating electric field of 0.8 kV/cm at 100 kHz to
fuse biological cells together. It is noted that the alternating
current provides a series of electrical pulses all of which have
the same duration, the same magnitude, and the same interval
between pulses.
[0013] In an article by Okamoto et al entitled "Optimization of
Electroporation for Transfection of Human Fibroblast Cell Lines
with Origin-Defective SV40 DNA: Development of Human Transformed
Fibroblast Cell Lines with Mucopolysaccharidoses (I--VII)" in Cell
Structure and Function, Vol. 17, (1992), pages 123-128, there is a
disclosure that a variety of electric parameters were tested to
obtain optimum electroporation. The electric parameters included
voltage, pulse-duration, the number of pulses, and pulse shape. It
is noted that for any particular set of electric parameters that
were selected, all of pulses with the selected parameters had the
same duration, the same magnitude, and the same interval between
pulses.
[0014] In an article by Orias et al entitled "Replacement of the
macronuclear ribosomal RNA genes of a mutant Tetrahymena using
electroporation" in Gene, Vol. 70, (1988), pages 295-301, there is
a disclosure that two different electroporation devices were
employed. It is noted that each electroporation device provided a
series of electrical pulses (pulse train) for each electroporation
run. For any particular electroporation run, all of the pulses in
the pulse train had the same duration, the same magnitude, and the
same interval between pulses.
[0015] In an article by Miller et al entitled "High-voltage
electroporation of bacteria: Genetic transformation of
Campylobacter jejuni with plasmid DNA" in Proc. Natl. Acad. Sci
USA, Vol. 85, February 1988, pages 856-860, there is a disclosure
that a variety of electric pulse parameters were tested to obtain
optimum electroporation. The electric pulse parameters included
field strength and time constant. It is noted that for any
particular set of pulse parameters that were selected, all of
pulses with the selected parameters had the same field strength and
the same time constant.
[0016] In an article by Ogura et al entitled "Birth of normal young
after electrofusion of mouse oocytes with round spermatids" in
Proc. Natl. Acad. Sci USA, Vol. 91, August 1994, pages 7460-7462,
there is a disclosure that oocytes were electroactivated by
exposures to AC (2MHz, 20-50 V/cm for 10 seconds) and DC (1500 V/cm
for 80 microsec.) pulses in Dulbecco's PBS medium. Following
electroactivation, the cells were removed from the Dulbecco's PBS
medium and placed in a Hepes/Whitten medium and injected with
single spermatids. The oocyte-spermatid pairs were placed in fusion
medium and exposed to, in sequence, an AC pulse (2 MHz, 100 V/cm,
for 15-30 seconds), a fusion DC pulse (3750-4000 V/cm for 10
microsec.), and a subsequent AC pulse (2 MHz, 100 V/cm for 15-3.0
seconds). The time interval between application of the oocyte
activation pulse and the oocyte-spermatid fusion pulse was 15-40
minutes. Several points are noted with respect to the disclosures
in the Ogura et al article. First, electroporation is not
conducted; instead, electrofusion is conducted. Moreover, entry of
the spermatid into the oocyte is by injection, not electroporation.
Second, only two DC pulses are employed. Neither of the AC pulses
has a sufficient voltage level to provide an electroporation
threshold, e. g. 200 V/cm. The sequence of two DC pulses are not
disclosed as having induced pore formation. Pore formation is not
utilized in this method of cell fusion. No provision is made to
sustain pores formed or to maintain viability of cells treated.
[0017] In an article by Andreason et al entitled "Optimization of
electroporation for transfection of mammalian cell lines" in Anal.
Biochem., Vol. 180, No. 2, pages 269-275, Aug. 1, 1989, there is a
disclosure that transfection by electroporation using square wave
pulses, as opposed to exponentially decaying pulses, was found to
be significantly increased by repetitive pulses. Different pulse
amplitudes were tried in different experimental runs to determine
the effects of different electric field strengths. For a given
experimental run, each DC pulse has the same voltage and same
duration as each other DC pulse.
[0018] In an article by Vanbever et al entitled "Transdermal
Delivery of Metoprolol by Electroporation" in Pharmaceutical
Research, Vol. 11, No. 11, pages 1657-1662, (1994), there is a
disclosure that electroporation can be used to deliver drugs across
skin tissues. The article discloses a series of electroporation
experiments for the purpose of determining optimum electroporation
conditions. An electroporation apparatus was employed that could be
programmed to produce three types of pulses based on exponentially
decaying capacitive discharge: a single HV pulse (ranging from 3500
V to 100 V; a single LV pulse (ranging from 450 V to 20 V); and a
twin pulse consisting of a first HV pulse, and interpulse delay (1
second), and a second LV pulse. If more than one pulse were
applied, they were separated by 1 minute. It is noted that none of
the pulses applied are rectangular in shape. In actual experiments
run, using a twin pulse, the second LV pulse had a pulse amplitude
of 100 volts (see FIGS. 1 and 2 on page 1659). As a result of
comparisons made between the results of actual experiments
conducted, it was concluded in the second column on page 1659 that
"single pulse was as efficient as the twin pulse to promote
metoprolol permeation, indicating that twin pulse application was
not necessary". Moreover, a further conclusion beginning in the
same column of the same page and extending to the first column of
page 1660 states "long pulse (621.+-.39 ms) at a low voltage was
much more efficient than a high voltage pulse with a short pulse
time (3.1.+-.0.1 ms) to promote metoprolol permeation". Beginning
in the first paragraph of the first column on page 1660, the
authors state "The short high voltage pulses used in this study
hardly had any effect, while pulses of hundreds of volts and a few
ms time constants were reported to have dramatic effect on
transdermal permeation". Clearly, Vanbever et al teach away from
using a pulse train having pulses of different amplitudes.
Moreover, nothing in the Vanbever et al article relates to the
issue of cell viability for cells undergoing electroporation.
[0019] In an abstract of an article by Bahnson et al entitled
"Addition of serum to electroporated cells enhances survival and
transfection efficiency" in Biochem. Biophys. Res. Commun., Vol.
171, No. 2, pages 752-757, Sep. 14, 1990, there is a disclosure
that serum rapidly reseals the membranes of electroporated cells
and that timely addition of serum following electroporation can
improve cell survival and transfection efficiency. Rather than
require the use of serum, it would be desirable is an electrical
way were provided to improve cell survival and transfection
efficiency.
[0020] In an abstract of an article by Knutson et al entitled
"Electroporation: parameters affecting transfer of DNA into
mammalian cells" in Anal. Biochem., Vol. 164, No. 1, pages 44-52,
July 1987, there is a disclosure of an instrument which permits the
high-voltage discharge waveform to be varied with respect to rise
time, peak voltage, and fall time. Tests were done in which the
mammalian cells were exposed to multiple voltage discharges, but
the multiple exposures did not improve DNA transfer. It is noted
that with the use of multiple pulses, each pulse has the same
voltage and same duration as each other pulse.
[0021] In an abstract of an article by Kubiniec et al entitled
"Effects of pulse length and pulse strength on transfection by
electroporation" in Biotechniques, Vol. 8, No. 1, pages 16-20,
January 1990, there is a disclosure that the relative importance of
pulse field strength E and pulse length tau 1/2 (half decay time of
an exponential decay pulse) were investigated for HeLa or HUT-78
cells. In the abstract, there is no disclosure of using a plurality
of DC pulses for carrying out the electroporation.
[0022] Throughout the years a number of innovations have been
developed in the fields and electroporation, electrofusion,
dielectrophoresis, and the U.S. patents discussed below are
representative of some of those innovations.
[0023] U.S. Pat. No. 4,441,972 discloses a device for
electrofusion, cell sorting, and dielectrophoresis which includes
specially designed electrodes which provide a non-uniform
electrical field. The non-uniform electric fields are used for
sorting cells. More specifically, at least one of the electrodes
has a surface which includes a plurality of substantially
concentric grooves. Because preparation of such
concentric-groove-containing electrodes may be expensive and time
consuming, it would be desirable if an electrofusion device could
be provided that provides variations in electric fields applied to
living cells without the use of electrodes having a plurality of
concentric grooves.
[0024] The device in U.S. Pat. No. 4,441,972 can be used for cell
sorting by dielectrophoresis. For cell sorting, the frequency and
intensity of an AC voltage that is applied to the electrodes may be
varied so that the cells which are desired for collection will
arrive at a predetermined radial distance from an opening port and
then later be collected and withdrawn through an exit port when the
field is relaxed. DC electrical pulses are not used for cell
sorting.
[0025] The device in U.S. Pat. No. 4,441,972 can also be used for
electrofusion. With this manner of use, a low AC voltage is applied
to the electrodes in order to allow the cells to contiguously align
between the electrodes. Typically a mild AC field of about 10 volts
rms at about 250 Khz may be utilized. Then, a single brief pulse of
about 10 to about 250 volts DC for about 50 microseconds may be
applied to cause fusion of the aligned cells. The patent discloses
that the frequency, voltage and duration of impulse may be adjusted
depending on the type and size of cells to be sorted or fused or
upon the type of carrier stream used. In the patent, there is
disclosure that various devices, including computers, can be used
to control input frequency and voltage to the electrode. However,
with particular attention being paid to electrofusion, in U.S. Pat.
No. 4,441,972, there is no disclosure of using more than a single
DC pulse for carrying out the electrofusion. It is noted that U.S.
Pat. No. 4,476,004 is closely related to U.S. Pat. No. 4,441,972
and has a similar disclosure.
[0026] U.S. Pat. No. 4,695,547 discloses a probe for electrofusion,
electroporation, and the like. A suitable source of high voltage
pulses is disclosed as being capable of providing output voltage
pulses in the range of 25-475 DC at currents up to 1 amp and
durations of 1-990 ms. There is no disclosure of using a plurality
of DC pulses for carrying-out electrofusion or electroporation.
[0027] U.S. Pat. No. 4,750,100 discloses a high-voltage and
high-amperage switching device capable of delivering an exponential
decay pulse or a rectangular wave pulse for electroporation. There
is no disclosure of using a plurality of DC pulses for carrying out
electroporation or transfection.
[0028] U.S. Pat. No. 4,832,814 discloses an electrofusion cell that
is used for conducting electrofusion of living cells. An electrical
power source provides a series of three DC pulses, each of 12
microsecond and of 68 volts with a 1 second separation between
pulses. It is noted that each DC pulse has the same voltage and
same duration as each other DC pulse.
