U.S. patent application number 09/957891 was filed with the patent office on 2002-06-06 for electroporation apparatus for control of temperature during the process.
This patent application is currently assigned to GENETRONICS, INC.. Invention is credited to Hofmann, Gunter A., Laverdiere, Rejean, Nanda, Gurvinder Singh.
Application Number | 20020068338 09/957891 |
Document ID | / |
Family ID | 22643130 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068338 |
Kind Code |
A1 |
Nanda, Gurvinder Singh ; et
al. |
June 6, 2002 |
Electroporation apparatus for control of temperature during the
process
Abstract
An electroporation method and apparatus generating and applying
an electric field according to a user-specified pulsing and
temperature profile scheme. The apparatus includes a cuvette holder
with a Peltier device forming part of the electrode structures that
form part of the holder. Advantageously, one such pulse includes a
low voltage pulse of a first duration, immediately followed by a
high voltage of a second duration, immediately followed by a low
voltage of a third duration. The low voltage electroporation field
accumulates molecules at the surface of a cell, the appropriately
high voltage field creates an opening in the cell, and the final
low voltage field moves the molecule into the cell. The molecules
may be DNA, portions of DNA, chemical agents, the receiving cells
may be eggs, platelets, human cells, red blood cells, mammalian
cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi,
yeast, sperm, or other suitable cells. The molecules are placed in
close proximity to the cells, either in the interstitial space in
tissue surrounding the cells or in a fluid medium containing the
cells.
Inventors: |
Nanda, Gurvinder Singh; (San
Diego, CA) ; Laverdiere, Rejean; (San Diego, CA)
; Hofmann, Gunter A.; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
GENETRONICS, INC.
|
Family ID: |
22643130 |
Appl. No.: |
09/957891 |
Filed: |
September 20, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09957891 |
Sep 20, 2001 |
|
|
|
09702166 |
Oct 30, 2000 |
|
|
|
09702166 |
Oct 30, 2000 |
|
|
|
09176136 |
Oct 21, 1998 |
|
|
|
Current U.S.
Class: |
435/173.6 ;
435/283.1; 435/285.2 |
Current CPC
Class: |
C12M 35/02 20130101 |
Class at
Publication: |
435/173.6 ;
435/283.1; 435/285.2 |
International
Class: |
C12N 013/00 |
Claims
We claim:
1. An electroporation apparatus, the apparatus comprising:
receptacle means for providing a) an interface to a voltage of
specified output pulsing, and b) temperature control of materials
undergoing electroporation disposed within; at least two spaced
apart electrodes, at least one of the electrodes contains a Peltier
device; at least one opening to provide transfer of the materials;
a source of energy for energizing the at least two electrodes; and
a controller to receive user specification of an integrated output
electroporation pulsing event with a corresponding temperature
profile superimposed therewith, the controller outputs gating
signals to the source of energy to generate the specified output
pulsing and required polarity and magnitude to the Peltier device
at the at least two electrodes, whereby the at least two electrodes
enable temperature control and electroporation.
2. The apparatus of claim 1, the controller including a
microprocessor and the user specification of the electroporation
pulsing event and required temperature of the materials undergoing
electroporation by superposition of the gating signals to power
supplies for electroporation and temperature control.
3. The apparatus of claim 1, further comprising a user interface
coupled to the controller for inputting the temperature
profile.
4. The apparatus of claim 1, the electrodes comprising plate
electrodes.
5. The apparatus of claim 1, further comprising: a gap sensor to
sense a distance between the electrodes and provide a
representative electronic gap distance output signal.
6. The apparatus of claim 1, wherein the controller and the energy
source for generating the user specified electroporation pulse
shape comprising: a first power supply to provide a first output
voltage; a second power supply to provide a second output voltage,
the first and second power supplies are separate sources of
electrical energy; a transformer having electromagnetically coupled
primary and secondary windings, the secondary winding being
interposed between a pair of terminals; a first switch coupled to
the first power supply and the primary winding and being responsive
to a first gating signal to apply the first output voltage from the
first power supply to the primary winding; a second switch coupled
to the second power supply and the secondary winding and responsive
to a second gating signal to apply the second output voltage from
the second power supply to the secondary winding; and a controller
to receive user specification of an output pulse shape and provide
the first and second gating signals to generate the specified
output pulse shape at the terminals.
7. The apparatus of claim 5, the controller further being
programmed to perform steps comprising: receiving an input
specifying a desired electric field; receiving an input specifying
an electrode gap distance; and calculating a first voltage to
provide the specified electric field across the specified electrode
gap distance.
8. The apparatus of claim 6, further comprising: a pair of
electrodes electrically connected to predetermined contacts of the
transformer; a gap sensor to sense an electrode gap distance
between the electrodes and provide a representative electrode gap
distance output signal; wherein the controller is further
programmed to perform steps comprising: receiving an input of a
desired electric field; receiving the electrode gap distance output
signal; and computing the first voltage to provide the input
desired electric field across the electrodes.
9. The apparatus of claim 1, wherein the controller and the energy
source for generating the user specified temperature state during
the electroporation pulsing event includes means for controlling
polarity of electrical energy at the at least two electrodes from
the controller that controls heat flow using the Peltier
device.
10. The apparatus of claim 6, wherein the controller and the energy
source includes means for controlling polarity and power magnitude
to the at least two electrodes thereby controlling direction of
heat transfer from the Peltier device, the controller includes a
temperature sensor for closed loop control of the Peltier
device.
11. The apparatus of claim 1, wherein the receptacle means
comprises a cuvette with at least one integral electrode contact
surface attached to external surfaces of the cuvette, the at least
one cuvette electrode is configured to slidably interface with and
maintain positioning in a holder device, the holder device includes
the at least one Peltier device in a complementary electrode
structure that interfaces with at least one of the electrodes
forming part of the cuvette, the holder has terminals with means
for connection to the controller.
12. The apparatus of claim 11, wherein the cuvette is a non-flow
type containing device.
13. The apparatus of claim 11, wherein the cuvette is a flow
through containing device.
14. A receptacle device and complementary holder device for
electroporation in kit form, the kit comprising: the receptacle
device has at least two electrode structures for providing an
interface to a specified output voltage pulsation waveform and heat
transfer control for materials contained within the device; at
least one Peltier device forming a junction with at least one of
the electrode structures; and means for providing positional
stability for the receptacle device and connecting the at least two
electrode structures to a connectable external controller.
15. The kit of claim 14, the electrodes comprising plate
electrodes.
16. The kit of claim 14, further comprising: a gap sensor to sense
a distance between the electrodes and provide a representative
electronic gap distance output signal.
17. The kit of claim 14, wherein the receptacle device provides for
liquid flow through the receptacle device that includes: the
receptacle device including an elongated flow through chamber
having an inlet and an outlet at opposite ends thereof; and a pair
of elongated spaced apart parallel internal electrodes disposed in
and extending along opposite sides of the chamber between the inlet
and the outlet for fluid to flow between.
18. The kit of claim 17, wherein the receptacle device comprises a
generally elongated nonconductive bar member having a rectangular
cross section and an elongated through slot intermediate the ends
thereof, the electrodes are elongated flat conductive members
sealingly applied to opposite sides of the bar member closing the
slot and defining the elongated flow through chamber.
