Pretreatment method, molecule introduction method, method for producing molecule introduced cell, molecule introduction efficiency improving agent, and use as molecule introduction efficiency improving agent

Abstract

Provided is a pretreatment method that is performed before introducing a molecule into a target cell, said pretreatment method comprising a pretreatment step for promoting expression of at least one type of protein selected from the group consisting of Caveolin 1, Caveolin 2, and Cavin 1 of the target cell. In the pretreatment step, the target cell is brought into contact with at least one chemical agent selected from the group consisting of insulin and an insulin analog.

Description

Pretreatment method, molecule introduction method, method for producing molecule-introduced cells, molecule introduction efficiency improver, and use as molecule introduction efficiency improver

[0001] The present invention relates to a pretreatment method, a molecule introduction method, a method for producing molecule-introduced cells, a molecule introduction efficiency improver, and use of the molecule introduction efficiency improver. This application claims priority to Japanese Patent Application No. 2023-169641, filed on September 29, 2023, the contents of which are incorporated herein by reference.

[0002] With the decoding of the human genome and mouse genome, when conducting molecular biology-related research and development or drug development in fields such as medicine or pharmacology, there is an increasing need 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 molecules into target cells: chemical methods, physical methods, and biological methods. Chemical methods involve using transfection reagents such as cationic polymers, cationic lipids, or calcium phosphate to allow molecules to be taken up by target cells through endocytosis. Physical methods involve directly introducing molecules 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 in viral particles into target cells using the infectivity of the virus.

[0004] Non-Patent Document 1 describes that when a gene is introduced into hematopoietic stem cells using a retroviral vector, the addition of fibronectin improves the efficiency of gene introduction.

[0005] Non-Patent Document 2 describes that when gene transfer into blood cells using a retroviral vector is performed, the transfer efficiency is improved by adding a recombinant protein (RetroNectin (registered trademark), recombinant human fibronectin CH-296) containing three functional domains of human fibronectin: the cell adhesion domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 site.

[0006] Patent Document 1 describes a method for introducing an exogenous gene into target cells using a retroviral vector, which includes the steps of: (a) placing a liquid containing a retroviral vector carrying an exogenous gene into a culture vessel onto which a retrovirus-binding substance such as fibronectin or a fibronectin fragment CH-296 [RetroNectin] has been immobilized, and then incubating the culture vessel at a temperature below 25°C for 4 hours or more to obtain a culture vessel to which the retroviral vector has bound; and (b) placing target cells into the culture vessel obtained by step (a) and incubating the culture vessel.

[0007] Patent Document 2 describes a method for producing a cell population into which a desired gene has been introduced, comprising: (1) a step of culturing a cell population containing T cells and / or T cell precursors in a container containing fibronectin or a fragment thereof and a CD3 ligand; and (2) a step of adding a vector carrying the desired gene to the container of step (1).

[0008] International Publication No. 2012 / 002452 International Publication No. 2009 / 119793

[0009] Moritz, T. et al., "Fibronectin Improves Transduction of Reconstituting Hematopoietic Stem Cells by Retroviral Vectors: Evidence of Direct Viral Binding to Chymotryptic Carboxy-Terminal Fragments," Blood, August 1, 1996, Vol. 88, No. 3, pp. 855-862; Hanenberg, H. et al., "Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells," Nature Medicine, August 1996, Vol. 2, No. 8, pp. 876-882

[0010] There is a need for a technology that can improve the efficiency of molecule introduction into target cells. An objective of the present invention is to provide a technology that can improve the efficiency of molecule introduction into target cells.

[0011] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that the efficiency of molecule introduction into target cells can be improved by promoting endocytosis via the caveolae pathway before introducing a molecule into the target cells. In another aspect, the present inventors have found that the efficiency of molecule introduction into target cells can be improved by contacting the target cells with insulin or an insulin analog. The present invention includes the following aspects.

[0012] [1] A pretreatment method carried out before introducing a molecule into target cells, comprising a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2, and Cavin 1 in the target cells. [2] The pretreatment method according to [1], comprising a pretreatment step of contacting the target cells with at least one drug selected from the group consisting of insulin and insulin analogs. [3] The pretreatment method according to [2], wherein the target cells are cultured in a medium containing the drug in the pretreatment step. [4] The pretreatment method according to any one of [1] to [3], wherein the target cells are vertebrate cells. [5] The pretreatment method according to [4], wherein the vertebrate cells are human-derived cells. [6] The pretreatment method according to [5], wherein the human-derived cells are at least one type of cultured cell selected from the group consisting of blood-derived cells, HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, and iPS cells. [7] The pretreatment method according to any one of [1] to [5], wherein the target cells are CD3-positive cells derived from blood. [8] The pretreatment method according to any one of [2] to [7], wherein the contact time between the target cells and the drug in the pretreatment step is 20 to 48 hours. [9] The pretreatment method according to any one of [2] to [8], wherein the concentration of the drug when contacting the target cells with the drug in the pretreatment step is 5 to 150 μg / mL.

[10] The pretreatment method according to any one of [2] to [9], wherein the contact time between the target cells and the drug in the pretreatment step is 2 to 8 hours.

[11] The pretreatment method according to any one of [2] to

[10] , wherein the concentration of the drug when contacting the target cells with the drug in the pretreatment step is 100 to 200 μg / mL.

[0013]

[12] A molecule introduction method for introducing a molecule into a target cell, comprising: a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2, and Cavin 1 in the target cell; and a molecule introduction step of contacting the target cell with a liquid containing the molecule to be introduced, thereby introducing the molecule into the target cell.