[0029] U.S. Pat. No. 4,882,281 discloses a probe for electrofusion,
electroporation, and the like. Just as disclosed in U.S. Pat. No.
4,695,547 described above, a suitable source of high voltage pulses
is disclosed as being capable of providing output voltage pulses in
the range of 25-475 DC at currents up to 1 amp and durations of
1-990 ms. There is no disclosure of using a plurality of DC pulses
for carrying out electrofusion or electroporation.
[0030] U.S. Pat. No. 4,910,140 discloses a method for the
electroporation of prokaryotic cells by applying high intensity
electric fields of short duration. This patent discloses that the
pulse will normally consist of a single pulse comprising the entire
desired period. Alternatively, the pulse may consist of a plurality
of shorter pulses having a cumulative time period coming with
desired 2 to 20 msec range. Such a series of pulses must be spaced
sufficiently close to one another so that the combined effect
results in permeabilization of the cell wall. Typically, such
pulses are spaced apart by 5 msec or less, more preferably being
spaced apart by 2 msec or less.
[0031] U.S. Pat. No. 4,955,378 discloses electrodes for delivering
pulses to animal or human anatomical sites for carrying out in vivo
electrofusion. It is disclosed that, generally, electrofusion is
preferably accomplished under constant voltage conditions by
applying to the electrode 3 square waves of 20 volts amplitude and
of 20 microsecond duration at a pulse rate of 1 pulse per second.
It is noted that each DC pulse has the same voltage and same
duration as each other DC 5 pulse.
[0032] U.S. Pat. No. 5,007,995 discloses a device for electrofusion
of living cells. Instead of using DC pulses, AC pulses were
employed. A series of studies were conducted among the variables of
AC frequency, AC voltage applied, and the time duration of the AC
pulse. In each study, two of the three variables were held
constant, and one variable was varied by setting the variable at
different incremental settings. There is no disclosure of using a
plurality of DC pulses for carrying out electrofusion.
[0033] U.S. Pat. No. 5,019,034 discloses a method for
electroporating tissue for the purpose of transporting large
molecules into the tissue. Frog skin is used as an example. In
carrying out the electroporation, the shape, duration, and
frequency of the pulse are selected. The peak voltage is also
placed at an initial setting. The pulse is gradually cycled to
higher voltages until electroporation occurs. At that point, the
pulse shape, duration, frequency, and voltage are maintained until
a desired amount of molecular transfer has occurred.
[0034] U.S. Pat. No. 5,124,259 discloses a method of
electroporation using a specifically defined buffer in which the
chloride ion is eliminated. There is a disclosure that, in carrying
out the electroporation, the voltage may be 100-1000 V/cm and the
time for applying the voltage may be 0.1-50 msec. There is no
disclosure of using a plurality of DC pulses for carrying out the
electroporation.
[0035] U.S. Pat. No. 5,128,257 discloses several chambers and
electrodes used for electroporation. Power supplies provide a
voltage range of 200 to 2000 volts. The pulse width is in a range
from 0.1 to 100 milliseconds, preferably 1 to 10 milliseconds.
There is no disclosure of using a plurality of DC pulses for
carrying out the electroporation.
[0036] U.S. Pat. No. 5,134,070 discloses a specially coated
electrode on which cells are cultivated. The cells on the electrode
are subjected to electroporation. In carrying out the
electroporation, a device for measuring electrical field intensity
is appropriately interfaced to a micro-processor so that an
"intelligent" electroporation device is provided which is capable
of applying an ever increasing electrical potential until the cells
have porated and which is capable of sensing at what field
intensity the cells have porated. Since the device measures the
conditions required to induce poration, and detects when poration
occurs, substantial reductions in current mediated cell death will
be realized since only enough energy to induce poration is
introduced into the system. However, it is noted that there is no
disclosure of using a plurality of DC pulses for carrying out
electroporation. In addition, the electroporation device is capable
of recording information concerning the poration potential required
for various cell lines and the effects of various media
compositions on the types and sizes of porations that may occur. It
is noted, however, that provisions are not made to sustain pore
formation that has been induced.
[0037] U.S. Pat. No. 5,137,817 discloses an apparatus and method
for electroporation using specially designed electrodes for
conducting electroporation in vivo. In carrying out the
electroporation, a single DC voltage pulse is applied to the host
cells. There is no disclosure of using a plurality of DC pulses for
carrying out electroporation.
[0038] Each of U.S. Pat. Nos. 5,173,158 and 5,283,194 discloses an
apparatus and methods for electroporation and electrofusion in
which an electrode is employed that selectively admits cells of a
certain size and excludes others. A single pulse generates an
electric field which causes electroporation. There is no disclosure
of using a plurality of DC pulses for carrying out either
electroporation or electrofusion.
[0039] U.S. Pat. No. 5,186,800 discloses, as does U.S. Pat. No.
4,910,140 discussed above, a method for the electroporation of
prokaryotic cells by applying high intensity electric fields of
short duration. U.S. Pat. No. 5,186,800 also discloses that an
applied pulse will normally consist of a single pulse comprising
the entire desired period. Alternatively, the pulse may consist of
a plurality of shorter pulses having a cumulative time period
coming with desired 2 to 20 msec range. Such a series of pulses
must be spaced sufficiently close to one another so that the
combined effect results in permeabilization of the cell wall.
Typically, such pulses are spaced apart by 5 microsec. or less,
more preferably being spaced apart by 2 microsec. or less. A series
of experiments were conducted to ascertain method parameters which
provided maximum cell transformation. In each of the experiments, a
single electrical pulse was used to bring about electroporation.
Experimental parameters included a number of parameters of the
electrical pulse, concentration of the host cells, concentration of
the transforming material, and post-shock incubation period. It was
observed that the viability and transformability of the cells
undergoing electroporation were very sensitive to the initial
electric field strength of the pulses. A conclusion reached was
that cell survival declines steadily with increasing field
strength; and in each of the experiments conducted, the maximum
transformation efficiency is reached when 30 to 40% of the cells
survive the pulse. There is no disclosure of using a plurality of
DC pulses for carrying out electroporation. In view of the above,
it would be desirable if a method of electroporation were provided
in which the maximum transformation efficiency were achieved when
greater than 40% of cells survive the pulse effecting
electroporation.
[0040] U.S. Pat. No. 5,211,660 discloses a method for performing an
in vivo electrofusion. Details relating to electrical parameters of
a direct current electrical charge that is utilized are not
disclosed.
[0041] U.S. Pat. No. 5,232,856 discloses an electroporation device
which employs specially designed electrodes. A number of
electroporation experiments were conducted using a number of
different host cells and different transforming material. In each
experiment, only a single DC pulse was applied to the host cells.
There is no disclosure of using a plurality of DC pulses for
carrying out electroporation.
[0042] U.S. Pat. No. 5,273,525 discloses a syringe based
electroporation electrode for drug and gene delivery. In using the
electroporation electrode, a conventional power supply is employed
which provides from one to one hundred consecutive pulses having a
constant pulse amplitude, a constant pulse width, and a constant
pulse interval.
[0043] Each of U.S. Pat. Nos. 5,304,120 and 5,318,514 discloses an
electrode for in vivo electroporation of living cells in a person.
In applying electrical energy for bringing about electroporation, a
power supply preferably applies electric fields repeatedly, and the
amplitude and duration of the electric fields make the walls of the
living cells sufficiently permeable to permit drugs or genes to
enter the living cells without killing them. The power supply
includes a unipolar oscillating pulse train and a bipolar
oscillating pulse train. It is noted that, for a chosen pulse
train, each pulse rises to the same voltage and has the same
duration as each other pulse in a pulse train.
[0044] Having discussed a number of theoretical considerations and
a number of prior art disclosures, attention is now returned to a
further discussion of certain theoretical concepts relating to
induction of pore formation in biological cells. It is understood,
however, that none of the theoretical concepts discussed herein are
intended to limit the scope of the invention. Instead, the scope of
the invention is limited only by the claims appended hereto and
equivalents thereof.
[0045] It has been discovered by the inventors of the present
invention that changing pulse parameters during a pulse session
reduces damage to cells while maintaining or improving
electroporation and electrofusion efficiency. The reduced cell
damage can be related to reduced energy applied to the cell. More
specifically, two parameters determining total energy applied per
pulse are pulse amplitude and pulse width. Variation of pulse width
would have different effects than variation of pulse amplitude.
Reduction of pulse width following application of a wider pulse
would permit application of an above threshold voltage while
reducing the total energy in a series of pulses.
[0046] Furthermore, for theoretical reasons described below,
maintenance of pores already formed in a cell should require less
energy than the energy required to initiate a new pore. Variation
of pulse width while maintaining an above threshold voltage would
be particularly useful in those instances where very small pores
are initiated by a wider pulse. Narrower pulses could assist pore
expansion in a controlled manner. The ideal condition for any
particular type of cell would be to find a set of electrical pulse
parameters that would cause pore expansion to a size large enough
for permit a foreign molecule (such as a small organic molecule or
DNA) to enter the cell without expanding the pore size to one
beyond recovery. The pulse parameters to accomplish this goal would
have to be experimentally determined for each cell type.
[0047] Variation of pulse amplitude would permit application of a
below threshold maintenance pulse. Once a pulse of sufficient
energy with an above threshold voltage is applied to a cell, a
transient decrease in electrical resistance across the cell
membrane occurs. Because of the decreased electrical resistance of
the cell membrane, pulse voltages below threshold should be
sufficient to maintain a cell pore induced by an above threshold
pulse.
[0048] Thus, while the foregoing body of prior art indicates it to
be well known to use electrical pulses to induce electroporation,
the prior art described above does not teach or suggest a method of
treating materials with pulsed electrical fields which has the
following combination of desirable features: (1) provides a process
for application of a series of electrical pulses to living cells
wherein the electrical pulses produce reduced cell lethality; (2)
provides an operator of electrical pulse equipment a process for
maximum operator control of an applied pulse series; (3) provides a
process for changing pulse width during a series of electrical
pulses; (4) provides a process for changing pulse voltage during a
series of electrical pulses; (5) provides a machine for control of
the process; (6) provides a pulse protocol that sustains induced
pores formed in electroporation; (7) provides a pulse protocol
which provides three or more pulses to allow more time for
materials to enter cells undergoing electroporation; (8) provides
an electrical way to improve cell survival and transfection
efficiency; and (9) provides a method of electroporation in which
maximum transformation efficiency is achieved when greater than 40%
of cells survive the pulse effecting electroporation. The foregoing
desired characteristics are provided by the unique method of
treating materials with pulsed electrical fields of the present
invention as will be made apparent from the following description
thereof. Other advantages of the present invention over the prior
art also will be rendered evident.
