Method for introducing molecules

Abstract

To provide a method for introducing a molecule into a cell with improved introduction efficiency.SOLUTION: (A) preparing cells into which molecules containing nucleic acids are to be introduced, (B) mixing the cells obtained in the step (A) with the molecules containing nucleic acids, and (C) introducing the molecules containing nucleic acids into the cells by endocytosis of the cells, wherein the step (A) is a step of adjusting cell cycle of the cells so that the cells enter G2 phase before M phase, which is mitotic phase.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a molecule introduction method. [Background technology]

[0002] With the decoding of the human genome and mouse genome, there is an increasing need in the fields of medicine and pharmacy for molecular biology-related research and development or drug development to introduce molecules such as polynucleotides such as DNA or RNA or their derivatives, high molecular weight compounds such as proteins such as signal transduction proteins or transcriptional regulators or their derivatives, low molecular weight physiologically active substances, or drug candidates into target cells and test the function of genes or the physiological activity of physiologically active molecules within the target cells.

[0003] There are three main methods for introducing genes into target cells: chemical, physical, and biological. Chemical methods involve using transfection reagents such as cationic polymers, cationic lipids, or calcium phosphate to introduce genes into target cells through endocytosis. Physical methods involve directly introducing genes into target cells through physical manipulations such as microinjection, sonoporation, laser irradiation, or electroporation. Biological methods involve using viral vectors such as retroviruses, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses to transfer nucleic acids within viral particles into target cells using the infectivity of the virus.

[0004] Non-Patent Document 1 discloses that CAR-mRNA is introduced into T cells by electroporation. [Prior art documents] [Non-patent literature]

[0005] Non-Patent Document 1: R. Kitte et al., Lipid nanoparticles outperform electroporation in mRNA-based CAR T cell engineering, Mol. Ther. Methods Clin. Dev., Oct. 18, 2023 Summary of the Invention [Problem to be solved by the invention]

[0006] When molecules are introduced into cells by the electroporation method disclosed in Non-Patent Document 1, the efficiency of molecule introduction may be low.

[0007] An object of the present invention is to provide a method for introducing a molecule into a cell with improved introduction efficiency. [Means for solving the problem]

[0008] [1] A molecule introduction method comprising: step A of preparing cells into which a molecule containing a nucleic acid is to be introduced; step B of mixing the cells obtained in step A with the molecule containing a nucleic acid; and step C of introducing the molecule containing a nucleic acid into the cells by endocytosis of the cells, wherein step A is a step of adjusting the cell cycle so that the cells enter the G2 phase, which precedes the M phase, which is the division phase. [2] The molecule introduction method according to [1], wherein step C is a step of irradiating a mixture of the cells and the molecules containing the nucleic acid with plasma. [3] The step A is 0.5 × 10 4 ~2.5×10 4 cells / cm 2 The molecule introduction method according to [1] or [2], wherein the step of seeding cells in the medium is carried out in the presence of a phosphate buffer solution, and culturing the cells for more than 24 hours but less than 72 hours. [4] The molecule introduction method according to any one of [1] to [3], wherein the step A is a step of preparing a cell population having a high proportion of the cells in the G2 phase. [5] The molecule introduction method according to any one of [1] to [4], wherein the cells are stem cells. [6] The molecule introduction method according to any one of [1] to [5], wherein the molecule is at least one molecule selected from the group consisting of DNA and RNA. [Effects of the Invention]

[0009] According to the present invention, a method for introducing a molecule into a cell with improved introduction efficiency can be provided. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a cross-sectional view of a molecule introduction device that can be used in the molecule introduction method of the present invention. [Figure 2] FIG. 2 is a cross-sectional view of the irradiating body 14 of FIG. [Figure 3] 1 is a graph showing the molecule transfer efficiency and cell survival rate in Experimental Examples 1 to 8. [Figure 4] 1 is a graph showing the molecule transfer efficiency and cell survival rate in Experimental Examples 9 to 16. [Figure 5] FIG. 1 shows the results of Goterm analysis. [Figure 6] 1 is a graph showing the molecule introduction efficiency in Experimental Examples 17 and 18. [Figure 7] 1 is a graph showing the mRNA transfection efficiency in Experimental Examples 19 and 20. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the present invention, the symbol "to" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0012] [Gene transfer method] The molecule introduction method of the present invention comprises the steps of: step A of preparing cells into which a molecule containing a nucleic acid is to be introduced; step B of mixing the cells obtained in step A with the molecule containing a nucleic acid; and step C of introducing the molecule containing a nucleic acid into the cells by endocytosis of the cells, wherein step A is a step of adjusting the cell cycle so that the cells enter the G2 phase, which precedes the M phase, which is the division phase.

[0013] <Cell preparation process A> Cells are prepared for the introduction of molecules containing nucleic acids. More specifically, the cell cycle is adjusted so that the cells are in the G2 phase, which precedes the M phase, the division phase. Methods for adjusting the cell cycle to the G2 phase include appropriately controlling the seeding density and the number of days of cell culture.