[13] The molecule introduction method according to

[12] , wherein the pretreatment step involves contacting the target cell with at least one drug selected from the group consisting of insulin and insulin analogues.

[14] The molecule introduction method according to

[12] or

[13] , wherein the molecule introduction step involves irradiating the target cell with plasma.

[15] The molecule introduction method according to any of

[12] to

[14] , wherein the target cell is a vertebrate cell.

[16] The molecule introduction method according to

[15] , wherein the vertebrate cell is a human-derived cell.

[17] The molecule introduction method according to

[16] , wherein the human-derived cells are at least one type of cultured cell selected from the group consisting of blood-derived cells, HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, and iPS cells.

[18] The molecule introduction method according to any of

[12] to

[16] , wherein the target cells are blood-derived CD3-positive cells.

[19] The molecule introduction method according to any of

[13] to

[18] , wherein the contact time between the target cells and the drug in the pretreatment step is 20 to 30 hours.

[20] The molecule introduction method according to any of

[13] to

[19] , wherein the concentration of the drug when contacting the target cells with the drug in the pretreatment step is 5 to 150 μg / mL.

[21] The molecule introduction method according to any of

[13] to

[20] , wherein the contact time between the target cells and the drug in the pretreatment step is 2 to 8 hours.

[22] The molecule introduction method according to any one of

[13] to

[21] , wherein in the pretreatment step, the concentration of the drug when contacting the target cells with the drug is 100 to 200 μg / mL.

[0014]

[23] A method for producing molecule-introduced cells, comprising: a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2, and Cavin 1 in target cells; and a molecule introduction step of contacting the target cells with a liquid containing a molecule and introducing the molecule into the cells.

[24] The method for producing molecule-introduced cells according to

[23] , wherein the pretreatment step involves contacting the target cells with at least one drug selected from the group consisting of insulin and insulin analogues.

[25] The method for producing molecule-introduced cells according to

[23] or

[24] , wherein the molecule introduction step involves irradiating the target cells with plasma.

[26] The method according to any one of

[23] to

[25] , wherein the liquid containing the molecule to be introduced contains a transfection reagent containing the molecule.

[26] ' The method according to any one of

[23] to

[25] , wherein the target cells are vertebrate cells.

[27] The method for producing molecule-introduced cells according to

[26] ', wherein the vertebrate cells are human-derived cells.

[28] The method for producing molecule-introduced cells according to

[27] , wherein the human-derived cells are at least one type of cultured cell selected from the group consisting of blood-derived cells, HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, and iPS cells.

[29] The method for producing molecule-introduced cells according to any of

[23] to

[27] , wherein the target cells are blood-derived CD3-positive cells.

[30] The method for producing molecule-introduced cells according to any of

[24] to

[29] , wherein the pretreatment step involves contacting the target cells with the drug for 20 to 48 hours.

[31] The method for producing molecule-introduced cells according to any of

[24] to

[30] , wherein the pretreatment step involves contacting the target cells with the drug at a concentration of 5 to 150 μg / mL.

[32] The method for producing molecule-introduced cells according to any of

[24] to

[31] , wherein the pretreatment step involves contacting the target cells with the drug for 2 to 8 hours.

[33] The method for producing molecule-introduced cells according to any one of

[24] to

[32] , wherein in the pretreatment step, the concentration of the drug when contacting the target cells with the drug is 100 to 200 μg / mL.

[0015]

[34] A molecule transfer efficiency improver for improving the efficiency of molecule transfer into target cells, comprising at least one drug selected from the group consisting of insulin and insulin analogs as an active ingredient.

[0016]

[35] Use of at least one agent selected from the group consisting of insulin and insulin analogues as an agent for improving molecule introduction efficiency, for improving the introduction efficiency of a molecule into a target cell.

[0017]

[36] A method for treating blood cancer, comprising: a collection step of collecting T cells from a patient; a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2, and Cavin 1 in the T cells; a gene transfer step of introducing a chimeric antigen receptor gene into the T cells; a culture step of culturing the T cells introduced with the chimeric antigen receptor gene; and an administration step of administering the cultured T cells to the patient, wherein the T cells are irradiated with plasma in the gene transfer step.

[37] The method of treatment according to

[36] , wherein the T cells are contacted with at least one drug selected from the group consisting of insulin and insulin analogues in the pretreatment step.

[38] The method of treatment according to

[36] or

[37] , wherein the blood cancer is at least one selected from the group consisting of B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, transformed low-grade non-Hodgkin's lymphoma, high-grade B-cell lymphoma, and follicular lymphoma.

[39] The method of treatment according to

[37] or

[38] , wherein the contact time between the T cells and the drug in the pretreatment step is 20 to 30 hours.

[40] The method of treatment according to any of

[37] to

[39] , wherein the concentration of the drug when contacting the T cells with the drug in the pretreatment step is 5 to 150 μg / mL.

[41] The method of treatment according to any of

[37] to

[40] , wherein the contact time between the T cells and the drug in the pretreatment step is 2 to 8 hours.

[42] The method of treatment according to any one of

[37] to

[41] , wherein in the pretreatment step, the concentration of the drug when the T cells are brought into contact with the drug is 100 to 200 μg / mL.

[0018] According to the present invention, a technique can be provided that can improve the efficiency of molecule introduction into target cells.

