A means of controlling intracellular reactions using needle-shaped structures

Abstract

To provide a new technique and method for controlling an intracellular reaction that enables introduction of a prescribed factor into a large number of cells through a simple, efficient operation.SOLUTION: Provided is a method for controlling an intracellular reaction, the method including a step for bringing a needle-shaped body having an intracellularly introduced factor immobilized thereto into contact with a cell having a weight bound thereto and inserting a portion of the needle-shaped body into the cell to introduce the intracellularly introduced factor into the cell, and a step for withdrawing the portion of the needle-shaped body from the cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for simply and efficiently introducing intracellular transfection factors immobilized on needle-shaped structures into a large number of cells, and to a means for controlling intracellular reactions using the same. [Background technology]

[0002] Today, various technologies and methods for controlling intracellular reactions have been developed and reported. However, most of these utilize genetic recombination or genome editing technologies, which generally involve cumbersome procedures and significant costs, placing a heavy burden on those working on them. For this reason, new technologies and methods for controlling intracellular reactions remain in high demand in this field.

[0003] Patent document 1 discloses a needle-shaped material for cell insertion onto which antibodies against intracellular or intercellular protein antigens are immobilized, and describes that by aligning the needle-shaped material with a cell and inserting it into the cell by moving it up and down, it is possible to quantify, evaluate, etc. intracellular or intercellular proteins in living cells.

[0004] Patent documents 2 and 3 disclose a cell insertion device in which a large number of nanoneedles are arranged on a support, formed using photolithography, dry etching, and wet etching methods. They describe that by immobilizing a substance that binds to a gene or a substance involved in gene expression, or a marker substance such as an antibody, on each nanoneedle, a large number of nanoneedles can be inserted simultaneously into a large number of cells arranged on a substrate, allowing the gene expression state to be analyzed in a large number of cells while the cells are still alive, or target cells to be selectively lifted.

[0005] However, when inserting these needle-shaped materials or nanoneedles into cells or the like, the positioning and up-and-down movement can be complicated, placing a heavy burden on the operator. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-246731 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-183706 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-166884 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide new techniques and methods for controlling intracellular reactions that enable the introduction of a specific factor into a large number of cells through simple and efficient manipulations. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the present inventors have found that when a cell to which a weight is bound is brought into contact with a needle-shaped structure, the needle-shaped portion of the needle-shaped structure is inserted into the cell more efficiently than when a cell to which a weight is not bound is brought into contact. They have also found that by immobilizing an intracellular introduction factor on a needle-shaped structure and bringing it into contact with a cell to which a weight is bound, the introduction factor can be introduced into a large number of cells simply and efficiently, and intracellular reactions can be controlled and analyzed.

[0009] The present invention is based on these new findings and includes the following inventions. [1] A step of contacting a needle-shaped structure on which an intracellular introduction factor is immobilized with a cell to which a weight is attached, and inserting a part of the needle-shaped structure into the cell to introduce the intracellular introduction factor into the cell; and a step of extracting a portion of the needle-shaped structure from the cell; A method for producing modified cells, comprising: [2] The method according to [1], wherein the weight is one or more materials selected from the group consisting of resin, metal, magnetic material, and combinations thereof. [3] The method according to [1] or [2], wherein the weight has a bead-like shape. [4] Any of the methods [1] to [3], wherein the step of inserting a portion of the needle-shaped structure into a cell and / or extracting it from the cell is carried out using one or more external forces selected from the group consisting of centrifugal force, magnetic force, water flow, water pressure, and electrostatic interaction. [5] Any of the methods [1] to [4], wherein the intracellular introduction factor is a conjugate capable of binding to an intracellular molecule, and the method comprises extracting the needle-shaped body from the cell and extracting the intracellular molecule bound to the conjugate within the cell together with the needle-shaped body. [6] The method according to [5], wherein the conjugate is a nucleic acid, a protein, a peptide, or a low molecular weight compound. [7] The method of [5] or [6], wherein the conjugate is an antibody or a fragment thereof. [8] Any of the methods [1] to [4], wherein the intracellular introduction factor is a physiologically active substance, and the method comprises removing the needle-shaped structure from the cell and leaving the intracellular introduction factor within the cell. [9] The method of [8], wherein the physiologically active substance is one or more substances selected from the group consisting of nucleic acids, peptides, proteins, sugars, polysaccharides, fatty acids, cholesterol, lipids, signal transduction substances, ligand substances, hormone substances, cytokines, ions, metal particles, magnetic particles, inorganic compounds, quantum dots, organic compounds, and drugs.

[10] The method according to [8] or [9], wherein the bond between the physiologically active substance and the needle-shaped structure is a bond that can be separated within a cell.