19. The kit of claim 18, wherein the inlet and the outlet are
formed in one of the electrodes and communicate with opposite ends
of the slot.
20. The kit of claim 19, wherein the receptacle device further
comprises a generally U-shaped housing formed of a non-conducting
material and having a pair of parallel side walls with an opening
therebetween for removably receiving the flow through chamber.
21. The kit of claim 20, wherein the U-shaped housing includes
slots in one side thereof for accommodating tubing connected to the
inlet and the outlet.
22. The kit of claim 17, wherein the electrodes extend
substantially the full length of the flow through chamber.
23. The kit of claim 22, wherein the flow through chamber comprises
an elongated nonconductive tubular member; a header on one end of
the tubular member defining one of the inlet and the outlet; and a
pair of elongated conductive bars extending along opposite sides of
the tubular member defining the electrodes.
24. The kit of claim 17, wherein the flow through chamber comprises
an elongated conductive tubular member defining one of the
electrodes, and a pair of headers on the ends of the tubular member
defining the inlet and the outlet; and a conductive rod extending
coaxially of the tubular member defining the other of the
conductors.
25. The kit of claim 17, wherein the flow through chamber
substantially rectangular in cross section.
26. The kit of claim 25, wherein the flow through chamber comprises
an elongated nonconductive tubular member; a header on one end of
the tubular member defining one the inlet and the outlet; and a
pair of elongated conductive bars extending along opposite sides of
the tubular member defining the electrodes.
27. A method for regulating temperature of an electroporation pulse
apparatus that includes a receptacle means for containing and
controlling temperature of a material contained within, the
receptacle means has an electrode structure that includes a Peltier
device; receiving user input specifying an output pulse pattern of
at least one output pulse for effectuating electroporation of the
material, the user input specifying a duration for each pulse
wherein the pulse pattern defines an event; receiving a second user
input specifying an output pulse pattern for controlling
temperature of the material during the event.
28. The method of claim 27, the material comprising cells removed
from a living being.
29. A method of electroporation using a cuvette holder comprising:
positioning a pair of electrodes containing a Peltier device
relative to a region of cells; applying at least one voltage pulse
to the electrodes of specified polarity and delivering a
predetermined implant agent to the region of cells at a specified
temperature; moving molecules of the implant agent toward the cells
by applying at least one pulse at a second voltage to the
electrodes for a second predetermined time; creating pores in a
plurality of the cells; and moving molecules of the implant agent
into a plurality of the pores while controlling temperature.
30. The method of claim 29, the second predetermined magnitude of
voltage providing a resultant electric field at the electrodes in
the range of 300-3000 V/cm.
31. The method of claim 29, further comprising the steps of
computing the second predetermined magnitude of voltage by
multiplying a desired electric field by measurement of a gap
existing between the electrodes.
32. An article of manufacture comprising a data storage medium
tangibly embodying a program of machine-readable instructions
executable by a digital processing apparatus to perform method
steps for generating an electroporation pulse pattern in an
electroporation pulse apparatus that includes a electrode structure
containing Peltier material, the method comprising: receiving user
input specifying an output pulse pattern of one or more output
pulses, the user input specifying a of an integrated output
electroporation pulsing event with a corresponding temperature
profile; and providing gating signals to generate the specified
output pulsing at the electrode structure.
33. The article of manufacture of claim 32 wherein the data storage
medium tangibly embodying a program of machine-readable
instructions executable by a digital processing apparatus to
perform method steps for electroporation and temperature control,
the method further comprising: (a) applying an electric field of a
first predetermined magnitude to a region of cells for a first
predetermined duration; (b) increasing the electric field to a
second predetermined magnitude greater than the first predetermined
magnitude; and (c) reducing the electric field to a third
predetermined magnitude less than the second predetermined
magnitude.
34. An apparatus for programmable control of an electroporation
device, comprising: a receptacle for containing a material
undergoing electroporation, the receptacle includes at least two
electrodes for effectuating the electroporation process of the
material when disposed in the receptacle; programmable means for
controlling a excitation of a pulsing scheme at the electrodes for
controlling the magnitude and polarity of the electric field of a
Peltier means connected to the electrodes for regulating the
temperature of the material within the receptacle and the
electroporation process.
35. The apparatus as recited in claim 34, wherein the programmable
controlling means comprises: a control board electrically connected
to each electrode for controlling magnitude, polarity and duration
of electrical energy supplied to each the electrode.
36. The apparatus as recited in claim 34, wherein the Peltier means
comprises: within each electrode having an internal and external
surface, the internal surface configured for containing the
material whereby the internal surface is a thermally conductive
sheet; a Peltier device having an internal and external surface,
whereby the internal surface of the cell is attached to the
external surface of each the plate; and a control board
electrically connected to each cell for inputting electrical input
to the cell which, in response to the electrical input, the cell
transfers heat to and from the material.
37. The apparatus as recited in claim 34 wherein the control board
is a microprocessor and memory-based controller with programmable
pulse generator.
38. An apparatus for programmable control of an electroporation
process comprising: means for containing a material undergoing
electroporation; programmable means for controlling a magnitude of
an electric field acting upon the material; a controllable Peltier
device for controlling heat flow to the material; and control means
for controlling the programmable means, the Peltier device,
independent of one another.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of Invention
[0002] The present invention generally relates to electro-cell
manipulation. More particularly, the invention concerns an
electroporation apparatus and method for generating and applying an
electric field to a material while controlling temperature of the
process using a Peltier device for effective molecular introduction
into cells and minimize damage to cellular tissue.
[0003] 2. Description of Related Art
[0004] A cell has a natural resistance to the passage of molecules
through its membranes into the cell cytoplasm. Scientists in the
1970s first discovered "electroporation," where electrical fields
are used to create pores in cells without causing permanent damage
to them. Electroporation was further developed to aid in the
insertion of various molecules into cell cytoplasm by temporarily
creating pores in the cells through which the molecules pass into
the cell.
[0005] Electroporation has been used to implant materials into many
different types of cells. Such cells, for example, include eggs,
platelets, human cells, red blood cells, mammalian cells, plant
protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and
sperm. Furthermore, electroporation has been used to implant a
variety of different materials, referred to herein as "implant
materials," "implant molecules," "implant agents." Namely, these
materials have included DNA, genes, and various chemical
agents.
[0006] Electroporation has been used in both in vitro and in vivo
procedures to introduce foreign material into living cells. With in
vitro applications, a sample of live cells is first mixed with the
implant agent and placed between electrodes such as parallel
plates. Then, the electrodes apply an electrical field to the
cell/implant mixture. Examples of systems that perform in vitro
electroporation include the Electro Cell Manipulator ECM 600
product, and the Electro Square Porator T820, both made by the BTX
Division of Genetronics, Inc. In San Diego, Calif.
[0007] Known electroporation techniques for both in vitro and in
vitro applications apply a brief high voltage pulse to electrodes
positioned around the effectuating region. The electric field
generated between the electrodes causes the cell membranes to
temporarily become porous, whereupon molecules of the implant agent
enter the cells. In known electroporation applications, this
electric field comprises a single square wave pulse on the order of
1000 V/cm, of about 100 .mu.s duration. Such a pulse may be
generated, for example, in known applications of the Electro Square
Porator T820, made by Genetronics, Inc.
