Methods for the selection, separation and fusion of cells

• US 5,589,047 A
• Filed: 04/05/1994
• Issued: 12/31/1996
• Est. Priority Date: 09/05/1991
• Status: Expired due to Term

• Current Assignee: Apollo Life Sciences Limited
• Original Assignee: Fucell Pty., Limited
• Inventors: Coster, Hans G. L., Ashcroft, Robert G., Mahaworasilpa, Tohsak
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Wong, Edna
• Application Number:
  US08/204,253
• Time in Patent Office: 1,001 Days
• Field of Search: 204/180.1, 204/186, 204/183.1, 204/299 R, 435/173.4, 435/173.5, 435/173.6
• US Class Current: 204/450
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: B03C 5/005 Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
• CPC Class Codes: B03C 5/026 using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: C07D 239/34 One oxygen atom
• CPC Class Codes: C07D 239/36 as doubly bound oxygen atom or as unsubstituted hydroxy radical
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 413/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/02 Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion monoclonal antibodies C07K16/00; apparatus for cell fusion C12M
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Methods are described for the separation of cells of different types using dielectrophoresis; for the electrochemical selection of cells by the dielectrophoretic attraction of ligand bearing cells into contact with an electrode bearing a complimentary ligand; and for the fusion of an individually isolated cell with another cell or a vector using dielectrophoresis to manipulate the cells or the cell and the vector into juxtaposition so that they may be fused.

Claims

1. A method for the selective recovery of cells, vectors, particles or molecules expressing or presenting a defined ligand, comprising the steps of physically or chemically binding to a surface of a solid and immobile target a ligand or ligands complementary to the defined ligand, introducing the target into a suspension or solution of cells, vectors, particles or molecules in an electrically conductive liquid in which some of the cells, vectors, particles or molecules express or present the defined ligand, causing at least some of the cells, vectors, particles or molecules expressing or presenting the defined ligand to migrate into contact with the target and to bind with the complementary ligand or ligands by establishing between a pair of electrodes in the suspension or solution an alternating electric field of such a frequency, intensity and inhomogeneity that at least some of the cells, vectors, particles, ligands or molecules expressing or presenting the defined ligand will move towards the target, and separating the cells, vectors, particles or molecules which have bound to the target due to a dielectrophoretic force induced by the said alternating electric field from at least some of the other cells, vectors, particles or molecules in the suspension or solution.

2. A method as claimed in claim 1 in which the complementary ligand or ligands, is or are, attached to one of the electrodes which itself constitutes the target.

3. A method as claimed in claim 1 in which the at least one of the frequency and intensity and inhomogeneity of the electric field is altered after the cells, vectors, particles or molecules have become bound to the ligand or ligands on the target so as to cause at least some of the bound cells, vectors, particles or molecules to migrate away from the target.

4. A method as claimed in claim 1 in which vibration, stirring or other mechanical means is used to detach at least some of the bound cells, vectors, particles or molecules from the target.

5. A method as claimed in claim 1 in which the cells, vectors, particles or molecules which are bound to the target and the other cells, vectors, particles or molecules in the suspension or solution are separated by physically flushing unbound cells, vectors, particles or molecules away from the target or by removing the target from the vicinity of the unbound cells, vectors, particles or molecules.

6. A method as claimed in claim 1 in which in addition to the alternating electric field applied to the suspension or solution of cells, vectors, particles or molecules between the electrodes a direct current electric field is simultaneously applied to the cells, vectors, particles or molecules therein.

7. A method as claimed in claim 1 in which the ligand bound to the target is a chemical ligand which is not part of a cell or vector.

8. A method as claimed in claim 1 in which the ligand bound to the target is a part of a cell or vector.

9. A method as claimed in claim 1 in which the method is used for the separation of cells.

10. A method as claimed in claim 2 in which the complementary ligand or ligands, is or are bound to an electrode by non-specific binding of the ligand or ligands to the electrode induced by the application of an electric field between the electrodes in the solution or suspension containing the ligand.

