JP2011083215A - Method for inducing gene by gas flow and induction device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which makes it possible to safely induce intracellular induction substances such as DNA, RNA and proteins into a target site in a cell without cumbersome operations and special devices, and a device therefor. <P>SOLUTION: There are disclosed an intracellular induction method for intracellular induction substance which makes it possible to easily, flawlessly, efficiently and intracellularly induce intracellular induction substances such as DNA, RNA and proteins by blowing gas 1 without requiring a specific device, and a spray device for using the intracellular induction method for intracellular induction substance in which a nozzle 2 can be operated by computing control. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method capable of safely and simply introducing intracellular substance introduction into cells, a cell into which an intracellular substance introduced by using the method, and an intracellular introduction apparatus using the method.

Many technological developments have been made on methods and devices for introducing intracellularly introduced substances into cells.
For example, as a method for introducing a substance such as a gene into a cell, a method using a virus or a method using a reagent has been proposed. However, in this method, there are restrictions on the biohazard and place of use of the virus, and since the reagent has a large cytotoxicity and influence on the cell itself, a different method is required for functional analysis.

  There is electroporation as a physical method without such influences, but there are drawbacks in that the operation is complicated and the cell damage is too great. Similarly, the particle gun method is a method in which a substance to be introduced is applied to metal fine particles and driven at a high speed by a gas flow (see, for example, Patent Document 1). Although this method can be used for many cell types, the operation of attaching the substance to be introduced into the microparticles is complicated and requires consumables such as a rupture disk.

  By developing this method, using gas pressure, spraying liquid at high speed (see, for example, Patent Document 2) and electrospray phenomenon, the suspension containing particles is passed through the tip of the capillary. There is a method in which a high voltage is applied and the cells are sprayed (see, for example, Patent Document 3). However, although it has been improved, there are significant cell damage and operational complexity. Moreover, since fine particles fly, there exists a danger that a user may inhale the metal as an aerosol. Furthermore, since the substance to be introduced into cells is made into an aerosol, there is a risk of accidental injection and inhalation to the user.

  As a method free from such a risk of misfiring or inhalation, a method of electrospraying a liquid that does not contain a substance to be introduced into cells has been proposed (see, for example, Patent Document 4). Although this method is simple and has a simple structure, since a liquid is added to the cells, there is a risk that the influence of the liquid will occur on the cells.

  The method using a particle gun has been realized by implanting a substance to be introduced into a cell. However, there is a need for a method that does not require the use of such fine particles and that does not generate an aerosol with a biohazard hazard. There is also a need for a method that does not leave residual fine particles or liquid that has an effect on cells. Furthermore, there is a need for a method that can introduce a gene or the like at a target location for cell function analysis, but this method is difficult. In addition, the device structure is required to be simple and inexpensive.

U.S. Pat. No. 4,945,050 JP 2004-236657 A US Pat. No. 6,093,557 WO2007 / 1322891

  The problem to be solved by the present invention is to solve the above-mentioned problems of the prior art, and to provide a method and apparatus capable of safely and simply introducing intracellular introduction substances such as DNA, RNA, and protein into a targeted place in the cell. That is.

As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have introduced a substance introduced into a cell with a simple apparatus by blowing gas at an appropriate rate in a state where the substance introduced into the cell and the cell coexist. Has been found to be safely and rapidly introduced into cells at a high introduction rate, and the present invention has been completed.

According to the present invention, there is provided a means capable of safely introducing a substance introduced into a cell such as DNA, RNA, and protein into a targeted place in a cell without requiring a complicated operation or a special apparatus, and an apparatus for the same. To do.