[0014] For example, 0.5 x 10 4 ~2.5×10 4 cells / cm 2 It is preferable to seed the cells at 100°C and culture them for more than 24 hours but less than 72 hours to prepare cells in the G2 phase.

[0015] Here, "adjusting the cell cycle to be in the G2 phase" means adjusting the proportion of cells in the G2 phase in the cultured cell population to be higher. For example, "adjusting the cell cycle to be in the G2 phase" means adjusting the expression of proteins encoding genes associated with the G2 phase in the cultured cells to be 1.4 times or more compared to before adjustment.

[0016] Whether the cell cycle is adjusted to the G2 phase can be confirmed by the proteome analysis described in the Examples. Since the time from cell seeding to adjustment to the G2 phase may vary depending on the cell type, proteome analysis may be performed in advance to determine an appropriate seeding density and culture period for adjusting the cell cycle to the G2 phase.

[0017] (cell) In this embodiment, the term "cell" refers to a cell that is a target for introducing a molecule containing a nucleic acid, and is not limited to a particular type of cell. Specific examples of such cells include animal cells, including human cells, non-human animal cells, cells collected from human individuals, cells within human individuals, human individuals, plant cells, and microbial cells. These cells may be used in a single type or in a mixture of two or more types.

[0018] The above-mentioned cells collected from human individuals include cells that are not intended to be returned to human individuals and are used for pharmaceutical research and development, etc., and cells that are intended to be returned to human individuals and are used for regenerative medicine, etc. The above-mentioned cells collected from human individuals also include cells cultured from cells collected from human individuals.

[0019] Furthermore, the non-human animal cells and plant cells mentioned above include cells present within an individual, cells present within a tissue, cells collected from an individual or tissue, and cells cultured from cells collected from an individual or tissue.

[0020] From another perspective, the cells used in the present invention include prokaryotic cells such as Escherichia coli, actinomycetes, and Bacillus subtilis; and eukaryotic cells such as yeast, non-human animal cells, cells collected from human individuals, cells contained in human individuals, and plant cells.

[0021] The cells are preferably at least one type selected from the group consisting of blood-derived cells, adherent cells, and iPS cells. Examples of the blood-derived cells include T cells. Examples of the adherent cells include stem cells such as HEK293 cells, mesenchymal stem cells, and adipose-derived stem cells. The cells are preferably adipose-derived stem cells. T cells are a type of lymphocyte, differentiated and matured from precursor cells produced in the bone marrow through selection in the thymus. T cells have a characteristic T cell receptor (TCR) on their surface. They account for 70 to 80% of lymphocytes in peripheral blood. T cells collected from peripheral blood or cultured T cells can be used. T cells are included in CD3-positive cells. Examples of CD3-positive cells include Jurkat cells in addition to T cells. T cells can be cultured using commercially available T cell medium according to known protocols.

[0022] HEK293 cells are a cell line established by transforming human embryonic kidney cells with the E1 gene of adenovirus. HEK293 cells are easy to culture and transduce genes into, and are used for a variety of purposes, including recombinant protein production and as a host for recombinant adenovirus production and amplification. They are available commercially.

[0023] Mesenchymal stem cells (MSCs) are a type of stem cell present in the adult body and have the ability to differentiate into bone, cartilage, blood vessels, and cardiac muscle cells, which are tissues derived from the mesoderm. They were first discovered in bone marrow in the 1960s, and then in adipose tissue in the 2000s, and both are called "mesenchymal stem cells." In recent years, it has been reported that they can also differentiate into nerve cells and glial cells (which have functions such as supporting nerve cells) derived from the ectoderm, and liver cells derived from the endoderm.

[0024] Adipose-derived stem cells are a type of mesenchymal stem cell (MSC) that can be extracted from adipose tissue. Compared to bone marrow-derived MSCs, adipose-derived MSCs have the following advantageous characteristics: they are found in greater quantities in the body and can be extracted in large quantities from adipose tissue throughout the body; they produce large amounts of growth factors (regenerative factors) such as HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) that contribute to organ repair; they have strong immunosuppressive properties; and they can proliferate without problems even when extracted from the adipose tissue of elderly people.

[0025] iPS cells (induced pluripotent stem cells) are cells that, by introducing four types of genes into somatic cells, have the pluripotency to differentiate into a large number of cells, like ES cells (embryonic stem cells), and the ability to self-replicate, maintaining this ability even after division and proliferation.

[0026] Furthermore, the cells used in the present invention also include those having a lipid bilayer structure, such as erythrocyte ghosts and liposomes.

[0027] The cells used in the present invention may be untreated, but in order to improve the efficiency of gene transfer, they may be treated as competent cells, a process commonly used in gene transfer. A specific example is competent Escherichia coli cells that have been treated with calcium chloride to change the structure of their cell membranes and make DNA molecules more easily dialyzable.

[0028] In the present invention, genes can be introduced into multiple cells simultaneously. In this case, the multiple cells may be cells of the same species or cells of different species. Alternatively, the multiple cells preferably include cells of different species. Furthermore, the cells are preferably one or more types selected from the group consisting of non-human animal cells, non-human animal individuals, cells collected from human individuals, cells within human individuals, human individuals, plant cells, and microbial cells.