[0049] Turning to another aspect of the science of electroporation,
wherein treating substances are added to materials being
electroporated, in the article "Electrochemotherapy: Transition
from Laboratory to the Clinic", by Gunter A. Hofmann, Sukhendu S.
Dev, and Gurvinder S. Nanda, in IEEE Engineering in Medicine and
Biology, November/December 1996, pages 124-132, there is a
disclosure of a mechanical switching arrangement for changing the
directions of electrical fields applied to a set of electrodes
arrayed around an in vivo organ. The mechanical switching
arrangement is in the form of a mechanical rotating switch which
mechanically selects electrodes in a six-needle array of
electrodes. Such a mechanical switching arrangement bears a close
relationship to an automobile distributor for distributing energy
to spark plugs. Such a mechanical switching arrangement does not
permit a wide variation in selectable sequences of pulse patterns
for electrodes.
[0050] Aside from the field of electroporation, the concept of
reversing the direction of electrical fields has been employed in a
number of areas, such as cardiac cardioversion and defibrillation,
gel electrophoresis and field inversion capillary
electrophoresis.
[0051] With respect to cardiac applications, U.S. Pat. No.
5,324,309 of Kallok discloses a method and apparatus for
cardioversion and defibrillation. In using this method, a plurality
of pulses are directed to a plurality of electrodes placed in an
array of locations around an animal heart. A microprocessor
controlled switching device directs pulses to predetermined
electrodes. The purpose of the Kallok method and apparatus is to
modify the electrical system in the heart so that fibrillation or
other electrical conduction problems are corrected. Kallok does not
disclose adding any treating substances to the heart. In this
respect, Kallok does not disclose that changing electrical fields
aid in the uptake of the treating substances by the heart.
[0052] As a matter of interest, with gel electrophoresis,
electrical fields from different directions are applied over
relatively long periods of time (e. g. 0.6-125 seconds) and with
relatively low voltages (e. g. 3.5-20 volts/cm.). Also, as a matter
of interest, with field inversion capillary electrophoresis, the
reverse-direction electrical fields are applied over relatively
long periods of time (e. g. 2 seconds) and with relatively low
voltages (e. g. 50 volts/cm). Moreover, with gel electrophoresis
and field inversion capillary electrophoresis, membrane-containing
materials (such as cells, tissues, organs and liposomes) do not
undergo treatment.
Disclosure of Invention
[0053] Electrical pulse sequences are almost infinite in their
potential variability. A great variety of pulse sequences are used
in the areas of electrical communications and radar. For example, a
pulse sequence can be continuous. A continuous pulse sequence can
be unipolar or bipolar. A pulse sequence can include rectangular
waves or square waves (a special case of rectangular waves). A
predetermined number of rectangular pulses, either unipolar or
bipolar, can be provided in a gated or burst pulse sequence. In a
pulse sequence, pulses can be provided at different levels of
amplitude (pulse amplitude modulation); this form of pulse train is
used commonly in modems and computer to computer communications.
Pulses can be provided with different pulse widths or durations
(pulse width modulation); in such a case, a constant pulse interval
can be maintained. Pulses can be provided with different pulse
intervals (pulse interval modulation); in such a case, a constant
pulse width can be maintained.
[0054] A specific category of electrical pulse sequences is known
as an "agile pulse sequence". For purposes of the present patent,
by definition, an agile pulse sequence has the following
characteristics: (1) the number of pulses in the sequence can range
from 2 to 1,000; (2) the pulses in the sequence are rectangular in
shape; (3) each pulse in the sequence has a pulse width; (4) there
is a pulse interval between the beginning of a pulse and the
beginning of a succeeding pulse in the sequence; (5) pulse
amplitude for pulses in the sequence is greater than 100 volts and
preferably greater than 200 volts; and (6) pulse polarity can be
unipolar or bipolar for pulses in the sequence. Another
characteristic in an agile pulse sequence that may be present is
that the "on" time of a rectangular pulse in the sequence is less
than 10% of the pulse interval.
[0055] Although agile pulse sequences have been employed in
communications and radar applications, agile pulse sequences have
not been employed to treat materials. More specifically, the prior
art does not disclose, and the subject invention provides, the use
of agile pulse sequences to treat materials to provide very well
controlled intense electric fields to alter, manipulate, or serve
as a catalyst to cause well defined and controlled, permanent or
temporary changes in materials.
[0056] More specifically, in accordance with the invention, a
method is provided for treating materials, especially organic
materials, with pulsed electrical fields, wherein the method
includes the step of applying an agile pulse sequence having at
least three pulses to a material, wherein the agile pulse sequence
has one, two, or three of the following characteristics: (1) at
least two of the at least three pulses differ from each other in
pulse amplitude; (2) at least two of the at least three pulses
differ from each other in pulse width; and (3) a first pulse
interval for a first set of two of the at least three pulses is
different from a second pulse interval for a second set of two of
the at least three pulses.
[0057] More specifically, in accordance with the invention, a
method is provided for treating materials, especially organic
materials, with pulsed electrical fields and includes the step of
applying an agile pulse sequence having at least three pulses to a
material, wherein at least two of the at least three pulses differ
from each other in pulse amplitude.
[0058] In accordance with the invention, a method is provided for
treating materials, especially organic materials, with pulsed
electrical fields and includes the step of applying an agile pulse
sequence having at least three pulses to a material, wherein at
least two of the at least three pulses differ from each other in
pulse width.
[0059] In accordance with the invention, a method is provided for
treating materials, especially organic materials, with pulsed
electrical fields and includes the step of applying an agile pulse
sequence having at least three pulses to a material, wherein a
first pulse interval for a first set of two of the at least three
pulses is different from a second pulse interval for a second set
of two of the at least three pulses.
[0060] In accordance with another broad aspect of the invention, a
method is provided for treating biological cells with pulsed
electrical fields to induce pore formation in the cells. The method
includes the step of applying an agile pulse sequence having at
least three pulses to the cells, wherein the agile pulse sequence
has one, two, or three of the following characteristics: (1) at
least two of the at least three pulses differ from each other in
pulse amplitude; (2) at least two of the at least three pulses
differ from each other in pulse width; and (3) a first pulse
interval for a first set of two of the at least three pulses is
different from a second pulse interval for a second set of two of
the at least three pulses, such that induced pores are sustained
for a relatively long period of time, and such that viability of
the cells is maintained.
[0061] More specifically, in accordance with the invention, a
method is provided for treating biological cells with pulsed
electrical fields to induce pore formation in the cells. The method
includes the step of applying an agile pulse sequence having at
least three pulses to the cells, wherein at least two of the at
least three pulses differ from each other in pulse amplitude, such
that induced pores are sustained for a relatively long period of
time, and such that viability of the cells is maintained.
[0062] Further, in accordance with the invention, a method is
provided for treating biological cells with pulsed electrical
fields to induce pore formation in the cells. The method includes
the step of applying an agile pulse sequence having at least three
pulses to the cells, wherein at least two of the at least three
pulses differ from each other in pulse width, such that induced
pores are sustained for a relatively long period of time, and such
that viability of the cells is maintained.
[0063] Also, in accordance with the invention, a method is provided
for treating biological cells with pulsed electrical fields to
induce pore formation in the cells. The method includes the step of
applying an agile pulse sequence having at least three pulses to
the cells, wherein a first pulse interval for a first set of two of
the at least three pulses is different from a second pulse interval
for a second set of two of the at least three pulses, such that
induced pores are sustained for a relatively long period of time,
and such that viability of the cells is maintained.
[0064] It is clear from the above description that an agile pulse
sequence is a class of sequences of non-sinusoidal electrical
pulses. In this respect, in accordance with the principles of the
invention, other categories of non-sinusoidal electrical pulse
sequences can employed for treating materials aside from agile
pulse sequences.
[0065] In this respect, in accordance with a broader aspect of the
invention, a method is provided for treating material with pulsed
electrical fields and includes the step of applying a sequence of
at least three non-sinusoidal electrical pulses, having field
strengths equal to or greater than 100 V/cm and preferably equal to
or greater than 200 V/cm, to the material. The sequence of at least
three non-sinusoidal electrical pulses has one, two, or three of
the following characteristics: (1) at least two of the at least
three pulses differ from each other in pulse amplitude, (2) at
least two of the at least three pulses differ from each other in
pulse width, and (3) a first pulse interval for a first set of two
of the at least three pulses is different from a second pulse
interval for a second set of two of the at least three pulses.
Preferably, the material is an organic material.
[0066] In accordance with another broad aspect of the invention, a
method is provided for treating biological cells with pulsed
electrical fields to induce pore formation in the cells and
includes the step of applying a sequence of at least three
non-sinusoidal electrical pulses, having field strengths equal to
or greater than 100 V/cm and preferably equal to or greater than
200 V/cm, to biological cells. The sequence of at least three
non-sinusoidal electrical pulses has one, two, or three of the
following characteristics (1) at least two of the at least three
pulses differ from each other in pulse amplitude, (2) at least two
of the at least three pulses differ from each other in pulse width,
and (3) a first pulse interval for a first set of two of the at
least three pulses is different from a second pulse interval for a
second set of two of the at least three pulses, such that induced
pores are sustained for a relatively long period of time, and such
that viability of the cells is maintained.
[0067] In accordance with still another aspect of the invention, a
method of treating membrane-containing material with an added
treating substance and with electrical fields is provided in which
the directions of electrical fields are changed. In the field of
electroporation, an important aspect is that the greatest effect is
at sites on the cell where the electrical field is perpendicular to
the tangent of the cell surface. Where electroporation is greatest,
entry of the added treating substance into the cell is facilitated.
