JP5111367B2 - Intracellular introduction of substances - Google Patents

Description

The present invention relates to a method capable of continuously and conveniently introducing an intracellularly introduced substance into cells, a cell into which an intracellularly introduced substance obtained by using the method is introduced, and an intracellularly introduced substance using the method. The introduction device In more detail, when the intracellularly introduced substance is taken into the cell, droplets are generated by electrospraying a liquid that does not contain the intracellularly introduced substance, and the droplets are formed in the presence of the intracellularly introduced substance. Or after the droplets are sprayed onto the cells, the intracellular introduction method of the substance to be brought into the cell by bringing the intracellular introduction substance into contact with the cells for the first time, and the intracellular obtained by using the method The present invention relates to a cell into which a substance to be introduced is incorporated, and a device for introducing a substance into the cell using the method.
A method for introducing a gene such as DNA or RNA into a cell, a protein such as an enzyme or an antibody, or a chemical substance such as a pharmaceutical, and a cell obtained by the method and a device using the method are used in medicine, pharmacy, agriculture. This is a useful tool in research and development situations in bio-related fields such as gene therapy and clinical situations such as targeting therapy for cancer cells.

Many methods and apparatuses have been developed for use in the operation of introducing an intracellular introduction substance into a cell, particularly the gene recombination operation of introducing a gene such as DNA or RNA into the cell.
For example, competent cells are often used in bacteria and yeasts having cell walls containing peptide glucan, cellulose and the like (see, for example, Non-Patent Document 1). This method is a method in which a cell is changed so that the substance is easily taken up, and then a heat shock or the like is given to the cell to take up the substance. However, in this method, it is necessary to strictly control the culture conditions of the cells to be used, and the problem is that the cells must be treated with a freezing treatment or a solution containing metal ions without being lethal. It has a point (for example, refer nonpatent literatures 2 and 3).
Animal cells usually have a slower growth rate than microorganisms such as bacteria, and are greatly different in both cell structure and cell membrane structure. For this reason, it is said that animal cells are generally difficult to introduce substances into cells as compared to microorganisms, and furthermore, very delicate handling is required. Therefore, it is difficult to introduce intracellular introduction substances, for example, genes such as DNA and RNA into animal cells.

Several methods for animal cells have been proposed. For example, there is a calcium phosphate method (see, for example, Non-Patent Document 4). In this method, calcium phosphate particles containing DNA are produced and incorporated by endocytosis. However, this method has the disadvantages of complicated operation and low introduction efficiency. As a method for improving this, a method of incorporating a substance into cells using a reagent such as a cationic polymer or a liposome has been proposed (see, for example, Non-Patent Documents 5 and 6). However, this method has a problem that a very complicated operation is required when the reagent and the cell are mixed, and the reagent to be used is expensive. In addition, these reagents may be lethal to cells depending on the dose, and the concentration range that can be used is limited.
In addition to these methods, there is a method of introducing a substance into cells using a virus (for example, see Non-Patent Document 7). However, when a virus is used, a complicated purification operation must be taken, and there is a risk of introducing a substance into cells or tissues that are not intended because it is a virus. Moreover, since it is a virus, it is necessary to consider the risk of biohazard.

  Electroporation is well known as a method for incorporating intracellularly introduced substances into cells (see, for example, Patent Document 1). In this method, a high voltage is applied in a pulsed manner to a solution in which a gene and cells are suspended, and the gene contained in the solution is taken into the cell. This method has a wide range of application and high gene transfer efficiency. However, since there is a proportional relationship between the gene transfer efficiency and the cell lethality, it cannot be introduced regardless of cell lethality. That is, if the pulse conditions are inappropriate, not only is the target substance difficult to be introduced into the cell, but there is a drawback that the cell dies. In order to prevent discharge into the solution called towing, the electrical conductivity of the cell suspension must be lowered when a voltage is applied. Therefore, an operation for separating and washing cells using centrifugation or the like is necessary, and the cells may die during the operation.

  Further, there is a particle gun as a method for putting a substance into a cell (for example, see Patent Document 2). This method is a method of implanting fine particles such as gold or tungsten with a gene attached thereto into cells. By carrying the intracellularly introduced substance on the fine particles and increasing the overall mass, the cells are made to collide with the cells with high energy and pass through the cell membrane. This method shows high introduction efficiency for cells hit by fine particles, but when there are many cells, the fine particles do not hit uniformly, and the introduction efficiency as a whole is difficult to increase. In addition, this method requires an operation for attaching the gene to fine particles and the like, and has the disadvantage that consumables such as carrier fine particles and a rupture disk for releasing the pressure are expensive. Furthermore, it is necessary to use plasma explosion, explosives, and cylinder gas to inject fine particles, which makes the device large, the cells scatter due to the momentum of the gas, and the particles remain in the cells, causing damage to the cells. It tends to occur, and there are drawbacks such as accumulation of particles in cells when it is repeatedly driven. Particularly in gene therapy, it is not preferable that microparticles remain and accumulate in cells because the possibility of cell death increases. In addition, since the size of the fine particles used is 0.5 to 3 μm, it is not a problem in the case of relatively large cells such as plants and animals, such as 10 to 100 μm. However, for example, about 1 μm like bacteria. For very small cells, it is very difficult to apply.