[0029] The cells may be contained in tissue. In the present invention, tissue containing cells may be referred to as target tissue. The target tissue is not limited to a particular type of tissue. Specific examples of such target tissue include organs from donors used for transplantation, tissues such as skin and tooth roots reconstructed using regenerative medicine methods, and tissues constructed by callus culture prior to differentiation into plants. These target tissues may be used alone or in combination of two or more types.

[0030] <Mixing process B> In step B, the cells obtained in step A are mixed with molecules containing nucleic acids. The molecules containing nucleic acids are preferably dispersed in a liquid such as a solution or dispersion liquid, such as a buffer solution. The buffer solution is not particularly limited, but is preferably one that has no or little effect on the efficiency of introducing the molecules containing nucleic acids into the cells and has no or little cytotoxicity to the cells. The cells may be contained in the medium used in step A.

[0031] Examples of molecules containing nucleic acids include cDNA (complementary DNA), DNA encoding miRNA (microRNA), DNA encoding shRNA (small hairpin RNA), and liposomes containing these.

[0032] cDNA contains the sequence of the region of a gene that is translated into protein, from the start codon to the stop codon. In cells into which the gene is introduced, it is transcribed into mRNA, which is then translated by the ribosome, allowing the expression of proteins such as enzymes and fluorescent proteins.

[0033] The size (number of bases) of the gene is not particularly limited, but is preferably 10 bp to 10 kbp, more preferably 500 bp to 6 kbp, and even more preferably 1 kbp to 3 kbp.

[0034] miRNAs are functional nucleic acids that undergo a multi-step production process starting with transcription, ultimately becoming small RNAs of 20 to 25 bases in length. miRNAs are classified as functional ncRNAs (non-coding RNAs) and play an important role in biological phenomena by regulating the expression of other genes.

[0035] shRNAs are hairpin-shaped RNA sequences used for gene silencing by RNA interference.

[0036] <Introduction process C> The introduction step C is a step of introducing a molecule containing a nucleic acid into a cell by endocytosis of the cell. Methods for introducing a molecule containing nucleic acid into a cell include plasma introduction, lipofection, and electroporation, but introduction by plasma introduction is preferred. The method for introducing a molecule containing nucleic acid into a cell may also be lipofection by plasma introduction.

[0037] (Plasma introduction method) In the introduction step C, the method for introducing nucleic acid-containing molecules into cells is preferably a plasma introduction method, in which cells are irradiated with plasma to activate cellular activity and then nucleic acid-containing molecules are introduced into the cells. The plasma introduction method is a method for introducing nucleic acid-containing molecules into cells by irradiating cells with discharge plasma. This method has the advantages of not using viruses, thereby reducing the risk of carcinogenesis, and not using chemicals, thereby reducing the risk of cellular dysfunction. Furthermore, since no current is passed directly through the cells, there is little damage to the cells, and the survival rate of gene-introduced cells is high.

[0038] In this specification, plasma is defined as satisfying the following (1), (2), and (3). (1) A collection of charged particles of opposite polarity that is almost electrically neutral as a whole. (2) A group of at least one type of charged particle in (1) above is undergoing irregular thermal motion. (3) The charged particles in (1) and (2) above have dimensions greater than the Debye length.

[0039] In a narrow sense, plasma may refer to an ionized gas, but the plasma in the present invention is not limited to plasma generated by discharge in the gas phase, but also includes plasma generated by discharge in a liquid. Furthermore, "irradiating a target with plasma" does not only mean directly contacting the target with the plasma, but also includes causing the target to be acted upon by activated species generated by the reaction of the plasma with a surrounding gas or liquid, or electromagnetic waves such as ultraviolet rays, electrons, or heat generated by the plasma.

[0040] In the introduction step C, plasma is generated in the vicinity of the mixture of cells and the nucleic acid-containing molecules. The plasma irradiation conditions will be described in detail in the description of the gene introduction device below.

[0041] Plasma irradiation induces endocytosis in cells, resulting in the introduction of molecules containing nucleic acids into the cells. Plasma irradiation may be performed on cells after the cell preparation step A or after the mixing step B.

[0042] The molecule transfer method of this embodiment may further include the step of adding a substance that improves the transfer efficiency of molecules containing nucleic acids (hereinafter, sometimes referred to as a transfer efficiency improver).

[0043] The transfer efficiency improver is preferably at least one selected from the group consisting of cytokines and recombinant forms thereof. Examples of cytokines include interleukins (hereinafter sometimes referred to as "IL"), interferons, chemokines, colony-stimulating factors, tumor necrosis factors, and growth factors. ILs are more preferred, ILs produced by activated T cells are even more preferred, and interleukin-2 (hereinafter sometimes referred to as "IL2") and interleukin-15 (hereinafter sometimes referred to as "IL15") are even more preferred.

[0044] The introduction efficiency improver may be added in a state of being mixed with a liquid. The liquid is not particularly limited, but examples thereof include a liquid in which the introduction efficiency improver is dissolved or dispersed in a buffer solution. The buffer solution is not particularly limited, but it is preferable that it has no or little effect on the efficiency of molecule introduction into cells and has no or little cytotoxicity to cells.

