Electroporation device

• US 5,232,856 A
• Filed: 07/30/1990
• Issued: 08/03/1993
• Est. Priority Date: 06/25/1990
• Status: Expired due to Fees

• Current Assignee: Kevin L. Firth
• Original Assignee: Kevin L. Firth
• Inventors: Firth, Kevin L.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Trembley, T. A.
• Application Number:
  US07/559,669
• Time in Patent Office: 1,100 Days
• Field of Search: 435/287, 435/172.1, 435/172.2, 435/172.3, 435/173, 435/817, 935/52, 935/85, 204/180.1, 204/180.3, 204/299 R, 204/403
• US Class Current: 435/285.2
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

An electroporation apparatus provides for the electroporation of cells attached to an electrode. The electrode may be transparent to allow cell viewing. If the electrode exhibits a voltage drop a constant electrical field may be obtained over the cell layer by controlling the position or character of the opposed electrode. Alternately, cells may be electroporated under an electrical field which varies in a continuous, geometrically uniform manner over the cell layer.

Claims

1. An electroporation apparatus for electroporating living cells with molecules comprising:

(a) first and second electrodes, respectively having, first and second mutually opposed electrode surfaces, said first and second mutually opposed electrode surfaces being positioned to create an electric field there-between when connected to a source of electrical potential, said first electrode being only partially conductive and exhibiting a voltage drop across its said first electrode surface upon the passage of current therethrough;

(b) an electrolyte positioned between said first and second said electrolyte containing molecules to be deposited within living cells by electroporation and being in contact with said first and second electrode surfaces;

(c) living cells attached to said first electrode surface and being in contact with said electrolyte, and

(d) containment means positioned to contain the electrolyte and living cells between said first and second electrodes,

wherein by reason of the spacing between opposed portions of said first and second mutually opposed electrode surfaces, said second electrode, in combination with said first electrode, provides a local electrical field over said first electrode surface which is of substantially equal strength over said first electrode surface.

2. An apparatus as in claim 1 wherein said first and second mutually opposed electrode surfaces are planar.

3. An apparatus as in claim 2, wherein said second electrode surface is angled with respect to said first electrode surface to create along said first electrode surface said local electrical field of substantially equal strength.

4. An apparatus as in claim 1, 2 or 3 wherein;

(a) said first electrode comprises a non-conductive substrate;

(b) said first electrode surface comprises a partially conductive layer bonded to said non-conductive substrate, and

(c) said non-conductive substrate and partially conductive layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

5. An apparatus as in claim 1, 2 or 3 wherein said first electrode comprises a, partially conductive planar layer deposited on a non-conducting substrate and wherein said containment means comprises an encircling, elevated boundary wall formed on said layer, partially conductive planar delimiting an area on the surface of said partially conductive planar layer for containment of said electrolyte.

6. An apparatus as in claim 5 wherein said non-conducting substrate and partially conductive planar layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

7. An electroporation apparatus for electroporating living cells with molecules comprising:

(a) first and second electrodes, mutually having, first and second mutually opposed electrode surfaces, said first and second mutually opposed electrode surfaces being positioned to create an electric field there-between when connected to a source of electrical potential, said first electrode being only partially conductive and exhibiting a voltage drop across its said first electrode surface upon the passage of current therethrough;

(b) an electrolyte positioned between said first and second electrodes, said electrolyte containing molecules to be deposited within living cells by electroporation and being in contact with said first and second mutually opposed electrode surfaces;

(c) cells attached to said first electrode surface and being in contact with said electrolyte, and

(d) containment means positioned to contain the electrolyte and living cells between the said first and second electrodes,

wherein said second electrode surface is angled with respect to said first electrode surface whereby said second electrode, in combination with said first electrode, provides a local electrical field over said first electrode surface which is of a strength which varies in a continuous, increasing, regular manner when proceeding in a given direction along said first electrode surface.

8. An apparatus as in claim 7 wherein said first and second electrode surfaces are planar.

9. An apparatus as in claim 7 or 8 wherein;

(a) said first electrode comprises a non-conductive substrate;

(b) said first electrode surface comprises partially conductive layer bonded to said non-conductive substrate, and

(c) said non-conductive substrate and partially conductive layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

10. An apparatus as in claim 7 or 8 wherein said first electrode comprises a partially conductive planar layer deposited on a non-conducting substrate and wherein said containment means comprises an encircling, elevated boundary wall formed on said partially conductive planar layer, delimiting an area on the surface of said partially conductive planar layer for containment of said electrolyte.