There is an electrospray method as a method of spraying a liquid at a high speed. In the electrospray method, a high voltage is applied to the spray tube to collect charges at the tip of the tube, and liquid is passed through the tip of the tube where the charge is collected, thereby charging the liquid with fine droplets. It is a method of spraying at high speed on the target. In addition, this method is a method especially used as an ionization method for mass spectrometry (see, for example, Non-Patent Documents 8 and 9).
As one of the particle guns using this electrospray, there is a method in which a high voltage is applied to a suspension containing particles through the tip of a capillary to spray the cells. (For example, refer to Patent Document 3). This method is a method of accelerating the particles with intracellular introduction substance or the mixture containing intracellular introduction substance and particles by electrospray. In any case, this method uses a suspension containing particles. For this reason, there is a drawback in that the capillary tip is clogged with particles. In addition, there is a drawback that solids remain in the cells as in the case of a normal particle gun. Furthermore, there is no particular problem when introducing the same type of intracellularly introduced substance, but when changing the intracellularly introduced substance, it is necessary to carry out a loading operation for each intracellularly introduced substance. Moreover, it is necessary to wash and replace the liquid flow system of the suspension each time, and the problem of requiring complicated operation and time cannot be solved. Moreover, since the suspension containing the substance to be introduced is sprayed, the inside of the apparatus may be contaminated. In addition, as the dissolved mass contained in the spray solution increases, the risk of spark discharge between the tube and the cells increases.
Because of the circumstances related to the background art, it is not necessary to support the intracellularly introduced substance on a carrier such as particles, and the intracellularly introduced substance can be simply and continuously introduced into the cell as it is, It has been desired to provide a method and apparatus capable of continuously introducing various types of intracellular introduction substances into various types of cells in a short time.

US Pat. No. 4,945,050 Japanese Patent No. 2606856 US Pat. No. 6,093,557 H. Inoue, H. Nojima, H. Okayama, Gene, 1990, vol. 96 (1), p23-28. J. Haensler, F. C. Szoka Jr, Bioconjugate Chem., 1993, vol. 4, p374-379. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, 1147766570484_0. '); 1987 vol. 84 (21), p7413-7. F. L. Graham, A. J. Van Der Eb, Virology, 1973, vol. 52, p446-467. J. Haensler, F. C. Szoka Jr, Bioconjugate Chem., 1993, vol. 4, p374-379. P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, 1147765309078_0. '); 1987 vol. 84 (21), p7413-7. D. Yu, T. Shioda, A. Kato, M. K. Hasan, Y. Sakai, Y. Nagai, 1147765309078_1. '); 1997 vol. 2 (7), p457-66 J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Science, 1989, vol.246, p64-71. Z. Takats, J. M. Wiseman, B. Gologan, R. G. Cooks, Science, 2004, vol. 306, p471-473.

  The problem of the present invention is that it is not necessary to perform complicated loading operations or mixing operations with particles when introducing intracellularly introduced substances, for example, high molecular weight physiologically active substances such as genes such as DNA and RNA into cells. In addition, there is no need to wash and replace the liquid transfer system each time the introduced substance is changed, and the substance can be continuously and easily introduced into the cell. It is an object of the present invention to provide a method capable of easily introducing a substance to be introduced, a cell obtained by using this method, and a device for introducing a substance into the cell using this method.

As a result of intensive studies to solve the above-mentioned problems, the present inventors surprisingly, when introducing an intracellularly introduced substance into cells, droplets generated by electrospraying a liquid not containing this substance As a result, it was found that various kinds of cell-introducing substances can be efficiently introduced into many kinds of cells by bringing them into contact with cells, and the present invention has been completed.
That is, the present invention provides a method for introducing a substance into a cell, which comprises electrospraying a liquid containing no substance introduced into the cell as shown in the following (1) to (9), and the method: And a device for introducing a substance into the cell using the method.
(1) When introducing an intracellularly introduced substance into a cell, a droplet is generated by electrospraying a liquid not containing the substance, and the droplet is brought into contact with the cell. Substance introduction method.
(2) The method for introducing a substance into a cell according to (1), wherein the droplet is brought into contact with the cell in the presence of the cell and the substance introduced into the cell.
(3) The method for introducing a substance into a cell according to (1), wherein the cell and the intracellularly introduced substance are contacted for the first time after the droplet is brought into contact with the cell.
(4) The method for introducing a substance into a cell according to (3), wherein the time from when the droplet is brought into contact with the cell until the cell and the substance introduced into the cell are brought into contact for the first time is within 30 minutes.
(5) The method for introducing a substance into a cell according to (1), wherein the potential difference between the liquid and the cell is 0.1 kV to 100 kV.
(6) The method for introducing a substance into cells according to (1), wherein the liquid to be electrosprayed is water that does not contain an intracellular introduction substance or an aqueous solution that does not contain an intracellular introduction substance.
(7) The method for introducing a substance into a cell according to (1), wherein the substance introduced into the cell is a gene.
(8) A cell into which an intracellularly introduced substance is introduced using the method according to any one of (1) to (7).
(9) An apparatus for introducing a substance into a cell using the method according to any one of (1) to (7).

