Methods for introducing substances into cells

Abstract

To efficiently incorporate a substance to be introduced into a cell when an anionic introduced substance such as nucleic acid is treated with a device for making a hole in the cell membrane and is introduced into the cell.SOLUTION: Provided is a method for introducing an anionic substance to be introduced into a cell, characterized by comprising: allowing a cationic compound to act on a cell to obtain a cationic compound-treated cell; adding an anionic substance to be introduced to a liquid containing the cationic compound-treated cell to obtain a liquid for introduction; and treating the solution to be introduced with a device for making a hole in the cell membrane to make a hole in the cell membrane of the cell.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This invention relates to a method for introducing a substance into cells. [Background technology]

[0002] Technologies for introducing substances, including genes, into cells are known. Various methods have been developed for introducing substances into cells, such as chemical methods using cationic compounds and liposomes, biological methods using viruses, physical methods such as electroporation (which is expected to have low toxicity) and gene guns, and microinjection methods, which offer high selectivity for the introduced substance and high reliability of introduction.

[0003] In recent years, with the emergence of cell therapies and induced pluripotent stem cells, which involve introducing substances such as genes into cells to modify their properties, there is a growing demand for more efficient methods of introducing substances.

[0004] Patent Document 1 describes a method for introducing a substance into cells using a microfluidic channel. In this method, cells are dispersed in a liquid containing the substance to be introduced, and this liquid is passed through a microfluidic channel. This applies shear force to the cells, temporarily creating holes in the cell membrane, and introducing the extracellular substance into the cells.

[0005] Patent Document 2 demonstrates that by using a liquid dispensing device that applies the configuration of an image recording device, it is possible to effectively introduce a target substance into cells by generating pressure and shear force in a micro-sized space. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 2018 / 0003696 [Patent Document 2] Patent No. 5645657 [Overview of the project] [Problems that the invention aims to solve]

[0007] Generally, cell surfaces have a negative charge. Genes also have a negative charge. When attempting to introduce a gene into a cell, a charge repulsion occurs between the cell surface and the gene, making it difficult to efficiently introduce the gene into the cell. Patent Document 1 describes introducing substances into cells using microfluidic channels. However, no measures are taken regarding the negative charge present in cells and genes. In Patent Document 2, the gene is pre-mixed with a cationic compound to form a complex of the gene and the cationic compound. Then, the complex is dispensed with cells from a liquid dispensing device, and the gene is introduced into the cells. However, the complex is larger than the gene. When introducing a substance into a cell using pressure and shear force with a liquid dispensing device, we found that the larger the substance to be introduced, the more difficult it is to introduce it into the cell. In other words, we found that while the charge repulsion between the cell surface and the gene can be suppressed by making a complex of the gene and the cationic compound, the size of the complex becomes larger, which is disadvantageous when introducing the substance into the cell by creating a hole in the cell membrane. In view of the above problems, the present invention aims to provide a method for introducing a substance into a cell while suppressing the charge repulsion of the substance. [Means for solving the problem]

[0008] The present invention In vitro or in non-human cells, cells Plasmid DNA A method for introducing, To the aforementioned cells Polyethyleneimine Apply the action, Polyethyleneimine processed cells The first liquid contains The process of obtaining A step of recovering the polyethyleneimine-treated cells from the first liquid by centrifugation, The recovered polyethyleneimine-treated cells are suspended in an aqueous medium to obtain a second liquid. The second of the above The liquid Plasmid DNA The process of adding to obtain an introduction solution, and The step of treating the introduction liquid with a device for creating holes in the cell membrane to create holes in the cell membrane of the cells A method characterized by including this is provided.

Advantages of the Invention

[0009] In order to allow a cationic compound to act on cells, it is not necessary to form an anion introduction substance into a complex, and the charge repulsion between the cell surface and the introduction substance can be suppressed without increasing its size. As a result, an anion introduction substance can be efficiently introduced into cells using a device for creating holes in the cell membrane.