[0008] U.S. Pat. No. 5,442,272 teaches of a quick connect suction
electrode assembly for electroporation that includes a temperature
regulating element. However, there is no suggestion to integrate
temperature control with an electroporation control for efficient
and effective implant material processing.
[0009] U.S. Pat. No. 5,185,071 teaches of a programmable
electrophoresis apparatus using temperature controlling Peltier
devices attached to the sides of a buffer chamber. This disclosure
teaches only of electrophoresis applications.
[0010] Although known methods of electroporation may be suitable
for certain applications, the electric field may actually damage
the electroporated cells in some cases. For example, an
uncontrolled electric field and generated heat may damage the cells
by creating permanent pores in the cell walls. In extreme cases,
the electric field may completely destroy the cell caused by
overheating during an electroporation event.
[0011] Thus, existing electroporation systems may not be suitable
for certain applications due to imprecise temperature control of
implant agent materials and host cells during electroporation.
Furthermore, many existing electroporation systems lack sufficient
control over the parameters of the electric field pulses such as
amplitude, duration, number of pulses during this process while
simultaneously controlling the temperature of the implant
materials.
SUMMARY OF THE INVENTION
[0012] Broadly, the present invention concerns an electroporation
method and apparatus for generating and applying an electric field
while controlling temperature according to a user-specified control
scheme. An exemplary electroporation temperature controlling
receptacle device includes a cuvette, a holder and a Peltier device
for controlling temperature, the receptacle is configured for
either static or flow conditions. The invention also provides for
integrated control of the electric field for electroporation to
move the molecule into the cell while controlling temperature of
the implant agent and host cellular material. The implant agent
molecular material may be genes or drugs such as DNA, portions of
DNA, chemical agents or any other molecule. The molecular material
is placed in close proximity to the cells in a fluid medium
containing the cells.
[0013] Accordingly, one aspect of the present invention concerns a
method of generating and applying an electric field according to a
user-specified temperature control scheme integrated with an
electroporation pulsing scheme for more efficient introduction of
implant agents into cells and minimize damage to the cellular
material.
[0014] The present invention provides a number of distinct
benefits. Generally, the invention is useful to introduce molecules
of an implant agent into cells with significantly increased
effectiveness. The implant agent, for example, may include drugs
for treating cancer, kaposi's sarcoma, and a number of other
diseases and conditions.
[0015] In addition, by using electroporation to open cells for
receipt of molecules of an implant agent, the invention increases
the efficacy of the agent. Consequently, less of the implant agent
is needed, thereby being more economical. The invention also
provides a number of other benefits, as discussed in greater detail
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The nature, objects, and advantages of the invention will
become more apparent to those skilled in the art after considering
the following detailed description in connection with the
accompanying drawings, wherein:
[0017] FIGS. 1A, 1B, 1C, 1D, 1E and 1F are diagrams illustrating
various features of an electroporation cuvette and flow through
chamber holders that include Peltier devices for temperature
control for a contained or flow-through condition during the
electroporation process.
[0018] FIG. 2A and 2B show the electroporation and temperature
control hardware and interconnections to the receptacle holders in
perspective and block diagram form pursuant to the present
invention;
[0019] FIG. 3 is a diagram illustrating an electroporation waveform
known in the art;
[0020] FIG. 4 is a diagram of an exemplary hardware components and
interconnections of an electroporation pulse controller and
generator subsystem pursuant to one aspect of the present
invention;
[0021] FIG. 5 is a diagram of an exemplary article of manufacture,
comprising a data storage medium, in accordance with one aspect of
the present invention;
[0022] FIG. 6 is a flowchart illustrating an exemplary sequence of
method steps in accordance with one aspect of the present
invention; and
[0023] FIG. 7 is a drawing illustrative of an electroporation
pulse, pursuant to the invention.
[0024] FIGS. 8-11 are drawings of illustrative electroporation
pulsing schemes with temperature control, pursuant to the
invention; and
[0025] FIG. 12 is a flowchart illustrating an exemplary sequence of
method steps in accordance with one example of the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hardware Components & Interconnections
[0026] As mentioned above, one aspect of the present invention
concerns an electroporation apparatus and method for processing
implant agents into cells by generating and applying an electric
field to these materials while controlling temperature during the
processing with a user selected control scheme of temperature for
efficient and effective introduction of these materials to minimize
damage to cellular tissue. Precision temperature control of the
processing is provided by an integrated Peltier device with the
electroporation apparatus using processor control.
[0027] Receptacle:
[0028] FIG. 1A illustrates a first preferred exemplary embodiment
of the invention showing in cross-section a cuvette 5 which is a
receptacle for both host cells and implant materials undergoing
electroporation. FIG. 1B illustrates an alternate exemplary
embodiment using a flow through cuvette 5a, which comprises a clear
plastic rectangular housing defining an enclosure 9 having a round
opening at the upper end. This is similarly taught in U.S. Pat. No.
5,676,646
[0029] entitled "Flow Through Electroporation Apparatus," which is
incorporated by reference. Both of these receptacles can be in
disposable-form. An example of the cuvette 5 is BTX brand cuvette,
model number 640 made by Genetronics, Inc. of San Diego, Calif. The
cuvette 5 is portable and can be insertable into a cuvette holder
10. The cuvette holder 10 is a platform, which supports the cuvette
5 for electroporation processing and electrically interfaces with
electroporation and temperature controller 100.
[0030] This opening is closed by a push-on plastic cap 9. The
tubing segment 130 extends snugly through a hole in the middle of
the cap 9 at one end of the chamber. The tubing segment 132 extends
through a hole in the lower end of the enclosure 2 which is sealed
with a fitting 6. The enclosure 2 is preferably molded with a pair
of embedded elongated electrodes 7 and 8 which that interface with
the electrodes 40, 42 of the holder in a preferred form that carry
the electrical signal from the electroporation and temperature
control assembly 100. The electrodes 7 and 8 are uniformly spaced
apart and extend parallel, substantially the full length of the
receptacle 5a, between the inlet and outlet to enable fluid to pass
therebetween. The electrodes (7 and 8) and (40 and 42) may be of
any suitable conductive material such as stainless steel or
aluminum and may be gold plated.
[0031] Receptacle Holder:
[0032] The holder 10 (FIG. 1A) in exemplary form is an electrically
insulated platform with terminals for connecting at least two
independent conductors 11, 13 for supplying the electrical energy
for both the electroporation pulses and causing the Peltier devices
44 to transfer heat energy to or from the receptacle and holder
assembly 20. The holder device 10 is typically made of delrin or
other nonconducting material with a screw clamping member for
placement of a cuvette 5 or 5a in the holder 10. The electrodes 7,8
preferably interface with a pair of electrodes 40, 42 attached to
the holder 10, but can be integrated in a single unit if required.
An example of such a cuvette holder without components for
regulating temperature is a BTX brand Safety Stand model 630-B made
by Genetronics, Inc. of San Diego, Calif. Such a holder can also
include an electrical safety interlock feature incorporated into a
hood covering member to prevent electrical shock to personnel when
operated.
