JP5471003B2 - Intracellular introduction method of intracellular introduction substance - Google Patents

Description

  The present invention relates to a method for introducing an intracellularly introduced substance into a cell and an apparatus therefor, and in particular, after bringing a cell into contact with the intracellularly introduced substance, the cell is caused to collide with a droplet containing no intracellularly introduced substance. Relates to a method for introducing an intracellularly introduced substance into a cell. A method that can efficiently introduce DNA, RNA, and protein into cells in an intact form is a very useful technical means in research and application fields related to medical treatment, agriculture, and the like.

  As a typical 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, but there are restrictions on the biohazard and place of use of the virus. Different methods are required for functional analysis due to its large toxicity and its own impact on cells.

  There is electroporation as a method that does not have such an effect, but there is a drawback 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 sprayed on fine particles and shot at a high speed, resulting in large cell damage and complicated operation. By developing this method, gas pressure is used to spray a liquid at high speed (Patent Document 1) and the electrospray phenomenon is used to apply a high voltage to the suspension containing particles when passing through the tip of the capillary. There is a method of applying and spraying on cells (for example, see Patent Document 2). Since these methods use aerosol as a substance to be introduced into cells, there is a risk of accidental injection and inhalation to the user.

  Unlike particle guns, instead of directly shooting the substance to be introduced, the cell and the intracellular transgene are contacted and then electrosprayed onto the cells and the intracellular transgene with a liquid that does not contain the intracellular transgene. In addition, methods and devices for continuously and simply introducing a variety of intracellular transgenes into cells have been developed (see, for example, Patent Documents 3 and 4).

  As described above, the gene introduction method using electrospray has a very excellent feature in that the gene can be introduced into the cell by a physical means of applying a high voltage to the spray liquid and spraying. In order to form a potential difference between the spray nozzle and the cell, a ground must be provided. Further, since charged droplets fly due to an electric field, there is a drawback that it is easily affected by the shape of the spray target. In addition, high voltage power supplies are expensive, and there is a need for a method that does not require them.

JP 2004-236657 A US Pat. No. 6,093,557 International Publication No. 2007/132891 Pamphlet JP 2008-220298 A

  An object of the present invention is to provide a method capable of safely and simply introducing intracellular introduction substances such as DNA, RNA, and protein into cells.

The present inventor examined a method capable of introducing an intracellularly introduced substance into cells at a high introduction rate. As a result, a droplet having a specific size not containing the intracellularly introduced substance was transferred to a specific speed without using an electrospray. It was found that the substance introduced into the cell can be easily introduced into the cell by colliding with the cell, and the present invention shown in the following (1) to (3) has been completed.
(1) In a state where the intracellularly introduced substance is present in the vicinity of the cell, a droplet having a sphere equivalent diameter of 0.001 to 10 mm and containing no intracellularly introduced substance is discharged at a speed of 0.5 without using electrospray. To introduce a substance to be introduced into the cell into the cell at 50 m / s.
(2) The intracellular introduction method of the intracellular introduction substance according to (1), wherein the cells to be used are animal cells.
(3) The method for intracellularly introducing an intracellularly introduced substance according to (1), wherein the intracellularly introduced substance is one or more substances selected from DNA, RNA, and protein.

  By using the method of the present invention, it is possible to introduce an intracellularly introduced substance into cells with a simple and inexpensive apparatus and procedure. Since an electric field is not used when the droplets collide, it is less affected by the shape of a cell aggregate or the like into which the substance to be introduced into the cell is introduced than in the method using electrospray, and more uniform introduction can be performed. Furthermore, since the liquid that collides with the cells as droplets (hereinafter referred to as the collision liquid) can be used with a safe substance that does not contain intracellular introduction substances such as water, it contains the genes to be introduced. Aerosols are less likely to form and there is no risk of the experimenter being exposed to the mist. For this reason, the intracellularly introduced substance can be safely and rapidly introduced into the cell at a high introduction rate. Furthermore, when the method of the present invention is used, since a droplet that does not contain a substance introduced into the cell such as a gene can be used using a safe substance such as water, an aerosol containing the gene to be introduced, etc. Is less likely to form and the risk of the experimenter being exposed to the mist is eliminated. As described above, by using the method of the present invention, it becomes possible to introduce the intracellularly introduced substance into the cell safely and rapidly at a high introduction rate.