Brief Description of the Drawings

[0010] [Figure 1] Perspective view of the ejection head (a) and diagram of the liquid ejection device (b) [Figure 2] Cross-sectional view of the ejection head [Figure 3] Process diagram [Figure 4] Appearance when a cationic compound is added to a cell suspension and then an anionic introduction substance is added [Figure 5] Process diagrams of Example 1, Comparative Example 1, and Comparative Example 2

Modes for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described in detail. The present invention is a method for introducing an anionic introduction substance into cells, comprising: a step of allowing a cationic compound to act on the cells to obtain cells treated with the cationic compound, a step of adding the anionic introduction substance to the liquid containing the cells treated with the cationic compound to obtain an introduction liquid, and a method characterized by including a step of treating the introduction liquid with a device for creating holes in the cell membrane to create holes in the cell membrane of the cells.

[0012] (Cells) In the present invention, cells include eukaryotic cells and prokaryotic cells without limitation, but are preferably mammalian cells. Mammals are not limited and include humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, cats, dogs, and all other mammals. Furthermore, the types of cells are not limited and include, for example, somatic cells, germ cells, hematopoietic cells, nerve cells, cancer cells, stem cells, etc. Cells also include primary cultured cells, cultured cells, cell lines, cancerous cells, chimeric cells, transformed cells, etc.

[0013] (Introduced substance) In this specification, "introduced substance" refers to a substance intended to be introduced into a cell. In this invention, the introduced substance is an anionic substance. Examples of introduced substances include nucleic acids, proteins, and low-molecular-weight compounds.

[0014] Examples of nucleic acids include DNA (deoxyribonucleotides), RNA (ribonucleotides), single-stranded DNA, double-stranded DNA, oligonucleotides, polynucleotides, probes, primers, EST or SAGE tags, exons, introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant DNA, plasmids, vectors, and other isolated DNA or RNA of any sequence, methylated DNA, parts thereof, and mixtures thereof. When the introduced substance is a nucleic acid, it is preferably a single-stranded or double-stranded nucleic acid corresponding to 5 to 100 kilobases, more preferably 1 to 40 kilobases.

[0015] Proteins are used without particular limitations, but examples include various enzymes, antibodies, receptors, recombinant proteins, signaling molecules, membrane proteins, blood components, cytokines, hormones, allergens, alloproteins, heteroproteins, some of these, and mixtures thereof.

[0016] Small molecule compounds are used without particular limitations, but examples include fluorescent molecules, dyes, drugs, analogs, antagonists, agonists, inhibitors, antagonists, substrates, signaling molecules, and functional compounds.

[0017] The introduced substance is, in particular, nucleic acid. In recent years, in fields such as medicine, pharmacy, regenerative medicine, and cell engineering, there has been a demand for superior methods of introducing nucleic acids into cells, as their function and characteristics can be controlled, modified, and altered by introducing them into cells.

[0018] (Cationic compounds) Cationic compounds are used to suppress the charge repulsion between the introduced substance and the cell surface. For example, when a cationic compound is applied to a material such as a gene, a complex is formed between the gene and the cationic compound, and its size increases. It has been reported in a paper (Mol Pharm 10(11):4120-4135(2013)) that the size of the complex increases over time after mixing the gene and the cationic compound. It is thought that the size increases exponentially if there are many positively charged cationic compounds around a negatively charged gene, and then many more negatively charged genes around that, and then many more positively charged cationic compounds around that.

[0019] We have found that when introducing a substance into a cell by creating holes in the cell using pressure and shear force, as in the present invention, the larger the size of the introduced substance, the more difficult it becomes to introduce the substance into the cell. The method of mixing the introduced substance with a cationic compound to form a complex in order to suppress the charge repulsion between the introduced substance and the cell surface does not contribute to the introduction of the substance into the cell when introducing the substance by creating holes in the cell, but rather hinders it.