According to the present invention, it is possible to introduce a substance to be introduced into a cell, particularly a gene, without requiring a complicated pretreatment. That is, the substance to be introduced can be directly introduced into the cell without carrying out a complicated pretreatment operation in which the substance is previously supported on the carrier, and the carrier does not remain in the cell. In addition, since it is not controlled by the carrier particle size, it can be applied to small cells of submicron to several microns such as bacteria.
Furthermore, the apparatus of the present invention does not require expensive metal fine particles or rupturable plates, and the processing cost per operation is reduced. In addition, since it is not necessary to replace the liquid to be sprayed, many kinds of genes can be continuously introduced into many kinds of cells in a short time. As a result, the time required for the operation such as gene recombination can be greatly reduced as compared with the conventional technique, and a large amount of specimen can be processed.

Hereinafter, embodiments of the present invention will be described in detail.
The cell used in the present invention is not particularly limited, and may be any animal, plant, or microbial cell. Further, not only cells but also tissues, organs, and living bodies may be used. Naturally, germ cells such as eggs, sperm, pollen, spores, and seeds can be targeted.
The present invention is particularly effective for animal cells. The specific definition of animal cells is intended for organisms belonging to the animal kingdom and organisms belonging to protozoa, or cells derived from them based on the five-world theory. The organisms belonging to the animal kingdom are multicellular organisms consisting of eukaryotic cells, which are heterotrophic without photosynthesis ability. Sponge animals, coelenterates, protozoa, pelagic animals, annelids, mollusks, arthropods, fur animals, echinoderms, protozoa, and vertebrates belong to this category. Protozoa are unicellular organisms consisting of eukaryotic cells belonging to the protozoan kingdom, and are heterotrophic.
In the case of cells with cell walls such as bacteria, yeast, filamentous fungi, archaebacteria cells, plants, etc., the efficiency of introduction can be improved by using cells with damaged cell walls or by removing cell walls and exposing the plasma membrane. I can expect to go up. Alternatively, a spraying method may be employed in the presence of the substance that creates the above state. Specifically, cells with weakened cell walls such as competent cells and protoplasts, and Ca ions, Li ions, Rb ions, Cs ions, Mg ions, Mn ions, Zn ions, etc. for making these cells, or Cellulase, pectinase, etc. may coexist. In addition, a polymer such as polyethyleneimine, polyallylamine, polyspermine, polylysine and polyethylene glycol, and a surfactant such as liposome and micelle may coexist. As a method of coexistence, a method of adding these substances in advance into the cell solution to be sprayed or mixing them at the spray stage may be appropriately selected.

The present invention is characterized in that, when an intracellularly introduced substance is introduced into a cell, droplets generated by electrospraying a liquid not containing the substance are brought into contact with the cell. More specifically, when the intracellular introduction substance is taken into the cell, a liquid not containing the intracellular introduction substance is electrosprayed into the cell while the cell is in contact with the intracellular introduction substance, or the cell is intracellularly injected. It is characterized in that, after electrospraying a liquid not containing an introduction substance, the cells are brought into contact with the intracellular introduction substance for the first time so that the cells introduce the intracellular introduction substance.
In the electrospray as used in the present invention, a liquid passes through a thin tip of a tube applied with a high voltage to form liquid droplets, and further, liquid fine particles refined by repulsion of electric charges on the surface of the liquid droplets are targeted at high speed. To be injected into cells. FIG. 1 shows a case where, as one embodiment of the present invention, a liquid that does not contain an intracellular introduction substance is electrosprayed on the cell while the cell and the intracellular introduction substance are in contact with each other.
In FIG. 1, a cell (Cell) and an intracellularly introduced substance (Material A) coexist. Here, a liquid (Material B) containing no intracellular introduction substance is electrosprayed. Thereby, a cell (Material A in Cell) into which a cell introduction substance is introduced is obtained. The substance is introduced into the cell by electrospraying a liquid that does not contain the intracellular introduction substance in the presence of the cell and the intracellular introduction substance. That is, the liquid is not intended to be directly introduced into the cell, but is used to allow the intracellularly introduced substance to be taken into the cell.

Next, FIG. 2 shows another case in which an intracellularly introduced substance is taken into a cell by bringing the cell into contact with the intracellularly introduced substance only after the cell is electrosprayed with a liquid not containing the intracellularly introduced substance.
In FIG. 2, a liquid (Material B) that does not contain an intracellular introduction substance is sprayed on the cells (Cell). Thereafter, an intracellular introduction substance is added. Thereby, the intracellularly introduced substance can be introduced into the cell. It is easier to introduce cells by bringing them into contact with the intracellularly introduced substance in a short time after spraying. Although it varies depending on the state, type and spraying conditions of the cells, it is desirable to contact within 30 minutes, preferably within 15 minutes, more preferably within 3 minutes. The intracellularly introduced substance is preferably brought into contact with the cells in the form of a solution, but it is not disturbed to contact the substance in the form of a powder containing the intracellularly introduced substance as long as it does not affect the cells.