The schematic diagram which concerns on this invention. Scanning device. Fluorescence micrograph Scanning device 2 for flowing saturated water vapor Fluorescence micrograph 2

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to a method and an apparatus for introducing an intracellularly introduced substance into a cell. Examples of the substance to be introduced into cells in the present invention include nucleobases such as DNA, RNA, and similar compounds and derivatives thereof. These are provided in the form of a plasmid, phage, virus, viroid, oligo DNA, oligo RNA, or microRNA. The size of the base sequence is not particularly noted. The base may be either double-stranded or single-stranded. Further, nucleic acid analogs having different main chains or nucleic acids with artificial bases may be used. It can also be used when introducing proteins other than genes, proteins such as amyloid and prion, and peptides into cells. Of course, it can also be used when introducing relatively low molecular weight substances such as sugars, lipids, agricultural chemicals, antibacterial agents, metal ions, fluorescent labeling reagents, or isotope labeling reagents into cells.

In this way, substances introduced into cells can be used as such solutions or solids, but in combination therewith liposomes, cationic polymers, and calcium salts that are generally used as reagents for introducing genes into cells are coexistent. It is not a problem to use. These substances are provided in a solid or solution, in the kendak state.

There are no particular restrictions on the cells, tissues, and living bodies that are the subject of the present invention, such as animals, plants, and microorganisms. In particular, cell types that can be used effectively are animal cells, and both adherent cells and suspension cells can be used.
In the present invention, the intracellularly introduced substance can be introduced into the cell by coexisting the cell and the intracellularly introduced substance and blowing gas.

  As schematically shown in FIG. 1, gas 1 is ejected from nozzle 2, cells and an intracellular introduction substance are made to coexist in a container such as petri dish 3, and gas is blown there to introduce the cells into the cell. Substances are introduced.

  In the present invention, it is necessary that the gas to be blown is in the range of 10 to 3000 m / s, more preferably a speed of 30 to 1000 m / s is recommended. More preferably, a speed of 100 to 400 m / s is recommended.

Regarding the type of gas, it is preferable that the gas does not have great toxicity to the human body, and in particular, it may be nitrogen, oxygen, carbon dioxide, argon, neon, helium, water vapor, dimethyl ether, chlorofluorocarbon, petroleum gas alone or as a mixture. preferable. In particular, the mixture is important because air, which is a mixture of nitrogen and oxygen, and those added with carbon dioxide, and those added with water vapor, are the environment in which actual cells grow. In particular, it is important for water vapor to be contained in the gas until it is saturated to prevent the cells from drying and to increase the survival rate.

The gas can be continuously blown over the cells, but can also be blown temporarily or in pulses.

The volume of gas used is derived from the flow rate and time and is not particularly limited. The flow rate may be an amount necessary to produce the above wind speed.

The gas blowing time is not particularly limited, but is preferably from 0.01 s to 30 minutes. If it is longer than this, it is not preferable because most of the cells are killed and the throughput of the treatment is not increased.

The gas may be supplied from a cylinder or a pump, and there is no restriction on the supply.

The operating temperature of the gas is not particularly limited as long as the cells do not die, but it is generally -80 to 100 ° C, particularly preferably -20 to 50 ° C.

  Next, as a representative method, introduction of a gene into a cell will be described according to an operation procedure. The cells remove the medium and expose the cells. A solution containing an intracellular transgene is placed in contact with the cells. Next, gas is blown, and the introduction of the intracellular transgene into the cell is completed. After completion, the medium is added and the cells subjected to the gene introduction treatment are cultured.

The structure of the device should satisfy the above conditions. The simplest structure is a liquefied gas spray can.
Mechanization is effective to promote uniformity and automation. It can be uniformly introduced by moving the gas ejection nozzle. Furthermore, it becomes possible to enter the target position by controlling the moving speed, the shape of the nozzle, and the gas speed.

As a specific automated device structure, it is desirable that gas is supplied from a cylinder or a pump, the pressure is made constant through a regulator, and then adjusted so as to have a constant gas velocity through a flow meter. This gas is blown onto the cells through a nozzle. This nozzle may be blown against a petri dish or a plate with many holes by computer control.
Since the inner diameter of the nozzle is related to the wind speed, it may be determined to be within the range of the present invention. However, in actual use, the inner diameter is 0.01 to 90 mm. Is difficult to use due to excessive gas flow.