Schematic diagram of intracellular introduction method for intracellularly introduced substances Example device (manual spraying) Fluorescence micrograph of Example 1 (photograph of fluorescent cells photographed with an objective lens 10 times using a NIKON microscope TE-2000S, a light source with a high-pressure mercury lamp and a fluorescence filter NIKON GFP block) Example device (droplets collide by gravity drop)

  Hereinafter, embodiments of the present invention will be described in detail.

  The present invention relates to a method for introducing an intracellularly introduced substance, such as a gene, into a cell. That is, the intracellularly introduced substance can be introduced into the cell by causing the intracellular substance to be present in the vicinity of the cell and causing a droplet not containing the intracellularly introduced substance to collide with the cell (FIG. 1).

  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 limited. 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.

  In the present invention, the cell into which the intracellular substance is introduced is not particularly limited as long as it can collide with a droplet such as an isolated cell, a cell existing on the surface of a tissue, a cell existing on the surface of a living body, and the like. There are no particular restrictions on the origin of cells such as plants and microorganisms. In particular, cell types that can effectively introduce intracellular substances are animal cells, which may be adherent cells or suspension cells.

  In the present invention, with respect to the size of a droplet that collides with a cell, the sphere equivalent diameter needs to be 0.001 mm or more, and preferably 0.01 mm or more. The equivalent sphere diameter is usually 10 mm or less, but preferably 5 mm or less. Furthermore, it is desirable that the sphere equivalent diameter of the droplet is larger than the major axis of the cell. If the droplet is too large, it may lead to cell death, and if it is less than 0.001 mm, the efficiency of introduction decreases. In addition, when a droplet is made to collide using a spray device such as a manual spraying device, the droplet size often has a distribution, and in the present invention, the droplet having the above diameter is 5% or more in volume. It is preferably included. The sphere equivalent diameter of the droplet can be obtained by placing the droplet in silicon oil and measuring the diameter of the spherical droplet.

  The speed of the droplets needs to be 0.5 m / s or more, preferably 2 m / s or more. If the speed is low, the efficiency of introduction is significantly reduced. The speed of the droplet is usually 50 m / s or less, preferably 20 m / s. If the speed is too high, cells may die, which is not preferable. The velocity also has a distribution, but if it is contained as a volume of 5% or more like the diameter, it is considered to work effectively.

  The collision liquid does not contain an intracellular introduction substance. Furthermore, it is necessary to show no toxicity because it is used for cells. Therefore, it is preferably water or an aqueous solution. In the case of an aqueous solution, the components that can be used are usually those used as a medium, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate and potassium phosphate, organic acid salts such as sodium acetate and sodium ascorbate Sugars such as sucrose, sorbitol, trehalose, glucose and fructose, water-soluble polymers such as polyethylene glycol, and nutritional components such as fetal bovine serum and amino acids can be used.

  Although there is no restriction | limiting in particular regarding the pH of a collision liquid, pH 4-pH11 considered that the inhibitory effect with respect to survival and proliferation of a cell stops in a slight range is preferable, and pH 5-pH9 are more preferable.

  In order to cause the droplets to collide, various methods and apparatuses that satisfy the above conditions can be used. For example, a spray device can be used. The simplest structure as a spray device is a manual sprayer. A type of spray that pushes liquid at the nozzle outlet by manual compression is suitable for the present invention because the amount of liquid to be sprayed is small. Particularly preferred is a spray bottle with a single spray amount of 0.1 to 200 μL. Naturally, a pressurized spray can also be used. As another method in which the apparatus has a simple structure, a method using gravity drop can be used, and the speed can be controlled by the height. Further, a piezoelectric element often used in a printer and a heater heating type droplet forming technique can be used as a matter of course. Although a technique for forming droplets using a gas flow can be used as a mist formation technique, the gas flow is not preferable because it has a high risk of drying and blowing off cells.