  The voltage applied to the tip of the tube may be used by obtaining an optimum value from the distance from the cell, the size of the tube, the flow rate and properties of the electrosprayed liquid, and the like. The voltage is preferably 0.1 kV to 100 kV, more preferably 1 kV to 20 kV, in terms of the potential difference between the liquid at the tip of the tube and the cells. When the voltage is lower than 0.1 kV, the introduction efficiency of the target substance into the cell is lowered. Moreover, even if the voltage is higher than 100 kV, a large difference is not observed in the efficiency of introducing the substance into the cell, and conversely, the risk of causing the cell to die increases, which is not preferable.

  The inner diameter of the tube tip is preferably 0.01 μm to 10 mm, more preferably 1 μm to 5 mm. When the tube tip is smaller than 0.01 μm, the flow path becomes too narrow, and an excessive pressure is required for liquid passage. On the other hand, when the inner diameter is larger than 10 mm, the liquid to be sprayed may not form droplets and may not become electrospray.

  Electrospraying is preferably performed at a distance between the tube tip and the cell of 0.1 μm to 2000 mm. If the distance is shorter than this, the dielectric breakdown is likely to occur, and if the distance is longer than this, the voltage to be applied increases, which is not realistic. More preferably, it is 1 μm to 500 mm.

The amount of liquid electrosprayed on the cells may be changed according to the amount, type and shape of the cells. Along with this, the electrospray time changes.
As a specific example, when electrospraying to adherent animal cells in a petri dish having a diameter of 3.5 cm, the amount of liquid to be sprayed is preferably 1 μl to 4 ml, and more preferably 10 μl to 2 ml.
When electrospraying on a colony of microorganisms in a petri dish having a diameter of 6 cm, the amount of liquid to be sprayed is preferably 1 μl to 8 ml, more preferably 10 μl to 4 ml.

  In order to achieve the object of the present invention, it is required that the liquid to be electrosprayed does not contain a substance that adversely affects cells. Such a liquid is preferably water or an aqueous solution. The water may be any of ion exchange water, distilled water, or sterilized water. Further, the aqueous solution may contain a metal ion, an organic solvent, a polymer compound, a culture medium, a buffer substance and the like. As a more detailed example, examples of metal ions contained in water are preferably group 1 to group 15 metal ions, and group 1 to group 3 or group 6 to 12 metal ions in the periodic table. In particular, aqueous solutions of ions of Li, Na, K, Cs, Mg, Ca, La, Cr, Mo, W, Mn, Fe, Co, Ni, Cu, and Zn are often used in biochemical experiments and are easy to use. These metal ions may be provided in any form such as halide, organic acid salt, phosphate, nitrate, sulfate, borate, carbonate, silicate and the like.

  Examples of organic solvents contained in the liquid to be electrosprayed include methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, glycerin, polyethylene glycol, polypropylene glycol, acetone, methyl ethyl ketone, ethyl acetate, dimethyl sulfoxide, sulfolane, ethanolamine , Diethanolamine, triethanolamine, propanolamine, dipropanolamine, tripropanolamine, chloroform, methylene chloride, acetamide, methylacetamide, dimethylacetamide, N-methylpyrrolidone, formamide, methylformamide, dimethylformamide, polyethyleneimine, polyallylamine, Hexane, triacetin or the like can be used.

  Examples of the polymer compound include polyethyleneimine, polyallylamine, polyethylene glycol, polyvinyl alcohol, spermidine, agarose, starch, cellulose, polyvinylpyrrolidone, alginic acid, carrageenan and the like.

  Examples of the medium contained in the liquid to be sprayed include LB medium, Sauton medium, SOB medium, ham medium, Eagle medium, modified Eagle medium, RPMI medium, Fischer medium, MCDB medium, white medium, and Murashige-Skoog medium. In addition to these, amino acid used as a medium component, sucrose, sorbitol, peptone, yeast extract, surfactant, serum and the like may be included.

  As an equilibrium buffer, EDTA aqueous solution, Tris (hydroxymethyl) aminomethane hydrochloride buffer (Tris buffer), Tris-EDTA buffer (TE buffer), phosphate buffer (PBS), Tris-acetic acid-EDTA buffer (TAE buffer), A mixture used in biochemical experiments such as Hank's Balanced Salt Solution (HBSS) and Eagle's Balanced Salt Solution (EBSS) can be used.

  The concentration of the aqueous solution used in the present invention is not particularly limited. However, it is better to adjust if the osmotic pressure of the cells to be used differs greatly. In order to increase the introduction efficiency, it is preferable to coexist a gene introduction reagent such as a liposome or a basic polymer that can be commonly used for target cells.

In the present invention, the substance to be introduced into the cell is not particularly limited, and a gene is mentioned as an example having particularly high utility. Specific examples include DNA or RNA, or an analogous compound or derivative thereof. These are provided in the form of a plasmid, phage, virus, viroid, oligo DNA, oligo RNA, micro RNA and the like. The size of the base sequence is not particularly noted. The base may be either double-stranded or single-stranded.
In the present invention, a substance smaller than a gene can generally be more easily incorporated. Examples thereof include proteins, peptides, sugars, lipids, agricultural chemicals, antibacterial agents, metal ions, fluorescent labeling reagents, isotope labeling reagents and the like.