[0045] The concentration of the introduction efficiency improver in the liquid containing the introduction efficiency improver is not particularly limited, but in the case of IL2 or IL15, the concentration in the system where the cells exist, for example, in the medium in contact with the cells, is 10 to 2000 ng / mL.

[0046] The timing of addition of the introduction efficiency improving agent is not particularly limited. For example, the introduction efficiency improving agent may be added to a solution containing cells, such as a culture medium. For example, cells may be cultured in a culture medium containing the introduction efficiency improving agent, and then a molecule containing a nucleic acid may be introduced into the cells. Alternatively, the introduction efficiency improving agent may be added to a liquid containing a molecule containing a nucleic acid.

[0047] [Method of producing transfected cells] The present invention also provides a method for producing molecule-introduced cells using the above-mentioned molecule introduction method. The method for producing molecule-introduced cells of the present invention comprises the steps of: Step A: preparing cells into which a molecule containing a nucleic acid is to be introduced; Step B: mixing the cells obtained in Step A with the molecule containing the nucleic acid; and Step C: introducing the molecule containing the nucleic acid into the cells by endocytosis of the cells, wherein Step A is a step of adjusting the cell cycle so that the cells enter the G2 phase, which precedes the M phase, which is the division phase.

[0048] [Gene transfer device] Hereinafter, a gene transfer device that can be suitably used in the molecule transfer method of the present invention will be described with reference to one embodiment. The molecule introduction device of this embodiment has a first electrode, a second electrode, and an irradiation field located between the first electrode and the second electrode. The molecule introduction device introduces molecules containing nucleic acids into cells by generating plasma from a first electrode and irradiating the plasma onto an irradiation target in which cells and molecules containing nucleic acids coexist, thereby introducing the molecules containing nucleic acids into the cells.

[0049] The molecule introduction device 1 of FIG. 1 includes a first electrode 10, a second electrode 20, a power supply part 30, and a container 40.

[0050] The second electrode 20 is spaced apart from the first electrode 10 and is located below the lower end (tip) of the first electrode 10. That is, the second electrode 20 is located beyond the tip of the first electrode 10, and the first electrode 10 and the second electrode 20 face each other. The power supply unit 30 is connected to the first electrode 10 and the second electrode 20 . The container 40 is located between the first electrode 10 and the second electrode 20. The container 40 is opposed to the second electrode 20 and is spaced apart from the first electrode 10.

[0051] The first electrode 10 has an electrode body 12 and an irradiation body 14 provided at the tip of the electrode body 12. In the present invention, the first electrode 10 may be composed of only the irradiation body 14. In this embodiment, the first electrode 10 is a high voltage electrode.

[0052] The electrode body 12 is a rod-shaped body that extends in one direction. The electrode body 12 may have a hollow structure such as a cylindrical or polygonal tube, or may have a solid structure such as a columnar or polygonal pillar. In particular, when the electrode body 12 has a solid structure, durability is increased and manufacturing is easy. Examples of materials for the electrode body 12 include metals such as stainless steel, copper, and tungsten, and carbon.

[0053] The thickness (outer diameter) R12 of the electrode body 12 is, for example, 1 to 50 mm. When the electrode body 12 is in the shape of a polygonal cylinder or polygonal column, the outer diameter R12 is the diameter of a circumscribed circle of the cross section of the electrode body 12.

[0054] The irradiator 14 has a flat substrate 15 and a wall 16 hanging down from the periphery of the substrate 15. The substrate 15 and the wall 16 are connected. FIG. 2 shows the end face of the irradiator 14 when the first electrode 10 is viewed from the lower end (i.e., the direction toward the second electrode 20). As shown in FIG. 2, the wall 16 is hollow and cylindrical with an open lower end (tip). The wall 16 is a single cylinder with a tube axis O1 extending from the first electrode 10 toward the second electrode 20. The wall 16 is not limited to a cylindrical shape, and may be a polygonal tube. Reference numeral 17 denotes the inner surface of the wall 16. Reference numeral 18 denotes the outer surface of the wall.

[0055] Examples of materials for the substrate portion 15 include metals such as stainless steel, copper, and tungsten, and carbon. Examples of materials for the wall portion 16 include metals such as stainless steel, copper, and tungsten, and carbon. The base plate portion 15 and the wall portion 16 may be integrally molded, or may be individually molded and then joined together.

[0056] When the electrode body 12 has a hollow structure, the substrate portion 15 may or may not have a through-hole that connects the inside of the electrode body 12 to the inside of the wall portion 16 .