11. A method as claimed in claim 10 in which the electric field is an alternating electric field and a direct current electric field in combination so selected that the ligands will be deposited on the other electrode in a variety of orientations.

12. A method for the separation of cells, vectors, particles or molecules in a suspension or solution of cells, vectors, particles or molecules of at least two different species thereof in an electrically conductive liquid, comprising the steps of establishing between two electrodes in the liquid an alternating electric field of such frequency, intensity and inhomogeneity as to selectively cause the cells, vectors, particles or molecules of one specie to migrate through the liquid towards the electrodes due to a dielectrophoretic force induced by the said alternating electric field while simultaneously or sequentially causing the cells vectors, particles or molecules of the other specie to migrate through the liquid away from the electrodes due to a dielectrophoretic force induced by the said alternating electric field and separating the cells, vectors, particles or molecules which have migrated towards the electrodes from the cells, vectors, particles or molecules that have migrated away from them the electrodes.

13. A method as claimed in claim 12 in which the alternating electric field is maintained until some or all of the cells, vectors, particles or molecules of the one specie is in contact with at least one of the electrodes and remaining cells, vectors, particles or molecules are then separated from the cells, vectors, particles, or molecules in contact with the electrode or electrodes.

14. A method as claimed in claim 13 in which a surface of at least one of the electrodes carries a ligand or ligands to which cells, vectors, particles or molecules of the one specie will bind specifically.

15. A method as claimed in claim 13 in which the alternating electric field is such that the cells, vectors, particles or molecules of the one specie is caused to migrate through the liquid towards the electrodes while simultaneously the cells, vectors, particles or molecules of the other specie migrates away from the electrodes.

16. A method as claimed in claim 13 in which the method is applied for the separation of cells of at least two species.

17. A method for fusing a selected first cell with a selected second cell to form a single fusate cell, comprising the steps of:

i) physically or chemically binding at least one first cell to a first of a pair of electrodes between which an alternating electric current of an appropriate frequency, intensity and inhomogenicity subsists such that each first cell is drawn into direct contact with one of the electrodes, the physical or chemical binding being so achieved that when a plurality of fusate cell are formed there is only a monolayer of cells present on said one of the electrodes;

ii) physically manipulating a single second cell into sufficiently close proximity with the first cell or one of the first cells such that the second cell is drawn into juxtaposition with the said first cell by a local dielectrophoretic field of the said first cell induced by the alternating electric current between the electrodes; and

iii) causing the first and second cells to fuse.

18. A method as claimed in claim 17 in which said single second cell is physically or chemically bound to a second of the pair of electrodes and is then brought into proximity with the said first cell by physical manipulation of one of the electrodes.

19. A method as claimed in claim 18 in which the first and second cells are released from at least one of the electrodes before they are fused.

20. A method as claimed in claim 19 in which the first and second cells are released from both of the electrodes before they are fused.

21. A method as claimed in claim 17 in which the juxtaposed cells are fused by a fusagenic agent selected from the group comprising application of an intense electric field, a fusagenic chemical, viral mediation and a combination of at least two of the foregoing.

22. A method as claimed in claim 18 in which said second cell is attached to the second electrode, another second cell is attached to the second cell already attached to the second electrode and this other second cell is released from the second electrode in close proximity to the first cell by changing the alternating electric field so that the other second cell is separated from the second electrode, withdrawing the second electrode from proximity with the other second cell and again changing the alternating electric field so that the other second cell is caused to move into juxtaposition with the first cell.

23. A method as claimed in claim 22 in which the electric field is changed so that the other second cell on the second electrode is repelled from the second cell on the second electrode, without necessarily withdrawing the second electrode, towards the first cell on the first electrode, and then changing the electric field when the other second cell has reached a position between the two electrodes, so that the other second cell is moved into juxtaposition with the first cell.

24. A method as claimed in claim 17 in which one of the first and the second cell is replaced by a vector.