  The mechanism by which substances are introduced into cells by the method of the present invention is not clear, but is expected as follows. When the liquid becomes electrosprayed, a large amount of fine charged droplets are generated. This is sprayed on the cells and collides. As a result, the electric field becomes imbalanced inside and outside the cell, so that a hole is formed on the cell membrane, and the intracellularly introduced substance existing in the vicinity is taken into the cell through this hole. In addition, it seems that this hole closes with time after electrospraying and is restored in about 30 minutes.

As the atmosphere for spraying the liquid, a gas that does not have a lethal action on cells such as air, nitrogen, carbon dioxide, argon, SF6, and has no danger of explosion can be used. In that sense, air and nitrogen are common and can be used easily.
The pressure is not particularly limited as long as it is a condition that allows the cells to be processed without dying, but is easy to use in the range of 1 pa to 1 Mpa, particularly atmospheric pressure.
In order to prevent contamination such as contamination with other bacteria when spraying, it is very important to carry out aseptic conditions. Specifically, contamination can be prevented by spraying in a case or using a clean booth or the like. Further, according to the present method, the liquid can be sprayed in the direction opposite to the gravity, so that the operation can be performed while the sprayed cell is placed on the upper portion and the entry of the contamination source falling is prevented.
There is no particular limitation on the temperature when electrospraying. It is sufficient that the cell does not die, and room temperature is easy to use.

FIG. 3 shows, as an example, a schematic structure of an apparatus for introducing a substance into a cell using the method for introducing a substance into cells according to the present invention, in which electrospraying is performed in the presence of the cell and the substance introduced into the cell.
The apparatus is composed of a high voltage power supply 1, a tube 2, a liquid (Material B) 3, a cell and an intracellular introduction substance (Cell + Material A) 4. The liquid 3 to which voltage is applied is sprayed on the cells and the intracellularly introduced substance 4 through the tube 2. At this time, a voltage is applied to the liquid 3 by the high voltage generator 1. As a result, the liquid 3 is charged and sprayed from the tube 2 onto the cells and the intracellularly introduced substance 4. More specifically, the high voltage generator 1 for applying a voltage to the liquid 3 is connected to the tube 2 with a cable or the like. Further, the tube is present at a position where it can be sprayed onto the cells and the intracellularly introduced substance 4.
At the tip of the tube to which a voltage is applied by a high voltage generator to create an electrospray state, the liquid is charged into droplets. When a voltage is applied, the repulsion of the surface charge is larger than the surface tension of the droplet, the droplet is miniaturized, and the charged droplet is sprayed toward the cell at a high speed by the electric field. Note that the electrospray has a device structure that can be appropriately adjusted to suit the purpose because it varies depending on the voltage, the distance from the cell, the tube size, the liquid flow rate, the liquid properties, and the like.
This apparatus can send a liquid quantitatively to a tube by having a pump. When the pump is not provided, the substance is sent to the tube using gravity and pressure, but the liquid can be sent quantitatively more easily by having the pump. The pump can be a tube pump, a plunger pump, a diaphragm pump, a syringe pump, or the like, and may be selected according to the purpose of use.

Further, the structure of the apparatus will be specifically described. The high voltage power supply generates a potential difference between the liquid to be sprayed and the target cell. In the present invention, the amount of current flowing is small and is not particularly limited. A maximum of 1 mA is sufficient. The high voltage power supply and the tube are connected. The connection may be made with a high voltage cable.
The voltage applied to the liquid to be sprayed may be positive or negative. In the present invention, discharging may be accompanied when droplets are sprayed on cells, but it is naturally not preferable to discharge for a long time such that damage occurs in the cells.
The material of the tube is not particularly limited, such as metal, plastic, glass and ceramic. However, when a tube made of a material having no electrical conductivity is used, it is necessary to use a tube having an electrode in contact with the liquid therein or a tube coated with a conductive material on the surface. FIG. 4 schematically shows an example of connection between the tube and the high-voltage power source.
When a conductive material such as a metal shown in A is used for the tube, an electrode can be connected to the tube to charge the liquid coming inside. B is a tube using an insulator such as glass. In this case, a conductive electrode such as a metal is placed inside to charge the liquid. C is a case where a conductive pair is coated inside the electrode, and the inside of the tube is connected to a high voltage power source to charge the liquid.
The shape of the tube tip is not limited to a straight shape, and may be a shape having a taper. Further, the tube may have a double tube structure, and a gas may flow along with the liquid. The tube may be an electrospray tube used in mass spectrometry or the like. Furthermore, a nanospray that can flow a minute amount of liquid can be used as long as the introduction efficiency of the intracellular introduction substance is not lowered.
At least one tube is needed for spraying, and if there are many tubes, the liquid can be sprayed on the surface of cells more widely. In addition, a plurality of specimens can be processed at the same time.

