I CLAIM :
1. An apparatus for applying electric field pulses across a liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: a non-conductive, non-magnetic hollow chamber for holding said liquid suspension, means cooperative with said chamber for the insertion and removal of said liquid suspension, and means operative on said chamber for magnetically producing said electric field pulses in a uniform homogeneous electric field across said chamber containing said liquid suspension to treat said vesicles.
2. An apparatus for applying electric field pulses across a liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: a hollow chamber for holding said liquid suspension, means in said chamber for the insertion and removal of said liquid suspension, a magnetic core, means operative with said magnetic core and said chamber for releasably holding said chamber near said magnetic core, and means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said magnetic core, said magnetic core when energized being capable of providing a uniform electrical field across said chamber containing said liquid suspension having said
vesicles when said chamber is held near said magnetic core by said holding means so that said electrical field pulses are applied to said vesicles for said treatment .
3. The apparatus of Claim 2 wherein said chamber is formed of non-conductive material, said chamber being capable of being disposed of after said vesicles are treated.
4. The apparatus of Claim 2 wherein said insertion and removal means is a formed hole in said chamber .
5. The apparatus of Claim 2 wherein said magnetic core is a closed loop.
6. The apparatus of Claim 5 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
7. An apparatus for applying electric field pulses across an electrolytic liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: an electrically non-conductive hollow chamber for holding said electrolytic liquid suspension, means in said chamber for the insertion and removal of said electrolytic liquid suspension containing said vesicles, a magnetic core.
means operative with said magnetic core and said chamber for releasably holding said chamber orthogonal to said magnetic core, and means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said magnetic core, said magnetic core when energized being capable of generating a uniform electrical field across said chamber containing said electrolytic liquid suspension carrying said vesicles when said chamber is held near said magnetic core by said holding means so that said electrical field pulses are applied to said vesicles for said treatment.
8. The apparatus of Claim 7 wherein said chamber is toroidal in shape, said chamber being capable of being disposed of after said vesicles are treated.
9. The apparatus of Claim 7 wherein said insertion and removal means is a formed hole in said chamber.
10 . The apparatus of Claim 7 wherein said magnetic core is a closed loop .
11 . The apparatus of Claim 10 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
12. An apparatus for applying electric field pu l s e s acros s an e lectrolytic liquid suspension containing vesicles , said vesicles to be treated by
said electric field pulses in said apparatus, said apparatus comprising: an electrically non-conductive hollow chamber for holding said electrolytic liquid suspension, said chamber being formed in a toroidal shape and having a center annulus region, means in said chamber for the insertion and removal of said electrolytic liquid suspension containing said vesicles, a closed loop magnetic core, said magnetic core comprising:
(a) a C-shaped conductive member, and
(b) a closure arm having one end pivotal ly connected to one end of said C-shaped member, said closure arm having its other end releasably connectable to the other end of said C-shaped member, said annulus of said chamber being capable of sliding on and off over said closure arm when said closure arm is disconnected from said C-shaped member, said chamber being orthogonal to said closure arm when said chamber is slid over said closure arm, and said closure arm when connected to said C-shaped member completing a magnetic path around said magnetic core, means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said closure arm so that said electrical field pulses are applied across said chamber containing said electrolytic liquid suspension carrying said vesicles when said chamber is
oriented over said closure arm and when said closure arm is connected to said C-shaped member.
13. The apparatus of Claim 12 wherein said chamber is formed of non-conductive material, said chamber being capable of being disposed of after said vesicles are treated.
14. The apparatus of Claim 12 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
15. An apparatus for applying electric field pulses across an electrolytic liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: an electrically non-conductive hollow chamber for holding said electrolytic liquid suspension, said chamber being formed in a toroidal shape and having a center annulus region, said chamber being capable of being disposed of after said vesicles are treated, means in said chamber for the insertion and removal of said electrolytic liquid suspension containing said vesicles, a closed loop magnetic core, said magnetic core comprising:
(a) a C-shaped conductive member, and
(b) a closure arm having one end pivotally connected to one end of said C-shaped member, said closure arm having its other end releasably connectable to the other end of said C-shaped member,
said annulus of said chamber being capable of sliding on and off over said closure arm when said closure arm is disconnected from said C-shaped member, said chamber being orthogonal to said closure arm when said chamber is slid over said closure arm, and said closure arm when connected to said C-shaped member completing a magnetic path around said magnetic core, (c) two half wind current sheaths would around said C-shaped member and said closure arm, and means connected to said current sheaths wound on said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said closure arm so that said electrical field pulses are applied across said chamber containing said electrolytic liquid suspension carrying said vesicles when said chamber is oriented over said closure arm and when said closure arm is connected to said C-shaped member.
16. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous electric field across said chamber containing said liquid suspension,
means for generating a high voltage, high current charge of predetermined value, means connected to said generating means for selectively delivering said charge at a high speed rise time to said providing means so that immediate application of said charge to said providing means is obtained, said delivering means being selected from a group consisting of a moving spark gap, a gas filled spark gap, an ignition, a hot cathode hydrogen " thyratron, and a cold cathode hydrogen thyratron, means connected to said delivering means for producing a predetermined time length, said producing means being capable of high speed termination of said charge to said providing means, said predetermined time length being sufficient to permit said charge to produce said stable pores in and through said membrane walls of said vesicles in said suspension.
17. The apparatus of Claim 16 wherein said providing means comprises a pair of parallel electrodes connected to said delivering means, said electrodes being spaced a predetermined distance apart on opposing ends of said chamber.
18. The apparatus of Claim 17 wherein each of said electrodes has the side facing the opposing electrode coated with an inert stable material to minimize electrolytic decomposition of said suspension containing said vesicles.
19. The apparatus of Claim 16 wherein said providing means comprises:
means connected to said delivering means for creating magnetic flux proportional to said charge, and means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
20. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous electric field across said chamber containing said liquid suspension, means for generating a high voltage, high current charge of predetermined value, means connected to said generating means for selectively delivering said charge at a high speed rise time through a triggered ionization breakdown to said providing means so that immediate application of said charge to said producing means is obtained, and means connected to said delivering means for producing a predetermined time length, said producing means being capable of high speed termination of said charge to said providing means, said predetermined time length being sufficient to permit said charge to produce said stable pores in and through said membrane walls of said vesicles in said suspension.
21. The apparatus of Claim 20 wherein said providing means comprises a pair of parallel electrodes connected to said delivering means, said electrodes being spaced a predetermined distance apart on opposing ends of said chamber .
22. The apparatus of Claim 21 wherein said delivering means comprises:
(a) two opposing electrodes, one of said electrodes being connected to said generating means, the opposing electrode being connected to said producing means , and
(b) means for moving said electrodes together to cause said charge to jump from said one electrode to said opposing electrode.
23. The apparatus of Claim 20 wherein said providing means comprises: means connected to said delivering means for creating magnetic flux proportional to said change, and means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
24. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles,
means in said chamber for the insertion and removal of said liquid suspension, a pair of parallel electrodes in said chamber spaced a predetermined distance apart on opposing ends of said chamber for providing a homogenous electric field throughout said chamber containing said liquid suspension, each of said electrodes having the side facing the opposing electrode coated with an inert stable material to minimize electrolytic decomposition of said suspension containing said vesicles, means for generating a high voltage, high current charge of predetermined value, means connected to said generating means for selectively delivering said charge at a high speed rise time to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained, and means connected to said delivering means for producing a prede ermined time length, said producing means being capable of high speed shorting of said charge at the end of said predetermined time length thereby terminating said high voltage, high current charge between said electrodes, said time length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension, the size of said pores being dependent upon (i) the value of said high voltage, (ii) said predetermined time length, and (iii) said high speed of said rise time and said termination of said charge across said electrode.
25. The apparatus of Claim 24 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
26. The apparatus of Claim 24 further comprising means connected to said chamber for minimizing the inductance of said chamber and of said electrodes and matching the impedance of said chamber.
27. The apparatus for Claim 26 wherein said inductance minimizing means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes, each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
28. The apparatus of Claim 24 wherein a fluid tight seal exists between each of said electrodes and said dielectric material.
29. The apparatus of Claim 24 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and (b) a charge accumulator connected to said h igh v o lt ag e pow e r s u p p l y f o r generating said high voltage , high current charge.
30. The apparatus of Claim 24 wherein said delivering means comprises:
(a) a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to said producing means, and
(b) means for moving said switch electrodes together to cause said charge to jump from said one switch electrode to said opposing switch electrode.
31. The apparatus of Claim 24 wherein said delivering means is selected from the group consisting of a moving spark gap, a gas filled spark gap, a vacuum spark gap, an ignitron, a hot cathode hydrogen thyratron and a cold cathode hydrogen thyratron.