  In the apparatus used in the present invention, it is preferable to use a material that can be sterilized in order to prevent contamination of microorganisms or the like in the portion that comes into contact with the collision liquid. As the sterilization treatment, there are an autoclave, a heat sterilization, a sterilizing agent, and a radiation treatment, but it is simple and easy to use an autoclave and a sterilizing agent.

  The amount of liquid that collides with cells as droplets may be changed according to the amount and purpose of the cells, and is not particularly limited. As a typical example, when it is desired to uniformly introduce the intracellular introduction substance into the adherent animal cells in the 3.5 cm petri dish, it is preferably sprayed from 10 μl to 2 ml, more preferably from 30 to 200 μl. If the amount to be sprayed is small, it will not be uniform, and if it is too large, there is a risk of causing damage to the cells and scattering of the cells.

  Next, the case where the intracellularly introduced substance is a gene will be described according to the operation procedure as a typical method. The cells remove the medium and expose the cells. An aqueous solution containing a gene to be introduced into the cell is put therein, and the cell is brought into contact with the gene to be introduced into the cell. Next, a collision liquid that does not contain a gene to be introduced into the cell is collided with the cell as a droplet. This completes the introduction of the gene to be introduced into the cell. After completion, the medium is added and the cells subjected to the gene introduction treatment are cultured.

  The reason why the method of the present invention is effective is predicted as follows. The size of the droplet used in the method of the present invention is preferably larger than the size of the cell, and is not a mechanism that has hitherto been considered, such as a particle gun, a particle smaller than a cell punching out the film, but a large droplet. It is thought that the cell is compressed by hitting the cell, and a temporary hole that increases the substance permeability is formed by releasing the cell. 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 the present invention is used, it is possible to use a collision liquid that is composed only of a safe substance such as water and does not contain an intracellular introduction substance such as a gene, so that an aerosol containing a gene to be introduced is formed. It is difficult to do so and there is no risk of the experimenter being exposed to the mist. As described above, by using the method of the present invention, it becomes possible to introduce the intracellularly introduced substance into the cell safely and rapidly at a high introduction rate.

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

Method for measuring the equivalent diameter of a sphere of a droplet The measurement of the equivalent diameter of a sphere of a droplet is performed by putting silicon oil in a petri dish and causing the droplet to collide with the silicon oil surface under the same conditions as when the droplet collides with cells Droplets made spherical in silicon oil were made by comparing with the scale with the naked eye or microscope from above.

Method for Measuring Droplet Speed Droplet speed is measured using a high-speed camera (High-speed EXILIM FH-20 manufactured by Casio Computer Co., Ltd.) (1000 sheets / Second).

Method of measuring introduction efficiency Introduction efficiency, that is, the efficiency with which a gene encoding a green fluorescent protein is introduced was measured using a Nikon microscope TE-2000S, a high-pressure mercury lamp as the light source, and a fluorescent filter Nikon GFP block. Cells were photographed with a lens 10 times, the total number of photographed cells and the number of cells emitting fluorescence were counted, and the ratio of cells emitting fluorescence relative to the total number of cells was defined as introduction efficiency.

Example 1
Experimental apparatus The structure of the spray apparatus used in the experiment is shown in FIG.

  The collision liquid 1 enters a tank 2 having a capacity of 3 ml and is sucked up by a plastic tube 3. The head part from which the liquid droplets are ejected has a push-type spray spray structure 4. When the head part is pushed by hand, the liquid droplets are ejected from the nozzle 5. The amount of liquid discharged as a droplet each time the head part was pressed was 50-60 μl. The average diameter of the droplets was 0.11 mm, the minimum value was 0.006 mm, and the maximum value was 1.025 mm. In addition, in this spray apparatus, the speed of the liquid droplet was 5-10 m / s from 2 cm to 12 cm in height.

  Chinese hamster ovary cells (CHO cells), which are adherent cells, were used as the cells into which the intracellular introduction substance was introduced. The size of the cell was 0.01 to 0.03 mm in major axis.

As a medium, α-MEM + 10% fetal calf serum was used. 100 μL of CHO cells at a concentration of 20 × 10 ⁴ cells / mL was added to a 3.5 cm polystyrene dish coated with poly-L-lysine together with 2 mL of medium. Four days later, the medium was removed, and a plasmid DNA (10 μg) having a size of 4.7 kbp encoding a green fluorescent protein gene dissolved in 100 μL of water was added.