There are no particular limitations on the container in which the cell and the substance to be introduced into the cell are placed so long as the liquid sprayed from the tube is applied, and the same applies to the case of spraying on a cell aggregate such as a colony, tissue or individual. For example, when targeting cells, containers such as petri dishes and test tubes are easy to use.
In order to spray the liquid so that the substance can be efficiently introduced into the cells, the charge may be removed by connecting to the ground so that the potential difference between the cells and the container can be in a preferable range. Specifically, when spraying a cell contained in a petri dish or the like and a substance introduced into the cell, it is preferable that the cell is connected to the ground via a solution or gel that contacts the cells inside the petri dish. There is no problem even if the cells move on the stage so that they can be rotated or selected in order to receive a liquid droplet spray uniformly.
By introducing an electromagnetic optical system into the device, it is possible to control the location of the spray and the spread of the droplet spray area. For example, a droplet can be sprayed more locally by placing a part having a mask, a lens, and a collimator function before the spraying position. Furthermore, it becomes possible to enable the position of the spray, scanning, etc. with a quadrupole lens or the like. As the mask function, plate-like (film) parts that determine the spray range made of organic polymer materials, metals, and ceramics can be used. In addition, a charged metal mask or dielectric mask component can be used for the lens and collimator functions.

In the present invention, even when the type of cell and the substance to be introduced are changed, it is not necessary to change the liquid to be electrosprayed, and the same liquid can be used. Accordingly, it is possible to continuously introduce different substances into different cells for each well using a container such as a 96-well microplate. For example, if different cells and different genes are placed in each well, efficient gene transfer can be performed in a short time.
Specifically, for example, by attaching an electrospray tube to an arm that can move in the XY direction, a large number of samples can be processed continuously. It is also possible to move the cells on the XY stage so that the cells can move. Furthermore, it is also possible to use a combination such that the tube is moved along the X axis and the stage is moved along the Y axis. The moving mechanism may be either an XY type or a scalar type, and the power of this movement may be any of electricity, air, and human power. In addition, it is possible to automate the operation by giving a mechanical position control function to them, thereby saving the labor of processing operations.
FIG. 5 shows a specific moving mechanism. D is a mechanism for moving the stage in the XY direction, E is a mechanism for moving the tube in the XY direction, and F is a mechanism for moving the tube in the X direction and the stage in the Y direction.

In the present invention, it is possible to change the substance to be introduced into a cell or the type of cell to be introduced without changing the liquid to be electrosprayed. Moreover, the change can be performed more efficiently by introducing the mechanism as described above. Further, even if the cells are changed, it is not necessary to change the liquid to be electrosprayed, and efficient introduction can be performed. Furthermore, the consumable of the present invention is mainly a liquid to be electrosprayed and may be water, so that the substance can be introduced at a very low cost.
Moreover, it is possible to easily introduce biochemically important substances such as genes into cells by electrospraying liquids and bringing cells into contact with intracellularly introduced substances without requiring complicated operations. Become. Further, before electrospraying, an operation of removing the medium used for culturing the cells or washing the cells with a washing solution may be added as necessary.

EXAMPLES Hereinafter, although this invention is demonstrated in detail with an Example and a comparative example, this invention is not limited by these examples.
Example I-1
1. FIG. 6 shows an apparatus diagram used for the experimental apparatus experiment.
The high voltage power source 1 is connected to the tube 2 with a high voltage cable. At the same time, it is connected to the ground. The tube 2 is made of stainless steel and is a luer lock type having an outer diameter of 0.36 mm and an inner diameter of 0.18 mm. This is connected to the sample inlet 6. The liquid 3 to be sprayed is introduced from this sample inlet. The pump 5 sends air with a tube pump. The liquid 3 charged with the pressure of the air is pushed to the tube. At this time, air is supplied through a filter 8 made of cellulose acetate. Animal cells and intracellularly introduced substance 4 are contained in a 60 mm polystyrene dish. The petri dish is installed on the jack 10. The jack is connected to the ground. The jack is connected to the inside with a stainless steel ribbon 9 so that the inside of the petri dish is connected to the ground. The spraying part is installed in an aluminum frame acrylic container with an internal size of 272 x 260 x 370 mm. The HEPA clean unit has an air flow rate of 0.5 m ³ / min at an air blowing speed of about 0.25 m / s at the top of the container. 7 is installed. Clean air flows from the top to the bottom, and a blowout opening is provided at the bottom of the container.

2. The above-mentioned apparatus 1 was used as an experimental apparatus for gene introduction into animal cells .
As a substance to be introduced into the cell, a green fluorescent protein (GFP) gene integration plasmid (4.7 kbp) was used. The cells used were fibroblasts (CHO cells) biopsied from the ovaries of Chinese hamsters. CHO cells were plated at a concentration of 1.0 × 10 ^{4 in a} 35 mm dish, and cultured in an α-MEM medium + 10% FBS medium at 37 ° C. for 4 days in a CO 2 incubator. The cell culture medium was removed, and 100 μl of a plasmid concentration of 50 μg / ml was added thereto.
100 μl of water was sprayed from a distance of 2 cm between the tube tip and the jack. At this time, minus 7 kV was applied to the tube. Water was electrosprayed from the tube and sprayed onto the animal cells in the petri dish. After spraying, the medium was placed in a petri dish and further cultured for 4 days.
By fluorescence microscope observation, cells showing green fluorescence were observed, and gene transfer was confirmed. FIG. 7 shows a fluorescence micrograph of CHO cells into which the GFP gene has been introduced.