  The reason why spraying the gas of the present invention is effective for cells is expected as follows. The method of the present invention is not expected to work in a mechanism for punching a film such as a particle gun. The expected mechanism is that when the gas hits the cell, the cell is compressed vertically, and when it is released, it tries to return to its original size. At this time, the force applied to the cells varies depending on the horizontal and horizontal components. For this reason, a non-uniform state is formed in the membrane, resulting in cracks on the surface of the cells and a temporary increase in substance permeability. It is thought that a hole is formed. The speed defined in the present invention is believed to impart substance permeability to the cell without causing the cell to die.

  When the method of using the gas of the present invention is used, it does not contain an intracellularly introduced substance such as a gene, so that it is difficult to form an aerosol containing the introduced gene and the like, and there is a risk that the experimenter is exposed to mist. Disappear. Also, there is no residue and the after-effect of the treatment with respect to cell culture is reduced. As described above, by using the method and apparatus of the present invention, it is possible to introduce the intracellularly introduced substance into the cells safely and rapidly with a high introduction rate with a simple apparatus.

  Hereinafter, the contents of the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the present invention is not limited to these examples.

<Example 1>
The structure of the apparatus used for the experiment is shown in FIG.
The pressure of the gas is adjusted by a cylinder 4 with a regulator, and the gas is supplied to the nozzle 2 with a constant flow rate of 1/16 inch outer diameter and 0.25 mm inner diameter by a flow meter 5 with a needle valve. The nozzle 2 is connected to the three-axis dispenser robot 7 and can operate in the horizontal direction and the height direction. The stage 6 on which the petri dish 3 rides operates in the vertical direction. The apparatus was used under clean air environment 8 that passed through a hepa filter.
Chinese hamster ovary cells (CHO cells), which are adherent cells, were used.
The medium used was α-MEM + 10% fetal calf serum. 100 μL of CHO cells at a concentration of 20 × 10 ⁴ cells / mL was added to a 3.5 cm poly-L-lysine-coated polystyrene dish together with 2 mL of medium. Four days later, the medium is removed, and a 4.7 kbp plasmid DNA (10 μg) encoding the green fluorescent protein gene in 100 μL of water is added.
After 5 minutes, the petri dish was scanned with a nozzle from a height of 1 cm at a moving speed of 5 mm / s at intervals of 3 mm using the above apparatus. Air was used and sprayed at a flow rate of 0.4 L / min and a speed of 136 m / s. The medium was immediately added and cultured. One day later, a NIKON microscope TE-2000S was used, the light source was photographed with a high-pressure mercury lamp using a fluorescent filter NIKON GFP block at a magnification of 10 times, and cells expressing the fluorescent protein were observed.

A fluorescence micrograph is shown in FIG. The gene was introduced where the gas hit, and the cells expressing GFP were lined up in a straight line, confirming that they were introduced at a specific location.

<Example 2>
An apparatus similar to that in Example 1 was used. CHO cells were added at a concentration of 1 × 10 ⁶ cells / mL and 100 μL was added to a 3.5 cm poly-L-lysine-coated polystyrene petri dish together with 2 mL of medium, and cultured for 2 days. The gas used was carbon dioxide and sprayed at a speed of 136 m / s while scanning the entire petri dish while repeating the stopping operation at intervals of 0.25 seconds. The introduction efficiency was 5.3%.

<Example 3>
In Example 1, air was changed to a flow rate of 0.05 L / min and a speed of 17 m / s, and the same operation as in Example 1 was performed. When the cells into which the gene was introduced were confirmed with a fluorescence microscope, the introduction efficiency was 0.1% or less.

<Example 4>
In Example 1, the same operation as in Example 1 was performed except that the air was changed to a flow rate of 0.2 L / min and a speed of 68 L / min. When the cells into which the gene was introduced were confirmed with a fluorescence microscope, the introduction efficiency was 0.1% or less.

