Foreign substance introduction device and method of producing cells with introduced foreign substance

Patent metadata

Field	Value
Publication number	US-10066199-B2
Application number	US-201314650560-A
Country	US
Kind code	B2
Filing date	Dec 12, 2013
Priority date	Dec 12, 2012
Publication date	Sep 4, 2018
Grant date	Sep 4, 2018

Abstract

An exogenous substance transfer apparatus that introduces an exogenous substance into a small amount of cells by electrical action and a method for manufacturing exogenous substance-bearing cells at low cost are provided.
In the introducing an exogenous substance like a gene into a cell, a cell suspension liquid that contains the cell and the exogenous substance is supplied to a container through an opening portion in an amount that is prepared such that a liquid droplet that is formed will not come into contact with a pair of electrodes simultaneously. The liquid droplet is formed in an oil stored in the container, without the cell suspension liquid mixing with the oil. When a direct current voltage is supplied to the electrodes from a power supply, the liquid droplet moves there-between, and the exogenous substance is introduced into the cell within the liquid droplet by electrical action.

First claim

The invention claimed is:

1. An exogenous substance transfer apparatus configured to introduce an exogenous substance into a cell from outside the cell by electrical action, comprising:
at least one storage vessel having an opening through which a substance can be supplied from the outside;
at least one electrode portion including a pair of electrodes, the at least one electrode portion disposed inside the at least one storage vessel, the pair of electrodes being separated from one another extending in a direction that is intersectional to a horizontal plane, each of the pair of the electrodes having an inside portion being disposed inside the at least one storage vessel, the inside portion of each of said pair of electrodes comprising a minimum length extending in a direction that is orthogonal to the horizontal plane being greater than a maximum length of the inside portion extending in a direction that is orthogonal to an opposite side wall of the storage vessel, and the minimum length of the inside portion extending in a direction that is orthogonal to the horizontal plane being greater than a diameter of a liquid droplet of a cell suspension liquid;
an electric field generating portion configured to generate an electric field by applying a direct current voltage to the at least one electrode portion for a specified length of time, and
the electric field generating portion being configured, when applying the direct current voltage to the at least one electrode portion, to cause the liquid droplet to move toward an opposite polarity electrode and to cause the liquid droplet to reverse its direction of movement by causing the liquid droplet to come into contact with the opposite polarity electrode, the opposite polarity electrode being one of the pair of the electrodes whose polarity is an opposite of a polarity of the liquid droplet, the direct current voltage being applied in a state in which the cell suspension liquid has been supplied between the pair of the electrodes in each of the at least one storage vessel storing an insulating liquid to form the liquid droplet in the insulating liquid, the cell suspension liquid containing an exogenous substance and a cell, the cell suspension liquid not being capable of being mixed with the insulating liquid, and the diameter of the liquid droplet being smaller than a gap between the pair of the electrodes.

2. The exogenous substance transfer apparatus according to claim 1 , wherein:
the at least one storage vessel has a bottom face portion;
the pair of the electrodes are separated from the bottom face portion in the direction that is orthogonal to the horizontal plane;
the minimum length of the inside portion extending in a direction that is orthogonal to the horizontal plane being substantially equal to a height of the opposite side wall of the storage vessel, the height of the opposite side wall extending in a direction that is orthogonal to the horizontal plane; and
the liquid droplet is separated from the bottom face of the storage vessel in the state in which the cell suspension liquid has been supplied between the pair of the electrodes.

3. The exogenous substance transfer apparatus according to claim 2 , wherein:
the one of the pair of the electrodes includes a first sheet portion, the first sheet portion being a sheet portion that extends in a direction that is substantially orthogonal to the horizontal plane, the first sheet portion being a sheet portion formed to be curved into a substantially circular arc in which a side that faces the other electrode is dented.

4. The exogenous substance transfer apparatus according to claim 3 , wherein:
the other of the pair of the electrodes includes a second sheet portion, the second sheet portion being a sheet portion that extends in a direction that is substantially orthogonal to the horizontal plane, the second sheet portion being a sheet portion formed to be curved into a substantially circular arc in which a side that faces the one of the electrodes is dented.

5. The exogenous substance transfer apparatus according to claim 1 , wherein:
at least one of the pair of the electrodes includes one of concave portion and convex portion.

6. The exogenous substance transfer apparatus according to claim 1 , wherein:
the gap between the pair of the electrodes is not greater than one centimeter, and
the electric field generating portion is configured to move the liquid droplet that is formed between the pair of the electrodes back and forth between the pair of the electrodes a plurality of times by repeating a cycle in which the electric field generating portion causes the liquid droplet to move toward the opposite polarity electrode and causes the liquid droplet to reverse its direction of movement by causing the liquid droplet to come into contact with the opposite polarity electrode.

7. The exogenous substance transfer apparatus according to claim 1 , wherein the pair of electrodes extends to at least the opening portion.

8. The exogenous substance transfer apparatus according to claim 1 , wherein:
the at least one storage vessel is a plurality of the storage vessels arrayed in parallel, the opening portion being formed in a top face of each one of the plurality of the storage vessels, one of the electrode portion being provided in each one of the plurality of the storage vessels,
each one of the pair of the electrodes extends to at least the opening portion,
the exogenous substance transfer apparatus further comprises:
a first connecting portion that electrically connects to one of the pair of the electrodes in the opening portion of each one of the plurality of the storage vessels; and
a second connecting portion that electrically connects to the other of the pair of the electrodes in the opening portion of each one of the plurality of the storage vessels, and
the electric field generating portion applies the direct current voltage to the one electrode portion in each one of the plurality of the storage vessels through the first connecting portion and the second connecting portion.

9. The exogenous substance transfer apparatus according to claim 1 , wherein:
each one of the at least one storage vessel contains a plurality of the electrode portions, which are disposed with gaps between each of the plurality of the electrode portions,
each one of the pair of the electrodes extends to at least the opening portion,
the exogenous substance transfer apparatus further comprises:
a first connecting portion that electrically connects to one of the pair of the electrodes in the opening portion of each one of the at least one storage vessel; and
a second connecting portion that electrically connects to the other of the pair of the electrodes in the opening portion of each one of the at least one storage vessel, and
the electric field generating portion applies the direct current voltage to the plurality of the electrode portion in each one of the at least one storage vessel through the first connecting portion and the second connecting portion.

10. A method for manufacturing an exogenous substance-bearing cell using the exogenous substance transfer apparatus according to claim 1 , the method comprising:
a first process that, by supplying the cell suspension liquid that contains the cell and at least one type of the exogenous substance to the insulating liquid, forms the liquid droplet in the insulating liquid, the insulating liquid being stored in the storage vessel, and the insulating liquid being not capable of being mixed with mix with the cell suspension liquid; and
a second process that introduces the at least one type of into the cell in the liquid droplet by applying the direct current voltage for

Assignees

• National Univ Corporation Toyohashi Univ Of Technology

Inventors

• Mizuno Akira
• Numano Rika
• Kurita Hirofumi

Classifications

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12M35/02 Primary

  Electrical or electromagnetic means, e.g. for electroporation or for cell fusion · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title