Since cells are roughly round, the pores are therefore normally
formed at the poles of the cells nearest the electrodes. The effect
of the pulse decreases as the angle of the cell surface tangent
becomes more parallel to the electrical field. The effect of
electroporation becomes zero (on a perfect sphere) 90 degrees away
from the site of maximal effect. With these considerations in mind,
it has been realized that a change of electrical field direction
would expose different areas of cells to maximal electrical fields,
whereby entry of the added treating substance into the cells would
be facilitated. This change of direction of electrical fields would
induce the formation of more pores in the cell and, therefore,
increase electroporation efficiency.
[0068] The method of the invention, in which the directions of
electrical fields are changed, is comprised of the following steps.
A plurality of electrodes are arranged in an array of locations
around the membrane-containing material to be treated. The
electrodes are connected to outputs of an electrode selection
apparatus. Inputs of the electrode selection apparatus are
connected to outputs of an agile pulse sequence generator. A
treating substance is added to the membrane-containing material.
Electrical pulses are applied to the electrode selection apparatus.
The applied pulses are routed through the electrode selection
apparatus in a predetermined sequence to selected electrodes in the
array of electrodes, whereby the material is treated with the added
treating substance and with electrical fields of sequentially
varying directions.
[0069] The routing of applied pulses through the electrode
selection apparatus in a predetermined sequence to selected
electrodes in the array of electrodes can be done in a number of
ways with the invention.
[0070] In one type of pulse routing, a first pulse can be applied
to a first selected group of electrodes. Then, a second pulse can
be applied to a second selected group of electrodes. Then, a third
pulse can be applied to a third selected group of electrodes. This
type of pulse routing can be generalized by stating that a sequence
of N pulses can be routed to N selected groups of electrodes in
sequence.
[0071] As an example, there can be eight electrodes arrayed around
an in vivo tissue. Pulses applied can be high voltage agile pulse
sequences of at least three non-sinusoidal electrical pulses,
having field strengths equal to or greater than 100 V/cm, wherein
the sequence of at least three non-sinusoidal electrical pulses has
one, two, or three of the following characteristics: (1) at least
two of the at least three pulses differ from each other in pulse
amplitude; (2) at least two of the at least three pulses differ
from each other in pulse width; and (3) a first pulse interval for
a first set of two of the at least three pulses is different from a
second-pulse interval for a second set of two of the at least three
pulses. A first pulse of an agile pulse sequence can be routed to
the first, third, fifth, and seventh electrodes. A second pulse of
an agile pulse sequence can be routed to the second, fourth, sixth,
and eighth electrodes. A third pulse of an agile pulse sequence can
be routed to the first, second, fourth, and fifth electrodes. The
possible number of variations of selected groups of only eight
electrodes from pulse to pulse is a very large number with the
apparatus and method of the invention. Using even more electrodes,
would provide even a greater number of possible variations. This
type of pulse routing can be generalized by stating that N pulses
which comprise an agile pulse sequence can be routed to N selected
groups of electrodes in sequence. An even broader statement can be
made to the effect that, with the invention, N pulses which
comprise an agile pulse sequence can be routed to N selected groups
of electrodes in any combination and in any order.
[0072] In another type of pulse routing for in vivo tissues, a
first high voltage agile pulse sequence pattern can be applied to a
first selected group of electrodes. Then, a second pulse pattern
can be applied to a second selected group of electrodes. Then, a
third pulse pattern can be applied to a third selected group of
electrodes. This type of pulse routing can be generalized by
stating that N pulses patterns can be routed to N selected groups
of electrodes in sequence.
[0073] As another example, there can still be eight electrodes
arrayed around an in vivo tissue. A first pulse pattern can be
routed to the first, third, fifth, and seventh electrodes and
returned by way of the second, fourth, sixth, and eighth
electrodes. A second pulse pattern can be routed to the first,
second, fourth, and fifth electrodes and returned by way of any one
or more of the remaining electrodes. The possible number of
variations of selected groups of only eight electrodes from pulse
pattern to pulse pattern is a very large number with the apparatus
and method of the invention. Using even more electrodes, would
provide even a greater number of possible variations.
[0074] When the object subjected to high voltage agile pulse
electrical pulse treatment is an in vivo object, e. g. an animal
(including human) or plant tissue or organ, in which cells are
being electroporated, the direction of the electric fields in a
high voltage agile pulse sequence of pulses applied to the
electrodes can be rotated, whereby electroporation into the in vivo
body part is improved.
[0075] In accordance with another aspect of the invention, a method
is provided for treating membrane-containing material in vitro with
electrical fields. The membrane-containing material can include
cells, tissue, organs, and liposomes. This in vitro method of the
invention includes the steps of:
[0076] arranging the in vitro material in an array of
locations;
[0077] arranging a plurality of electrodes in an array of locations
corresponding to the array of in vitro material locations;
connecting the electrodes to outputs of an electrode selection
apparatus;
[0078] connecting inputs of the electrode selection apparatus to
outputs of an electrical pulser apparatus;
[0079] applying electrical pulses to the electrode selection
apparatus; and
[0080] routing applied pulses through the electrode selection
apparatus in a predetermined sequence to selected electrodes in the
array of electrodes, whereby the in vitro material in the array of
locations is treated with electrical fields sequentially in the
array of locations.
[0081] In one manner of carrying out the in vitro method of the
invention, at least two successive pulses applied to a selected
electrode are reversed in electric field direction. In another
manner of carrying out the in vitro method of the invention, the
electrodes are arrayed as M pairs of electrodes associated with M
wells in a testing plate. In another manner of carrying out the in
vitro method of the invention, the electrodes are arrayed as a pair
of electrodes associated with a cuvette. In another manner of
carrying out the in vitro method of the invention, the electrodes
are arrayed as P electrodes associated with a cuvette, wherein P is
greater than two.
[0082] When the electrodes are arrayed as M pairs of electrodes for
M wells (cuvettes) in a testing plate, high voltage pulses/electric
fields may be routed to any of these cuvettes in any order by
computer control of the electrode selection apparatus. This can be
used to run a large number of experiments, for example in a
sensitivity analysis greatly reducing the time to run the
experiments by using one cuvette at a time.
[0083] When the object subjected to electrical pulse treatment is
an in vitro object, e. g. a cuvette, in which cells are being
electroporated, the direction of the electric fields in a sequence
of pulses applied to the electrodes can be rotated, whereby
electroporation is improved.
[0084] When the electrodes are arrayed as two electrodes located on
opposite sides of a cuvette in which biological cells are being
subject to electroporation, the direction of the electric fields in
a sequence of pulses applied to the electrodes can be sequentially
reversed, whereby electroporation is improved.
[0085] In accordance with yet another aspect of the present
invention, an apparatus is provided for treating materials with
electrical pulses. The subject apparatus includes an agile pulse
sequence generator and an electrode selection apparatus
electrically connected to the agile pulse sequence generator The
electrode selection apparatus includes a set of input connectors
electrically connected to the agile pulse sequence generator, a set
of at least two output connectors, and an array of selectable
switches connected between the input connectors and the output
connectors. A set of electrodes is electrically connected to at
least two of the output connectors of the electrode selection
apparatus. Preferably, the selectable switches are programmable
computer controlled switches. Alternatively, the selectable
switches can be manually operated switches.
[0086] The above brief description sets forth rather broadly the
more important features of the present invention in order that the
detailed description thereof that follows may be better understood,
and in order that the present contributions to the art may be
better appreciated. There are, of course, additional features of
the invention that will be described hereinafter and which will be
for the subject matter of the claims appended hereto.
[0087] In view of the above, it is an object of the present
invention is to provide a method of treating materials with pulsed
electrical fields which provides a process for application of a
series of electrical pulses to living cells wherein the electrical
pulses produce reduced cell lethality.
[0088] Still another object of the present invention is to provide
a method of treating materials with pulsed electrical fields that
provides an operator of electrical pulse equipment a process for
maximum operator control of an applied pulse series.
[0089] Yet another object of the present invention is to provide a
method of treating materials with pulsed electrical fields which
provides a process for changing pulse width during a series of
electrical pulses.
[0090] Even another object of the present invention is to provide a
method of treating materials with pulsed electrical fields that
provides a process for changing pulse voltage during a series of
electrical pulses.
[0091] Still a further object of the present invention is to
provide a method of treating materials with pulsed electrical
fields which provides a machine for control of the process.
[0092] Yet another object of the present invention is to provide a
method of treating materials with pulsed electrical fields that
provides a pulse protocol that sustains induced pores formed in
electroporation.
[0093] Still another object of the present invention is to provide
a method of treating materials with pulsed electrical fields which
provides a pulse protocol which provides three or more pulses to
allow more time for materials to enter cells undergoing
electroporation.
[0094] Yet another object of the present invention is to provide a
method of treating materials with pulsed electrical fields that
provides an electrical way to improve cell survival and
transfection efficiency.
[0095] Still a further object of the present invention is to
provide a method of treating materials with pulsed electrical
fields that provides a method of electroporation in which maximum
transformation efficiency is achieved when greater than 40% of
cells survive the pulse effecting electroporation.
[0096] Yet another object of the invention is to provide a method
and apparatus for selectively applying electrical pulses to an
array of electrodes, either in vivo or in vitro.
[0097] Still another object of the invention is to provide a method
and apparatus for rotating an electrical field applied to either a
tissue or organ in vivo or a cuvette in vitro.
[0098] Yet another object of the invention is to provide a method
and apparatus for sequentially applying and reversing electrical
fields applied to a cuvette in vitro.
[0099] These together with still other objects of the invention,
along with the various features of novelty which characterize the
invention, are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages and the
specific objects attained by its uses, reference should be had to
the accompanying drawings and descriptive matter in which there are
illustrated preferred embodiments of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0100] 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:
[0101] FIG. 1 is a block diagram of the overall apparatus used to
carry out the method of the invention of treating materials with
pulsed electrical fields and with a programmable electrode
selection apparatus.
[0102] FIG. 2 is a block diagram, partially in schematic form,
showing major functional units of the interface-control assembly
shown in FIG. 1.