32. An apparatus for the high speed application of high voltage, high current electric field pulses in a homogeneous electric field across a liquid suspension containing vesicles having membrane walls, said electric field pulses being capable of producing stable pores in and through said membrane walls of said vesicles, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, said chamber being capable of enduring autoclave temperatures and comprising: (a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for
providing said homogeneous electric field throughout said chamber, each of said electrodes having the side facing the opposing electrode coated with an inert stable material to minimize electrolytic decomposition of said suspension containing said vesicles,
(b) dielectric material separating said electrodes for forming the sides of said chamber, and
(c) means in said dielectric material for the insertion and removal of said liquid suspension, means connected to said chamber for minimizing the inductance of said chamber and said electrodes and matching the impedance of said chamber when said electric field pulses are applied, and means connected to said minimizing means and to said electrodes for applying said electric field pulses, said applying means comprising:
(a) means for generating a high voltage, high current charge of predetermined value,
(b) means connected to said generating means for selectively delivering said charge at a high speed rise time to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained, (c) means connected to said delivering means for producing a pulse signal of predetermined time length, said producing means being capable of high speed shorting of said charge at the
end of said predetermined time length thereby terminating the application of said high voltage, high current charge between said electrodes by providing a high speed fall time to said charge, said predetermined time length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension, the size of said pores being dependent upon (i) the value of said high voltage, (ii) said time length, and (iii) said speed of rise time and said fall time of said charge across said electrodes.
33. The apparatus of Claim 32 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
34. The apparatus for Claim 32 wherein said inductance minimizing means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes, each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
35. The apparatus of Claim 32 wherein a fluid tight seal exists between each of said electrodes and said dielectric material.
36. The apparatus of Claim 32 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and
(b) a charge accumulator connected to said high voltage power supply for generating said high voltage, high current charge.
37. The apparatus of Claim 32 wherein said delivering means comprises :
(a) a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to said producing means, and
(b) means for moving said switch " electrodes together to cause said charge to jump from said one switch electrode to said opposing switch electrode.
38. The apparatus of Claim 32 wherein said delivering means is selected from the group consisting of a moving spark gap, a gas filled spark gap, a vacuum spark gap, an ignitron, a hot cathode hydrogen thyratron and a cold cathode hydrogen thyratron.
39. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising:
a chamber for holding said liquid suspension containing said vesicles, said chamber comprising:
(a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing a homogeneous electric field throughout said chamber,
(b) dielectric material separating said electrodes for forming the sides of said chamber, and
(c) means in said dielectric material for the insertion and removal of said liquid suspension, means for generating a high current, high voltage charge of predetermined value, a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means , the opposing switch electrode being connected to one of said parallel electrodes, means connected to said high speed switch for moving said switch electrodes together to cause said charge to jump from said one switch electrode to said opposing switch electrode, and means connected to said opposing electrode of said high speed switch for producing a predetermined time length commencing with said jump of said charge, said producing means being capable of high speed shorting of said charge at the end of said predetermined time length thereby terminating said charge to said parallel electrodes, said time length being sufficient to permit said charge between said parallel electrodes to produce stable pores in and
through said membrane walls of said vesicles in said suspension.
40. A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: placing the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis , and applying a second uniform, homogeneous electric field pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field across each aligned vesicle to produce said stable pores in said vesicles.
41. An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, means operative with said chamber for
providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of aligning said vesicles along either their major or minor axis, and second means connected to said providing means for generating a second uniform, homogeneous electric field at a high voltage, high current charge of predetermined value for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential across each aligned vesicle, said predetermined value and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles.
42. A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: placing the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across in said chamber for a first predetermined time interval, said preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis, and applying a second uniform, homogeneous electric field pulse at a preselected high voltage and high current across said chamber for a second
predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field across each aligned vesicle to produce said stable pores in said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.
43. A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: placing the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together, applying a second uniform, homogeneous electric field pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of
creating pores at contiguous adjacent points in and through the membranes of said cell pairs, and waiting a predetermined period of time to permit the paired cells to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.
44. A method for fusing ellipsoidal, electrically or dielectrically aspherical vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of:
(a) applying a first uniform, homogeneous electric field at a first preselected frequency across said chamber for a first predetermined time interval, said first preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis and to produce initial cell pairing,
(b) applying a second uniform, homogeneous electric field pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of alignment, said electric field pulse being capable of creating pores in and through contiguous membranes of said vesicles,
(c) repeating steps (a) and (b) above until poration of said vesicles is completed,
(d) applying a third uniform, homogeneous electric field pulse at a second preselected frequency across said liquid suspension for a third predetermined time interval in response to the step of poration, said second preselected frequency being capable of pairing said porated vesicles together at said pores, and
(e) waiting a predetermined period of time to permit the paired cells to fuse into a single vesicles in response to the step of applying said third uniform pulse. 45. An apparatus for producing stable pores in and through membrane walls of vesicles during poration or electrofusion contained in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous electric field across said chamber containing said liquid suspension, means for generating a high power charge of greater than one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than fifty nanoseconds to said providing means so that immediate application of said charge to said producing means is obtained, and means connected to said delivering means for producing a predetermined time length, said producing means being capable of high speed termination of said charge to said providing means, said predetermined time length being sufficient to permit said charge to produce said stable pores in and through said membrane walls of said vesicles in said suspension.