[11] The method of

[10] , wherein the bond between the intracellular introduction factor and the needle-shaped structure is at least one selected from the group consisting of an electrostatic bond, a bond due to hydrophobic interaction, a chelate bond, a covalent bond that is cleaved within the cell, a bond via a photocleavable linker, and a bond via an enzyme-cleavable linker.

[12] The method according to any one of [1] to

[11] , wherein the needle-shaped bodies are needle-shaped particles.

[13] The method according to

[12] , wherein the length of the needle-shaped part of the acicular particles is 1 to 50 μm.

[14] The method of

[12] or

[13] , wherein the acicular particles are zinc oxide.

[15] The method according to any one of [1] to

[14] , wherein the needle-shaped structure is immobilized on a substrate.

[16] The method according to

[15] , wherein the substrate has a binder on its surface, and a portion of the needle-shaped structure is fixed to the substrate by the binder.

[17] The method of

[16] , wherein the binder is made of a protein-non-adsorbing material.

[18] The method according to

[15] , wherein the needle-shaped structure is manufactured using one or more methods selected from the group consisting of photolithography, dry etching, wet etching, and combinations thereof.

[19] The method according to any one of [1] to

[18] , wherein the surface of the substrate having the needle-shaped structures has a fine uneven structure capable of accommodating cells.

[20] A method for analyzing the function of a factor introduced into cells, comprising a step of analyzing the state of a modified cell produced by any one of the methods [1] to

[19] .

[21] The method according to

[20] , wherein the step of analyzing the state of the modified cells comprises analyzing one or more selected from the group consisting of cell proliferation rate, survival rate, metabolism, quantification of reactive oxygen species, and gene expression. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide new techniques and methods for controlling intracellular reactions that enable the introduction of a predetermined factor into a large number of cells through simple and efficient manipulations. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing a method for producing a composite substrate for cell treatment on which acicular particles are immobilized. [Figure 2] FIG. 2 is a photograph showing acicular particles (Panatetra) immobilized on a cell treatment composite substrate. [Figure 3] Figure 3 is a graph showing the percentage of activated cells (CD25+ or CD137+) in PBMCs obtained by inserting and removing the needle of a Panatetra coated with anti-Cbl antibody. (A) shows the results after 1 day and (B) shows the results after 3 days of culture. [Figure 4] FIG. 4 is a graph showing the cell viability (ie, the efficiency of insertion of the needle of Panatetra into the cells) when the cells were repeatedly treated with the cell treatment composite substrate. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present invention, the term "needle-shaped structure" means a fine structure having at least one needle-shaped portion.

[0013] The "needle-shaped portion" is elongated, has a tip and width that are sufficiently small, and when inserted into a cell, causes little or no damage to the cell, preferably no damage at all. As long as it has the above-mentioned shape, the needle-shaped portion may have a shape such as, but not limited to, a cylindrical, conical, tubular (e.g., prismatic), or pyramidal shape, and its tip may or may not be sharp. From the viewpoint of minimal invasiveness to cells and insertion efficiency, a cylindrical or conical shape is preferred. The length of the needle-shaped portion is 0.5 to 100 μm, preferably 1 to 50 μm, more preferably 5 to 40 μm, and even more preferably 10 to 30 μm (e.g., 10 μm, 20 μm, etc.). The aspect ratio of the needle-shaped portion is not particularly limited, but can be about 5:1 to 50:1, preferably 10:1 to 40:1.

[0014] The "needle-shaped body" is made of a material that has little or no toxicity to cells, and preferably no toxicity at all. Examples of such materials include (but are not limited to) metal oxides (e.g., zinc oxide, etc.), inorganic materials (e.g., quartz, nickel oxide, silica, alumina, diamond, titania, zirconia, etc.), metals (e.g., gold, silver, copper, platinum, aluminum, etc.), metal crystals (e.g., tungsten, titanium, silicon crystal, zirconium, etc.), glass, resins (e.g., polyethylene, polypropylene, cyclic polyolefin, cyclic olefin copolymer, polyester, polystyrene, polymethyl acrylate, polylactic acid, polyether ether ketone, fluororesin, etc.), silicon nitride, silicon, etc., and needle-shaped solid materials can be suitably used.

[0015] The shape of the "needle-shaped body" is not particularly limited as long as it allows the needle-shaped portion to be inserted into and removed from cells, but for example, the "needle-shaped body" can have the form of a needle-shaped particle or one immobilized on a substrate.