[0103] FIGS. 2A-2O are circuit diagrams relating to the
interface-control assembly of FIG. 2. More specifically, FIG. 2A is
a circuit diagram of Status to microprocessor (IN). FIG. 2B is a
circuit diagram of control from microprocessor (OUT). FIG. 2C is a
circuit diagram of Chip Select Decoder. FIG. 2D is a circuit
diagram of Pulse Router Interface. FIG. 2E is a circuit diagram
relating to Sinewave Amplitude. FIG. 2F is a circuit diagram
relating to Sinewave Frequency. FIG. 2G is a circuit diagram of HV
Power Supply Vprog. FIG. 2H is a circuit diagram of Pulse Current
Monitor (P-IMON). FIG. 2I is a circuit diagram of HV Power Supply
VMON. FIG. 2J is a circuit diagram of Pulse Width Counter. FIG. 2K
is a circuit diagram of Pulse Out Drivers. FIG. 2L is a circuit
diagram of Bus Connectors. FIG. 2M is a circuit diagram of Power
and Reset. FIG. 2N is a circuit diagram of RS232. FIG. 2O is a
circuit diagram of Non Volatile Static RAM.
[0104] FIG. 3 is a circuit diagram showing major components of the
high voltage assembly shown in FIG. 1.
[0105] FIGS. 3A-3I are circuit diagrams relating to the high
voltage assembly shown in FIG. 3. More specifically, FIG. 3A is a
circuit diagram of HV Control and PS VMON. FIG. 3B is a circuit
diagram of the Reservoir Capacitor. FIG. 3C is a circuit diagram of
the HV Switch. FIG. 3D is a circuit diagram of Power Supply and
Pulse Disable with FAULT. FIG. 3E is a circuit diagram of HV Power
Supply Program Voltage and Discharge Control. FIG. 3F is a circuit
diagram of Pulse Voltage Monitor (P-VMON). FIG. 3G is a circuit
diagram of Pulse Control Monitor (P-IMON). FIG. 3H is a circuit
diagram of Signal Bus Interface. FIG. 3I is a circuit diagram of
Fault Monitor Circuits.
[0106] FIG. 4 is a block diagram, partially in schematic form,
showing major components of the programmable pulse switches shown
in FIG. 1.
[0107] FIG. 4A is an electrical schematic diagram of one of the
three-state switches shown in FIG. 4.
[0108] FIG. 5 illustrates non-sinusoidal pulses wherein at least
two of the at least three pulses differ from each other in pulse
amplitude.
[0109] FIG. 6 illustrates non-sinusoidal pulses wherein at least
two of the at least three pulses differ from each other in pulse
width. It is noted that FIG. 6 also corresponds with Group A in
Table I hereinbelow.
[0110] FIG. 7 illustrates non-sinusoidal pulses wherein a first
pulse interval for a first set of two of the at least three pulses
is different from a second pulse interval for a second set of two
of the at least three pulses.
[0111] FIG. 8 illustrates a single cuvette equipped with two
electrodes connected to an electrode selection apparatus of the
invention.
[0112] FIG. 9 illustrates a single cuvette equipped with eight
electrodes distributed peripherally at different locations around
the cuvette and connected to an electrode selection apparatus of
the invention.
[0113] FIGS. 10A-10F illustrate a first exemplary pattern of moving
electric fields through the single cuvette wherein a two-pole
electric field is rotated through the cuvette.
[0114] FIGS. 11A-11F illustrate a second exemplary pattern of
moving electric fields through the single cuvette wherein a
four-pole electric field is rotated through the cuvette.
[0115] FIGS. 12A-12C illustrate a third exemplary pattern of moving
electric fields through the single cuvette wherein the electric
field changes from a four-pole field to a three-pole field to a
five-pole field.
[0116] FIG. 13 illustrates a test plate that has eight test wells,
each of which has an electrode connected to a respective terminal
on the electrode selection apparatus.
MODES FOR CARRYING OUT THE INVENTION
[0117] This invention involves a process for applying electrical
pulses to living cells for the purpose of electroporation or
electrofusion. The parameters of pulse width and pulse voltage can
be changed from pulse to pulse by prior hardware or software
programming. An important object of applying pulses of changing
voltage and width is to maximize the desired result of
electroporation or electrofusion and minimize lethal damage to
cells. This object may be achieved by optimizing the energy applied
to cells through reduction of applied energy after an initial
higher energy pulse.
[0118] Conventional theory in the fields of electroporation and
electrofusion teaches that a threshold voltage must be exceeded to
achieve cell electroporation or electrofusion. In implementing the
conventional theory, a single pulse is employed by applying a pulse
with a voltage above threshold. Moreover, the single pulse concept
is extended in conventional theory to include a series of pulses
without accounting for the changes in cell membrane resistance
induced by the first above-threshold pulse. The inventors of the
subject invention described herein have realized that changes in
cell membrane resistance induced by the first above-threshold pulse
should be taken into consideration in applying subsequent
pulses.
[0119] It also is accepted in conventional theory that the energy
of a pulse is as important as the voltage of a pulse. Within
limited parameters, decreasing pulse width has the same effect as
decreasing pulse voltage. Again, conventional wisdom does not take
into consideration the altered electrical resistance following the
first pulse when sequential pulses of equal energy are applied.
[0120] The diameter of a pore induced in a cell is increased by
increasing energy. Beyond a critical energy level that is dependent
upon cell type and size, a pore is created that destroys the cell
by unlimited expansion. Cell structures such as the cell
cytoskeleton, limit the expansion of cell pores. Maximum poration
is achieved by a maximum number of pores of a size as close to but
not larger than the pore size that results in unlimited pore
expansion.
[0121] It is understood that the metes and bounds of the subject
invention are not bound by theoretical considerations. However, for
purposes of better understanding of the use and operation of the
subject invention, a brief theoretical explanation may be helpful.
More specifically, in accordance with new theoretical
considerations set forth by the inventors herein, if an applied
pulse initiates pore formation in a cell and that pulse is followed
by a pulse of lesser energy, the second pulse would have the effect
of expanding the pore at a slower rate than a pulse of full initial
energy. Pulses of continually decreasing energy would have the
effect of even slower pore expansion thus allowing a greater
control of pore expansion nearer the critical maximum pore
size.
[0122] As stated above, conventional theory relating to
electroporation does not discuss an occurrence of decreased cell
membrane resistance with continually expanding pore size. However,
it is appreciated by the inventors herein that this decreased
resistance may actually result in less effect of the applied
voltage because the local voltage decreases in proportion with the
decreased local resistance. This would result in additional
attenuation of the tendency to expand pore size. In this respect,
conventionally applied pulse trains may expand pores too rapidly to
take advantage of this natural attenuation of pore expansion. It is
the inventors' appreciation that approaching maximum pore size
through the application of stepwise decreasing or continually
decreasing the pulse energy in a train of pulses would permit
maximum usage of the natural attenuation of pore size expansion
through decreased cell membrane resistance.
[0123] Electroporation of a cell is a heterogenous process for
several reasons. First, cells are roughly round and the electrical
force upon the cell membrane is proportional to the angle of the
cell membrane relative to the direction of current. The greatest
force is at the site of the cell where the cell membrane is
perpendicular to the current. Second, cell membranes are irregular
in shape. Some cells have projections that have cell membrane
sections perpendicular to the current at sites distant to the site
nearest to the electrode. Irregularities is cytoskeleton contribute
to heterogenous electroporation.
[0124] Irregular electroporation makes maximization of
electroporation difficult because if only one pore expands beyond
rupture, the cell will die. This makes it imperative to develop a
technique that gently expands pores after pore initiation. The
subject invention satisfies this need.
[0125] With reference to the drawings, apparatus for carrying out
the method of treating materials with pulsed electrical fields
embodying the principles and concepts of the present invention are
illustrated.
[0126] The apparatus employed for carrying out the method of
treating material with pulsed electrical fields of the invention
includes the Model PA-4000 Electroporation System of Cyto Pulse
Sciences, Inc., Columbia, Md., USA, shown in FIG. 1. The Model
PA-4000 Electroporation System is designed to accomplish a wide
range of electroporation tasks, many of which are not possible with
existing equipment. Some of the new tasks that can be carried out
by the Model PA-4000 Electroporation System include: changing pulse
width from one pulse to the next; changing pulse amplitude from one
pulse to the next; changing pulse interval from one pulse to the
next; producing a high fidelity pulsed electric field, effectively
independent of load; providing a pulse amplitude monitor which
gives a very accurate replica of high voltage pulses; providing a
pulse current monitor which gives a very accurate replica of pulse
current; providing a computer-generated agile pulse sequence; and
providing automatic data logging and recall of each pulse sequence
used. As a result, the Model PA-4000 Electroporation System
provides a sequence of very finely controlled, high fidelity,
pulsed electric fields to electroporate a wide variety of materials
including plant and mammalian cells.
[0127] The Model PA-4000 Electroporation System includes three
major components: a high voltage agile pulse sequence generator 7
(known as Pulse Agile (.TM.) generator); a combined control
computer and computer interface 4; and a cuvette apparatus. The
cuvette apparatus operates with standard 0.4 cm, 0.2 cm and 0.1 cm
cuvettes. Custom interfaces are available for other cuvette holders
and delivery systems. The Model PA-4000 Electroporation System
specifications are contained in the Specifications Table presented
below.
1 Specifications Table for Model PA-4000 Electroporation System
Field Strength vs Cuvette Used PULSE PARAMETERS 0.4 cm 0.2 cm 0.1
cm Voltage-Low Range: (step size 1 volt) Minimum 10 volts 25 v/cm
50 v/cm 100 v/cm Maximum 255 volts 637 v/cm 1275 v/cm 2550 v/cm
High Range: (step size 5 volts) Minimum 50 volts 125 v/cm 250 v/cm
500 v/cm Maximum 1100 volts 2750 v/cm 5500 v/cm 11000 v/cm Time
Required to Decrease Amplitude <50 milliseconds (previous pulse
to next pulse) Maximum Pulse Current 120 amps Pulse Droop <5% at
pulse width of 100 Microsecs into 20 ohms Width 1 Microsec to 1000
Microsecs Width Step Size 1 Microsec Rise Time <100 ns Interval:
Minimum 0.1 seconds (a function of amplitude change) Maximum 4,000
seconds Interval Step Size 1 millisec MODES Rectangular wave up to
99 times 9 pulses Agile Pulse Sequence 9 Groups; Pulse Parameters
Constant Within Group 1 to 100 pulses per group FRONT PANEL STATUS
LED'S Cuvette Holder Open Fault Pulser Ready Process On High
Voltage Enabled High Voltage Off Zero Pulsing WINDOWS OPERATOR
INTERFACE Set-up of Pulse Sequence Automatic Save of Sequence File
with Unique File Name Automatic Data Logging SAFETY Pulser will not
operate when cuvette holder open Front Panel Sequence Stop Button
Load resistance check before high voltage turned on High Voltage
Pulsing Shut Down on Detection of Load Fault High Voltage Pulsing
Shut Down on Detection of Internal Fault
[0128] More specifically with respect to the agile pulse sequencer
(known as Pulse Agile (.TM.) generator) the overview is shown in
FIG. 1. This Figure shows the PA-4000 system configured with the
Programmable Pulse switch. The cuvette Holder may also be connected
at the output of the Programmable Pulse Switch or if the
Programmable Pulse Switch is not used the Cuvette Holder may be
connected directly to the Pulsed high voltage out 11. Also
available are pulse voltage and current monitor ports, 10. An
oscilloscope may be connected to these port to view replicas of the
pulse signals. A control cable 9 is used to control the
Programmable Pulse Switch. The programmable Pulse Switch has N
output which are set to one and only one of three states, pulse
out, pulse return, no connection 13.