[0020] This invention is characterized by the fact that, rather than reacting a cationic compound with an introduced substance, a cationic compound is reacted with cells to obtain cationic compound-treated cells. Because the cell surface has a negative charge, cationic compounds tend to adhere to the cell surface. On the other hand, cells do not aggregate in a snowball-like manner via the cationic compound. This is thought to be because cells are sufficiently large in size compared to cationic compounds. The inventors mixed cells with a cationic compound, incubated them for a certain period of time, and then collected the cells by centrifugation. The collected cells resuspended, and no phenomenon of cells adhering to each other via the cationic compound was observed.

[0021] A cationic compound may have one or more positively charged cationic groups, but preferably, it is preferable that the cationic compound contains primary, secondary, and tertiary amines within its molecule. Furthermore, it is advantageous for the cationic compound to adhere to the cell surface at multiple points rather than at a single point for the cationic compound to cover the cell surface. It is preferable that the cationic compound has two or more cationic groups per molecule, and it is also preferable that it contains two or more primary, secondary, and tertiary amines within its molecule. To further increase the number of cationic groups per molecule of cationic compound, it is more preferable that the cationic compound is a polymer. Even more preferably, it is a polymer with a weight-average molecular weight of 1,000 to 100,000.

[0022] Preferred examples of cationic compounds include polyethyleneimine, poly-L-lysine, poly-L-arginine, polyvinylpyrrolidone, and polyamidoamine dendrimers. Polyethyleneimine is even more preferred because it is water-soluble, unaffected by serum in the culture medium, has low cytotoxicity, and is low-cost.

[0023] Alternatively, a cell-affinity polymer may be used as the main chain, with compounds such as spermine and spermidine introduced as side chains to form a cationic compound. Examples of cell-affinity polymers include sugars, proteins, and lipids.

[0024] (A device that creates holes in cell membranes) A device for creating holes in cell membranes is a device capable of creating at least one or more holes in a cell membrane, at least temporarily. Methods for creating holes in cell membranes include using lasers, sonoporation using ultrasound, and applying pressure to a liquid to pass cells through an orifice.

[0025] This section describes a method for creating holes in cell membranes using a simple setup, by applying pressure to a liquid and passing cells through an orifice. In this method, a temporary hole can be created in the cell membrane by applying pressure to the liquid containing the cells and simultaneously applying shear force to the cells.

[0026] Means of applying pressure to a liquid include various pumps such as syringe pumps and peristaltic pumps, compressors, vacuum, syringes, pipettes, pistons, gravity, capillaries, or means of generating pressure in a micro-sized space, such as dispensing means in a liquid dispensing head. Dispensing means in a liquid dispensing head include dispensing energy generating elements, and more specifically, heaters in thermal-type liquid dispensing heads and piezoelectric elements (piezo elements) in piezoelectric-type liquid dispensing heads.

[0027] An orifice can also be called an opening. An orifice should be large enough to exert a shear force on a cell as it passes through it with the liquid. The orifice should be slightly larger than the cell, allowing it to pass through. The direction of liquid flow is the Z direction, and the directions perpendicular to the Z direction and perpendicular to each other are the X and Y directions. The shape of the orifice in the XY plane is not particularly limited, but depending on the shape of the cell to be perforated, or the ease of constructing the device, examples include quadrilaterals, squares, rectangles, ellipses, circles, irregular shapes, and shapes approximating these. The size of the orifice in the XY plane is determined according to the diameter of the cell used, and it is preferable that the minimum diameter is 10 times or less the cell diameter. A larger diameter in the XY plane of the orifice reduces the shear force experienced by the cell as it passes through the orifice. The XY plane of the orifice is 1 μm. 8000 μm or more 2 It is preferably 8000 μm or less, and the minimum diameter is preferably 1 μm or more and 100 μm or less. There is no particular limitation on the length of the orifice in the Z direction, that is, the distance of the orifice through which the cells pass, but it is preferably 1 μm or more and 200 μm or less. If the length of the orifice in the Z direction is longer than 200 μm, the pressure for passing the cells through the orifice becomes too large. The shape and area of the XY plane may vary depending on Z. For example, the area of the XY plane (S(Z ent =Z ent ) at the entrance (Z = Z exit ) is larger than the area of the XY plane (S(Z exit ) at the exit (Z = Z ent ), or S(Z exit ) is smaller than S(Z ent ), or the shape of the XY plane at Z exit is square and the shape of the XY plane at Z