46. The apparatus of Claim 45 wherein said chamber holds volumes of said suspension greater than one milliliter.
47. The apparatus of Claim 45 wherein said delivering means comprises means for adjusting the rise time of said charge.
48. The apparatus of Claim 45 wherein said providing means comprises: means connected to said delivering means for creating magnetic flux proportional to said change, and means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
49. The apparatus of Claim 45 wherein said delivery means comprises means connected to said chamber for minimizing the inductance of said chamber and for matching the impedance of said chamber.
50. An apparatus for producing stable pores in and through membrane walls of vesicles during poration or electrofusion contained in a liquid suspension, said apparatus comprising: a chamber for holding said liquid suspension containing said vesicles, means in said chamber for the insertion and removal of said liquid suspension, means for generating a high power charge of greater than one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than fifty nanoseconds to said providing means in said chamber so that immediate
application of said charge to said providing means is obtained, said delivering means being capable of minimizing the inductance of said chamber and matching the impedance of said chamber, and means connected to said delivering means for producing a predetermined time length, said time length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension, the size of said pores being dependent upon (i) the value of the voltage of said charge, (ii) said predetermined time length, and (iii) said high speed of said rise time of said charge across said providing means.
51. The apparatus of Claim 50 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
52. The apparatus of Claim 51 wherein said inductance minimizing means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes , each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
53. The apparatus of Claim 50 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and
(b) a charge accumulator connected to said high voltage power supply for generating said high voltage, high current charge.
54. A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis , and applying a second uniform, homogeneous electric field pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field across each aligned vesicle to produce said stable pores in said vesicles.
55. An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising:
a chamber for holding at least one milliliter of said liquid suspension containing said vesicles , means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of aligning said vesicles along either their major or minor axis, and second means connected to said providing means for generating a second uniform, homogeneous electric field having a power greater than one megawatt with a rise time of fifty nanoseconds or less for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential across each aligned vesicle, said predetermined value and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles.
56. A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: placing more than one milliliter of the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across in said chamber for a first predetermined time interval, said preselected frequency being capable of
electrically aligning said vesicles along either their major or minor axis, and applying a second uniform, homogeneous electric field pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field across each aligned vesicle to produce said stable pores in said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.
57. A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together,
applying a second uniform, homogeneous electric field pulse having a power greater than one megawatt with a rise time less than 50 nanoseconds across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said cell pairs, and waiting a predetermined period of time to permit the paired cells to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.
58. A method for fusing ellipsoidal, electrically or dielectrically aspherical vesicles together, said vesicles being placed in a liquid suspension in a chamber, said method comprising the steps in the order of:
(a) applying a first uniform, homogeneous electric field at a first preselected frequency across said chamber for a first predetermined time interval, said first preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis and to produce initial cell pairing,
(b) applying a second uniform, homogeneous electric field pulse having a power greater than one megawatt with a rise time less than fifty nanoseconds across said chamber for a second predetermined time interval in response to the step of alignment, said electric field pulse being capable of creating pores in and through contiguous membranes of said vesicles,
(c) repeating steps (a) and (b) above until poration of said vesicles is completed,
(d) applying a third uniform, homogeneous electric field pulse at a second preselected frequency across said liquid suspension for a third predetermined time interval in response to the step of poration, said second preselected frequency being capable of pairing said porated vesicles together at said pores, and
(e) waiting a predetermined period of time to permit the paired cells to fuse into a single vesicles in response to the step of applying said third uniform pulse.
AMENDED CLAIMS
[received by the International Bureau on 02 November 1987 (02.11.87); original claims 1-58 replaced by new claims 1-63 (33 pages)]
1. An apparatus for treating vesicles contained in a liquid suspension by forming pores in said: vesicles, fusing said vesicles, providing
5 rotational prealignment to said vesicles and for providing dielectrophorectic bunching to said vesicles, said apparatus comprising: a non-conductive, non-magnetic hollow chamber for holding said liquid suspension,
ID means cooperative with said chamber for the- insertion and removal of said liquid suspension, and means operative on said chamber for magnetically producing electric field pulses in a
15 uniform homogeneous electric field across said chamber containing said liquid suspension to treat said vesicles by forming pores in said vesicles, fusing said vesicles, providing rotational prealignment to said vesicles and for providing
20. dielectrophorectic bunching to said vesicles.