[0057] The thickness (outer diameter) R16 of the wall portion 16 can be determined appropriately taking into consideration the size of the container 40, etc. The outer diameter R16 is preferably, for example, more than 1 mm, more preferably 3 to 50 mm, and even more preferably 5 to 10 mm. When the outer diameter R16 is equal to or greater than the above-mentioned lower limit, the plasma can be irradiated over a wider area, further increasing the efficiency of gene transfer into cells (hereinafter sometimes simply referred to as "transfer efficiency"). When the outer diameter R16 is equal to or less than the above-mentioned upper limit, stable discharge can be easily generated. Furthermore, it is preferable that the outer diameter R16 is larger than the outer diameter R12. If the outer diameter R16 is large, the plasma can be irradiated over a wider range, and the introduction efficiency can be further improved. When the wall portion 16 is in the shape of a polygonal cylinder, the outer diameter R16 is the diameter of the circumscribed circle of the cross section of the wall portion 16. When the irradiation body 14 has two or more wall portions 16, the outer diameter R16 of the wall portion 16 is the outer diameter of the wall portion 16 located on the outermost side.

[0058] The inner diameter r16 of the wall portion 16 is preferably, for example, 0.1 mm or more. When the wall portion 16 is a polygonal cylindrical shape, the inner diameter r16 is the diameter of the inscribed circle of the cross section of the wall portion 16. When the irradiation body 14 has two or more wall portions 16, the inner diameter r16 of the wall portion 16 is the inner diameter of the wall portion 16 located on the innermost side.

[0059] The angle θ1 between the lower end surface of wall 16 and the outer surface of wall 16 is preferably, for example, 60° or less, more preferably 30° or less, and even more preferably 10° or less. The intersection of the lower end surface of wall 16 and the outer surface of wall 16 is the so-called edge. If angle θ1 is equal to or less than the above upper limit, the electric field is concentrated at the edge, making it easier to generate plasma between the electrodes. The angle formed between the lower end surface of wall portion 16 and the inner surface of wall portion 16 is the same as angle θ1. The angle formed between the lower end surface of wall portion 16 and the inner surface of wall portion 16 may be the same as angle θ1 or may be different.

[0060] The thickness t16 of the wall portion 16 is preferably, for example, 1 mm or less, and more preferably 0.1 mm or less. When the thickness t16 is equal to or less than the above upper limit, the strength of the electric field at the end can be further increased.

[0061] The height h16 of the wall portion 16 is not particularly limited, and is preferably, for example, 1 to 10 mm.

[0062] The second electrode 20 may be any electrode that functions as a ground electrode, and may be, for example, a flat electrode. Examples of materials for the second electrode 20 include metals such as stainless steel, copper, and tungsten, and carbon.

[0063] In a plan view, the area of ​​the second electrode 20 is larger than the area of ​​the first electrode 10. When the area of ​​the second electrode 20 is larger, the plasma can be irradiated from the first electrode 10 to the irradiation target over a wider range.

[0064] The power supply unit 30 is only required to apply a voltage between the first electrode 10 and the second electrode and generate plasma between the two electrodes. The power supply unit 30 switches on and off the supply of electricity to the electrodes. The power supply unit 30 also adjusts the voltage and frequency applied to the electrodes. An example of the power supply unit 30 is a circuit connected to an external power source and including an inverter. Another example of the power supply unit 30 is a secondary battery.

[0065] In this embodiment, the container 40 functions as an irradiation field. The irradiation field may be any place where an irradiation target is positioned in a molecule introduction method described later. Therefore, if the container 40 is not a part of the configuration of the molecule introduction device 1, the second electrode 20 on which the container 40 is placed may serve as the irradiation field. The container 40 may be any container that can accommodate an irradiation target, such as each well of a microplate. The inner bottom surface of the container 40 is spaced apart from the tip of the first electrode 10 (that is, the irradiation body 14).

[0066] The material of the container 40 may be a conductive material or an insulating material. However, an insulating material is preferable from the viewpoint of preventing localized discharge due to high voltage. Examples of insulating materials include resin, ceramics, and glass. Examples of resins include polystyrene, polyolefin, and polyester.

[0067] The control unit 50 controls the power supply unit 30 and a liquid addition unit (not shown) by a computer. The computer of the control unit 50 includes a processing unit (CPU), a main memory, a storage area, and an input / output (I / O) circuit, and reads out a program recorded in the storage and performs processing by the CPU using the memory as a working area. The control unit 50 controls a liquid injection step in which the liquid addition unit injects a liquid containing molecules including nucleic acids into the container 40, a voltage application step in which the power supply unit 30 applies a voltage between the first electrode 10 and the second electrode 20, and the timing of execution of the liquid injection step and the voltage application step.

[0068] The liquid adding unit injects a liquid containing molecules including nucleic acids into the container 40 in response to a command from the control unit 50 .

[0069] The molecule introduction device 1 of this embodiment may further include an image acquisition unit (not shown) that acquires an image of the cells in the container 40. In this case, the control unit 50 analyzes image data from the image acquisition unit and controls the liquid injection step in which the liquid addition unit injects a liquid containing molecules including nucleic acids into the container 40, the voltage application step in which the power supply unit 30 applies a voltage between the first electrode 10 and the second electrode 20, and the timing of execution of the liquid injection step and the voltage application step.