[0028] As a device having both a means for applying pressure to the liquid and an orifice through which the liquid passes, a liquid ejection device or a liquid ejection head in the liquid ejection device can be cited.

[0029] The liquid ejection head and the liquid ejection device for controlling the liquid ejection head will be described. In this specification, among the liquid ejection devices, the part excluding the liquid ejection head may be referred to as the liquid ejection device main body or the liquid ejection device. Also, the liquid ejection device may be called a printer, and the part excluding the liquid ejection head from the printer may be referred to as the printer main body or the printer.

[0030] FIG. 1(a) is a perspective view showing one form of the liquid ejection head. The liquid ejection head 1 has a pressure element substrate 2, a space 3 capable of holding the liquid to be ejected, and an electrical connection portion 4 for sending power and signals from the liquid ejection device main body to the pressure element substrate.

[0031] Figure 1(b) shows an example of a liquid dispensing device. The liquid dispensing device 100 includes a liquid dispensing head holder 101 to which a liquid dispensing head can be attached, a drive motor 102, and a drive belt 103 connected to the drive motor 102. The drive motor 102 allows the liquid dispensing head 1 to be moved to any location within the transportable area. The liquid dispensing head 1, filled with liquid, is attached to the liquid dispensing head holder 101. When the liquid dispensing head 1 comes into contact with the suction recovery mechanism 104, the suction motor 105 is activated, allowing the liquid inside the liquid dispensing head 1 to be discharged. The liquid generated by suction and discharge is discharged outside the system through the waste liquid tube 106. The suction recovery mechanism 104 can also be used as a cap to prevent the head from drying out during non-introduction processing. The liquid containing cells etc. that is filled in the liquid dispensing head 1 is discharged from the liquid dispensing head 1 toward a container 108 such as a culture dish on the processing stage 107. The intake port 109 and exhaust port 110, each equipped with a fan and filter, can reduce mist and dust within the device. This reduces contamination.

[0032] Figure 2(a) is a cross-sectional view of the A-A plane in Figure 1. Figure 2(b) is an enlarged view of the pressure element substrate in Figure 2(a). The pressure element substrate consists of a discharge port 5 for discharging liquid, a flow path 6 for supplying liquid to the discharge port 5, a discharge energy generating element 7 that generates energy for discharging liquid, and an electronic circuit element (not shown) that controls the discharge energy generating element. The flow path 6 is connected to a space 3 that can hold the liquid to be discharged. The discharge energy generating element 7 generates flow in the liquid. A shear force is applied to the cells at the discharge port 5 or the flow path 6. The liquid containing the cells and the cells themselves come out of the discharge port 5.

[0033] If the device that creates holes in the cell membrane is a liquid discharge head, then the discharge energy generating element 7 corresponds to the means of applying pressure to the liquid, and the discharge port 5 corresponds to the orifice.

[0034] Specifically, the introduction liquid obtained in the second step described later is filled into the space 3 that can hold the liquid discharged by the liquid discharge head 1. Then, a portion of the introduction liquid is introduced into the flow path 6. Power from the liquid discharge device body is supplied to the discharge energy generating element 7 on the pressure element substrate via the electrical connection part 4 and the electronic circuit element that controls the discharge energy generating element 7. Then, the introduction liquid is heated and bubbles are generated, and the introduction liquid is discharged through the discharge port 5, which is an orifice. At that time, a shear force is applied to the cells contained in the introduction liquid, holes are made in the cells, and the introduced substance is introduced into the cells.