2. An apparatus for treating vesicles contained in a liquid suspension by forming pores in said vesicles, fusing said vesicles, providing rotational prealignment to said vesicles and for
255 providing dielectrophorectic bunching to said vesicles, said apparatus comprising: a hollow chamber for holding said liquid suspension, means in said chamber for the insertion 30: and removal of said liquid suspension, a magnetic core, means operative with said magnetic core
and said chamber for releasably holding said chamber near said magnetic core, and means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said magnetic core, said magnetic core when energized being capable of providing a uniform electrical field across sair" chamber containing said liquid suspension having said vesicles when said chamber is held near said magnetic core by said holding means so that electrical field pulses treat said vesicles by forming pores in said vesicles, fusing said vesicles, providing rotational prealignment to said vesicles and for providing dielectrophorectic bunching to said vesicles.
3. The apparatus of Claim 2 wherein said chamber is formed of non-conductive material, said chamber being capable of being disposed of after said vesicles are treated.
4. The apparatus of Claim 2 wherein said insertion and removal means is a formed hole in said chamber.
5. The apparatus of Claim 2 wherein said magnetic core is a closed loop.
6. The apparatus of Claim 5 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
7. An apparatus for treating vesicles contained in a liquid suspension by forming pores in
said vesicles, fusing said vesicles, providing rotational prealignment to said vesicles and for providing dielectrophorectic bunching to said vesicles, said apparatus comprising: an electrically non-conductive hollow chamber for holding said liquid suspension, means in said chamber for the insertion and removal of said liquid suspension containing said vesicles, a magnetic core, means operative with said magnetic core and said chamber for releasably holding said chamber orthogonal to said magnetic core, and means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said magnetic core, said magnetic core when energized being capable of generating a uniform electrical field across said chamber containing said liquid suspension carrying said vesicles when said chamber is held near said magnetic core by said holding means so that electrical field pulses treat said vesicles by forming pores in said vesicles, fusing said vesicles, providing rotational prealignment to said vesicles and for providing dielectrophorectic bunching to said vesicles.
8. The apparatus of Claim 7 wherein said chamber is toroidal in shape, said chamber being capable of being disposed of after said vesicles are treated.
9. The apparatus of Claim 7 wherein said insertion and removal means is a formed hole in said chamber.
10. The apparatus of Claim 7 wherein said magnetic core is a closed loop.
11. The apparatus of Claim 10 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
12. An apparatus for applying electric field pulses across an liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: an electrically non-conductive hollow chamber for holding said liquid suspension, said chamber being formed in a toroidal shape and having a center annulus region, means in said chamber for the insertion and removal of said liquid suspension containing said vesicles, a closed loop magnetic core, said magnetic core comprising:
(a) a C-shaped conductive member, and
(b) a closure arm having one end pivotally connected to one end of said C-shaped member, said closure arm having its other end releasably connectable to the other end of said C-shaped member, said annulus of said chamber being capable of sliding on and off over said closure arm when said closure arm is disconnected from said C-shaped member, said chamber being
orthogonal to said closure arm when said chamber is slid over said closure arm, and said closure arm when connected to said C-shaped member completing a magnetic path around said magnetic core, means connected to said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said closure arm so that said electrical field pulses are applied across said chamber containing said liquid suspension carrying said vesicles when said chamber is oriented over said closure arm and when said closure arm is connected to said C-shaped member.
13. The apparatus of Claim 12 wherein said chamber is formed of non-conductive material, said chamber being capable of being disposed of after said vesicles are treated.
14. The apparatus of Claim 12 wherein said magnetic core comprises two half wind current sheaths wound on said core to generate said magnetic flux.
15. An apparatus for applying electric field pulses across an liquid suspension containing vesicles, said vesicles to be treated by said electric field pulses in said apparatus, said apparatus comprising: an electrically non-conductive hollow chamber for holding said liquid suspension, said chamber being formed in a toroidal shape and having a center annulus region, said chamber being capable
of being disposed of after said vesicles are treated, means in said chamber for the insertion and removal of said liquid suspension containing said vesicles, a closed loop magnetic core, said magnetic core comprising:
(a) a C-shaped conductive member, and
(b) a closure arm having one end pivotally connected to one end of said C-shaped member, said closure arm having its other end releasably connectable to the other end of said C-shaped member, said annulus of said chamber being capable of sliding on and off over said closure arm when said closure arm is disconnected from said C-shaped member, said chamber being orthogonal to said closure arm when said chamber is slid over said closure arm, and said closure arm when connected to said C-shaped member completing a magnetic path around said magnetic core,
(c) two half wind current sheaths would around said C-shaped member and said closure arm, and means connected to said current sheaths wound on said magnetic core for selectively energizing said magnetic core thereby causing magnetic flux to flow through said closure arm so that said electrical field pulses are applied across said chamber containing said liquid suspension carrying said vesicles when said chamber is oriented
over said closure arm and when said closure arm is connected to said C-shaped member.
16. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension so that materials can be loaded into said vesicles or unloaded therefrom, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous and uniform electric field across said chamber containing said liquid suspension, means connected to said chamber for generating a high voltage, high current poration charge, means connected to said generating means for selectively delivering said charge at a high speed rise time to said providing means so that immediate application of said charge to said providing means is obtained in less than 50 nanoseconds so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, said delivering means being selected from a group consisting of a moving spark gap, a gas filled spark gap, an ignition, a hot cathode hydrogen thyratron, and a cold cathode hydrogen thyratron.
means connected to said delivering means for producing a predetermined time length, said producing means being capable of high speed termination of said charge to said providing means, said predetermined time length being sufficient to permit said charge to produce said stable pores in and through said membrane walls of said vesicles in said suspension.
17. The apparatus of Claim 16 wherein said providing means comprises a pair of parallel electrodes connected to said delivering means, said electrodes being spaced a predetermined distance apart on opposing ends of said chamber.
18. The apparatus of Claim 17 wherein each of said electrodes has the side facing the opposing electrode coated with an inert stable material to minimize electrolytic decomposition of said suspension containing said vesicles.
19. The apparatus of Claim 16 wherein said providing means comprises: means connected to said delivering means for creating magnetic flux proportional to said charge, and means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
20. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension so that materials can be
loaded into said vesicles or unloaded therefrom, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said
5 vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and, removal of said liquid suspension,
HD means operative with said chamber for providing a homogeneous and uniform electric field acrjo.sis said chamber containing said liquid suspension so that all of said vesicles throughout said' suspension in said chamber are subjected to the
15 same electric field potential, means connected to said chamber providing means for generating a high voltage, high current rectangular wave poration charge, means connected to said generating means
20 for selectively delivering said charge at a high speed, rise time through a triggered ionization breakdown to said providing means so that immediate application of said charge to said providing means in less than 50 nanoseconds is obtained so that all
25. of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time., means connected to said delivering means for adjusting said rise time of said charge, and
3D- means connected to said delivering means for producing a variable pulse length to said charge, said producing means being capable of high speed termination of said charge to said providing means, said pulse length being sufficient to permit
35 said charge to produce said stable pores in and
through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles.
21. The apparatus of Claim 20 wherein said providing means comprises a pair of parallel electrodes connected to said delivering means, said electrodes being spaced a predetermined distance apart on opposing ends of said chamber.
22. The apparatus of Claim 21 wherein said delivering means comprises:
(a) two opposing electrodes, one of said electrodes being connected to said generating means, the opposing electrode being connected to said producing means, and
(b) means for moving said electrodes together to cause said charge to jump from said one electrode to said opposing electrode.
23. The apparatus of Claim 20 wherein said providing means comprises: means connected to said delivering means for creating magnetic flux proportional to said change, and means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
24. An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension so that materials can be
loaded into said vesicles or unloaded therefrom, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said
5 vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension,
ICE a pair of parallel electrodes in said chamber spaced a predetermined distance apart on opposing ends of said chamber for providing a homogenous and uniform electric field throughout said chamber containing said liquid suspension so
15 that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, each of said electrodes having the side facing the opposing electrode coated with an inert stable material to
20 minimize electrolytic decomposition of said suspension containing said vesicles, means connected across said parallel electrodes for generating a high voltage, high current rectangular wave poration charge having a
25 power greater than one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, means connected to said generating means for selectively delivering said charge at a high
30 speed rise time of less than 50 nanoseconds to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained, means connected to said delivering means
35 for adjusting said rise time of said charge, and
means connected to said delivering means for producing a variable pulse length to said charge, said producing means being capable of high speed shorting of said charge at the end of said pulse length thereby terminating said high voltage, high current charge between said electrodes, said pulse length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles, the size of said pores being dependent upon (i) the value of said high voltage, (ii) said pulse length, and (iii) said high speed of said rise time and said termination of said charge across said electrode.
25. The apparatus of Claim 24 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
26. The apparatus of Claim 24 further comprising means connected to said chamber for matching the impedance of said chamber with said delivering means.