[0019] FIG. 1 is a cross-sectional view of a molecule introduction device according to one embodiment of the present invention. FIG. 2 is an end view of the irradiator 14 of FIG. 1. FIG. 3 is a graph showing the results of Experimental Example 1. FIG. 4 is a graph showing the results of Experimental Example 2. FIG. 5 is a graph showing the results of Experimental Example 3. FIG. 6 is a graph showing the results of Experimental Example 4. FIG. 7 is a graph showing the experimental results (expression levels of cell membrane-associated genes) for T cells in Experimental Example 5. FIG. 8 is a graph showing the experimental results (expression levels of cell proliferation-associated genes) for T cells in Experimental Example 5. FIG. 9 is a graph showing the experimental results (expression levels of cell membrane-associated genes) for Jurkat cells in Experimental Example 5. FIG. 10 is a graph showing the experimental results (expression levels of cell membrane-associated genes) for HEK293F cells in Experimental Example 5. FIG. 11 is a graph showing the experimental results (expression levels of endosome-associated genes) for HEK293F cells in Experimental Example 5. Figure 12 is a graph showing the experimental results (expression levels of cell proliferation-related genes) for HEK293F cells in Experimental Example 5. Figure 13 is a graph showing the molecule introduction efficiency with or without the addition of insulin. Figure 14 is an image showing the results of Western blotting of Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells. Figure 15 is a graph showing the molecule introduction efficiency by lipofection in Cav1-overexpressing cells, Cav2-overexpressing cells, Cavin1-overexpressing cells, and wild-type Jurkat cells. Figure 16 is a graph showing the molecule introduction efficiency by plasma irradiation in Cav1-overexpressing cells, Cav2-overexpressing cells, Cavin1-overexpressing cells, and wild-type Jurkat cells.

[0020] In the present invention, the symbol "to" indicating a numerical range means that the numerical values ​​before and after it are included as the lower and upper limits. In the present invention, the names and abbreviations of amino acids follow the nomenclature of IUPAC (International Union of Pure and Applied Chemistry) or IUB (International Union of Biochemistry). Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0021] [Pretreatment Method] A pretreatment method according to an embodiment of the present invention is a pretreatment method carried out before introducing a molecule into a target cell, and includes a pretreatment step of promoting the expression of at least one protein selected from the group consisting of Caveolin 1 (hereinafter referred to as Cav1), Caveolin 2 (hereinafter referred to as Cav2), and Cavin 1 in the target cell.

[0022] Target cells refer to cells into which molecules are introduced and are not limited to any particular type of cell. Specific examples of such target cells include eukaryotic cells, preferably animal cells, more preferably vertebrate cells, and even more preferably mammalian cells. Mammalian cells include cells derived from mammals, including humans, and cells derived from non-human mammals. Non-human mammals include mice, rats, chimpanzees, gorillas, orangutans, monkeys, cows, pigs, horses, dogs, and cats. A single type of target cell may be used, or two or more types may be mixed. The aforementioned human-derived cells include cells intended to be reintroduced into a human individual. The aforementioned human-derived cells also include cells cultured from cells harvested from a human individual. Furthermore, the aforementioned non-human animal cells and plant cells include cells present within an individual, cells present within a tissue, cells harvested from an individual or tissue, and cells cultured from cells harvested from an individual or tissue.

[0023] Suitable examples of target cells include blood-derived cells such as T cells, HEK293F cells, mesenchymal stem cells, adipose-derived stem cells, and iPS cells. Other suitable examples of target cells include blood-derived cells (e.g., macrophages, CD3-positive cells including T cells, B cells, NK cells, monocytes, dendritic cells, plasma cells, lymphocytes, etc.), mesenchymal stem cells, adipose-derived stem / progenitor cells, adipose-derived stem cells, iPS cells, HEK293F cells, Jurkat cells, and HuT-78 cells. It is preferable to use at least one type of target cell selected from the group consisting of these cells.

[0024] The molecule to be introduced into the target cell is not particularly limited, and examples thereof include molecules containing nucleic acids, such as complementary DNA (cDNA), DNA encoding microRNA (miRNA), or DNA encoding small hairpin RNA (shRNA), or liposomes containing these.

[0025] 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, and after translation in the ribosome, it can express proteins such as enzymes and fluorescent proteins.

[0026] 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.

[0027] 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.

[0028] shRNAs are hairpin-shaped RNA sequences used for gene silencing by RNA interference.

[0029] In the pretreatment step, the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 in the target cells is promoted. A preferred method for promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 is to contact the target cells with at least one agent selected from the group consisting of insulin and insulin analogs.

[0030] When substances are transported from the cell surface to the interior of the cell by endocytosis, they are transported in a vesicle-encapsulated state. Endocytosis can be divided into two pathways: the clathrin-mediated pathway, in which substances are transported via clathrin coating, and the caveolae pathway, in which substances are transported via caveolae.

[0031] Caveolae are formed when Cav1 and Cav2 bind to lipid rafts to form oligomers consisting of 14 to 16 molecules. Cavin 1 is a major regulator of caveolae formation. Promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin 1 in target cells activates endocytosis via the caveolae pathway, thereby improving the efficiency of molecule delivery into target cells.

[0032] A method for promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin 1 in target cells includes contacting the target cells with at least one agent selected from the group consisting of insulin and insulin analogs.

[0033] In particular, in cells such as Jurkat cells and T cells, which are CD3-positive cells with low efficiency of molecule introduction into cells, promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 activates endocytosis via the caveolae pathway, thereby improving the efficiency of molecule introduction.

[0034] The insulin is not particularly limited, but examples thereof include human insulin, bovine insulin, and porcine insulin. When the target cells are human-derived cells, human insulin is preferred. The insulin analog is preferably one obtained by modifying the amino acid sequence of human insulin, and commercially available products are available.

[0035] The method for contacting target cells with at least one drug selected from the group consisting of insulin and insulin analogs (hereinafter also simply referred to as "drug") is not particularly limited, but examples include a method of adding the drug to a medium used for pre-culture of target cells, or a method of directly contacting target cells with the drug.