[0070] In the introduction step C of the molecule introduction method of the present invention, plasma is irradiated into the container 40 while the cells, etc. 44 are in contact with the liquid 42 containing the molecules containing nucleic acid. Through plasma irradiation, the molecules containing nucleic acid are introduced into the cells, etc. 44. Note that in the introduction step (C), plasma may be irradiated into the container 40 while the cells, etc. 44 are in contact with the liquid 42 containing the molecules containing nucleic acid, thereby introducing the molecules containing nucleic acid into the cells, etc. 44.

[0071] In plasma irradiation, power is fed from the power feeder 30 to the first electrode 10 , and a voltage is applied between the first electrode 10 and the second electrode 20 . When a voltage is applied between the electrodes, an electric field is concentrated at the lower edge of the wall 16, and plasma is generated from the electric field concentration point toward the second electrode 20. At this time, because the wall 16 is cylindrical, the plasma can be irradiated over a wide area of ​​the target. By irradiating the plasma over a wide area, molecules containing nucleic acids can be introduced into cells, etc. over a wide area of ​​the target with a single plasma irradiation. This increases the amount introduced per unit time and further improves the introduction efficiency.

[0072] The distance L40 from the lower end of the first electrode 10 (the lower end surface of the wall portion 16) to the irradiation target (a liquid in which molecules containing nucleic acid and cells 44 coexist) is preferably 0.1 to 5 mm. When the distance L40 is within the above range, plasma can be generated more stably.

[0073] The AC voltage applied between the first electrode 10 and the second electrode 20 is preferably 1 to 30 kVpp, more preferably 5 to 20 kVpp. Setting the AC voltage to the above lower limit or above can further increase the introduction efficiency. Setting the AC voltage to the above upper limit or below can further reduce damage to cells, etc. The unit "Vpp (Volt peak to peak)" representing the AC voltage is the potential difference between the highest and lowest values ​​of the AC voltage waveform.

[0074] The frequency of the alternating current applied between the first electrode 10 and the second electrode 20 is preferably 1 to 500 kHz, more preferably 10 to 200 kHz. When the frequency is equal to or greater than the lower limit, the introduction efficiency can be further increased. When the frequency is equal to or less than the upper limit, damage to cells can be further reduced.

[0075] The plasma irradiation time in the irradiation step is, for example, preferably 10 nanoseconds to 1 second, and more preferably 100 nanoseconds to 0.1 second. If the irradiation time is equal to or greater than the above lower limit, the amount of molecules containing nucleic acid introduced into cells, etc. 44 can be further increased. If the irradiation time is equal to or less than the above upper limit, damage to cells, etc. can be further reduced.

[0076] In the irradiation step, the environment inside the container 40 is not particularly limited, but may be, for example, in the presence of CO2. The CO2 concentration is, for example, 1 to 10% by volume.

[0077] [Action and effect] The molecule transfer method of the present invention was able to achieve superior gene transfer efficiency compared to the case where step A of preparing cells was not performed. [Example]

[0078] The present invention will be described in more detail below with reference to experimental examples, but the present invention is not limited to these examples. Experimental Examples 1, 3, 17 and 19 are working examples, and Experimental Examples 2, 4 to 16, 18 and 20 are comparative examples.

[0079] [Transfection efficiency of molecules containing nucleic acids] Experimental Procedure (Nucleic acid-containing molecules) As a molecule containing a nucleic acid, pCMV-EGFP (a plasmid containing the eGFP gene sequence, which is a green fluorescent protein gene; SEQ ID NO: 1) was used.

[0080] (preculture) ADSCs (adipose-derived stem cells, manufactured by LONZA Corporation, PT-5006) were cultured in a CO2 incubator (manufactured by PHC Corporation, MCO-170AICUVD) at 37°C and 5% CO2 by volume using the cell culture medium shown in Table 1. 2 (Thermo Scientific) every 3 to 4 days, and the cells at the third passage after freezing and thawing were used for the molecular plasma introduction test.

[0081] [Table 1]

[0082] (cell seeding) <Experimental Example 1> (main culture) The pre-cultured ADSCs were collected and plated in a 48-well plate (Falcon® Cell Culture 48-well multiwell plate with flat bottom and lid, well area: 0.95 cm). 2 ) based on viable cells, 1.0 × 10 4 cells / well (1.1 × 10 4 cells / cm 2 After seeding, the cells were incubated (left stationary) for 48 hours under conditions of 37°C and 5% CO2 by volume.

[0083] (Molecular plasma introduction) After incubation, the medium was removed from the 48-well plate, and 5 μL of a liquid containing pCMV-EGFP adjusted to a concentration of 1 μg / μL was added to the center of each well and allowed to stand for 5 minutes. After incubation, the liquid containing pCMV-EGFP was removed, and plasma irradiation was performed. After incubation for 3 minutes, 300 μL of the cell culture medium shown in Table 1 was added, and the plate was incubated at 37°C and 5% CO2 by volume for 24 or 48 hours.