27. The apparatus of Claim 26 wherein said matching means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes, each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
28. The apparatus of Claim 24 wherein a fluid tight seal exists between each of said electrodes and said dielectric material.
29. The apparatus of Claim 24 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and (b) a charge accumulator connected to said high voltage power supply for generating said high voltage, high current charge.
30. The apparatus of Claim 24 wherein said delivering means comprises:
(a) a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to said producing means, and
(b) means for moving said switch electrodes together to cause said charge to jump from said one switch electrode to said opposing switch electrode.
31. The apparatus of Claim 24 wherein said delivering means is selected from the group consisting of a moving spark gap, a gas filled spark gap, a vacuum spark gap, an ignitron, a hot cathode
hydrogen thyratron and a cold cathode hydrogen thyratron.
32. An apparatus for the high speed application of high voltage, high current electric field pulses in a homogeneous electric field across a liquid suspension containing vesicles having membrane walls, said electric field pulses being capable of producing stable pores in and through said membrane walls of said vesicles, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber being capable of enduring autoclave temperatures and comprising:
(a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing said homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, each of said electrodes having the side facing the opposing electrode coated with an inert stable material to minimize electrolytic decomposition of said suspension containing said vesicles.
(b) dielectric material separating said electrodes for forming the sides of said chamber, and
(σ) means in said dielectric material for the insertion and removal of said liquid suspension, means connected to said chamber for matching the impedance of said chamber when said electric field pulses are applied, and means connected to said matching means and to said electrodes for applying said electric field pulses, said applying means comprising:
(a) means for generating a high voltage, high current rectangular wave poration charge having a power greater than one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time,
(b) means connected to said generating means for selectively delivering said charge at a high speed rise time of less than 50 nanoseconds to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained,
(c) means connected to said delivering means for producing a pulse signal of variable length, said producing means being capable of high speed shorting of said charge at the end of said variable length thereby terminating the application of said
high voltage, high current charge between said electrodes by providing a high speed fall time to said charge, said variable length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension, the size of said pores being dependent upon (i) the value of said high voltage, (ii) said variable length, and (iii) said speed of rise time and said fall time of said charge across said electrodes.
33. The apparatus of Claim 32 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
34. The apparatus for Claim 32 wherein said maximizing means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes, each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
35. The apparatus of Claim 32 wherein a fluid tight seal exists between each of said electrodes and said dielectric material.
36. The apparatus of Claim 32 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and
5 (b) a charge accumulator connected to said high voltage power supply for generating said high voltage, high current charge.
37. The apparatus of Claim 32 wherein said ID delivering means comprises:
(a) a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the
15 opposing switch electrode being connected to said producing means, and
(b) means for moving said switch electrodes together to cause said
20 charge to jump from said one switch electrode to said opposing switch electrode.
38. The apparatus of Claim 32 wherein said delivering means is selected from the group
25" consisting of a moving spark gap, a gas filled spark gap, a vacuum spark gap, an ignitron, a hot cathode hydrogen thyratron and a cold cathode hydrogen thyratron.
39. An apparatus for producing stable pores 30 in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising:
a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber comprising:
(a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing a homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential,
(b) dielectric material separating said electrodes for forming the sides of said chamber, and (c) means in said dielectric material for the insertion and removal of said liquid suspension, means for generating a high current, high voltage rectangular wave poration charge having a power greater than one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to one of said parallel electrodes, means connected to said high speed switch for moving said switch electrodes together to
cause said charge to jump from said one switch electrode to said opposing switch electrode in less than 50 nanoseconds, and means connected to said opposing 5 electrode of said high speed switch for producing a variable pulse length to said charge commencing with said jump of said charge, said producing means being capable of high speed shorting of said charge at the end of said pulse length thereby terminating said ice charge to said parallel electrodes, said pulse length being sufficient to permit said charge between said parallel electrodes to produce stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the
15 viability of said vesicles.
40. A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method
2Q comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- 25 electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency 3D being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a
preselected high voltage and high current having a power greater than one megawatt across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second field having a rise time less than 50 nanoseconds, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles.
41. An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said electric field, and second means connected to said providing means for generating a second uniform, homogeneous
electric field at a high voltage, high current rectangular wave poration charge rectangular wave poration having a power greater than one megawatt for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said second field having a rise time of less than 50 nanoseconds, said power and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles while maintaining the viability of said vesicles.
42. A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across
said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second pulse having a rise time less than 50 nanoseconds and a power greater than one megawatt, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said cells, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.