11. An apparatus as in claim 10 wherein said non-conducting substrate and partially conductive planar layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

12. An electroporation apparatus for electroporating living cells with molecules comprising:

(a) first and second electrodes, respectively having, first and second mutually opposed electrode surfaces, said first and second mutually opposed electrode surfaces being positioned to create an electric field there-between when connected to a source of electrical potential, said first electrode being only partially conductive and exhibiting a voltage drop across its said first electrode surface upon the passage of current therethrough;

(b) an electrolyte positioned between said first and second electrodes, said electrolyte containing molecules to be deposited within living cells by electroporation and being in contact with said first and second mutually opposed electrode surfaces;

(c) living cells attached to said first electrode surface and being in contact with said electrolyte;

(d) containment means positioned to contain the electrolyte and living cells between said first and second electrodes; and

(e) voltage control means for establishing differing voltage potentials at specific locations over said second electrode surface, said voltage control means thereby determining the local strength of said electric field over said first electrode surface according to the pattern of the voltage potential developed at such locations.

13. An electroporation apparatus as in claim 12 wherein said second electrode, in combination with said first electrode, provides a local electrical field over said first electrode surface which is of a substantially equal strength over said first electrode surface.

14. An electroporation apparatus as in claim 12 wherein said second electrode, in combination with said first electrode, provides a local electrical field over said first electrode surface which is of a strength which varies in a continuous, increasing regular manner when proceeding in a given direction along said first electrode surface.

15. An apparatus as in claims 12, 13, or 14 wherein:

(a) said first electrode comprises a non-conductive substrate;

(b) said first electrode surface is provided with a partially conductive layer bonded to said non-conductive substrate; and

(c) said non-conductive substrate and layer partially conductive layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

16. An apparatus as in claim 12, 13 or 14 wherein said first electrode comprises a partially conductive planar layer deposited on a non-conducting substrate and wherein said containment means comprises an encircling, elevated boundary wall formed on said partially conductive planar layer, delimiting an area on the surface of said partially conductive planar layer for containment of said electrolyte.

17. An apparatus as in claim 16 wherein said non-conducting substrate and partially conductive planar layer are sufficient transparent or translucent to allow viewing of the living cells on said exposed surface under a microscope by transmitted light.

18. An electroporation apparatus as in claims 12, 13 or 14 wherein said second electrode comprises segmented portions, each of said segmented portions being separated from adjacent segmented portions by insulation means, and each of said segmented portions being connected to a source of voltage potential through individual resistance means to thereby establish said voltage potential at specific locations over said second electrode surface.

19. An electroporation apparatus for electroporation apparatus living cells with molecules comprising:

(a) first and second electrodes, having respectively having, first and second mutually opposed electrode surfaces, said first and second mutually opposed electrode surfaces being positioned to create an electric field there-between when connected to a source of electrical potential, said first electrode being only partially conductive and exhibiting a voltage drop across its first electrode surface upon the passage of current therethrough;

(b) an electrolyte positioned between said first and second electrodes, said electrolyte containing molecules to be deposited within living cells by electroporation and being in contact with said first and second mutually opposed electrode surfaces;

(c) living cells attached to said first electrode surface and being in contact with said electrolyte, and

(d) containment means positioned to contain the electrolyte and living cells between said first and second electrodes,

wherein said first electrode is provided with a plurality of conductive strips that are distributed over a substantial portion of said first electrode surface whereby said plurality of conductive strips are able to provide current to said first electrode surface with a reduced degree of voltage drop across said first electrode surface over that which would occur in the absence of such plurality of conductive strips.

20. An electroporation apparatus in claim 19 wherein said second electrode, in combination with said first electrode, provides a local electrical field over the electrode surface of said first electrode which is of a substantially equal strength over said first electrode surface.

21. An electroporation apparatus as in claim 19 wherein said second electrode, in combination with said first electrode, provides a local electrical field over the electrode surface of said first electrode which is of a strength which varies in a continuously, increasing regular manner when proceeding in a given direction along the electrode surface of said first electrode.

22. An apparatus as in claim 19, 20 or 21 wherein:

(a) said first electrode comprises a non-conductive substrate;

(b) said first electrode surface is provided with a partially conductive layer bonded to said non-conductive substrate; and

(c) said non-conductive substrate and partially conductive layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.

23. An apparatus as in claims 19, 20 or 21 wherein said first electrode comprises a partially conductive planar layer deposited on a non-conducting substrate and wherein said containment means comprises an encircling, elevated boundary wall formed on said partially conductive planar layer, delimiting an area on the surface of said partially conductive planar layer for containment of said electrolyte.

24. An apparatus as in claim 23 wherein said non-conducting substrate and partially conductive layer are sufficiently transparent or translucent to allow viewing of the living cells on said first electrode surface under a microscope by transmitted light.