[0033] The holder 10 preferably uses a plate-shaped electrodes 40,
42 with Peltier devices 44 incorporated therein that allows for
rapid heating or cooling of host and implant materials in the
cuvette. The Peltier device 44 contained in the electrodes 40, 42,
control temperature of materials undergoing electroporation. The
holder preferably includes a temperature sensor 58 (FIG. 2B) that
is attached to or in close proximity to materials undergoing
electroporation. In particular, the temperature sensor 58 can be
attached to the holder 10 for maintaining close proximity to the
cuvette during processing to provide closed-loop processing
temperature control in real-time. The temperature sensor 58 can be,
for example an infrared temperature sensor that is attached to a
hood member of the holder 10 such that when closed, accurate
temperature measurement of the material undergoing electroporation
is achieved.
[0034] Before electroporation begins, the receptacle receives
cellular and implant materials for in vitro use, the materials are
cells and implant agent materials placed in either form of the
cuvette 5 or 5a. The operation entails having a technician inject a
liquid implant agent by pouring, eye-dropping, or otherwise
introducing the agent into the cuvette using automated equipment
for dispensing these materials.
[0035] Flow-Through Receptacle:
[0036] FIG. 1C illustrates another embodiment of the receptacle
device in a flow-through chamber 54 form. This receptacle device is
taught in U.S. Pat. No. 5,676,646 entitled "Flow Through
Electroporation Apparatus,". except for the means for controlling
temperature of the materials. The chamber 54 receptacle device
receives cellular and implant materials for in vitro use by
withdrawing fluids from a reservoir or human for processing during
transmission. The flow through receptacle and holder unit 20
comprises a rectangular outer housing 57 which encloses an
elongated flow through chamber 54. It includes a pair of uniformly
spaced apart elongated electrodes in the form of a cylindrical
conductive rod 55 concentrically mounted with a cylindrical vessel
57 defining opposed parallel electrode surfaces. Preferably, the
rod 55 and vessel 57 are made of stainless steel and may be gold
plated where desired. The ends of the electrode surface forming rod
55 and vessel 57 are mounted in hollow blocks 59 and 61 of
insulating, plastic material and sealed with high temperature
elasticomeric O-rings 63. The O-rings are seated within circular
groves machines inside the blocks 59 and 61. They are preferably
made of a high temperature resistant material such as that sold
under the trade mark of VITON.
[0037] The blocks 59 and 61 have cylindrical holes bored therein
for receiving the ends of the rod 55 and vessel 57. The blocks 59
and 61 have inlet and outlet ports, 65 and 67, respectively formed
therein so that the mixture of a blood and fluid medium can pass
through the flow through chamber between the opposed electrodes as
indicated by the arrows (FIG. 1C). Fittings 71 and 72 (FIG. 2A) are
screwed into the threaded walls of the inlet and outlet ports at 65
and 67 for coupling tubing segments 74 and 76 thereto. The tubing
segments 74 and 76 extend within the housing 52 and are in turn
connected to fitting 78 and 80 mounted on the front panel of the
housing. The tubing segments 130 and 132 are connected to the
fittings 78 and 80, respectively.
[0038] The electric cables 136 and 138 (FIG. 2A) from the
electroporation and temperature subsystem 100 have plugs that are
removably connected to jacks 82 and 84 on the front panel of the
housing 52 of the flow through chamber unit. These jacks are in
turn connected to wires 86 and 88 which connect to threaded shaft
and nut electrode assemblies 40 or 42 (FIG. 1C) as one electrode
and to a clamp around vessel 57 as the other electrode of the flow
through chamber 54. The electrode nut assembly 40 and 42 provide an
electrical connection to the rod electrode 55 while the assembly 42
provides an electrical connection to the vessel electrode 57. The
Peltier devices 44 are mounted to the external ends of the nut
electrode structures 40 and 42.
[0039] FIG. 1D shows another embodiment of a flow through chamber
receptacle holder 101 that can be used with a peristaltic pump (not
shown) with an input receiving tube 110. This is taught in U.S.
Pat. No. 5,676,646, except for components for controlling
temperature of the materials undergoing electroporation. This flow
through electroporation chamber receptacle includes a safety stand
116, having a generally U-shaped configuration with a slot 118 for
receiving the disposable chamber formed between opposing sides, or
thermally conducting panels 120 and 122. The side panel 122 is
provided with upper and lower slots 124 and 126, respectively, for
receiving the fluid or tubes 110 and 128 connected to the
chamber.
[0040] FIG. 1E shows the chamber, within which, the materials
undergoing electroporation pass through comprises a central
generally rectangular bar-shaped body member 128 having an
elongated centrally disposed through slot 130 formed therein as
shown in FIG. 1D. The slot 130 is enclosed on opposite sides of the
bar-shaped body member 128 by means of a pair of bar shaped
electrodes 40 and 42. The central body member 128 is preferably
constructed of a nonconductive material whereas the bar electrodes
40 and 42 are preferably constructed of conductive material that
can easily be gold-plated, at least along the surfaces, in
communication with the sides of the slot. The electrode 134 is
provided with upper and lower tube connections for attachment of
the tubes 110 and 128 for opening communications directly with the
upper and lower ends, respectively, the slot 130. The flow through
chamber receptacle is assembled with the electrodes 40 and 42 with
Peltier devices 44, sealingly engaging opposite sides of the bar
128 enclosing the slot 130. The electrodes are preferably,
sealingly bonded by suitable means to the opposite sides of the
central bar member. The assembled chamber then slides into the open
slot 118 in the safety stand 116 between a pair of opposing spring
contacts 136 and 138. These contacts are of a suitable conductive
material, such as a copper alloy and are mounted in conductive
holders 140 and 142, which are in turn attached to conductive
cables.
[0041] FIG. 1F shows the electrical cables in a top view 144 and
146 pass or extend through an opening 166 having a gromit 168 in
the backplate 152. A pair of conductive cables, 144 and 146,
include leads 148 and 150 connected such as by soldering to the
respective holders 140 and 142. Spring contacts 136 and 138 may
have any suitable construction, but in a preferred form is
constructed to have a somewhat louvered configuration for proper
thermal contact. A blower unit can also be incorporated if
required.
[0042] Variations of the cuvette can be use of a concentric annular
electrode structure wherein a center electrode would be at the
center of the cuvette and an outer electrode would be on the
surface of the cuvette. A variation of the holder 10 would include
an electrode that attaches through the top center and center of the
cuvette with an outer electrode interfacing with the cuvette which
would contain the Peltier device 44. At least one Peltier device
must be incorporated with the cuvette 5 or 5a and corresponding
holder 10. Preferably, the electrodes 40,42 provide heat flow
control using the integrated Peltier device in each of the
electrodes.
[0043] Electroporation and Temperature Subsystem:
[0044] An electrical pulsing field is placed across the material
between electrodes 40, 42 as discussed above in the receptacle and
holder assemblies 20. The electrode structures 40, 42 of these
above embodiments receive electrical input from a separate power
control 201 (FIG. 2B). Wires with low resistance are used to make
the connection to electrodes 40, 42. An electrical field is formed
between electrodes 40, 42 structures within the receptacle when the
power supply is controllably turned on from the subsystem 100 to
provide a pulsing electric field across the materials undergoing
electroporation. Control of the electroporation pulsing scheme by
an electric field which varies in both magnitude and duration
improves transfection of gene material. The preferred
electroporation pulsing scheme is taught in U.S. patent application
Ser. No. 08/709,615, entitled "Electroporation User Configured
Pulsing Scheme," which is hereby incorporated by reference.