[0035] (Process) The method of the present invention includes the following steps, as shown in Figure 3. Step 1: Step of applying a cationic compound to cells. Step 2: Step of adding an anionic substance Step 3: Processing using a device that creates holes in the cell membrane. Each of the above steps will be explained in detail below.

[0036] <Step 1: Step of applying a cationic compound to cells> In this process, cells are treated with a cationic compound to obtain cells treated with the cationic compound. Cells are suspended in an aqueous solvent to form a suspension. Suspension is a type of dispersion, and a suspension is also called a dispersion. Examples of aqueous solvents include phosphate-buffered saline (PBS), buffer solutions such as Hanks' Balance Salt Solution (HBSS), culture media such as Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute 1640 (RPMI1640), and physiological saline.

[0037] Cationic compounds can be mixed with a cell suspension as a solution to create a liquid containing cells and the cationic compound, thereby allowing contact between the cells and the cationic compound. The solvent used to dissolve the cationic compound can be the aqueous solvent mentioned above, or, to the extent that it does not affect the cells, water, other aqueous solvents, or organic solvents such as dimethyl sulfoxide. Alternatively, a solid cationic compound can be added to a cell suspension to dissolve the cationic compound in the cell suspension, creating a liquid containing cells and the cationic compound, thereby allowing contact between the cells and the cationic compound.

[0038] There are no restrictions on the concentration of cells or cationic compounds in a liquid containing cells and cationic compounds. If the amount of cationic compound relative to cells is too low, the negative charge of the cells cannot be sufficiently eliminated. If there is too much cationic compound, there is a high possibility that the excess cationic compound will form a complex with the introduced substance added in a later step. The cationic group relative to cells is 1 × 10⁶ cells. 6 For each individual, 1 × 10 -9 From 1 x 10 -5 moles are preferred.

[0039] To ensure sufficient cations are introduced to the cell surface, the cells may be incubated in a liquid state in contact with a cationic compound for an incubation period. For example, incubation can be performed at 37°C for 10 minutes.

[0040] <Step 2: Adding an anionic substance> In this process, an anionic introduction substance is added to a liquid containing cells treated with a cationic compound to obtain an introduction solution.

[0041] The introduced substance can be added to the solution containing cells and a cationic compound, which was prepared in the first step. In this case, a problem may arise in which the cationic compound remaining in the solution forms a complex with the introduced substance. To resolve this problem, the cationic compound-treated cells may be recovered from the solution containing cells and the cationic compound obtained in the first step by centrifugation or the like, the recovered cells may be suspended in another aqueous solvent, and the introduced substance may be added to this solution to use as the introduction solution, or the recovered cells may be suspended in an aqueous solvent containing the introduced substance to use as the introduction solution.

[0042] The number of cells in the introduction solution is 1 × 10⁶ 5 pieces ~1 × 10 7 pieces A aqueous solvent is preferred, and examples of aqueous solvents include phosphate-buffered saline (PBS), buffer solutions such as Hanks' Balance Salt Solution (HBSS), culture media such as Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute 1640 (RPMI1640), and physiological saline. The preferred concentration of the introduced substance in the introduction solution varies depending on the type and state of the cells and the type of introduced substance, but in the case of nucleic acids, for example, it can be between 0.1 μg / μl and 10 μg / μl.

[0043] The state of the introduction solution is schematically represented as shown in Figure 4. That is, the cationic compound 9 is attached to the cells 8, and the charge repulsion between the introduction substance 10 and the cell surface is suppressed.

[0044] <Step 3: Processing using a device that creates holes in the cell membrane> In this process, the introduction solution is treated with a device that creates holes in the cell membrane, thereby creating holes in the cell membrane of the cells.