[0036] The medium is not particularly limited as long as it can be used for culturing (pre-culturing) target cells.

[0037] The concentration of the drug when contacting the target cells with the drug is not particularly limited, but is preferably 5 to 150 μg / mL, more preferably 5 to 100 μg / mL, and even more preferably 5 to 50 μg / mL. In this case, the contact time between the target cells and the drug is not particularly limited, but is preferably 20 to 30 hours, and more preferably 24 to 27 hours.

[0038] The concentration of the drug when contacting the target cells with the drug may be 100 to 200 μg / mL, and in this case, the contact time between the target cells and the drug is preferably 2 to 8 hours, more preferably 3 to 6 hours.

[0039] Methods for promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 in target cells are not limited to those described above. For example, a gene that overexpresses at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 may be introduced into target cells. For example, a method may be used in which a plasmid encoding at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 is introduced into target cells.

[0040] [Molecule introduction method] The molecule introduction method of the present invention is a molecule introduction method for introducing a molecule into a target cell, and includes a pretreatment step of promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 in the target cell, and a molecule introduction step of contacting the target cell with a liquid containing the molecule.

[0041] The pretreatment step is the same as the pretreatment step in the pretreatment method described above, and therefore a description thereof will be omitted.

[0042] The method for introducing molecules into target cells in the molecule introduction step is not particularly limited, and includes chemical methods using transfection reagents such as lipofection reagents, cationic polymers, cationic lipids, or calcium phosphate to incorporate molecules into target cells via endocytosis; physical methods using physical manipulations such as microinjection, sonoporation, laser irradiation, or electroporation to directly introduce molecules into target cells; and biological methods using viral vectors such as retroviruses, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses to transfer nucleic acids within viral particles into target cells via the infectious ability of the virus. Plasma introduction methods can also be used, in which target cells are irradiated with plasma to activate their activity and then the molecules are introduced into the target cells. Plasma introduction methods are a relatively new method for introducing molecules into target cells by irradiating them with discharge plasma. This method has the advantages of eliminating the risk of carcinogenesis because it does not use viruses, and eliminating the risk of cellular dysfunction because it does not use chemicals. Furthermore, since no current is passed directly through the cells, there is minimal damage to the cells, and the survival rate of molecules-introduced cells is high.

[0043] By carrying out a pretreatment step that promotes the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 in target cells, endocytosis via the caveolae pathway is activated, thereby improving the efficiency of molecule introduction.

[0044] <Molecular Introduction Device> Hereinafter, a molecular introduction device that can be suitably used in the molecule introduction method of the present invention will be described with reference to one embodiment. A case will be described in which at least one drug selected from the group consisting of insulin and insulin analogs is contacted as a pretreatment step. The molecule introduction device of this embodiment has a first electrode, a second electrode, and an irradiation field located between the first and second electrodes. The molecule introduction device introduces a molecule into a target cell. The molecule introduction device generates plasma from the first electrode, irradiates an irradiation object in which the target cell and the molecule coexist, and introduces the molecule into the target cell.

[0045] 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.

[0046] The second electrode 20 is spaced apart from the first electrode 10 and 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 placed above the second electrode 20 and is spaced apart from the first electrode 10.

[0047] 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.

[0048] The electrode body 12 is a rod-shaped body extending in one direction. The electrode body 12 may have a hollow structure such as a cylindrical or polygonal tube, or a solid structure such as a columnar or polygonal pillar. In particular, if 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.

[0049] 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 a polygonal column, the outer diameter R12 is the diameter of a circumscribed circle of the cross section of the electrode body 12.

[0050] 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. Note that 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.

[0051] Examples of materials for the substrate 15 include metals such as stainless steel, copper, and tungsten, carbon, etc. Examples of materials for the wall 16 include metals such as stainless steel, copper, and tungsten, carbon, etc. The substrate 15 and the wall 16 may be integrally molded or may be individually molded and then joined together.

[0052] 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 .

[0053] 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 greater 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 improving the efficiency of molecule introduction into target cells (hereinafter simply referred to as "introduction efficiency"). When the outer diameter R16 is equal to or less than the above-mentioned upper limit, a stable discharge is more easily generated. Furthermore, the outer diameter R16 is preferably larger than the outer diameter R12. When the outer diameter R16 is larger, the plasma can be irradiated over a wider area, further improving the introduction efficiency. When the wall portion 16 is polygonal cylindrical, the outer diameter R16 is the diameter of the circumscribed circle of the cross section of the wall portion 16. When the irradiator 14 has two or more wall portions 16, the outer diameter R16 of the wall portion 16 is the outer diameter of the outermost wall portion 16.

[0054] 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 irradiator 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 innermost.

[0055] The angle θ1 between the lower end surface of the wall portion 16 and the outer surface of the wall portion 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 the wall portion 16 and the outer surface of the wall portion 16 is what is called an edge. If the angle θ1 is equal to or less than the above upper limit value, an electric field is concentrated at the edge, making it easier to generate plasma between the electrodes. The angle between the lower end surface of the wall portion 16 and the inner surface of the wall portion 16 is the same as the angle θ1. The angle between the lower end surface of the wall portion 16 and the inner surface of the wall portion 16 may be the same as or different from the angle θ1.

[0056] The thickness t16 of the wall portion 16 is preferably 1 mm or less, and more preferably 0.1 mm or less, for example. 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.

[0057] The height h16 of the wall portion 16 is not particularly limited, and is preferably, for example, 1 to 10 mm.