[0045] In addition to providing an electrical pulsing scheme that
is user-programmable, the present invention provides temperature
control of the material that is undergoing electroporation which
naturally warms or cools the materials contained within the
receptacle when electric fields are transmitted therethrough. The
receptacle and holder assembly 20 is equipped with a Peltier device
44 for the temperature control during the electroporation process.
The Peltier device 44 is electrically and thermally junctured at
the electrodes 40, 42. Peltier device 44 receives current from an
external, feedback regulated power supply forming part of the
subsystem 100. The Peltier device 44 controls temperature within
<.+-.0.1C. Controlling material temperature during the
electrical pulsing electroporation treatment provides an improved
material yield.
[0046] FIG. 2A shows an exemplary application of the apparatus that
is used with a peristaltic pump (not shown), and injection pump
(not shown), a flow through receptacle chamber receptacle assembly
89 and the electroporation and temperature control subsystem 100.
This is similarly taught in U.S. Pat. No. 5,676,646 entitled "Flow
Through Electroporation Apparatus," except for the means for
controlling material temperature. The apparatus includes a pair of
electric cables for connecting the subsystem 100 with the flow
through receptacle assembly 89.
[0047] FIG. 2B illustrates a block diagram showing the components
for controlling the pulsing field magnitude, field duration and
temperature control of the cuvette holder 10 with receptacle
device. Beginning with temperature control, the Peltier device 44
forms part of the electrode structures for receiving electrical
input from temperature control block 56. Upon receiving electrical
current from temperature control 56, the Peltier device transfers
heat from the adjacent material within the receptacle to its
external surface within the assembly 20. A temperature sensor may
be used to communicate with the material undergoing electroporation
to provide feedback signals to temperature control 56. Temperature
sensor 58, which can be an infrared type sensor, receives
temperature readings from the material in the electroporation
cuvette holder device 10 and sends corrective signals to
temperature control block 56 which then responds with an
appropriate electrical signal to Peltier device 44.
[0048] Temperature control block 56 functions to send an output
electric signal to device 44 in response to temperature sensor
input. The output of temperature sensor 58 is fed into an
amplifier. The amplifier, can be contained within the analog
portion of the temperature controller 56. Also, within temperature
controller 56 is a comparator which receives the amplified signal
and which functions to compare the amplified signal with an
operator-initial or a reference signal sent through a
digital-to-analog converter to the comparator. When the temperature
is above the reference, the output from the comparator forward
biases a power transistor with ratings of less than 100 amps and
100 volts which is connected in series with the Peltier device 44,
thereby allowing cooling of materials in the cuvette or flow
through receptacle. Conversely, heating can occur by reversing
directional current through the Peltier device 44 by having a
symmetrical connected type circuit attached to the electrode
structures. To provide information for the control board, an
amplified signal from temperature sensor 58 is sent to an analog to
a digital converter and is used for reporting. This signal could,
however, also be used for control through the user's program.
Temperature control 56 is regulated by control board 60 that is
digital-based. The control board 60 interfaces to the analog-based
control 56 via analog/digital converter 62 and digital/analog
converter 64 (FIG. 2B).
[0049] Control board 60 functions to control temperature of the
material undergoing electroporation. The control board 60 receives
its program from a controller 66 and, once the program is entered,
the keyboard and the computer are no longer necessary. Programming
can be in computer languages such as C or BASIC (registered trade
mark) if a personnel computer is used for the controller or
assembly language if a microprocessor is used for the controller
66. A user specified control of temperature is programmed in the
controller 66.
[0050] The controller 66 may comprise a computer, a digital or
analog processing apparatus, programmable logic array, a hardwired
logic circuit, an application specific integrated circuit ("ASIC"),
or other suitable device. In an exemplary embodiment, the
controller 66 may comprise a microprocessor accompanied by
appropriate RAM and ROM modules, as desired. The controller 66 is
coupled to a user interface 50 for exchanging data with a user. In
the illustrated example, the user may operate the user interface 50
to input a desired pulsing pattern and corresponding temperature
profile to be applied to the electrodes 40, 42 and Peltier device
44. The voltage polarity controls direction of heat flux to or from
the receptacle device and current output to the Peltier device 44
can be either, amplitude or pulse width modulated for precision
heat flux control.
[0051] As an example, the user interface 50 may include an
alphanumeric keypad, touch screen, computer mouse, push-buttons
and/or toggle switches, or another suitable component to receive
input from a human user. The user interface 50 may also include a
CRT screen, LED screen, LCD screen, liquid crystal display,
printer, display panel, audio speaker, or another suitable
component to convey data to a human user. Controller 66 used for
inputting and outputting signals to control board 60 may be any
type of ASCII terminal having an RS-232 or RS-485 port. The control
board which receives input from the computer and produces outputs,
can include an 8052-AH-BASIC microprocessor (8 Kb BASIC ROM, a
programmable pulse generator, built-in algorithm using EEPROMs and
EPROMs) 8 Kb of RAM, 8 Kb of battery-backed RAM, battery-backed
real-time clock and timer with 0.005 sec resolution, 8 Kb of ROM
command extensions, 8 Kb of EEPROM, 24 bits of programmable digital
input-output, and an RS/232 printer port with a programmable Baud
rate.
[0052] Control board 60 functions to receive controller 66 input
and is driven by the power supply 70. Power supply 70 is a
switching type which can have the following typical outputs: 12
volts with up to 20 amp output to the Peltier device 44, the RS-232
or RS-485 port, the digital to analog converters 62 and 64, and
temperature control block 56. Power supply 70 also provides -12
volts, 0.25 amps for the RS-232 port and the temperature control
block. Additionally, power supply 70 would typically supply 5 volts
and one amp to the control board 60 and the temperature control
56.
[0053] Electroporation Pulsing Subsystem:
[0054] FIG. 3 (PRIOR ART) shows a pulsing scheme for
electroporation as known in the art. To generate such a pulse,
Prior electroporation power supplies used electromechanical relay
to provide consecutive e first and second pulses, see S. I.
Sukharev et al., Biophys. J. Vol. 63, November 1992, pp. 1320-1327.
More particularly, Sukharev uses an electric field pulse 101. The
pulse scheme 101 includes (1) a first, narrow duration, high
voltage pulse 102, (2) a delay 103 of .DELTA.t, during which no
pulse is generated, then (3) a second, wide duration, low voltage
pulse 104. The first pulse 102 was intended to porate the membrane,
whereas the second pulse 104 was intended to electrophorese DNA
into the cell cytosol. Sukharev recognized that the delay 103
should not be excessive.
[0055] Although the Sukharev system may provide satisfactory
results in some applications, this system may not be completely
adequate for certain other applications. Some users may find, for
example, that Sukharev's electroporation does not effectively move
enough molecules of the implant agent into the target cells. This
results from an excessive delay 103 between Sukharev's first 102
and second 104 pulses, as recognized by the present inventor. The
pores of a cell, created by electroporation, stay open for a finite
time, largely depending upon the cell's temperature. Thus, the
effect of the first pulse may start to significantly decay (thereby
closing the cell's pores) during the delay between the first and
second pulses. IR some applications, this may be sufficient to
completely nullify the first pulse's effect upon the cell by the
time the second pulse occurs. As a result, the efficacy of
Sukharev's electroporation may be insufficient in some cases.