[0045] The introduction solution obtained in the second step is filled into a device that creates holes in cells. If the device that creates holes in the cell membrane is the liquid dispensing head 1, the introduction solution obtained in the second step is filled into the space 3 that can hold the liquid. The liquid dispensing head is connected to the liquid dispensing device body and operated to dispense the liquid. The introduction solution is then dispensed through the dispensing port 5, which is an orifice. The introduction substance is introduced into at least some of the dispensed cells. This is presumed to be because, during dispensing, one or more holes are created in the cells for a very short time, and the introduction substance in the introduction solution is introduced into the cells.

[0046] The present invention will be described in more detail below with reference to examples. (Consideration of the size of the introduced substance) After investigating the relationship between the size of the introduced substance and its introduction rate, we found that when introducing a substance into cells using a device that creates holes in cells, the introduction rate varies greatly depending on the size of the substance. This is explained below.

[0047] As introduced substances, we used calcein (Dojin Chemical Laboratories Co., Ltd., molecular weight 623) and flurecein-labeled dextran (Sigma-Aldrich, molecular weight 70,000, hereinafter referred to as FITC-Dex), which are relatively uncharged compounds with different molecular weights. Calcein is a fluorescent substance that does not pass through the cell membrane because it is not acetoxymethyl esterified. Each was dissolved in PBS, with calcein at a concentration of 0.25 mg / ml and FITC-Dex at 10 mg / ml. Cationic compounds were not used in this experiment.

[0048] Mouse macrophages, Raw264.7, were used as the cells. Cells that had reached approximately 80% confluence were treated with trypsin and detached. They were then centrifuged and the supernatant was removed. Next, the cells were resuspended in DMEM and 2.1 × 10⁶ 6 The concentration was adjusted to 1 / ml.

[0049] The solution containing the introduced substance and the solution in which the cells were resuspended were mixed in a ratio of 1:19 (vol:vol) to prepare the mixture. In this mixture, the cell concentration was 2.0 × 10⁻⁶. 6The concentration was set to 1 / ml. When calcein was used as the introduced substance, the concentration of the mixture was 0.0125 mg / ml, and when FITC-Dex was used, the concentration of the mixture was 0.5 mg / ml.

[0050] An inkjet printer (G1310, Canon Inc.) was selected as the device for creating holes in the cell membrane. The mixed solution was filled into the included black liquid ejection head and ejected into a culture dish. During ejection, a liquid ejection operation was performed to output a 1.5 cm x 1.5 cm solid image, and the cell-containing solution was ejected towards the culture dish 10 mm away from the ejection surface. This liquid ejection operation was repeated 40 times. DMEM medium containing 10% serum was added to the culture dish and incubated at 37°C in a 5% CO2 environment for 2 hours. Cells were detached using trypsin. They were centrifuged and washed. They were resuspended in PBS containing 2% serum and analyzed using flow cytometry (BD FACSMelody, Becton Dickinson Japan Co., Ltd.). For comparison, cells that were not mixed with or ejected with the introduced substance (normal cells) were also analyzed.

[0051] When FITC-Dex was used as the delivery substance, the delivery efficiency was lower compared to when calcein was used. This indicates a low percentage of cells that took up FITC-Dex. Even if pores are created in the cells by the liquid dispensing device, if the size of the introduced substance is large, it is difficult for the cell to take it up. This is thought to be because, after the pores are created in the cell, the introduced substance enters the cell by diffusion.

[0052] (Evaluation when the introduced substance and cationic compound are mixed) The introduced substance and a cationic compound were mixed to form a complex, and the size of the complex was evaluated.

[0053] Plasmid DNA (FresnoRFP expression plasmid, ATUM, hereafter pDNA) was used as the introduced material. A polyethyleneimine derivative (jetPEI, Polyplus transfection) was used as the cationic compound. The size of the plasmid DNA was evaluated using propidium iodide (Cellstain Double Staining Kit, Dojin Chemical Laboratories Co., Ltd.), which fluoresces only when intercalated into the double helix of pDNA.