[0129] The system consists of three cabinets, the compatible PC 1,
the PA-4000 cabinet 3 which contains the control microprocessor 4,
the Interface-Control Assembly 5, the High Voltage Assembly 6, the
High Voltage Power Supply 7, and the low voltage power supply 8 and
the Programmable Pulse Switch Cabinet 12.
[0130] The interface to the user is the PulseAgile software
installed on the compatible PC and operating under the MS Windows
operating system 1. The operator set the pulse parameter and
control the process start and stop. After the protocol is run a
data log report is presented on the computer screen. The PulseAgile
software in the compatible PC generates commands and sends the
commands over a standard RS 232 serial interface 2 to the Z-80
microprocessor in the PA-4000 cabinet 4. The Z-80 interprets the
commands and in turn controls all other assemblies. In addition the
Z-80 monitors the status of the circuits, and provides status
report back via the RS-232 link to the PulseAgile operator
interface software. Communicating over the serial interface can be
accomplished with any terminal program. However, the graphical
interface software in the PC makes the interface much more user
friendly.
[0131] The Interface Control Assembly contains, the microprocessor,
the analog to digital converts, digital to analog converters,
high/low control and status line, pulse generator and programmable
pulse switch interface, see FIG. 2
[0132] The microprocessor is connected to all other circuits via a
40 pin connector 19 Digital Bus 20. This bus contain 8 data line, 5
address line and 6 chip select lines. The data bus line transfer
information to other circuit bases on the state of the chip select
and address lines. A nonvolatile random access memory (NVRAM) part
18 is also connected to the Digital Bus 20. The NVRAM accumulates
information on how many time the discharge relay is opened and
closed and at what voltage it is opened and closed. This is
information is printed out at the end of each protocol run and is
used for failure analysis.
[0133] A Digital to Analog Converter (DAC) is used to convert the
microprocessor 8 bit word into an actual voltage to set the power
supply 22. This DAC output has a voltage range of 0 to 6 volts.
This 0 to 6 voltage is carried over the Signal Bus 24 to the high
voltage power supply. A 0 to 6 volt input to the high voltage
supply 7 produces a 0 to 1100 volt high voltage output. There are
two spare DAC connected to the Digital Bus.
[0134] An Analog to Digital Converter (A/D) 26 is used to convert
the high voltage power supply output voltage monitor signal level
of 0 to 5 volts into a digital word which is used by the
microprocessor. A 0 to 1100 volt high voltage supply output results
in the 0 to 5 volts monitor signal.
[0135] An Analog to Digital Converter (A/D) 28 is used to convert
the pre-pulse current monitor signal monitor signal level of 0 to 2
volts into a digital word which is used by the microprocessor. This
pre-pulse is generated by the microprocessor when the user starts
the protocol. Before the high voltage is turned on a 9 (s, 2 volts
pulses is generated. This pulse propagates through the load
(cuvette, etc.) . The current resulting from this pulse is
converted into the 0 to 2 volt signal.. This voltage is
proportional to the load resistance. If the load resistance is too
low (ionic concentration, etc. is too high) this can result in
excessive current which could damage the material being porated. If
the current is too high the microprocessor will not turn on the
high voltage and will issue a "Load Current Too High" message.
[0136] There are several high/low control signal lines produced by
the microprocessor 30. One is HVON (high voltage on) which is
carried over the Signal Bus to the High Voltage Assembly, 6.
Another control line, also carried over the Signal Bus to the High
Voltage Assembly 6 is the Fault Reset line. This line is used to
reset a latch if a fault occurs.
[0137] These are several status lines which are read by the
microprocessor and used for control 32. One is a line from the
Cuvette Holder 33. The Cuvette holder has magnet in the bottom. If
the handle is slid open exposing the cuvette, this will break the
contact. If this circuit is not closed, that is the handle push
fully in, the microprocessor display a "Cuvette Open" message an
the high voltage cannot be turned on, or if on will immediately be
turned off. A second status line 34 is a contact in the
Programmable Pulse switch. When the PPS parallel control cable is
plugged in this line is grounded. This low line is then read by the
microprocessor which indicated the PPS is available. A third line
34 is the Fault line. If a fault is detected in the High Voltage
Assembly, this line goes high. The microprocessor is constantly
monitoring this line and if it goes high a "Fault" message is
display and the high voltage and pulse generator are immediate
disabled.
[0138] The microprocessor control the low voltage pulse drive
circuit 36. This circuit has a pulse width generator which is
programmed by the microprocessor followed by a trigger signal which
generates the pulse, the-next pulse width is then set followed by a
trigger signal, etc. until all pulses have been generated.
[0139] The Programmable Pulse Switch interface 40 receives the
setup commands from the microprocessor and sends the setup
instructions via a special PPS parallel interface 9. This interface
consists of a set of eight line which go high of a Pulse Out is
programmed and a second set of eight line which goes high pulse
return is programmed. If switch I to remain not connected than both
line stay low.
[0140] The High Voltage Assembly contains the high voltage switch
circuit, the reservoir capacitor, power supply voltage monitor,
pulse voltage and pulse current monitors, discharge relay and fault
detection circuits, see FIG. 3.
[0141] When the high voltage is enabled the relay coil 44 is
energized and the discharge relay 45 is opened. The power supply
voltage program voltage is then set at the specified level. The
high voltage supply then charges the reservoir capacitor 40. The
floating high voltage switch 48 is then turned on by the pulse
drive signal 60 via the Signal Bus. The low voltage signal is
connected to the floating high voltage switch via an opto isolator
49. When the high voltage switch is turned on current from the
reservoir capacitor flows through the load 54, the pulse current
monitor viewing resistor 52 to System Ground 53 which is connected
to the power supply ground completing the current path. The
resistive voltage divider 42 produces a low voltage signal
proportional to the high voltage across the reservoir capacitor.
40. This scaled voltage 57 is connected to the Signal Bus and read
periodically by the microprocessor.
[0142] The scales version of the pulse current is produced by the
pulse current flowing though the current viewing resistor 52. This
voltage which is approximately 20 volts for 100 amps, is buffer by
amplifier 62. This is the Pulse Current Monitor Port. The scaled
pulse voltage is produced by the resistive voltage divider 50. This
voltage is then buffered by amplifier 85. The output is the Pulse
Voltage Monitor Port.
[0143] In the PA-4000 the pulse amplitude may be changed from pulse
to pulse, that is changed from 1100 voltage (maximum) to 50 volts
(minimum) on the next pulse which can occur is as little time as
125 milliseconds.. This is accomplished by the Discharge Relay
Circuit 44, 45, 46, 47, 56, 57, 58. A voltage comparitor 56 compare
the voltage between the high voltage power supply program voltage
64 and the scaled voltage on the reservoir capacitor 57. If the
high voltage power supply program voltage 64 is greater than or
equal to the capacitor scaled voltage 57 then the comparitor out 58
is low. When the output 58 is low and the high voltage is enabled,
the opposite side of the relay coil is high 66. This differential
voltage across the relay coil 44 energizes the coil 44 and opens
the relay 45. If the high voltage power supply program voltage 64
is lower than the scaled voltage on the reservoir capacitor 40, the
output of the comparitor 58 goes high. If 58 is high and the other
side of the relay coil 44 is high 66 (high voltage on) then there
is no voltage across the relay coil 44 and the relay closes 45.
When the relay 45 closes the reservoir capacitor 40 is connected to
system ground 53 through resistor 47. This total current during
discharge is limited by resistor 47 to prevent the relay contacts
from welding. The time constant between the reservoir capacitor 40
and resistor 47 can reduce the voltage across the capacitor by 95%
in 50 milliseconds which is half the time of the minimum pulse
interval. If the power supply program voltage 64 is set at voltage
which results in, for example 400 volts, out of the power supply 7
the comparitor will go high 58 when that voltage is reached and the
relay 45 will open and the Reservoir Capacitor 40 will not
discharge any further. Thus the voltage on the Reservoir Capacitor
40 may be set rapidly to any level between the maximum and minimum
within the shortest pulse interval.
[0144] There are three fault detection circuits in the high Voltage
assembly, high voltage switch 60, 83, 81, 76, discharge relay 75,
80, 74, 68, and load 84, 82, 78, 79. All three are connected to a
latch circuit 72. If any of one of the three line 68, 76, 79 go low
the latch out 35 will go high. If 35 goes high the program voltage
64 is disconnected via relay 65 and the pulse drive is disconnected
via relay 87 in hardware. The microprocessor constantly monitors
the latch out 35 and if it goes high a "Fault" message is
displayed. The system may be reset from the PC which momentarily
bringing the rest line high 31.
[0145] The relay discharge fault circuit 75, 80, 74, 68 monitors
the proper operation of the discharge relay 45. If the relay 45
fails and connects the power supply 7 and reservoir capacitor 40
through resistor 47 to ground 53 continuously, damage to the
internal circuits could occur. If the relay 45 does fail and
remains closed, current through viewing resistor 46 will produce a
voltage which will integrate up by RC combination 51, 52 until it
is 75 larger than the comparitor reference voltage 80. This takes a
few milliseconds if the high voltage power supply 7 is set to
maximum voltage.