Example I-2
The experiment was performed in the same manner as in Example I-1, except that 400 μl of medium was further added to the cells and the plasmid solution, and then 100 μl of water was sprayed. At this time, minus 7 kV was applied to the tube. As a result, cells showing green fluorescence were observed with a fluorescence microscope, and gene transfer was confirmed.

Example I-3
The experiment was performed in the same manner as in Example I-1, except that the voltage applied to the tube was minus 10 kV. As a result, cells showing green fluorescence were observed with a fluorescence microscope, and gene transfer was confirmed.

Example I-4
The experiment was performed in the same manner as in Example I-1, except that 100 μl of water was sprayed on the cells before adding the plasmid. At this time, minus 7 kV was applied to the tube, and the plasmid solution was added 1 minute after spraying. As a result, cells showing green fluorescence were observed with a fluorescence microscope, and gene transfer was confirmed.

Example I-5
The experiment was conducted in the same manner as in Example I-1. However, the container used was a 24-hole polystyrene plate with a metal ground attached to each hole. The voltage applied to the tube was minus 10 kV. As a result, in each hole, cells showing green fluorescence were observed with a fluorescence microscope, and gene transfer was confirmed.

Comparative Example I-1
The experiment was performed in the same manner as in Example I-1, except that electrospraying was not performed after plasmid mixing. After culture, the cells were observed with a fluorescence microscope, but no cells showing fluorescence were observed, and no gene was introduced.

Example II-1
1. FIG. 6 shows an apparatus diagram used for the experimental apparatus experiment. The high voltage power source 1 is connected to the tube 2 with a high voltage cable. At the same time, it is connected to the ground. The tube 2 is made of stainless steel and is a luer lock type having an outer diameter of 0.36 mm and an inner diameter of 0.18 mm. This is connected to the sample inlet 6. The liquid 3 to be sprayed is introduced from this sample inlet. The pump 5 sends air with a tube pump. The liquid 3 charged with the pressure of the air is pushed to the tube. At this time, air is supplied through a filter 8 made of cellulose acetate. The high-voltage power supply 1 used is equipped with a short-circuit prevention device and has a current limit of 1 mA. Cells and intracellularly introduced substance 4 are contained in a 60 mm polystyrene dish. The petri dish is installed on the jack 10. The jack is connected to the ground. The jack is connected to the inside with a stainless steel ribbon 9 so that the inside of the petri dish is connected to the ground. The spraying part is installed in an aluminum-frame acrylic container with an internal size of 272 x 260 x 370 mm. The HEPA clean unit 7 has an air flow rate of 0.5 m3 / min at an air blowing speed of about 0.25 m / s at the top of the container. Is installed. Clean air flows from the top to the bottom, and a blowout opening is provided at the bottom of the container.

2. Experiment for introduction of intracellular introduction substance 1.5% agarose-B medium was put in a polystyrene petri dish having a diameter of 6 cm, and Escherichia coli HB101 strain was spread on the whole surface and cultured at 25 ° C. for 2 days to generate colonies on the whole petri dish. .
100 μl of TE buffer solution (105 μg / ml) of plasmid pUC19 having ampicillin resistance gene was added thereto. Next, a stainless steel ribbon was connected to the agar medium in the petri dish so as to be ground, and 100 μl of water was sprayed on the E. coli colonies from a height of 2.5 cm. At this time, minus 7 kV was applied to the tube.
After spraying, the colony cells on the petri dish were scraped off and added to LB medium and centrifuged. The obtained bacterial cell precipitate was seeded in a 1.5% agarose-LB medium containing ampicillin and cultured at 30 ° C. for 2 days. As a result, colonies were formed, indicating that Escherichia coli acquired ampicillin resistance.

Example II-2
Except that the voltage was applied to minus 18 kV at the time of spraying, the same operation as in Example II-1 was carried out. The obtained centrifuged cells were seeded in 1.5% agarose-LB medium containing ampicillin and cultured at 30 ° C. for 2 days. As a result, colonies were formed, indicating that Escherichia coli acquired ampicillin resistance.

Example II-3
Electrophoresis experiments After ampicillin-resistant Escherichia coli obtained in Examples II-1 and II-2 and the HB101 strain used for spraying, a plasmid was taken out with a Qiagen plasmid purification kit, and then placed on an agarose gel for electrophoresis. It was.
As a result, the migration position of the ampicillin resistant strain plasmid obtained in Examples II-1 and II-2 is the same position as that of pUC19 used for gene transfer, and the substance introduced into the cell can be obtained by using the method of the present invention. It was found that it was introduced into the cell. The HB101 strain had no plasmid. (Fig. 8)

Example II-4
The nozzle part of a small dispenser robot equipped with a spray position moving mechanism was changed to a tube for electrospray. This robot has a mechanism that can change the position of the nozzle in the X-axis direction and move the stage in the Y-axis direction, and can spray by controlling the position two-dimensionally. A ground was attached to the chip-like plate having four holes with a metal wire, and spraying was performed. As the spray tube moved, all four holes could be sprayed.