Moreover, lacking an effective first pulse, the second pulse of
Sukharev's system may need to be increased to the point where it
permanently destroys cells.
[0056] The delay described above is inherent to the Sukharev system
due to the use of electromechanical relays. Sukharev uses
independent pulse generators, whose outputs are selectively coupled
to output electrodes by a relay. As known in the art, however, the
switching of an electromechanical relay typically takes a
significant amount of time, sometimes even 50-100 ms. Therefore,
the efficacy of the implant agent achieved by Sukharev may be too
low for some applications.
[0057] Electroporation Power Supply:
[0058] FIG. 4 shows the pulsing electroporation power subsystem 200
which receives an input voltage, such as 110V or 220 VAC, from a
power source 203. Preferably, the subsystem 200 includes a
comparable apparatus, which is disclosed in U.S. patent application
Ser. No. 08/709,615 and entitled "Electroporation User Configured
Pulsing Scheme.". The subsystem 200 has an electroporation power
supply 202 and driver control 204 which controls or changes voltage
magnitude, switch on or off, invert voltage polarity or provide
voltage pulsing from the power supply 202 to electrodes 40, 42.
Signals from control board 60 control provide the desired voltage
or current output. A power supply 202 provides a reliable source of
desired voltage levels for use by the electroporation power control
201. An example of such a source is available, for example, from
Electro Square Porator T820, made by the BTX Division of
Genetronics, Inc. Either independent from or associated with power
supply 70 is an electroporation power supply 202 which produces
sufficient voltage range, preferably up to 500 volts. The divider
204 converts the input voltage into multiple reference voltages. In
the illustrated embodiment, reference voltages of 500 V (D.C.)
reside on the divider output lines 204a-204b.
[0059] These voltages are provided to collectors 206b-207b of first
and second respective transistors 206-207. The transistors 206-207
are selectively gated to apply their input voltages to step voltage
nodes 208-209. The selective gating of the transistors 206-207 is
performed by respective comparators 212-213, which trigger gates
206a-207a of the transistors 206-207 when voltages at the step
voltage nodes 208-209 dips below voltages established on step
voltage input lines 216-217. For example, when the comparator 212
determines that the voltage on the step voltage node 208 is less
than the voltage on the preset input line 216, the comparator 212
activates the gate 206a of the transistor 206, causing the
transistor 206 to couple the input voltage of the divider 204
directly to the step voltage node 208. Thus, the transistors 206
maintain substantially constant voltages at the respective step
voltage nodes 208-209 in accordance with the step voltage input
lines 216-217.
[0060] Energy Reservoirs:
[0061] The subsystem 200 also includes energy reservoirs 220-221
coupled to respective step voltage nodes 208-209. Exemplary energy
reservoirs 220-221 may comprise capacitors, such as 3200 .mu.F, 500
V electrolytic capacitors. These capacitors are appropriate for
maximum step voltages 208-209 of 500 V (D.C.).
[0062] Transformer:
[0063] The subsystem 200 also includes a transformer 224, which
includes a primary winding 224a and a secondary winding 224b. The
transformer 224 preferably is designed with low leakage inductance
characteristics to provide a fast pulse rise time, i.e., several
microseconds. Preferably, the transformer 224 exhibits low
inductance, on the order of a few .mu.H. These features may be
provided by winding the transformer 224 with a single cable of
twelve separate, twisted conductors of which six are connected in
parallel for the primary, six are connected in series for the
secondary. This provides a 1:6 step-up ratio. In addition, a
separate low voltage D.C. bias winding around the core may be used
to employ the full flux swing of the transformer's core. As an
example, the transformer may utilize a core made of laminated
iron.
[0064] The transformer 224 may advantageously be constructed to
saturate if the pulse length exceeds a maximum prescribed value,
thereby protecting a patient from excessive electrical energy.
Preferably, the transformer 224 is capable of carrying 0.3 V-sec
(3000 V.times.100 .mu.sec) before saturation. Another advantage of
the transformer 224 is that its output is floating, and no
substantial current will flow if the patient is connected to ground
potential. The secondary winding 224b is coupled to output
connection nodes 230-231, which preferably connect to the cuvette
holder device 10.
[0065] The load between the electrodes 40, 42 is represented by the
in vitro implant agents and host material in the cuvette holder 10
which may contain platelets, human cells, red blood cells,
mammalian cells, plant protoplasts, plant pollen, liposomes,
bacteria, fungi, yeast, sperm, or other cells.
[0066] To protect the energy reservoir 220 and power supply 202, a
diode 236 may be placed between the energy reservoir 220 and the
connection 230. Likewise, to protect the energy reservoir 221 and
power supply 220, a diode 237 may be placed between the secondary
winding 224b and the connection 230.
[0067] Switches:
[0068] The subsystem 200 also includes switches 226-227 to
selectively enable current to flow through the primary and
secondary windings 224a-224b, respectively. In one exemplary
construction, each switch 226-227 may comprise an insulated gate
bipolar transistor ("IGBT"), such as a Fuji Electric brand
IMBI400F-060 bmodel IGBT.
[0069] The switch 226 and the energy reservoir 221 are coupled in
series, this series combination being attached in parallel with the
primary winding 224a. When voltage is applied to a gate 226a of the
switch 226, the collectors 226b and emitter 226c are electrically
connected. Thus, the energy reservoir 221 is effectively placed in
parallel with the primary winding 224a. This allows current from
the energy reservoir 121 to flow through the primary winding
224a.
[0070] Similarly, the switch 227 and energy reservoir 220 are
coupled in series, this series combination being attached in
parallel with the secondary winding 224b. When voltage is applied
to a gate 227a of the switch 227, the collectors 227b and emitter
227c are electrically connected. Thus, the energy reservoir 220 is
effectively placed in parallel with the secondary winding 224b.
This allows current from the energy reservoir 220 to flow through
the electrodes 40, 42.
[0071] Advantageously, none of the energy reservoirs 220-221 or
switches 226-227 grounds the windings 224a-224b. The windings
224a-224b, therefore electrically float. As a result, no
substantial current will flow through a patient or other load 234
that is connected to another earth or ground potential.
[0072] Electroporation Pulse Controller:
[0073] Another component of this example of the subsystem 200 is
the power control 201, which manages operation of the switches
226-227. Broadly, the controller 66 114 (FIG. 2) regulates the
on-times and off-times of the switches 226-227 in accordance a
specified schedule, thereby generating a predetermined pulsing
scheme at the electrodes 40, 42. When the control 201 triggers the
switch 227, the voltage of the energy reservoir 220 is applied to
the electrodes 40, 42. When the control 201 triggers the switch
226, the voltage of the energy reservoir 220 is applied to the
transformer 224, where it is multiplied by six and applied to the
connections 230-231. The controller 66 (FIG. 2B) may also trigger
both switches 226-227 to apply an additive voltage, comprising the
sum of the step voltages 208-209, to the electrode structures 40,
42.
[0074] Preferable Design Parameters:
[0075] The electrical requirements can be derived from the field
strength, which was determined efficacious from in vitro
experiments with tumor cells and drugs, typically 1200-1300 V/cm,
and a pulse length of about 100 .mu.sec. The maximum voltage of the
generator derives from the maximum cavity size used.