43. A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time
interval, said preselected frequency being capable of pairing said vesicles together at locations throughout the chamber, applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, said second pulse having a rise time less than 50 nanoseconds and a power greater than one megawatt, said second uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said cell pairs while maintaining the viability of said cell pairs, and waiting a predetermined period of time to permit the paired cells to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.
45. An apparatus for producing stable pores in and through membrane walls of vesicles during poration or electrofusion contained in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension.
means operative with said chamber for providing a homogeneous and uniform electric field across said chamber containing said liquid suspension so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, means connected to said providing means for generating a high power rectangular wave poration charge of greater than one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than fifty nanoseconds to said providing means so that immediate application of said charge to said producing means is obtained, and means connected to said delivering means for producing a variable pulse length to said charge, said producing means being capable of high speed termination of said charge to said providing means, said pulse length being sufficient to permit said charge to produce said stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles.
47. The apparatus of Claim 45 wherein said delivering means comprises means for adjusting the rise time of said charge.
48. The apparatus of Claim 45 wherein said providing means comprises: means connected to said delivering means for creating magnetic flux proportional to said change, and
means operative on said chamber for holding said chamber near said magnetic flux so that said chamber is oriented in said homogeneous electric field created by said magnetic flux.
49. The apparatus of Claim 45 wherein said delivery means comprises means connected to said chamber for matching the impedance of said chamber with said delivery means.
50. An apparatus for producing stable pores in and through membrane walls of vesicles during poration or electrofusion contained in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means to said chamber for generating a high power rectangular wave poration charge of greater than one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than fifty nanoseconds to said providing means in said chamber so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, said delivering means being capable of minimizing the inductance of said chamber and matching the impedance of said chamber, and means connected to said delivering means for producing a variable pulse length, said pulse
length being sufficient to permit said charge between said electrodes to produce said stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles, the size of said pores being dependent upon (i) the value of the voltage of said charge, (ii) said pulse length, and (iii) said high speed of said rise time of said charge across said providing means.
51. The apparatus of Claim 50 wherein said chamber is of cylindrical construction and wherein said electrodes are circular.
52. The apparatus of Claim 51 wherein said inductance minimizing means comprises an outer conductive cylinder around said chamber, said outer conductive cylinder being connected to one of said electrodes, each of said electrodes being coaxial to said outer conductive cylinder, said outer conductive cylinder being coaxially separated from said electrodes by said dielectric material.
53. The apparatus of Claim 50 wherein said generating means comprises:
(a) a high voltage power supply, said high voltage power supply being capable of being set to said predetermined value, and
(b) a charge accumulator connected to said high voltage power supply for generating said high voltage, high current charge.
54. A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- } electrolytic fluids, applying a first uniform, homogeneous electric field from a random function generator at least one preselected frequency across said chamber fox a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said first electric field, and applying a second uniform, homogeneous electric field rectangular poration pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, , said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles.
55. An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically
aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of aligning said vesicles along either their major or minor axis, and second means connected to said providing means for generating a second uniform, homogeneous rectangular wave electric field pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said predetermined value and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles while maintaining the viability of said vesicles.
56. A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a
liquid suspension, said method comprising the steps of in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis, and applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and
annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.
57. A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together, applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with a rise time less than 50 nanoseconds across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said cell pairs while maintaining the viability of said cell pairs, and waiting a predetermined period of time to permit the paired cells to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.
vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non- electrolytic fluids, applying a uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said uniform, homogeneous electric field pulse, and unloading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension.
60. A method for loading substances into vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: placing more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the
group consisting of electrolytic and non- electrolytic fluids, applying a uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with a rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.
61. Process for porating cells for loading and unloading substances to and from the cells of any origin, distinguished by the fact that the aforementioned cells are suspended in an appropriate suspension and subjected to a uniform electric field pulse of high intensity greater than one megawatt and fast rise time of less than 50 nanoseconds while maintaining the viability of the cell.
62. Process for loading cells of any origin with substances, distinguished by the fact that the aforementioned cells are suspended in an appropriate suspension and subjected to a uniform electric field pulse of high intensity and fast rise time, that the porated cells are separated from the suspension and placed in a medium carrying the desired substances to be loaded into the porated cells and that the loaded porated cells are then closed while maintaining the viability of the cell.
63. Process for porating ellipsoidal or electrically or dielectrically aspherical cells of any origin, distinguished by the fact that the aforementioned cells are suspended in an appropriate suspension and are then exposed to uniform electric fields at least one suitable frequency to prealign the cells and that the cells are then subjected to a uniform electric field pulse of high intensity and fast rise time while maintaining viability of the cell.