[0058] The second electrode 20 may be any electrode that functions as a ground electrode, such as a flat electrode. Examples of materials for the second electrode 20 include metals such as stainless steel, copper, and tungsten, and carbon.

[0059] 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.

[0060] The power supply unit 30 only needs 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 between starting and stopping 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.

[0061] In this embodiment, the container 40 functions as an irradiation field. The irradiation field may be any location where an irradiation target is positioned in the molecule introduction method described below. Therefore, if the container 40 is not 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 location 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 (i.e., the irradiator 14).

[0062] The container 40 may be made of either a conductive material or an insulating material. However, an insulating material is preferred 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.

[0063] The control unit 50 controls the power supply unit 30 and the liquid addition unit 60 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 work area. The control unit 50 controls a liquid injection step in which the liquid addition unit 60 injects a liquid containing a molecule introduction efficiency improver 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.

[0064] The liquid adding unit 60 injects a liquid containing a molecule introduction efficiency improving agent into the container 40 in response to a command from the control unit 50 .

[0065] The molecule introduction device 1 of this embodiment may further include an image acquisition unit (not shown) that acquires images of the target cells in the container 40. In this case, the control unit 50 analyzes image data from the image acquisition unit, and controls a liquid injection step in which the liquid addition unit 60 injects a liquid containing the molecule introduction efficiency improving agent 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.

[0066] As will be described later, the molecule transfer efficiency improver is an agent containing at least one drug selected from the group consisting of insulin and insulin analogs as an active ingredient.

[0067] [Method for producing molecule-introduced cells] In one aspect of the present invention, a method for producing molecule-introduced cells includes a pretreatment step of promoting expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1 in target cells, and a molecule introduction step of contacting the target cells with a liquid containing molecules. That is, one aspect of the present invention provides a method for producing molecule-introduced cells using the molecule introduction method described above. The pretreatment step and the molecule introduction step are the same as those in the molecule introduction method described above, and therefore their description will be omitted.

[0068] [Agent for improving molecule introduction efficiency and use as an agent for improving molecule introduction efficiency] At least one agent selected from the group consisting of insulin and insulin analogs can be used as an agent for improving molecule introduction efficiency during transfection of target cells. The agent for improving molecule introduction efficiency of the present invention contains at least one agent selected from the group consisting of insulin and insulin analogs as an active ingredient.

[0069] [Treatment method] The treatment method of the present invention is a method for treating blood cancer, comprising: a collection step of collecting T cells from a patient; a pretreatment step of promoting expression of at least one protein selected from the group consisting of Cav1, Cav2 and Cavin1 in the T cells; a gene transfer step of introducing a chimeric antigen receptor gene into the T cells; a culture step of culturing the T cells introduced with the chimeric antigen receptor gene; and an administration step of administering the cultured T cells to the patient, wherein the T cells are irradiated with plasma in the gene transfer step.

[0070] The pretreatment step is the same as the pretreatment step in the pretreatment method described above, and therefore a description thereof will be omitted.

[0071] The blood cancer is not particularly limited, but is preferably at least one type selected from the group consisting of B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, transformed low-grade non-Hodgkin's lymphoma, high-grade B-cell lymphoma, and follicular lymphoma.

[0072] In the pretreatment step, when the T cells are contacted with at least one drug selected from the group consisting of insulin and insulin analogs, the concentration of the drug is preferably 5 to 150 μg / mL, and in this case, the contact time between the T cells and the drug is preferably 20 to 48 hours. Furthermore, in the pretreatment step, the concentration of the drug when the T cells are contacted with the drug may be 100 to 200 μg / mL, and in this case, the contact time between the T cells and the drug is preferably 2 to 8 hours.

[0073] The treatment method of the present invention does not use a viral vector such as an adenovirus, and therefore does not involve the risk of side effects associated with the use of a viral vector directed at T cells in CAR-T therapy.

[0074] [Mechanism of Action] The mechanism of action is presumed to be as follows: (1) By promoting the expression of at least one protein selected from the group consisting of Cav1, Cav2, and Cavin1, membrane mobility is increased and endocytosis is activated (confirmed at the mRNA expression level). (2) Endosome activation. (3) As a result of the above (1) and (2), improved molecule introduction efficiency is achieved.

[0075] The present invention will be explained in more detail below with reference to experimental examples, but the present invention is not limited to the examples described below.

[0076] [Experimental Example 1] T cells 1.4 x 10 T cells (manufactured by LONZA) were placed in each well of a 48-well plate. ５ 400 μL of cells / well were seeded, and insulin was added to a final concentration of 0 μg / mL to 20 μg / mL. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 24 to 27 hours before the introduction of the molecule. ２ Prior to molecule introduction, the medium / supernatant was removed and a specified amount of EGFP plasmid (pCMV-EGFP) was added. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 48 hours. ２ The number of cells showing positive GFP fluorescence was measured using a flow cytometer, and the molecule introduction efficiency was expressed as an index, with the number of cells in the insulin-free group (insulin concentration 0 μg / mL) set at 100, to give the introduction efficiency. Figure 3 shows the relationship between insulin concentration (μg / mL) (horizontal axis) and introduction efficiency (%) (vertical axis).