After 5 minutes, the droplets were allowed to collide with the cells twice from a height of 2 cm using the device described above. The composition of the collision solution is a mixture of _{KH 2 PO 4 1.05g / L,} NaCl 45g / L, NaH 2 PO 4 · 7H 2 O 3.63g / L.

  Immediately after the droplets collided, the medium was added and cultured. One day later, the introduction efficiency was measured and found to be 12%. A fluorescence micrograph is shown in FIG.

Example 2-4
The same operation as in Example 1 was performed. What changed was the height, the number of times the spray was pressed, and the composition of the collision liquid. The results are shown in Table 1.

Example 5-10
In place of the spray apparatus of Example 1, an apparatus having the structure shown in FIG. 2 but having a tank volume of 30 ml and a volume of liquid discharged at a time of 130 to 150 μl was used. The average diameter of the droplets was 0.09 mm, the minimum value was 0.01 mm, and the maximum value was 0.86 mm. In this apparatus, the speed of the droplet is 5-30 m / s from 2 cm to 12 cm in height.
The results are shown in Table 2.

Example 11
In the experiment, the apparatus shown in FIG. 4 was used. A stainless 28G syringe needle 6 is connected to a 10 ml syringe 8 by a Teflon (registered trademark) tube 7. This syringe supplies a liquid to the syringe needle by a syringe pump 9 at a supply rate of 12 ml / h. The needle is placed at a height of 1.5 m from the petri dish 11 containing the cells and the substance introduced into the cell by the platform 10.

The medium used was α-MEM + 10% fetal calf serum. CHO cells (major axis 0.01-0.03 mm) were added at a concentration of 100 × 10 ⁴ cells / mL to 100 μL of poly-L-lysine-coated 3.5 cm polystyrene dishes together with 2 mL of medium. Two days later, the medium was removed, and a plasmid DNA (10 μg) having a size of 4.7 kbp encoding a green fluorescent protein gene dissolved in 100 μL of water was added.

After 5 minutes, using the above apparatus, the droplets were allowed to collide with the cells for 30 seconds while moving so that the droplets to be dropped uniformly applied to the entire petri dish. The composition of the collision solution was a mixture of _{KH 2 PO 4 0.63g / L,} NaCl 27g / L, NaH 2 PO 4 · 7H 2 O 2.385g / L. The droplet size was 5 mm and the velocity was 3.8 m / s. When the culture was performed in the same manner as in Example 1 after colliding the droplets, the introduction efficiency was 10%.

Comparative Example 1
Example 1 except that the colliding liquid is collided with the cell using the spray device in Example 1, except that the pipette (Pipetman P-200 manufactured by Gilson) is changed to an operation of dropping 100 μL from 1-2 cm in height. The experiment was conducted in the same manner as above. The introduction efficiency was 0%.

Comparative Example 2
The procedure was the same as Example 1 except that plasmid DNA was not added. As a result, the introduction efficiency was 0%.

Comparative Example 3
The same operation as in Example 11 was performed except that the height of the needle from the petri dish was changed to 2 cm. The droplet size at this time was 5 mm, and the speed was 0.44 m / s. As a result, the introduction efficiency was 0%.

  1 is a collision liquid, 2 is a tank, 3 is a tube, 4 is a head, 5 is a nozzle, 6 is a syringe needle, 7 is a Teflon (registered trademark) tube, 8 is a syringe, 9 is a syringe pump, 10 is a table, 11 Is a petri dish.

Claims (2)

The sphere equivalent diameter is 0.001 to 10 mm in a state where the intracellularly introduced substance, which is one or more substances selected from DNA, RNA and protein, is in contact with the isolated animal cell
By and the droplets do not contain intracellular introduction substance, without using electrospray, from the speed 0.5 intracellular introduction substance characterized by impinging on the isolated animal cells in 50 m / s single How to introduce into detached cells. The method for introduction into an isolated cell according to claim 1, wherein the aqueous solution has a pH of 4 to 11.