[0076] As an example, the material resistivity is assumed to be as
low as 100 Ohm.times.cm. With an electrode area of 3 cm.times.3
cm=9 cm.sup.2, the resistance is 22 Ohm. The internal impedance of
the generator should be at least a factor 10 lower than 22 Ohm so
that no substantial drop in voltage occurs between charging and
delivered voltage. With the maximum voltage of 3000 V and a load
impedance of 22 Ohm, the switching requirements from a partial
capacitor discharge to generate a square pulse are a very
substantial 400 kW.
[0077] The desired maximum permeation pulse length is 100 .mu.sec;
this results in an energy per pulse of 40 J. For the collection and
electrophoresis pulse parameters, a maximum voltage of 500 V and
maximum pulse length of 200 msec may be used.
[0078] The maximum load current is about 136 A, which translates
into a primary current of 6.multidot.136=816 A, which the switch
has to carry and turn on and off. The switches 226-227 can
preferably maintain continuous current 800 A for one msec. The
maximum voltage is 600 V. Transient spikes are limited to a maximum
of 550 V for a 10% safety margin. This required careful low
inductance mechanical assembly to reduce transients and to be able
to get as close as safely feasible to the maximum voltage limit of
the IGBT.
[0079] The load impedance of 22 Ohm is transformed to the primary:
22/6.times.6-0.61 Ohm. A total internal impedance of 0.055 Ohm was
achieved on the primary side of the transformer, which translates
to an equivalent impedance of 1.98 Ohm on the secondary. Such a low
impedance can lead to excessive currents in case of an arc or short
circuit and these would destroy the expensive switching IGBT. The
IGBT can be configured to contain a current limiting feature, which
turns the switch off within a few .mu.sec in case of excessive load
currents which might happen if an arc or a short circuit condition
occurs. By inducing an arc in the secondary, we measured a benign
shut down of the IGBT within 5 .mu.sec, as soon as the current
exceeds about 900 A in the primary, corresponding to 150 A in the
secondary.
[0080] The necessary capacitor size can be estimated from the
maximum allowable voltage drop across the load of 5%. The charge
conducted in the primary pulse is 100 .mu.s.times.816 A-0.08 Cb. If
this should be 5% of the capacitor bank, the bank needs to hold
20.times.0.08=1.6 Cb. At 500 V maximum, the required capacity is
C=Q/V=1.6/500=0.0032 F or 3200 .mu.F. The energy stored in these
capacitors is 400 Joule.
[0081] For the collection and electrophoresis pulse, a second
capacitor discharge circuit delivers the longer pulse lengths
(several 100 msec) and low voltage (500 V) without the pulse
transformer. The low voltage circuit and the high voltage circuit
are decoupled from each other by stacks of diodes 237 and 236.
[0082] In addition to the various hardware embodiments described
above, a different aspect of the invention broadly concerns a
method for generating a user-specified electric field pulsing
pattern to achieve improved electroporation.
[0083] Data Storage Media:
[0084] This method may be implemented, for example, by operating
the controller 66 to execute a sequence of machine-readable
instructions. These instructions may reside in various types of
data storage media. In this respect, one aspect of the present
invention concerns an article of manufacture, comprising a data
storage medium tangibly embodying a program of machine-readable
instructions executable by a digital data processor to perform
method steps to generate a user-specified electric field pulsing
pattern to achieve improved electroporation.
[0085] This data storage medium may comprise, for example, RAM
contained within the controller 66. Alternatively, the instructions
may be contained in another data storage medium, such as a magnetic
data storage diskette (FIG. 5). Whether contained in the controller
66 or elsewhere, the instructions may instead be stored on another
type of data storage medium such as DASD storage (e.g., a
conventional "hard drive" or a RAID array), magnetic tape,
electronic read-only memory (e.g., ROM), optical storage device
(e.g., WORM), paper "punch" cards, or other data storage media.
[0086] Operational Steps:
[0087] As mentioned above, one aspect of the invention broadly
concerns a method for generating a user-specified electric field
pulsing pattern to achieve improved electroporation. FIG. 6 shows a
sequence of method steps 400 to illustrate one example of this
aspect of the present invention. For ease of explanation, but
without any limitation intended thereby, the sequence of FIG. 6 is
described in the context of the subsystem 200 described above.
[0088] After the steps 400 are initiated in task 402, the
controller 66 in task 404 receives user input specifying an output
pulse pattern of one or more output pulses. As an example, this
user input may be received from the user interface 50. As an
alternative, the user input may be received from another electronic
device, or even a prestored record.
[0089] Preferably, the user input specifies a duration for each
pulse and also specifying either a "high" output voltage or a "low"
output voltage. Next, for each pulse of low predetermined voltage,
the pulse generator in task 404 generates the "low" predetermined
voltage at the output terminals 230 and 231 for the specified
duration. More particularly, the controller 66 may generate a low
voltage pulse by gating the switch 227, thereby permitting the
energy reservoir 220 to discharge through the electrodes 40,
42.
[0090] Also, in task 404, high voltage pulses are generated at the
secondary winding terminals by concurrently applying another
voltage to the primary winding terminals of the transformer for the
specified duration. More particularly, the high voltage pulse
involves generating the voltage as discussed above, while
concurrently triggering the switch 226 to permit the energy
reservoir 221 to discharge through the primary winding 224. As the
voltage of the reservoir 221 is multiplied by the transformer 224,
a high voltage is created at the electrodes 230-231. This voltage
is the additive sum of the voltages stored in the energy reservoirs
220-221. Alternatively, a lesser "high" voltage output may be
created solely by triggering the switch 226, without involving the
switch 227.
[0091] One or more of the above-mentioned pulses are therefore
generated in task 404 to produce the user-specified pulse pattern.
After the user-specified pulsing pattern is created completed in
task 404, the routine 400 ends in task 406.
[0092] Operation With Exemplary Pulsing Pattern:
[0093] As mentioned above, the pulse subsystem 200 provides a
user-specified pulse pattern comprising one or more pulses of
"high" and/or "low" output voltage.
[0094] Other exemplary pulse shapes, which may be used alone or in
combination to constitute the user-specified pulsing scheme is
taught in the U.S. patent application Ser. No. 08/709,615, as
discussed above. FIGS. 8-11 illustrate various exemplary pulse
shapes, which may be used alone or in combination to constitute the
user-specified pulsing scheme with associated temperature profile
in real-time for processing of materials. The time scale is not
necessarily linear as shown since the pulsing event occurs
temporally in microseconds and cooling and heating of materials is
an extrinsic factor of the apparatus used depending upon the
receptacle device's volume for containing the materials and thermal
design for effectuating heat transfer into or out of the receptacle
device.
[0095] Although each of the pulsing patterns of FIGS. 8-11 may
provide distinct advantages for different applications, the
following description highlights the features and operation of a
pattern 700 (FIG. 9) to illustrate the operation of the invention,
both in the electroporation pulse scheme and temperature control in
real-time. The polarity and current, which can be pulse width
modulated, through the Peltier device 44 directly correlates with
temperature in the receptacle device. In the cooling phase of the
materials undergoing electroporation, temperatures are typically
around four degrees centigrade at the lower extreme and can be
warmed to around 40 degrees centigrade. Desired material
temperatures associated with electroporation pulsing in real time
of particular cells and implant agents is functionally related to
the type of cells and implant materials used and the desired
outcome of the processing. Such control can be user specified and
stored in a programmable form on a data storage media such as that
shown in FIG. 5 and be implemented using the electroporation and
temperature controller 100.
