Method of introducing a substance into a cell, genetically modified cell and method of producing the same, and control program for apparatus for introducing a substance into a cell

A method of introducing a substance into a cell, the method comprising:

• a′)
• a′-1) seeding floating cells onto a substrate and layering a gel on the cells;
• a′-2) peeling the gel from the substrate to obtain a gel sheet with the cells transferred to a gel surface;
• a′-3) layering the gel sheet on a gel plate medium so that a cell transfer surface faces up, and setting a hardness of layered gel overall, by a hardness evaluation according to JIS K6503, to a breaking load of 2.0 N to 30 N, Young's modulus of 0.5×105 Pa to 7×105 Pa, or 1 mm pressing stress of 1.0 N to 8 N;
• b) filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolytic solution;
• c) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• d) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• e) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• f) retracting the nanopipette.

A method of producing a genetically modified cell, the method comprising:

• a) seeding floating cells on a gel plate medium having a hardness such that, by a hardness evaluation according to JIS K6503, a breaking load is 2.0 N to 30 N, Young's modulus is 0.5×105 Pa to 7×105 Pa, or 1 mm pressing stress is 1.0 N to 8 N;
• b) filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolytic solution;
• c) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• d) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• e) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• f) retracting the nanopipette.

A method of producing a genetically modified cell, the method comprising:

• a′)
• a′-1) seeding floating cells onto a substrate and layering a gel on the cells;
• a′-2) peeling the gel from the substrate to obtain a gel sheet with the cells transferred to a gel surface;
• a′-3) layering the gel sheet on a gel plate medium so that a cell transfer surface faces up, and setting a hardness of layered gel overall, by a hardness evaluation according to JIS K6503, to a breaking load of 2.0 N to 30 N, Young's modulus of 0.5×105 Pa to 7×105 Pa, or 1 mm pressing stress of 1.0 N to 8 N;
• b) filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolytic solution;
• c) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• d) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• e) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• f) retracting the nanopipette.

A genetically modified cell produced by a method comprising:

• a) seeding floating cells on a gel plate medium having a hardness such that, by a hardness evaluation according to JIS K6503, a breaking load is 2.0 N to 30 N, Young's modulus is 0.5×105 Pa to 7×105 Pa, or 1 mm pressing stress is 1.0 N to 8 N;
• b) filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolytic solution;
• c) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• d) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• e) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• f) retracting the nanopipette.

A genetically modified cell produced by a method comprising:

• a′)
• a′-1) seeding floating cells onto a substrate and layering a gel on the cells;
• a′-2) peeling the gel from the substrate to obtain a gel sheet with the cells transferred to a gel surface;
• a′-3) layering the gel sheet on a gel plate medium so that a cell transfer surface faces up, and setting a hardness of layered gel overall, by a hardness evaluation according to JIS K6503, to a breaking load of 2.0 N to 30 N, Young's modulus of 0.5×105 Pa to 7×105 Pa, or 1 mm pressing stress of 1.0 N to 8 N;
• b) filling the gel plate medium seeded with cells with an electrolytic solution and positioning a nanopipette filled with a substance at a cell-corresponding position in the electrolytic solution;
• c) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• d) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• e) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• f) retracting the nanopipette.

A control program for an apparatus for introducing a substance into a cell, the control program comprising instructions for executing processing comprising:

• a) positioning a nanopipette filled with a substance at a cell-corresponding position in an electrolytic solution in a gel plate medium of a specific hardness, the gel plate medium being seeded with floating cells and filled with the electrolytic solution;
• b) measuring a current between an inside of the nanopipette and the electrolytic solution and moving the nanopipette in a cell direction to a position at which a drop rate from a steady state current is a set current drop rate of 2% or more and 50% or less;
• c) moving the nanopipette in the cell direction by a set penetration distance of 1 μm or more and 50 μm or less to penetrate a cell;
• d) applying a voltage between the inside of the nanopipette and the electrolytic solution to eject the substance into the cell;
• and
• e) retracting the nanopipette.