[0054] A pDNA aqueous solution was added to 150 mM saline to make a 45 μg / ml pDNA solution. 5.4 μl of jetPEI was mixed with 44.6 μl of 150 mM saline. The pDNA solution and jetPEI solution were mixed to form a complex. 25 μl of 4.5 μmol / l propidium iodide solution was added to 25 μl of the solution containing the complex. The mixture was observed using a fluorescence microscope (BioZERO, KEYENCE Corporation) within 30 minutes of mixing the pDNA and jetPEI. For comparison, the pDNA solution was also observed using a fluorescence microscope after adding propidium iodide solution.

[0055] When a pDNA solution and a propidium iodide solution were mixed, only red fluorescence was observed, and no precipitates were seen. On the other hand, when propidium iodide was added after mixing pDNA with a cationic compound, precipitates several micrometers in size were observed. These precipitates also emitted red fluorescence. This is thought to be because the pDNA and the cationic compound formed a complex of several micrometers. It is thought that the positively charged jetPEI attached to the negatively charged pDNA, and then more pDNA attached to the jetPEI, causing the size of the complex to increase exponentially until it reached several micrometers in size, making it possible to photograph it with a fluorescence microscope.

[0056] When introducing a substance into cells using a device that creates holes in the cell membrane, as in the present invention, the larger the size of the substance, the more difficult it is to introduce into the cell. Therefore, this experiment suggests that a method of forming a complex by reacting the substance with a cationic compound beforehand to suppress the charge repulsion between the substance and the cell membrane leads to a decrease in the efficiency of uptake into cells.

[0057] (Examples 1 and 2 and Comparative Examples 1 and 2) In Examples 1 and 2, an anionic introduction substance was introduced into cells via steps 1, 2, and 3 described above. In contrast, in Comparative Example 1, the introduction solution was not treated with a device that creates holes in the cell membrane. Furthermore, in Comparative Example 2, a cationic compound was not used. The steps for Examples 1 and 2, Comparative Example 1, and Comparative Example 2 are shown in Figure 5.

[0058] Examples 1 and 2 are described below. 80% confluent Chinese hamster ovary cells, CHO-K1, were treated with trypsin and detached. After centrifugation, the supernatant was removed, and 1.1 × 10⁶ cells were obtained. 6 The individual cells were resuspended in 225 μl of Ham's F-12 Nutrient Mix (hereinafter referred to as F-12 medium).

[0059] As cationic compounds, polyethyleneimine (weight-average molecular weight 40,000) was dissolved in water in Example 1, and polyethyleneimine (weight-average molecular weight 300) was dissolved in water in Example 2. The solutions were then adjusted to pH 7.0 using a 1N sodium hydroxide aqueous solution and sterilized by filtration. The final concentration was 10 mg / ml. This polyethyleneimine was mixed with 150 mM saline solution in a ratio of 1:10 (volume ratio). 4.6 μl of the prepared polyethyleneimine solution was added to the cell suspension and mixed.

[0060] After incubation at room temperature for 10 minutes, 2 ml of F-12 medium containing 10% serum was added. Then, the mixture was centrifuged at 4°C and 90G for 10 minutes. After removing the supernatant, 500 μl of F-12 medium was added to prepare the suspension.

[0061] 475 μl of cell suspension was mixed with 25 μl of 1 μg / μl pDNA (FresnoRFP expression plasmid) to obtain a mixture. The cell concentration was 1 × 10⁻⁶. 6 The sample size was adjusted to 0.05 μg / μl, with a pDNA concentration of 0.05 μg / μl.

[0062] An inkjet printer (G1310, Canon Inc.) was selected as the device for creating holes in the cell membrane. The mixed solution was filled into the included black ink ejection head and ejected onto a culture dish. During ejection, a liquid ejection operation was performed to output a 1.5cm x 1.5cm solid image, and the cell-containing solution was ejected towards the culture dish, which was 10mm away from the ejection surface. This liquid ejection operation was repeated 40 times. F-12 medium containing 10% serum was added to the culture dish and incubated overnight at 37°C in a 5% CO2 environment.