[0084] For plasma irradiation, a molecular introduction device shown in Figure 1 was used. <Molecular introduction device> The following molecular introduction device was used. First electrode: One end of a 3 mm diameter stainless steel cylinder was covered with a 0.5 mm thick alumina cap. A 0.1 mm diameter through-hole was formed by laser processing on the surface of the alumina cap facing the second electrode to form the first electrode. Power frequency: 50kHz Discharge voltage: 14kV p-p Discharge time: 40ms Discharge points per well: 1 Distance from the center of the irradiation container to the plasma irradiation point: 1.0 mm DNA amount: pCMV-EGFP, 5 μg per well

[0085] One to two days after transfection, the medium was removed, the cells were washed with PBS, and trypsin / EDTA (LONZA, CC-5012) was added. The cells were then incubated at 37°C and 5% CO2 for 3 minutes to detach the cells. The cells were then diluted with an equal volume of trypsin neutralizing solution (TNS) (LONZA, CC-5002). The number of GFP-positive cells was measured using a flow cytometer (Thermo Scientific, Attune NxT flow cytometer, Model: AFC2). The transfection efficiency was expressed as an index, with the total number of viable cells set to 100. Viability was calculated by dividing the total number of viable cells in the plasma-unexposed group by 100 (denominator) and the number of viable cells in each plasma-exposed group by the numerator.

[0086] <Experimental Examples 2-3> Cell seeding was 0.1 x 10 4 cells / well (0.11 × 10 4 cells / cm 2 ) and 2.0 × 10 4 cells / well (2.1 × 10 4 cells / cm 2 The experiments were carried out in the same manner as in Experimental Example 1, except that the seeds were sown so that the diameter of the seedlings was 1 / 2.

[0087] <Experimental Examples 4-6> Experiments were carried out in the same manner as in Experimental Examples 1 to 3 except that molecular plasma was not introduced, and these were designated Experimental Examples 4 to 6, respectively.

[0088] <Experimental Examples 7-9> Cell seeding was 0.1 x 10 4 cells / well (0.11 × 10 4 cells / cm 2 ), 1.0×10 4 cells / well (1.1 × 10 4 cells / cm 2 ) and 2.0 × 10 4 cells / well (2.1 × 10 4 cells / cm 2 Experiments were carried out in the same manner as in Experimental Example 1, except that the incubation time after seeding was 24 hours. These experiments were named Experimental Examples 7 to 9.

[0089] <Experimental Examples 10-12> Experiments were carried out in the same manner as in Experimental Examples 7 to 9, except that molecular plasma was not introduced, and these were designated as Experimental Examples 10 to 12, respectively.

[0090] Figure 3 is a graph showing the molecule introduction efficiency and cell viability in Experimental Examples 1 to 6. Figure 4 is a graph showing the molecule introduction efficiency and cell viability in Experimental Examples 7 to 12. In Figures 3 and 4, the molecule introduction efficiency is represented by a bar, and the cell viability is represented by a dot. As shown in Figure 3, Experimental Examples 1 and 3 had high viability, with the molecule introduction efficiency reaching a high value of 5.8% or more. On the other hand, when the cell seeding density was 0.5 x 10 4 cells / cm 2 In Experimental Example 2, where the concentration was less than 100 ppm, the molecule introduction efficiency was low.

[0091] Furthermore, as shown in Figure 4, in Experimental Examples 7 to 12, in which the incubation time after seeding was 24 hours, the molecule introduction efficiency was low. As shown in Figures 3 and 4, in Experimental Examples 4 to 6 and 10 to 12, in which molecular plasma introduction was not performed, the molecule introduction efficiency was low.

[0092] [Proteome analysis] Proteome analysis was performed using the cells of Experimental Example 1 and the cells of Experimental Example 8, which had different efficiencies of molecule introduction into the cells. The cells used were collected after (main culture) in Experimental Example 1 and Experimental Example 8.

[0093] 5 x 10 per specimen 5 Cells were collected from multiple wells so that there were at least 100 cells (three samples were prepared for each of Experimental Examples 1 and 8). The collected cells were centrifuged at 200 g for 5 minutes, washed with PBS, and centrifuged again under the same conditions to form a pellet. The pellet was frozen in liquid nitrogen for 10 seconds and then sent to Promega's DIA proteome analysis service for deep DIA proteome analysis.

[0094] After peptide extraction, the samples were measured using nanoLC-MS / MS and analyzed using DIA-NN (1.8.1), and proteins and peptides were identified and quantified (performed by a contractor).

[0095] After Log2 transformation and missing value processing, a significance test (P<0.05) (two groups: Ttest) was performed (conducted by a contractor). From the results, proteins with a 2-fold or greater variation in the mean value of each group (Log2 (Experimental Example 1 / Experimental Example 8) > 1) and a difference between groups of P<0.05 or greater were extracted. GOterm analysis was performed using the gene symbols of the accession numbers listed in the Master Accession Numbers for the 82 proteins that were elevated 2-fold or greater in Experimental Example 1. The analysis was evaluated from the perspectives of biological process (BP), molecular function (MF), and cellular component (CC). Visualization was performed using the R package "clusterProfiler" (4.10.1).

[0096] In addition, proteins whose mean values ​​for each group varied by 1.4 times or more (Log2 (Experimental Example 1 / Experimental Example 8) > 0.5) and whose difference between groups was P < 0.05 or more were extracted, and using the gene symbols of the accession numbers listed in the Master Accession Number of the 293 proteins whose expression levels increased by 1.4 times or more in Experimental Example 1, evaluation was performed using KEGG_PATHWAY from the ENTERZ_GENE_ID of the genes with differential expression using DAVID's Functional Annotation Tool (v2023q4) (https: / / david.ncifcrf.gov / ).