Comparative Example II-1
The procedure was the same as in Example II-1 except that the spray treatment was not performed, and the obtained centrifuged cells were seeded in 1.5% agarose-LB medium containing ampicillin and cultured at 30 ° C. for 2 days. No colony formation was observed.

Comparative Example II-2
The procedure was the same as in Example II-1, except that no voltage was applied during spraying. The resulting centrifuged cells were seeded in 1.5% agarose-LB medium containing ampicillin and cultured at 30 ° C. for 2 days. However, no colony formation was observed.

Example III-1 (Example of Gene Introduction into Chicken Embryo)
1. FIG. 9 shows the electrospray apparatus used in the experimental apparatus experiment. In the apparatus of FIG. 9, the power source 13 uses a dry cell and supplies 3V. This 3V is made +10 kV by the booster circuit 11 and connected to the tank 12. The tank 12 is made of a conductive plastic, but is covered with an insulating plastic except for a portion in contact with a high voltage. The capacity of the tank is about 10 ml. The cylinder 17 is inside, and the shaft 18 is rotated by the motor 19 so that the liquid in the tank is introduced into a stainless steel tube 16 having an inner diameter of 0.1 mm having a luer lock connected to the luer lock joint 15. The liquid spray supply rate from the tube is 120 μl / min. In this device, the booster circuit 11, the tank 12, the power supply 13, the cylinder 17, the shaft 18, and the motor 19 are housed in the device housing 14, and a switch 20 provided on the outer surface of the device housing 14. Can be sprayed.

2. Phosphate buffer solution (PBS) used for gene transfer experiment to chick embryo is NaCl 8g / L, KCl 0.2g / L, NaHPO ₄ · 12H ₂ O 2.9g / L, KH ₂ PO ₄ 0.2g / L A solution having a composition of pH 7.3 was used.
A chicken embryo 1.5 days after the start of incubation (37.8 ° C.) was taken out and placed on an egg white agar mixed medium. 0.1 μl of a solution prepared by dissolving a green fluorescent protein (GEP) gene integration plasmid (4.7 kba) colored with 2% Fast Green in PBS at a concentration of 4.3 μg / μl was dropped onto the target region. After 30 seconds, the PBS was electrosprayed from a height of 10 cm to 15 seconds using the electrospray apparatus while observing under a microscope. The mixture was incubated at 37.8 ° C. and observed with a fluorescence stereomicroscope the next day. The results are shown in FIG. In FIG. 10, GFP expression was observed in the 1 mm region of the total length of 6 to 7 mm, and it was confirmed that the gene was introduced.

Schematic diagram when electrospraying cells in contact with intracellularly introduced substance The schematic diagram in the case of making a cell and an intracellular introduction | transduction substance contact after electrospraying. Schematic diagram of substance introduction device Schematic diagram of tube for substance introduction device Schematic diagram of a device for introducing a substance having a movement mechanism into a cell Device for introducing substances used in experiments into cells Fluorescence micrograph of CHO cells transfected with GFP gene Electrophoretic photograph of ampicillin resistant strain plasmid Schematic diagram of the intracellular introduction device used in Example III-1 Fluorescence stereomicrographs of GFP embryo-introduced chicken embryos

Explanation of symbols

Material A: Intracellularly introduced substance
Material B: Liquid that does not contain intracellular substances
Material A in Cell: Cell into which cell-introducing substance is introduced 1: High voltage power supply
2: Tube 3: Liquid that does not contain intracellular substances (Material B)
4: Cells and intracellular substances (Material A)
5: Tube pump 6: Liquid (Material B) inlet 7: Clean unit
8: Filter made of cellulose acetate 9: Stainless steel ribbon for ground 10: Jack 11: Booster circuit 12: Tank 13: Power supply 14: Device casing 15: Luer lock joint 16: Tube 17: Cylinder 18: Shaft 19: Motor 20: Switch A: When a conductive material is used for the tube B: When an insulator such as glass is used C: When a conductive pair is coated inside the electrode D: The stage has a mechanism for moving in the XY direction Case E: When the tube has a mechanism for moving in the XY direction F: When the tube has a mechanism for moving in the X direction and the stage in the Y direction Lane a: Size marker lane b: pUC19
Lane c: HB101 strain Lane d: Example II-1
Lane e: Example II-2

Claims (4)

A substance applied to cells in vitro, characterized in that when an intracellularly introduced substance is introduced into a cell, a droplet is generated by electrospraying a liquid not containing the substance, and the droplet is brought into contact with the cell. An introduction method ,
Contact the droplet with the cell in the presence of the cell and the intracellularly introduced substance, or contact the cell with the intracellularly introduced substance only after contacting the droplet with the cell.
The method in which the liquid to be electrosprayed is water containing no intracellular introduction substance or an aqueous solution containing no intracellular introduction substance . The method for introducing a substance into a cell according to claim 1 , wherein the time until the cell and the substance introduced into the cell are brought into contact for the first time after the droplet is brought into contact with the cell is 30 minutes or less.   The method for introducing a substance into a cell according to claim 1, wherein the potential difference between the liquid and the cell is 0.1 kV to 100 kV.   The method for introducing a substance into a cell according to claim 1, wherein the substance introduced into the cell is a gene.

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