Electromicroinjection of particles into living cells

• US 4,784,737 A
• Filed: 04/18/1986
• Issued: 11/15/1988
• Est. Priority Date: 04/18/1986
• Status: Expired due to Fees

• Current Assignee: United States Of America As Represented By The Department Of Energy
• Original Assignee: United States Department of Energy (Government of the United States of America)
• Inventors: Ray, F. Andrew, Galey, William R., Cram, L. Scott
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Hsing, Ben C.
• Application Number:
  US06/853,823
• Time in Patent Office: 942 Days
• Field of Search: 604/20, 604/21, 128/303.18, 128/784, 128/786, 435/296, 435/172.1, 435/172.2, 435/172.3, 204/180.1, 204/299 R, 204/403, 204/164, 204/165
• US Class Current: 435/455
• CPC Class Codes: A61N 1/306 Arrangements where at least part of the apparatus is introduced into the body
• 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: C12N 15/89 using microinjection

Abstract

Method and apparatus for introducing particles into living cells. Fluorescently-stained human chromosomes are introduced into cultured, mitotic Chinese hamster cells using electromicroinjection. The recipient cells frequently survived the physiological perturbation imposed by a successful chromosome injection. Successfully injected recipient cells maintained viability as evidenced by their ability to be expanded.

The technique relies on the surface charge of fluorescently stained chromosomes and their ability to be attracted and repelled to and from the tip of a micropipette. The apparatus includes a micropipette having a tip suitable for piercing the membrane of a target cell and an electrode inserted into the lumen thereof. The target cells and suspended particles are located in an electrically conducted solution, and the lumen of the micropipette is filled with an electrically conducting solution which contacts the electrode located therein. A second electrode is also located in the conducting solution containing the target cells and particles. Voltages applied to the electrode within the micropipette attract the particles to the region of the tip thereof. The particles adhere to the surface of the micropipette with sufficient force that insertion of the micropipette tip and attached particle through the membrane of a target cell will not dislodge the particle. By applying a voltage having the opposite polarity of the attraction voltage, the particles are expelled from the micropipette to which is then withdrawn from the cell body.

Claims

1. An apparatus for introducing individual particles suspended in an electrically conducting aqueous solution into recipient biological cells. the apparatus comprising in combination:

an injection micropipette having an exit orifice size for piercing the membrane of a target recipient cell and an entrance orifice;

an electrically conducting medium located within the lumen of said injection micropipette in contact with the exit orifice thereof;

first electrode means located within the lumen of said injection micropipette, said first electrode means having a first end positioned within said electrically conducting medium contained in the lumen of said injection micropipette, and a second end which emerges from the entrance orifice thereof;

second electrode means located in the electrically conducting aqueous solution containing the recipient biological cells; and

means for applying a first voltage and a subsequent second voltage between the second end of said first electrode means and said second electrode means both when the exit orifice of said injection micropipette is located in the conducting aqueous solution and when the exit orifice of said injection micropipette is located within the body of a recipient cell located in the conducting aqueous solution, the first voltage having a chosen magnitude and polarity for electrostatically attracting at least one particle to the region of the exit orifice of said injection micropipette, and for holding the particle in this location during the process of piercing the membrane of a cell with the exit orifice of said injection micropipette and insertion of the particle into the recipient cell body. and the second voltage having a polarity opposite to that of the first voltage and a chosen magnitude for electrostatically repelling the particle away from the injection micropipette.

2. The apparatus as described in claim 1, wherein the first voltage and the second voltage are pulsed voltages having a duration between about 1 and 300 ms.

3. The apparatus as described in claim 1, wherein the first voltage is a dc voltage and the second voltage is a pulsed voltage.

4. The apparatus as described in claims 1, 2 or 3, wherein the chosen magnitude of the first voltage and the chosen magnitude of the second voltage are between about 5 and 150 V.

5. The apparatus as described in claim 4, wherein said conducting medium comprises material to be injected into the target recipient cell.

6. A method for introducing individual particles suspended in an electrically conducting aqueous solution into recipient biological cells. the method comprising the steps of:

attracting at least one of the individual particles to the tip of a micropipette for piercing the membrane of a target recipient cell and located in the conducting aqueous solution containing the target recipient cells by applying a first voltage between the tip of the micropipette and the conducting aqueous solution;

inserting the micropipette tip into a target recipient cell, the at least one particle remaining attached thereto during said insertion step; and

releasing the at least one particle from the tip of the micropipette by applying a second voltage between the tip of the micropipette and the conducting aqueous solution. the second voltage having a polarity opposite to that of the first voltage.

7. The method as described in claim 6, wherein the first applied voltage is maintained during said insertion step.

8. The method as described in claim 6. wherein the first applied voltage and the second applied voltage are pulsed voltages.

9. An apparatus for introducing individual particles suspended in an electrically conducting aqueous solution into recipient biological cells, the apparatus comprising in combination:

an injection micropipette having an exit orifice size for piercing the membrane of a target recipient cell, the outside surface of said injection micropipette having a conducting material thereon in the region of the exit orifice;

first electrode means for providing electrical contact with the conducting material at a location away from the exit orifice;

second electrode means located in the electrically conducting aqueous solution containing the recipient biological cells; and

means for providing a first voltage and a second voltage between said first electrode means and said second electrode means both when the exit orifice of said injection micropipette is located in the conducting aqueous solution and when the exit orifice of said injection micropipette is located within the body of a recipient cell located in the conducting aqueous solution, the first voltage having a chosen magnitude and polarity for electrostatically attracting at least one particle to the region of the exit orifice of said injection micropipette, and for holding the particle in this location during the process of piercing the membrane of a cell with the exit orifice of said injection micropipette and insertion of the particle into the recipient cell body, and the second voltage having a polarity opposite to that of the first voltage and a chosen magnitude for electrostatically repelling the particle away from the injection micropipette.

10. The apparatus as described in claim 9, wherein said injection micropipette contains material to be injected into the target recipient cell.

11. An apparatus for introducing individual particles suspended in an electrically conducting aqueous solution into recipient biological cells. the apparatus comprising in combination:

a conducting microneedle having a tip size for piercing the membrane of a target recipient cell;

electrode means located in the electrically conducting aqueous solution containing the recipient biological cells; and

means for providing a first voltage and a second voltage between the tip of said conducting microneedle and said electrode means both when the tip of said conducting microneedle is located in the conducting aqueous solution and when the tip of said conducting microneedle is located within the body of a recipient cell located in the conducting aqueous solution, the first voltage having a chosen magnitude and polarity for electrostatically attracting at least one particle to the region of the exit orifice of said injection micropipette, and for holding the particle in this location during the process of piercing the membrane of a cell with the tip of said conducting microneedle and insertion of the particle into the recipient cell body, and the second voltage having a polarity opposite to that of the first voltage and a chosen magnitude for electrostatically repelling the particle away from the conducting microneedle.