[0077] Experimental Example 2 Jurkat Cells (1) 2.0 x 10 Jurkat cells (manufactured by ECACC (The European Collection of Authenticated Cell Cultures)) were placed in each well of a 6-well plate. ６ 500 cells / well / 5 mL were seeded, and insulin was added to a final concentration of 0 μg / mL to 150 μg / mL. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 24 to 27 hours before the introduction of the molecule. ２Prior to molecule introduction, the medium / supernatant was removed and a specified amount of EGFP plasmid (pCMV-EGFP) was added. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 48 hours. ２ The number of cells showing positive GFP fluorescence was measured using a flow cytometer, and the molecule introduction efficiency was expressed as an index, with the number of cells in the insulin-free group (insulin concentration 0 μg / mL) set at 100, to give the introduction efficiency. Figure 4 shows the relationship between insulin concentration (μg / mL) (horizontal axis) and introduction efficiency (%) (vertical axis).

[0078] Experimental Example 3 Jurkat cells (2) 2.0 x 10 Jurkat cells (manufactured by ECACC (The European Collection of Authenticated Cell Cultures)) were placed in each well of a 6-well plate. ６ 5 mL of cells / well were seeded and CO ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 20 hours. ２ Insulin was added to a final concentration of 0 μg / mL or 150 μg / mL. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 4 hours before the introduction of the molecule. ２ Prior to molecule introduction, the medium / supernatant was removed and a specified amount of EGFP plasmid (pCMV-EGFP) was added. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 48 hours. ２ The number of cells that showed positive GFP fluorescence was measured using a flow cytometer, and the molecule introduction efficiency was expressed as an index, with the number of cells in the insulin-free group (insulin concentration 0 μg / mL) set at 100, to give the introduction efficiency. Figure 5 shows the relationship between insulin concentration (μg / mL) (horizontal axis) and introduction efficiency (%) (vertical axis).

[0079] [Experimental Example 4] HEK293F cells 0.5 x 10 HEK293F cells (manufactured by ThermoFisher) were placed in each well of a 6-well plate. ６3 ml / well of cells were seeded, and insulin was added to a final concentration of 0 μg / mL to 20 μg / mL. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 24 to 27 hours before the introduction of the molecule. ２ Prior to molecule introduction, the medium / supernatant was removed and a specified amount of EGFP plasmid (pCMV-EGFP) was added. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 48 hours. ２ The number of cells showing positive GFP fluorescence was measured using a flow cytometer, and the molecule introduction efficiency was expressed as an index, with the number of cells in the insulin-free group (insulin concentration 0 μg / mL) set at 100, to give the introduction efficiency. Figure 6 shows the relationship between insulin concentration (μg / mL) (horizontal axis) and introduction efficiency (%) (vertical axis).

[0080] [Experimental Example 5] Quantitative PCR <RNA purification> After insulin treatment, CO ２ Culture in an incubator (37°C, 5% CO) for 24 to 27 hours. ２ The cells (concentration) were subjected to RNA purification as total RNA according to the protocol provided by NucleoSpin RNA (Takara Bio Inc., catalogue no. U0955S) (https: / / catalog.takara-bio.co.jp / com / tech_info_detail.php?mode=2&masterid=M100007234&unitid=U100006686).

[0081] <Reverse transcription reaction> To convert the purified RNA to cDNA, reverse transcription reaction was performed using CellAmp Direct RT Enzyme set (Takara Bio Inc.) according to the provided protocol (https: / / catalog.takara-bio.co.jp / PDFS / 3735a_j.pdf). The composition of the reaction solution (20 μL) per reaction is shown in Table 1.

[0082]

[0083] <qPCR reaction> The following reaction mixture was prepared using TB Green Fast qPCR Mix (RR430, Takara Bio) and qPCR was performed. The primers used were the set of qPCR primers (for evaluating mRNA expression) shown in Table 2, with the final concentrations of forward (Fw) and reverse (Rv) primers in each reaction mixture being 0.4 μM.

[0084]

[0085] In Table 2, HPRT1 (Homo sapiens hypoxanthine phosphoribosyltransferase 1) is an endogenous control gene used to standardize the expression level of each gene, and was manufactured by Takara Bio Inc. (Human Housekeeping Gene Primer Set, Catalog No. 3790).

[0086] The composition of the reaction solution (20 μL) per reaction is shown in Table 3.

[0087]

[0088] Next, qPCR was performed using a QuantStudio 3 real-time PCR system (ThermoFisher) under the following conditions: (1) Hold Stage: 50°C for 2 minutes, 95°C for 30 seconds; (2) PCR Stage: 95°C for 5 seconds, 60°C for 15 seconds, 40 cycles; and (3) Melt Curve Stage: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds.

[0089] The obtained qPCR data was normalized by the expression level of HPRT1, an endogenous control gene, and the expression level of each gene in the insulin-treated group was evaluated as "X" times the expression level in the insulin-untreated group, where the expression level was set to "1." The experimental results for T cells are shown in Figure 7 (cell membrane-associated genes) and Figure 8 (cell proliferation-associated genes), the experimental results for Jurkat cells are shown in Figure 9 (cell membrane-associated genes), and the experimental results for HEK293F cells are shown in Figure 10 (cell membrane-associated genes), Figure 11 (endosome-associated genes), and Figure 12 (cell proliferation-associated genes).