[0146] The high voltage switch fault circuit 60, 83, 81, 76 monitor
the proper operation of the high voltage switch. This circuit is to
insure that no high voltage appears at the load 54 if the high
voltage switch 48 fails shut continuously. If that happens and the
high voltage power supply 7 is turned on a voltage will appear at
the scale pulse voltage resistive divider 50. This voltage 83 is
compared to the presence of the drive pulse. Without a drive pulse
60 the output of the comparitor 81 goes low if 83 is more than 10
volts within 1 (s. This is vary rapid and the power supply does not
have time to turn on.
[0147] The load fault circuit 84, 82, 78, 79 monitor the current
through the load 54. If the load current becomes excessive while
the high voltage pulse is on pulse is on due to break down in the
cuvette, etc., the contents of the cuvette could vaporize or
internal circuits may be damaged. If the current becomes excessive
the voltage across the viewing resistor 52 increase above the
threshold vale 82. When the reference is exceed the output of the
comparitor 79 goes low in less than 1 (s.
[0148] The high voltage power supply 7 is a commercial unit which
delivery 110 ma at 1100 volts and is programmed with a 0 to 6 volt
signal. The low voltage power supply 8 provides voltage to all
control circuits, +5 volts, +12 volts, -12 volts. It is a
commercial unit.
[0149] The Programmable Pulse Switch 12 is connected to the PA-4000
by a high voltage cable 11 and parallel control cable 9. An
overview is presented in FIG. 4. There are may be N switches 93 in
the Programmable Pulse Switch, all of which operate identically.
The high voltage pulse 90 and return 91 is provide via coaxial
cable form the PA-4000 cabinet 5. Two control lines 95 one for
selecting pulse out and one for selection pulse return. If the
pulse out control line is high the pulse out will be connected to
the electrode 94, If the pulse return control line is high the
pulse return will be connected to the electrode 94. If neither is
high nothing will be connected to the electrode 94, If both are
high nothing is connected to the electrode 94.
[0150] A detailed circuit diagram of the Three State Switch is
present in FIG. 4A. Each switch has two inputs from the
microprocessor, pulse return select, 161 and pulse out select 162,
two inputs from the high voltage circuit, high voltage pulse, 164
and high voltage pulse return, 163, and one output port, 167 at
which is connected to high voltage pulse, high voltage return or
nothing at all depending on the command from the microprocessor.
There are also two indicator output which are connected to light
emitting diodes (LED), 166 (pulse), 165 (return) which indicate the
state of the switch.
[0151] The high voltage pulse and high voltage return from the
Agile Pulse generator are connected to all N switches.
[0152] There are two control line for each switch from the
microprocessor, select pulse, 162 or select return, 161. If the
select pulse control line goes high, the input to two NOR gates,
U1A and U1B go high. U1A pin 2 is then high and pin two should be
low causing pin 3 (out) to be high. The second NOR gates, U1B then
has pin 4 high and pin 5 high which causes pin 6 (out) to be low.
If pin 6 is low then the output of the non inverting, open
collector driver U7C pin 6 is low. When this pin is low pin 16 on
relay K2 is low energizing the relay coil and closing the relay K2.
When this relay is closed the high voltage pulse in is connected to
the output electrode. The second non-inverting driver is connected
to a LED which is illuminated if the relay is energized, 166.
[0153] The same procured occurs if the microprocessor selects
return. In that case relay K1 is closed connecting high voltage
pulse return to the output port, 167.
[0154] If the microprocessor inadvertently sets both the high
voltage pulse and high voltage pulse return lines high the NOR U1A
output goes low and neither relay is energized. This is a safety
feature to prevent the possibility of both relays being energized
at the same time resulting is a short circuit.
[0155] There are then four possible conditions at the-two lines
from the microprocessor.
[0156] Pulse Select line high--pulse high voltage is then connected
to the output port.
[0157] Pulse Return Select line high--Pulse return is then
connected to the output port.
[0158] Neither line high--neither pulse or pulse return is
connected to the output port.
[0159] Both lines high--neither pulse nor pulse return is connected
to the output port.
[0160] In summary, all N switches are operated independently via
the microprocessor and will present either a high voltage pulse, a
high voltage return or nothing at all at the single output port of
the switch.
[0161] FIG. 5 illustrates non-sinusoidal pulsed wherein at least
two of the at least three pulses differ from each other in pulse
amplitude.
[0162] FIG. 6 illustrates non-sinusoidal pulsed wherein at least
two of the at least three pulses differ from each other in pulse
width. It is noted that FIG. 6 also corresponds with Group A in
Table I hereinbelow.
[0163] FIG. 7 illustrates non-sinusoidal pulses wherein a first
pulse interval for a first set of two of the at least three pulses
is different from a second pulse interval for a second set of two
of the at least three pulses.
[0164] It is noted that the following terms herein are
substantially synonymous: pulse selector apparatus; electrode
selection apparatus 110; and Programmable Pulse Switch Cabinet
12.
[0165] The electrode selection apparatus 110 is manufactured and
sold as a PA-101 of Cyto Pulse Sciences, Inc., Columbia, Md., which
is the same company that manufactures and sells the Model PA-4000
Electroporation System described herein). The PA-101 is externally
mounted and may be purchased separately from the high voltage agile
pulse sequence generator. The PA-101 includes a set of eight
computer-controlled HV switches which can connect any electrode to
high voltage pulse, ground reference, or "float" the electrode
(disconnected from any reference). The electrode selection
apparatus is controlled by the same PA-4000 control software which
controls the PA-4000, described below. A DB-25 control cable
connects the PA-101 to the PA-4000.
[0166] As stated above, Model PA-4000 PulseAgile(.TM.)
Electroporation System is manufactured and sold by Cyto Pulse
Sciences, Inc. of Columbia, Md. The Model PA-4000 system runs all
standard square wave and many CD protocols. The Model PA-4000
system also runs advanced PulseAgile.TM. protocols. The Model
PA-4000 PulseAgile(.TM.) Electroporator accomplishes a wide range
of electroporation tasks, many of which are not possible with
existing equipment presently available. This system permits
researchers to use standard protocols and has the increased
flexibility of changing pulse parameters within a protocol. More
tools are available for optimization. The system provides very fine
control of the high fidelity pulsed electric fields to
electroporate a wide variety of materials including plant cells,
mammalian cells, and bacterial cells in aqueous solution and
tissue.
[0167] Turning to FIG. 8, this figure illustrates a single cuvette
112 equipped with two electrodes 113, 114. One electrode 113 is
connected to Terminal No. 1 of the electrode selection apparatus
110. The second electrode 114 is connected to Terminal No. 2 of the
electrode selection apparatus 110. The electrode selection
apparatus 110 can be programmed to sequentially reverse polarity of
pulses applied to the two electrodes, whereby the direction of the
electric field is reversed across the cuvette 113 for each polarity
reversal. Polarity reversal can be carried out for each successive
pulse, or, alternatively, for a predetermined pulse pattern.
[0168] FIG. 9 illustrates a single cuvette 133 equipped with eight
electrodes distributed peripherally at different locations around
the cuvette. Electrode Nos. 121-128 are connected to Terminal Nos.
1-8, respectively, of the electrode selection apparatus 110.
Positive/negative polarities can be applied first to Electrode Nos.
121 and 125, respectively. Then, positive/negative polarities can
be applied to Electrode Nos. 122 and 126, respectively. Then,
positive/negative polarities can be applied to Electrode Nos. 123
and 127, respectively. Then, positive/negative polarities can be
applied to Electrode Nos. 124 and 128, respectively.
[0169] At this point, polarity reversals can be applied. That is,
positive/negative polarities can be applied to Electrode Nos. 125
and 121, respectively. Then, positive/negative polarities can be
applied to Electrode Nos. 126 and 122, respectively. Then,
positive/negative polarities can be applied to Electrode Nos. 127
and 123, respectively. Then, positive/negative polarities can be
applied to Electrode Nos. 128 and 124, respectively.
[0170] It is clear from the above, that this manner of applying
pulses to Electrode Nos. 121-128 results in an electric field that
rotates around and through the cuvette 133.
[0171] Such a cyclic pattern of positive/negative polarity
application can be repeated as many times as desired.
[0172] FIG. 9 can also be thought of as illustrative of another
application of the invention. The large circle can be
representative of an in vivo organ, such as from an animal or
plant. The Electrode Nos. 121-128 can be placed around the organ in
three dimensions. Each of the electrodes is connected to a
respective terminal on the electrode selection apparatus 110. The
electrode selection apparatus 110 is connected to the high voltage
agile pulse sequence generator 2. In this way, high voltage,
non-sinusoidal agile pulses can be generated in the high voltage
agile pulse sequence generator 2 and routed through the electrode
selection apparatus 110 to the selected Electrode Nos. 121-128.
Substantially any desired agile pulse sequence from the high
voltage agile pulse sequence generator 2 can be routed to any
desired subset of Electrode Nos. 121-128 in any desired
programmable sequence.
[0173] FIGS. 10A-10F illustrate a first exemplary pattern of moving
electric fields through the single cuvette wherein a two-pole
electric field is rotated through the cuvette.
[0174] FIGS. 11A-11F illustrate a second exemplary pattern of
moving electric fields through the single cuvette wherein a
four-pole electric field is rotated through the cuvette.
[0175] FIGS. 12A-12C illustrate a third exemplary pattern of moving
electric fields through the single cuvette wherein the electric
field changes from a four-pole field to a three-pole field to a
five-pole field.
[0176] From just the few examples indicated above, it is clear that
a very large number of patterns of electrical field distribution
can be applied through the cuvette by changing the permutations and
combinations of electrodes that are selected by the electrode
selection apparatus in association with pulses that are provided by
the agile pulse sequence generator to the electrode selection
apparatus 110.
[0177] More specifically, with the electrode selection apparatus
110 of invention, to permit it to have such enormous flexibilty in
electric field distribution either in vivo or in vitro, at least
one of the selected electrodes serves as a pulse-receiving
electrode, and at least one of the selected electrodes serves as a
pulse-returning electrode. Moreover, any electrode, in any
combination of electrodes, must function as either a
pulse-receiving electrode, a pulse-returning electrode, or an
non-pulse-conveying electrode.