<Example 5>
In Example 1, the same operation as in Example 1 was performed except that carbon dioxide was changed to a flow rate of 0.05 L / min and 17 L / min. When the cells into which the gene was introduced were confirmed with a fluorescence microscope, the introduction efficiency was 0.1% or less.

<Example 6>
In Example 1, the same operation as in Example 1 was performed except that carbon dioxide was changed to a flow rate of 0.05 L / min and 17 L / min. When the cells into which the gene was introduced were confirmed with a fluorescence microscope, the introduction efficiency was 0.1% or less.

<Example 7>
The apparatus of FIG. 4 in which a gas washing bottle 9 containing 500 ml of water is placed in front of the nozzle of the same apparatus as in Example 1 so that the gas flow coming out of the nozzle becomes saturated water vapor, and the gas is made into saturated water vapor pressure. It was used.
CHO cells 100 × 10 ⁴ cell / ml 20 μl were planted in a petri dish and cultured for 3 days. After removing the medium and washing with PBS, 5 minutes after adding DNA, gas was blown at a speed of 136 m / s. At this time, air having a saturated water vapor pressure was blown from the fixed nozzle to the petri dish at a height of 1 cm for 1 minute at 0.4 L / min.

A fluorescence micrograph is shown in FIG. It was confirmed that 29% of the cells were fluorescent in the place where the gene was most expressed.

<Example 8>
The petri dish 3 was scanned using the apparatus of Example 7 while flowing air at a saturated water vapor pressure and a speed of 136 m / s. The gene transfer efficiency was 7.5%.

<Example 9>
A nozzle 2 having an inner diameter of 1 mm was attached to a spray can containing 90% dimethyl ether and 10% Freon HFC-152a.
The same operation as in Example 7, cells were used. Gas was blown from a height of 2 cm for about 2 seconds. At this time, the gas speed was 380 m / s at 29 L / min. The gene transfer efficiency was 4%.

<Comparative Example 1>
The same operation as in Example 1 was performed. However, no gas was blown, and the medium was added 6 minutes after adding plasmid DNA. As a result of observation with a fluorescence microscope, no cells exhibiting fluorescence were observed, and no cells were introduced with the gene.

<Comparative Example 2>
Gas was sprayed in the same manner as in Example 1 without adding plasmid DNA. As a result, no cells were introduced with the gene.

It is useful in research and development situations in bio-related fields such as medicine, pharmacy and agriculture, or in clinical situations such as gene therapy and targeting therapy for cancer cells.

1: Gas 2: Nozzle 3: Petri dish 4: Cylinder with regulator 5: Flow meter with needle valve 6: Stage 7: 3-axis dispenser robot 8: Clean air environment that passed through hepa filter 9: Air-washing bottle with water

Claims (7)

  A method for introducing an intracellularly introduced substance into a cell, in which gas is blown in a state where the intracellularly introduced substance and the cell coexist.   The method for intracellularly introducing an intracellularly introduced substance according to claim 1, wherein the wind velocity of the gas is 10 to 3000 m / s.   The method of introducing an intracellularly introduced substance into a cell according to claim 1, wherein the wind velocity of the gas is 100 to 400 m / s.   The intracellular introduction substance according to any one of claims 1 to 3, wherein the gas is nitrogen, oxygen, carbon dioxide, argon, neon, helium, water vapor, dimethyl ether, chlorofluorocarbon, or petroleum gas alone or in a mixture. Intracellular introduction method   The method for intracellularly introducing an intracellularly introduced substance according to any one of claims 1 to 4, wherein the intracellularly introduced substance is one or more substances selected from DNA, RNA, and protein.   A spray device for using the method for introducing an intracellularly introduced substance into a cell according to any one of claims 1 to 5. 7. The device according to claim 6, wherein the nozzle can be operated under computer control.