[0090] [Experimental Example 6] Molecule introduction device (1) 6-well scale The device was covered with an alumina cap. A through-hole with a diameter of 0.1 mm was formed by laser processing on the surface of the alumina cap facing the second electrode to form a first electrode. Second electrode: 3 mm thick copper flat plate. Power supply frequency: 50 kHz Discharge voltage: 16 kV ｐ－ｐ Discharge time: 40 ms Discharge points per well: 9 points r2 (distance from the center of the irradiation container to the plasma irradiation point): 14 mm Distance between the electrode edge and the top surface of the container: 1.5 mm Amount of DNA (GFP): 50 μg

[0091] (2) 48-well scale 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 create the first electrode. Second electrode: 3 mm thick copper plate. Power supply frequency: 50 kHz Discharge voltage: 16 kVp-p Discharge time: 80 ms Discharge point per well: 1 point at the center r2 (distance from the center of the irradiation container to the plasma irradiation point): None, as this was a single point Distance between the electrode end and the top surface of the container: 1.0 mm Amount of DNA (GFP): 4 μg

[0092] Experimental Example 7 Molecule Introduction Using a Transfection Reagent 2 x 10 Jurkat cells (manufactured by ECACC (The European Collection of Authenticated Cell Cultures)) were placed in each well of a 48-well plate. ５ 400 μL of cells / well were seeded, and insulin was added to a final concentration of 0 μg / mL or 15 μg / mL. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 18 to 24 hours before the introduction of the molecule. ２ concentration).

[0093] Solution 1 was prepared by adding 3.75 μL of Lipofectamine® 2000 Reagent (Thermo Fisher Scientific) to 125 μL of Opti-MEM (registered trademark, manufactured by Thermo Fisher Scientific).

[0094] To 125 μL of Opti-MEM (registered trademark) Medium (manufactured by Thermo Fisher Scientific), 1 μL of pCMV-EGFP adjusted to 1 μg / μL was added, and then 10 μL of P2000 (registered trademark) Reagent was added to prepare Solution 2.

[0095] Equal amounts of Solution 1 and Solution 2 were mixed and left to stand at room temperature for 10 to 15 minutes, and the resulting solution was dispensed into a well plate at 250 μL / well. The well plate was gently shaken up and down and left and right to mix, and then incubated at 37°C for 24 to 72 hours.

[0096] The number of cells showing positive GFP fluorescence was measured using a flow cytometer, and the molecule introduction efficiency was expressed as an index, with the number of cells in the insulin-free group (insulin concentration 0 μg / mL) set to 100. Figure 13 is a graph showing the molecule introduction efficiency with and without insulin addition. As shown in Figure 13, the addition of insulin increased the molecule introduction efficiency by 36%.

[0097] Experimental Example 8: Preparation of cells overexpressing Cav1, Cav2, and Cavin1 As the plasmid vector for Cav1 expression, pEF1-CAV1-puro (SEQ ID NO: 37), pEF1-CAV2-puro (SEQ ID NO: 38), and pEF1-Cavin1-puro (SEQ ID NO: 39) were used as the plasmid vector for Cav1 expression, Cav2 expression, and Cavin1 expression, respectively. As a control experiment, pRI-Pbase (SEQ ID NO: 40) was used.

[0098] pEF1-CAV1-puro, pEF1-CAV2-puro, pEF1-Cavin1-puro, or pRI-Pbase was introduced into Jurkat cells (manufactured by ECACC (The European Collection of Authenticated Cell Cultures)) using the following <Molecular introduction device>.

[0099] <Molecular introduction device> The molecular introduction device shown below was used. First electrode: One end of a stainless steel cylinder with a diameter of 3 mm was covered with an alumina cap with a wall thickness of 0.5 mm. A through-hole with a diameter of 0.1 mm was formed by laser processing on the surface of the alumina cap facing the second electrode to prepare the first electrode. Second electrode: A copper flat plate with a thickness of 3 mm. Power supply frequency: 50 kHz. Discharge voltage: 16 kV. ｐ－ｐ Discharge time: 40 ms Discharge points per well: 9 points r2 (distance from the center of the irradiation container to the plasma irradiation point): 12 mm Distance between the electrode edge and the top surface of the container: 2 mm DNA amount: 25 μg of any one of pEF1-CAV1-puro, pEF1-CAV2-puro, and pEF1-Cavin1-puro, and pRI-Pbase, 25 μg DNA solution: 50 μL

[0100] One day after plasmid vector introduction, 2 μg / mL puromycin was added. Five days after plasmid vector introduction, the medium was replaced with RPMI 1640 medium containing 5% serum (Gibco, fetal bovine serum, product number: 16140071), and the puromycin was removed. Culture was continued for 14 to 28 days after plasmid vector introduction, with medium changes, to obtain Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells.

[0101] To confirm whether the target protein was overexpressed in Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells, the molecular weight of the protein was measured by Western blotting. The Western blotting conditions were as follows: The electrophoresis sample was prepared by mixing an equal amount of 2x DTT-containing sample buffer (ATTO, AE-1430 EzApply) with the protein sample. Polyacrylamide gel: EHR-T10L (ATTO) Electrophoresis buffer: Electrophoresis buffer (x10) (Fujifilm Wako Pure Chemical Industries) Molecular weight marker: WSE-7020 EzProtein Ladder (ATTO) Staining solution: EzStain AQua (ATTO)

[0102] FIG. 14 is an image showing the results of Western blotting of Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells. As control examples, the results of Western blotting of wild-type Jurkat cells and HEK293F cells are also shown. In FIG. 14, Cav1-overexpressing cells are indicated as "Jarkat-pEF1-Cav1," Cav2-overexpressing cells as "Jarkat-pEF1-Cav2," Cavin1 as "Jarkat-pEF1-Cavin1," and the wild-type Jurkat overexpressing cells as "Jurkat-WT." Comparison with wild-type Jurkat cells confirmed that Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells overexpressed proteins with molecular weights corresponding to Cav1, Cav2, and Cavin1, respectively.

[0103] Furthermore, it was confirmed that the expression levels of proteins with molecular weights corresponding to clathrin heavy chains, which are involved in endocytosis via the clathrin-mediated pathway, were unchanged or decreased in Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells compared to wild-type Jurkat cells.