[0063] In Comparative Example 1, the cell suspension and polyethyleneimine solution were mixed and incubated in the same manner as in Example 1. After centrifugation, pDNA was added and mixed. The mixture was seeded into a culture dish and F-12 medium containing 10% serum was added. It was incubated overnight at 37°C in a 5% CO2 environment.

[0064] In Comparative Example 2, the procedure was the same as in Example 1, except that the cells were not mixed with the polyethyleneimine solution; that is, pDNA was added to cells that were not treated with the polyethyleneimine solution.

[0065] pDNA expression was evaluated as follows: Each cell, incubated overnight at 37°C in a 5% CO2 environment, was treated with trypsin and detached. After centrifugation and washing, the cells were suspended in PBS containing 2% serum. Flow cytometry was used to analyze and evaluate the pDNA expression rate. FresnoRFP emits fluorescence at 592 nm when excited at 553 nm.

[0066] Table 1 shows the evaluation results. In Comparative Example 1, where the cationic compound was applied to the cells but not dispensed using the liquid dispensing head, the gene expression rate was 0.06%. It is thought that even though the cationic compound suppressed the charge repulsion between the cells and pDNA, the pDNA could not pass through the cell membrane, resulting in a low gene expression rate. In Comparative Example 2, where the cationic compound was not applied to the cells but dispensing was performed using the liquid dispensing head, the gene expression rate was 1.7%. In Example 1, where the cationic compound was applied to the cells and dispensing was performed using the liquid dispensing head, the gene expression rate was 2.6%. Compared to the case where the cells were only dispensed using the liquid dispensing head without applying the cationic compound, the gene expression rate was 1.5 times higher when the cationic compound was applied to the cells and dispensing was performed using the liquid dispensing head. It is thought that the cationic compound suppressed the charge repulsion between the cells and pDNA, and the pDNA was taken up by the cells through the holes opened by the dispensing of the liquid dispensing head. In Example 2, the gene expression rate was slightly lower. It is thought that when the molecular weight of a cationic compound is low, the number of cationic groups per molecule that can attach to cells decreases, and therefore the compound cannot stably attach to cells.

[0067] [Table 1] [Explanation of Symbols]

[0068] 1. Liquid dispensing head 2. Pressure element substrate 3. Space to hold the liquid 4. Electrical connection 5.Discharge port 6. Flow path 7. Discharge energy generating element 8.Cells 9. Cationic compounds 10.Introduced substances 100.Liquid discharge device 101. Liquid dispensing head holder 102. Drive motor 103. Drive belt 104.Suction recovery mechanism 105. Suction motor 106. Waste liquid tube 107. Processing Stage 108.Container 109. Air intake 110. Exhaust port

Claims

1. A method for introducing plasmid DNA into cells in vitro or in vivo in non-human cells, A step of reacting the aforementioned cells with polyethyleneimine to obtain a first liquid containing polyethyleneimine-treated cells, A step of recovering the polyethyleneimine-treated cells from the first liquid by centrifugation, The recovered polyethyleneimine-treated cells are suspended in an aqueous medium to obtain a second liquid. A step of adding the plasmid DNA to the second liquid to obtain a solution for introduction, and The process involves treating the aforementioned introduction solution with a device that creates holes in the cell membrane, thereby creating holes in the cell membrane of the cells. A method characterized by including the following.

2. The method according to claim 1, wherein the apparatus for creating holes in the cell membrane comprises means for applying pressure to a liquid and an orifice through which the liquid passes.

3. The method according to 1 or 2, characterized in that the device for creating holes in the cell membrane is a liquid dispensing head.

4. The method according to any one of claims 1 to 3, characterized in that the polyethyleneimine is a polymer having a weight-average molecular weight of 1,000 to 100,000.

5. The method according to any one of claims 1 to 4, characterized in that the cells are mammalian cells.

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