Controlled electroporation and mass transfer across cell membranes

• US 7,955,827 B2
• Filed: 04/09/2010
• Issued: 06/07/2011
• Est. Priority Date: 07/21/1999
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Huang, Yong, Rubinsky, Boris
• Primary Examiner(s): Tate; Christopher R
• Assistant Examiner(s): Kosar; Aaron J
• Application Number:
  US12/757,595
• Publication Number:
  US 20100196984A1
• Time in Patent Office: 424 Days
• Field of Search: None
• US Class Current: 435/173.6
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10

Abstract

Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought. Electroporation is achieved by applying a voltage between the two chambers, and diffusive transport is achieved either by a difference in solute concentration between the liquids surrounding the cell and the cell interior or by a differential in concentration between the two chambers themselves. Electric current and diffusive transport are restricted to a flow path that passes through the opening.

Claims

1. A method for performing electroporation, comprising:

(a) applying a voltage across a plurality of biological cells;

(b) detecting a ratio of electric current through the cells to voltage across the cells as an indication of a degree of electroporation of the biological cells averaged over the plurality of cells; and

(c) adjusting the magnitude of said applied voltage in accordance with changes in the magnitude of the current-to-voltage ratio to achieve a controlled degree of electroporation.

2. A method in accordance with claim 1 in which step (b) comprises continuously detecting said current-to-voltage ratio as an indication of the onset of electroporation of said biological cell, and step (c) comprises adjusting the duration of said applied voltage in accordance with said current-to-voltage ratio to achieve a controlled degree of electroporation.

3. A method in accordance with claim 1, further comprising:

achieving a controlled averaged degree of electroporation in said plurality of biological cells.

4. A method for performing electroporation in biological tissue in a controlled manner, comprising:

(a) applying a voltage across the biological tissue;

(b) continuously detecting a ratio of electric current through the tissue to voltage across tissue as an indication of a degree of electroporation in said biological tissue; and

(c) adjusting a magnitude of the applied voltage in accordance with changes in a magnitude of the current-to-voltage ratio to achieve a controlled degree of electroporation in said biological tissue.

5. A method for the infusion of a biological cell with a chemical substance by electroporation in a manner that permits detection of the onset of and control of the electroporation, said method comprising:

(a) securing said biological cell in an electrical cell containing a liquid with said chemical substance dissolved therein, said electrical cell containing a barrier to electric current, said barrier arranged such that, when a voltage is imposed across said electrical cell, said barrier restricts electric current flow to a flowpath passing through said biological cell while permitting substantially no electric current to bypass said biological cell;

(b) imposing a voltage across said electrical cell and monitoring the relative values of current passing through said cell and of said imposed voltage as an indication of the occurrence of electroporation in said cell.