[0104] Experimental Example 9: Molecular introduction using expression-promoting cells with a transfection reagent Molecular introduction by transfection was carried out using the Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells obtained in Experimental Example 8. Molecule introduction by transfection was carried out in the same manner as in Experimental Example 7, except that insulin was not added. The experiment was also carried out using Lipofectamine 2000 (Thermo Fisher).

[0105] Figure 15 is a graph showing the efficiency of molecule introduction by lipofection in Cav1-overexpressing cells, Cav2-overexpressing cells, Cavin1-overexpressing cells, and wild-type Jurkat cells. The molecule introduction efficiency in Figure 15 was calculated by measuring the "number of live cells" from a gate estimated to be live cells using a flow cytometer, and the "number of GFP-positive cells" by measuring the number of cells showing positive GFP fluorescence, and then dividing the "number of GFP-positive cells" by the "number of live cells." It was found that lipofection using Lipofectamine® 2000 Reagent improved the efficiency of molecule introduction in Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells compared to wild-type Jurkat cells. The molecule introduction efficiency was particularly high in Cavin1-overexpressing cells.

[0106] [Experimental Example 10] Molecule introduction by plasma irradiation using expression-promoted cells Using the Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells obtained in Experimental Example 8, molecule introduction by plasma irradiation was carried out.

[0107] Jurkat cells (manufactured by The European Collection of Authenticated Cell Cultures (ECACC)) were placed in each well of a 48-well plate at a density of 2.0 × 10 ５ 0.4 mL of cells / well were seeded. ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 24 to 27 hours before the introduction of the molecule. ２Prior to molecule introduction, the medium / supernatant was removed and a specified amount of EGFP plasmid (pCMV-EGFP) was added. The molecule introduction was performed by plasma irradiation treatment under the following conditions, and CO ２ The mixture was cultured in an incubator (37°C, 5% CO ) for 48 hours. ２ Concentration). 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 prepare the first electrode. Second electrode: A 3 mm thick copper plate. Power supply frequency: 50 kHz Discharge voltage: 16 kV ｐ－ｐ Discharge time: 80 ms Discharge point per well: 1 point Distance between electrode edge and top surface of container: 1 mm Amount of DNA (GFP): 2 μg

[0108] Figure 16 is a graph showing the molecular introduction efficiency by plasma irradiation in Cav1-overexpressing cells, Cav2-overexpressing cells, Cavin1-overexpressing cells, and wild-type Jurkat cells. The molecular introduction efficiency in Figure 16 was calculated by measuring the "number of live cells" from a gate estimated to be live cells using a flow cytometer, and the "number of GFP-positive cells" (the number of cells showing positive GFP fluorescence), and then dividing the "number of GFP-positive cells" by the "number of live cells." It was found that molecular introduction by plasma irradiation improved the molecular introduction efficiency in Cav1-overexpressing cells, Cav2-overexpressing cells, and Cavin1-overexpressing cells compared to wild-type Jurkat cells. The molecular introduction efficiency was particularly high in Cav1-overexpressing cells.

[0109] According to the present invention, a technique can be provided that can improve the efficiency of molecule introduction into target cells.

[0110] 1 molecule introduction device, 10 first electrode, 12 electrode body, 14 irradiator, 16 wall portion, 20 second electrode, 30 power supply portion, 40 container, 42 molecule, 44 target cell, O1 tube axis, θ1 angle

Claims

1. A pretreatment method carried out prior to the introduction of a molecule into a target cell, the pretreatment method comprising a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2 and Cavnin 1 in the target cell.

2. The pretreatment method according to claim 1, wherein in the pretreatment step, the target cells are contacted with at least one drug selected from the group consisting of insulin and insulin analogues.

3. The pretreatment method according to claim 2, wherein in the pretreatment step, the target cells are cultured using a medium containing the drug.

4. The pretreatment method according to claim 2 or 3, wherein the target cells are blood-derived CD3-positive cells.

5. A pretreatment method according to claim 2 or 3, wherein in the pretreatment step, the contact time between the target cells and the drug is 20 to 48 hours.

6. The pretreatment method according to claim 5, wherein in the pretreatment step, the concentration of the drug when contacting the target cells with the drug is 5 to 150 μg / mL.

7. A method for producing molecule-introduced cells, comprising: a pretreatment step of promoting expression of at least one protein selected from the group consisting of Caveolin 1, Caveolin 2 and Cavin 1 in target cells; and a molecule-introducing step of contacting the target cells with a liquid containing the molecule to be introduced and introducing the molecule into the target cells.

8. The method for producing molecule-introduced cells described in claim 7, wherein in the pretreatment step, the target cells are contacted with at least one drug selected from the group consisting of insulin and insulin analogues.

9. A method for producing molecule-introduced cells as described in claim 7 or 8, wherein the target cells are irradiated with plasma in the molecule introduction step.

10. The method for producing a molecule-introduced cell according to claim 7 or 8, wherein the liquid containing the molecule to be introduced contains a transfection reagent containing the molecule.

11. The method for producing molecule-introduced cells described in claim 8, wherein the contact time between the target cells and the drug in the pretreatment step is 20 to 48 hours.

12. The method for producing molecule-introduced cells described in claim 11, wherein in the pretreatment step, the concentration of the drug when contacting the target cells with the drug is 5 to 150 μg / mL.

13. A molecule introduction efficiency improver for improving the efficiency of molecule introduction into target cells, comprising as an active ingredient at least one drug selected from the group consisting of insulin and insulin analogues.

14. Use of at least one drug selected from the group consisting of insulin and insulin analogues as a molecule introduction efficiency improving agent for improving the introduction efficiency of a molecule into a target cell.

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