[0097] Proteome analysis yielded results regarding the expression of genes encoding proteins related to the cell cycle. It was found that the cell group of Experimental Example 1 had relatively high expression of genes encoding proteins expressed in the G2 phase, specifically, CDK1 and CDC25C, compared to the cell group of Experimental Example 8. Because CDK1 and CDC25C are genes that are highly expressed in the G2 phase, it was suggested that the cell group of Experimental Example 1 had a high proportion of cells in the G2 phase. While CDK1 and CDC25C are genes that are expected to be expressed in the M phase as well, in many cells, the M phase lasts for half or less the time of the G2 phase, so it is believed that most of the cell group of Example 1 was in the G2 phase.

[0098] 5 shows the results of the Biological process of Goterm analysis. The results show that many of the genes detected in greater numbers in Experimental Example 1 than in Experimental Example 8 are genes involved in nuclear division.

[0099] [Molecule delivery into cells by lipofection] <Experimental Example 13> (Preparation of Liposomes) To 25 μL of Opti-MEM® I Reduced Serum Medium (Thermo Fisher Scientific, 31985062), 0.25 μL of a liquid containing pCMV-EGFP adjusted to a concentration of 1 μg / μL and 0.5 μL of TransIT®-2020 Transfection Reagent (mirus bio, V5400) were added and mixed. After allowing to stand for 15 minutes, the entire volume was added to the culture medium for the cultured cells.

[0100] (Liposome introduction) Molecular introduction into cells by lipofection was carried out using the cells of Experimental Example 1. The cells used were collected after (main culture) of Experimental Example 1. The experiment was carried out using the same procedure as in Experimental Example 1 (molecular plasma introduction), except that the above-mentioned liposome was used instead of pCMV-EGFP.

[0101] <Experimental Example 14> The experiment was carried out in the same manner as in Experimental Example 13, except that the cells in Experimental Example 8 were used instead of the cells in Experimental Example 1.

[0102] Figure 6 is a graph showing the molecule introduction efficiency in Experimental Examples 13 and 14. As shown in Figure 6, the molecule introduction efficiency was higher in Experimental Example 13, which is a cell group with a large number of cells in the G2 phase of the cell cycle, than in Experimental Example 14.

[0103] [Transfection of mRNA into cells] <Experimental Example 15> (mRNA introduction) The cells of Experimental Example 1 were used to introduce mRNA (CleanCap EGFP mRNA (5 moU), manufactured by TriLink BioTechnologies, Inc., L-7201-100) into the cells. The cells used were collected after the (main culture) of Experimental Example 1. The experiment was carried out using the same procedure as in Experimental Example 1 (molecular plasma introduction), except that the above-mentioned mRNA was used instead of pCMV-EGFP.

[0104] <Experimental Example 16> The experiment was carried out in the same manner as in Experimental Example 15, except that the cells in Experimental Example 8 were used instead of the cells in Experimental Example 1.

[0105] Figure 7 is a graph showing the mRNA transfection efficiency in Experimental Examples 15 and 16. As shown in Figure 7, the mRNA transfection efficiency was higher in Experimental Example 15, which is a cell group with a large number of cells in the G2 phase of the cell cycle, than in Experimental Example 16.

[0106] [Sequence List] Sequence number 1 / Sequence name: pCMV-EGFP / Sequence length: 6197 bp [Explanation of symbols]

[0107] 1 molecule introduction device, 10 first electrode, 12 electrode body, 14 irradiation body, 16 wall portion, 20 second electrode, 30 power supply portion, 40 container, 42 liquid containing molecules including nucleic acid, 44 cell, O1 tube axis, θ1 angle

Claims

1. Step A of preparing cells for introducing a molecule containing a nucleic acid; a step B of mixing the cells obtained in the step A with a molecule containing the nucleic acid; and step C of introducing the molecule comprising the nucleic acid into the cell by endocytosis of the cell, The molecule introduction method, wherein the step A is a step of adjusting the cell cycle so that the cells are in the G2 phase, which precedes the M phase, which is the division phase.

2. The molecule introduction method according to claim 1 , wherein step C is a step of irradiating a mixture of the cells and the nucleic acid-containing molecules with plasma.

3. The step A is 0.5 × 10 ４ ~2.5 x 10 ４ cells / cm ２ 3. The method for introducing a molecule according to claim 1, wherein the step of inoculating the cells is carried out in a manner similar to that described above, and culturing the cells for more than 24 hours but less than 72 hours.

4. The molecule introduction method according to claim 1 or 2, wherein step A is a step of preparing a cell population having a high proportion of cells in the G2 phase.

5. The molecule introduction method according to claim 1 or 2, wherein the cells are stem cells.

6. 3. The molecule introduction method according to claim 1, wherein the molecule is at least one molecule selected from the group consisting of DNA and RNA.

Images