[0178] Generally, N applied pulses can be routed to N groups of
electrodes in sequence. Furthermore, successive groups of
electrodes in the N groups of electrodes can be comprised of
different individual electrodes. Alternatively, successive groups
of electrodes in the N groups of electrodes can be comprised of the
same individual electrodes.
[0179] In FIG. 13, a test plate 135 is illustrated that has Test
Well Nos. 1-8. Each test well has a respective first electrode
141-148 connected to a respective Terminal No. 1-8 on the electrode
selection apparatus 110. Each test well also has a respective
second electrode 151-158 connected to Return Terminal No. 9 on the
electrode selection apparatus 110. Each test well can be pulsed in
a desired way either different from or the same as other test
wells.
[0180] Turning to results obtained by employing the above-described
apparatus for carrying out one aspect of the method of
electroporation of the invention, Table I set forth herein is a
tabulation of results of experiments which compare employing
principles of the invention and employing conventional principles
for carrying out electroporation. In carrying out the experiments
tabulated in Table I, specific details relating to the following
topics were taken into consideration: cells employed;
electroporation conditions; determination of percent of cells
porated; and determination of cells surviving electroporation.
Details relating to these topics follow.
[0181] Cells. CHO--K1 cells (ATCC) were maintained in complete
medium (CO.sub.2 Independent medium (Gibco) plus 10% heat
inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml
penicillin, 100 pg/ml streptomycin and 0.25 pg/ml amphotericin B).
Cells were grown in flat bottom T-150 flasks. For suspension
cultures, cells were scrapped from T-150 flasks with a cell
scraper. The cell suspension was added to a 100 ml spinner flask.
Complete medium was added to make a total volume of 100 ml. Spinner
flasks were maintained at 37.degree. C. with a stir speed of 80
rpm. Spinner cultures were fed by removing 90% of the cell
suspension and replacing the volume with complete medium. For the
electroporation, 50 ml of cell suspension was removed from a log
phase spinner culture. The cells were counted manually using a
hemocytometer. The cells were centrifuged at 400.times.g for 10
minutes. The cells were re-suspended in serum free medium (CO.sub.2
Independent medium without supplements) at a concentration of 5
million cells per ml.
[0182] Electroporation. A cell suspension volume of 250 .mu.l was
added to a sterile, disposable electroporation cuvette (Bio-Rad)
with a 2 mm electrode gap. If indicated, 50 .mu.l of either 1%
Trypan blue solution (Sigma) or a solution containing 10 .mu.g of
plasmid DNA was added to the cuvette. The cuvette was added to a
homemade cuvette holder. The pulser and computer control for the
electroporation were those described in this patent. The pulser was
turned on and the voltage was set. The pulse train was
programmed-into the attached lap top computer and the pulse train
executed by computer control.
[0183] Determination of Percent of cells Porated. Fifty microliters
of 1% Trypan blue dye solution (2.4 gm of 44% trypan blue dye added
to 100 ml distilled water) was added to the 250 .mu.l of cell
suspension in the electroporation cuvette. Before applying the high
voltage pulses, a 10 .mu.l sample was taken to determine the
percent of cells that take up dye (dead cells) prior to
electroporation. The pulses were applied to the cells as
programmed. After electroporation, a 10 .mu.l sample was taken to
determine the percent of cells electroporated. The cells were
counted manually on a hemocytometer. Blue cells were counted as
positive and clear cells negative. Actual electroporation was
calculated by subtracting background from both positive and
negative counts.
[0184] Determination of cells surviving electroporation. Cells
surviving electroporation were determined by the percent of cells
able to attach to a tissue culture plate. A 24 well plate was
prepared for the assay by adding 1 ml of complete medium to each
well. Cells were added to the electroporation cuvette as described.
A 10 .mu.l (20 .mu.l in some experiments) sample of cells was
removed from the cuvette and placed into a well of the 24 well
plate. Cells were rocked to evenly spread them across the plate.
After the pulse session was applied, an equal sample was taken from
the cuvette and placed into a different, adjacent well of the 24
well plate. Cells were cultured overnight at 37.degree. C. The next
day, cells were washed in PBS and fixed in 10% buffered formalin
for 1 hour. Cells were washed with PBS then distilled water. Cells
were stained with 1% Crystal Violet in distilled water by adding
400 .mu.l dye to each well. The cells were incubated for 5 min then
washed with distilled water until no dye was eluted from the plate.
The cells were air dried until reading of the plate. One ml of 70%
alcohol was added to each well and incubated for 5 min. The optical
density of the alcohol-dye mixture was measured at 592 nM with an
alcohol blank. Percent live cells was calculated as OD of sample
after electroporation divided by OD of the sample before
electroporation.
2TABLE I Comparison of percent poration and percent of cells
surviving poration. In accordance with the invention, multiple sets
of pulses having a 10 .mu.s pulse width and having a 400 volt pulse
amplitude were preceded by longer duration single pulses of either
40 .mu.s plus 20 .mu.s (for Group A) or 20 .mu.s alone (for Group
B). A prior art set of pulses is provided by Group C. Number of 10
.mu.s Pulses.sup.1,2 Group A.sup.3 % porated % live 0 25.51 81.98 1
55.62 87.91 2 55.62 86.94 4 81.51 85.84 8 88.29 95.14 16 96.31
76.99 Group B.sup.4 % por. % live 0 16.45 90.97 1 15.72 99.45 2
12.11 92.11 4 29.88 88.46 8 85.08 94.34 16 98 80.01 Group C.sup.5
(PRIOR ART) % por. % live 0 ND ND 1 5.25 94.05 2 12.03 87.75 4
28.48 77.2 8 70.52 77.36 16 83.96 70.59 .sup.1All pulse voltages
were 400 volts. .sup.2Pulse intervals were 0.1 second. .sup.3Group
A. In accordance with the invention, pulse trains of 10 .mu.
seconds were preceded by a single pulse of 40 .mu.s and a single
pulse of 20 .mu.s. .sup.4Group B. In accordance with the invention,
pulse trains of 10 .mu.s were preceded by a single pulse of 20
.mu.s. .sup.5Group C. As presented in the prior art, pulse trains
of 10 .mu.s were delivered without preceding pulses.
[0185] In interpreting the results of the experiments tabulated in
Table I, it is recalled that Group C data represent a prior art
pulse train of pulses having a constant pulse amplitude of 400
volts, having a constant pulse interval of 0.1 seconds, and having
a constant pulse width of 10 microsecs.
[0186] The data for Group A, with the exception of "0" additional
microsecond pulses, represent a pulse train in accordance with the
invention in which pulses have three different pulse widths. For
the pulses for Group A, the pulses have a constant pulse amplitude
and a constant pulse interval.
[0187] The data for Group B, with the exception of "0" additional
microsecond pulses, represent a pulse train in accordance with the
invention in which pulses have two different pulse widths. For the
pulse for Group B, the pulses have a constant pulse amplitude and a
constant pulse interval.
[0188] It is noted that, generally, the larger the number of
pulses, the larger the percentage of porated cells. This is true
for both the prior art pulse train (Group C) and the two pulse
trains of the invention (Groups A and B). The maximum percent
poration for the prior art pulse train is 83.96%. However, in sharp
contrast, the maximum percent poration for Group A pulse trains of
the invention is 96.31%. The maximum percent poration for Group B
pulse trains of the invention is 98%. Clearly, with the invention,
the percent poration exceeds the prior art percent poration.
[0189] With respect to viability, the average percent live for
Group C is 81.39%. The average percent live for Group A, excluding
the data from "0" additional 10 microsecond pulses, is 86.56%. The
average percent live for Group B, excluding the data from "0"
additional 10 microsecond pulses, is 90.87%. Clearly, the average
percent viability for the data which are encompassed by the method
of the invention in both Group A and Group B exceed the average
percent viability for the prior art data in Group C.
[0190] To derive further meaning from the data present in Table I,
Table II has been prepared. Table II relates to the fact that
success in electroporation depends upon both the number of cells
that are porated and the number of cells that remain alive. In
Table II, for each group of data, a product has been obtained by
multiplying the value of % porated by its corresponding value of %
live. Such products provide a composite number that represents both
the number of porated cells and the number of cells which survive
the electroporation process. Such a composite number is more
representative of the efficacy of electroporation that either %
poration or % live alone.
3TABLE II For each of the data in Groups A, B, and C, respectively,
in Table I, multiply % porated .times. % live. This product gives a
composite figure for the overall electroporation efficiency taking
into account both the extent of poration and the viability of the
cells. Number Group A Group B Group C of 10 .mu.s (% porated
.times. (% porated .times. (% porated .times. Pulses % live) %
live) % live) 0 2091 1493 -- 1 3218 1563 494 2 4836 1115 1056 4
6997 2643 2199 8 8400 8026 5455 16 7415 7841 5927
[0191] Clearly, the products for each of Groups A and B (the
invention) exceed the corresponding product for Group C (prior
art). Clearly, then, the overall electroporation efficiency, taking
into account both the extent of poration and the viability of the
cells, is greater with pulse trains of the invention than with the
prior art.
[0192] It is apparent from the above that the present invention
accomplishes all of the objects set forth by providing a method of
treating materials with pulsed electrical fields provides a process
for application of a series of electrical pulses to living cells
wherein the electrical pulses produce reduced cell lethality. With
the invention, a method of treating materials with pulsed
electrical fields provides an operator of electrical pulse
equipment a process for maximum operator control of an applied
pulse series. With the invention, a method of treating materials
with pulsed electrical fields is provided which provide a process
for changing pulse width during a series of electrical pulses. With
the invention, a method of treating materials with pulsed
electrical fields is provided which provide a process for changing
pulse voltage during a series of electrical pulses. With the
invention, a method of treating materials with pulsed electrical
fields provides a machine for control of the process. With the
invention, a method of treating materials with pulsed electrical
fields provides a pulse protocol that sustains induced pores formed
in electroporation. With the invention, a method of treating
materials with pulsed electrical fields provides a pulse protocol
which provides three or more pulses to allow more time for
materials to enter cells undergoing electroporation. With the
invention, a method of treating materials with pulsed electrical
fields provides an electrical way to improve cell survival and
transfection efficiency. With the invention, a method of treating
materials with pulsed electrical fields provides a method of
electroporation in which maximum transformation efficiency is
achieved when greater than 40% of cells survive the pulse effecting
electroporation.
[0193] While the present invention has been described in connection
with 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.
* * * * *