[0096] The pattern 700 comprises a "stepped pattern," in that it
provides first, second, and third voltage levels 702-704. One, two,
or all of these voltages may be the same, if desired. The pulses
have first, second, and third durations 706-708. In the present
example, the first and third voltages 706, 708 provide a 500 V
(D.C.), whereas the second voltage 707 provides 3000 V (D.C.). In
an alternative embodiment, the user may specify a desired magnitude
of electric field to be applied by the transformer 224, and a
measurement of the gap between the electrodes 40, 42. In this case,
the controller 66 may compute the appropriate voltage for the
transformer 224 to generate in order to apply the desired electric
field, for example by multiplying the electric field by the gap. In
one embodiment, the gap measurement may be input by the user
manually. Alternatively, the gap may be mechanically measured and
electronically fed to the controller 66 by automated means such as
shown in U.S. Pat. No. 5,439,440, which is hereby incorporated by
reference in its entirety.
[0097] The efficiency of the implant agents to cells during
electroporation processing is significantly dependent on the
duration of cellular membrane permeabilization which has been
discovered to be directly dependent on the temperature prior to,
during and after pulsing application. Cellular permeability is
modifiable by temperature changes. By raising the temperature, the
permeability of the cellular wall is lowered. The lowering the
ambient material temperature causes the permeability of the
cellular material to increase.
[0098] During initial processing, temperature should be lowered to
around ice cooled temperatures, e.g., 4 degrees centigrade. After
pulsing, usually several minutes, the temperature of the materials
should be raised to around 40 degrees centigrade to seal and anneal
the cellular membrane. However. some cellular materials are very
sensitive to temperature and cannot survive extreme temperature
changes. In such cases, cellular manipulations need to be carried
out at the optimal cell survival conditions.
[0099] The corresponding temperature profile in real time would
exist and be controlled by the Peltier device 44 in the materials
undergoing electroporation. The causal relationship of temperature
effects when materials undergo electroporation has been studied and
demonstrated that cellular membrane alterations do occur under
varying temperature and electrical fields. In particular, this is
taught in an article entitled "Studies on Electroporation of
Thermally and Chemically Treated Human Erythrocytes," by Nanda et
al. in Bioelectrochemistry and Bioenergetics, 34 (1994) 129-134.
Temperature is a significant factor during electroporation
processing of cells such as red blood cells with implant agents.
When temperature increases during electroporation processing,
effectiveness is significantly reduced. For example when using red
blood cells, Increase in the temperature during electroporation
from four to forty-three degrees Centigrade reduces electroporation
efficiency by approximately 50%. Post pulse incubation of red blood
cells at higher temperatures further reduces the electroporation
effectiveness.
[0100] FIG. 12 describes an illustrative sequence 2000 involved in
generating and applying the step pattern 700, and the effects
caused by application of the pattern 700 with corresponding control
of temperature. After the sequence begins in task 1002, the user
interface 50 receives user input in task 1004. In the illustrated
embodiment, the user input includes the user's specification of a
desired electroporation pulsing pattern, including a duration and
voltage level for each pattern portion and the corresponding
control of temperature.
[0101] Concurrently with task 1004, the power supply 202 generates
the reference voltages at the output nodes 208-209. In the present
example, the reference voltages 208-209 of 500 V (D.C.) are used.
Generation of the reference voltages in task 1008 charges the
energy reservoirs in task 1008.
[0102] After task 1008, an operator in task 1010 inserts molecules
of an implant agent to the receptacle device for processing. The
implant agent may comprise one or more types of DNA, genes, and/or
various chemical agents.
[0103] Step 1010 places the implant agent between the interstices
of the cells at the process site. Next, in task 1012 the Peltier
device 44 cools the materials in the receptacle device to around
ice temperatures for most processing applications. However, warming
of materials undergoing processing can be implemented.
[0104] After task 1012, the controller 66 in task 1014 gates the
switch 227, discharging the energy reservoir 220, thereby applying
the "low" voltage to the electrodes 40, 42. This step accumulates
molecules of the implant agent near the membranes of the cells in
the cell sample. As discovered by the present inventors, this step
may be adequately performed with a reduced voltage.
[0105] Accordingly, the "low" voltage of the energy reservoir 220
achieves this purpose, while still avoiding damage to the cells in
the sample and saving electrical energy.
[0106] FIG. 9 illustrates task 1014 as the voltage pulse 702. As
shown, this pulse preferably comprises a square wave having a
duration 706 of about 10-200 msec and a voltage of about 500 V
(D.C.). Depending upon the application, however, different
parameters may be substituted to define the pulse 702. A
corresponding cooling period occurs to allow greater permeability
of cellular materials undergoing processing.
[0107] After task 1014, the controller 66 in task 1016 gates the
switch 226 on (while continuing to gate the switch 227). This
creates a "high" voltage upon the electrodes 40, 42, corresponding
to the sum of the reference voltages 208-209. This high voltage is
sufficient to safely create small pores in the cells of the tissue
sample. FIG. 9 illustrates this step as the voltage pulse 703. As
illustrated, this pulse preferably comprises a square wave having a
duration 707 of about 100 .mu.sec and an electric field magnitude
of about 1200 V/cm. Depending upon the application, however,
different parameters may be substituted to define the pulse 702.
The temperature is maintained at the cool state during processing
to maintain high permeability of a cellular membrane undergoing
processing.
[0108] Advantageously, the subsystem 200 automatically limits
damage to cells of the tissue sample during this step. In
particular, when the voltage from the primary winding 224a
saturates the secondary winding 224b, the voltage presented by the
secondary winding 224b begins to decay, in accordance with known
principles of transformer operation. Thus, even if the voltage
applied to the primary winding 224a is applied for an excessive
length of time, the secondary winding 224b automatically limits the
tissue sample's exposure to this high voltage pulse.
[0109] Next, in task 1018 the controller 66 ceases gating of the
switch 226 while continuing to gate the switch 227. This step
permits the molecules of the implant agent to transit the cells'
permeable membranes, and enter the cells' cytoplasm.
[0110] FIG. 10 illustrates this step as the voltage pulse 704. As
illustrated, this pulse preferably comprises a square wave having a
duration 708 of about 1-200 msec and a voltage of about 500 V
(D.C.). Depending upon the application, however, different
parameters may be substituted to define the pulse 704.
[0111] After task 1018, the controller 66 releases gating of the
switch 227, ending the pulse 700. Then, the Peltier device 44
either warms or cools the material in the receptacle device in task
1022, and the sequence 2000 ends in task 1024. This step normally
takes on the order of several minutes where it is either desirable
to leave the cellular membrane open (keeping material cool) or
sealing or annealing the cellular membrane (keeping the material
warm).
[0112] Other uses of the invention provided herein include all
electroporation mediated processes such as electrofusion,
electroinsertion, electrochemotherapy, electrogene therapy and
electrostimulation. While the presently preferred embodiments of
the invention have been disclosed, it will be apparent to those
skilled in the art that various changes and modifications can be
made herein without departing from the scope of the invention as
defined by the appended claims.
* * * * *