[0016] Regarding the shape of the "acicular body," "acicular particle" refers to a fine particle having at least one needle-shaped portion. The shape of the acicular particle is formed by one or more needle-shaped portions arranged / connected at the center, and can be, for example, a rod shape, an L-shape, a V-shape, a T-shape, a Y-shape, or a radial shape (e.g., a tripod shape, a quatropod shape, a tetrapod shape, etc.), but is not limited to these. When multiple needle-shaped portions are included, each needle-shaped portion may have the same shape and / or length, or may have different shapes and / or lengths. At the center, the multiple needle-shaped portions may be directly connected to each other, or one or more needle-shaped portions may be connected to a core member. The size of the acicular particle varies depending on the length and number of the needle-shaped portions, but can be large enough to fit into a sphere with a radius of 0.5 μm to 100 μm.

[0017] In this specification, the numerical values ​​relating to the sizes of the "acicular bodies," "acicular particles," and "acicular portions" are expressed as average values.

[0018] Preferably, in the present invention, the acicular particles are made of zinc oxide having a plurality of needle-shaped portions, and more preferably have a shape in which needle-shaped portions made of zinc oxide single crystals having a length of 10 to 20 μm are arranged in a tetrapod shape. For example, Panatetra (registered trademark) WZ-0501 (manufactured by Amtec) can be suitably used as such acicular particles.

[0019] Furthermore, with regard to the form of the "needle-shaped structure," "immobilized on a substrate" refers to a substrate having a plurality of needle-shaped structures (or needle-shaped portions) on its surface. The "substrate" may be any material that can function as a support by immobilizing needle-shaped structures on its surface, and is not particularly limited. Examples include, but are not limited to, resins (polystyrene, polyethylene (e.g., linear low density polyethylene (LLDPE), very low density polyethylene (VLDPE / ULDPE), low density polyethylene (LDPE), or combinations thereof), polypropylene, polyester, polyacrylonitrile, styrene-butadiene copolymer, (meth)acrylic acid ester polymer, fluororesin, etc.), silica gel, cross-linked dextran, polysaccharides, polysaccharides such as agarose, glass, metal, magnetic substances, and combinations thereof. The shape of the substrate is not particularly limited as long as it can immobilize the needle-shaped structures, and can be any shape, such as a film, a flat plate, a tray, particles (beads), a sphere, a container (test tube, tube, microplate, microtube, cell, cuvette, dish, flask, bag), fiber, gel, etc. Specific examples include polyethylene film, polyethylene plate, magnetic beads, etc., but are not limited to these.

[0020] The needle-shaped structure can be fixed to the surface of the substrate by any means as long as the bond between the two can be maintained even after the needle-shaped portion is inserted into and removed from the cell. The needle-shaped structure can be fixed to the surface of the substrate by, for example, physical adsorption, covalent bonding, ionic bonding, embedding in the substrate, or using a binder (adhesive), but is not limited to these.

[0021] The "binder" that can be used in the present invention is not particularly limited as long as it can immobilize needle-shaped structures on the surface of a substrate, and examples include polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (Poly HEMA), polyvinylpyrrolidone, vinyl acetate resin, urethane resin, vinyl chloride resin, epoxy resin, vinyl chloride-vinyl acetate copolymer resin, modified silicone resin, 2-ethyl cyanoacrylate, polystyrene, chloroprene rubber, nitrile rubber, styrene-butadiene rubber, nitrocellulose, starch, dextrin, alginic acid, agarose, and gel-like proteins (gelatin, elastin, fibrin, etc.), and one or more combinations of these can be used. The binder is applied to a predetermined area on the surface of the substrate, and as the applied binder solidifies, it adheres the needle-shaped structures that are partially in contact with the binder, or preferably partially embedded in the binder, and immobilizes them on the surface of the substrate. Preferably, the binder has low binding or adsorption to the "intracellular transfer factor" described in detail below. Examples of such binders include PVA and PolyHEMA. By using such binders, when attempting to immobilize an intracellular transfer factor to a needle-shaped structure immobilized on a substrate, it is possible to suppress the binding or adsorption of the intracellular transfer factor to parts other than the needle-shaped structure (e.g., the substrate), thereby suppressing the loss of the intracellular transfer factor that is not immobilized on the needle-shaped structure. Furthermore, the binder can contain molecules that interact with cell surface proteins. Examples of such molecules include fibronectin, laminin, collagen, cadherin, or fragments thereof, and one or more of these can be used in combination. Such molecules can be incorporated by a simple method, for example, by premixing them with the binder component and then applying them to the substrate, or by applying them to the binder on the substrate. The use of such molecules can suppress deterioration of the cell condition caused by changes from the normal culture environment when treating cells.

[0022] Alternatively, in another embodiment, the needle-shaped structure can be immobilized on the surface of a substrate by integrally molding or processing the substrate and the needle-shaped structure (or needle-shaped portion) together, for example, by using one or more techniques selected from the group consisting of photolithography, dry etching, wet etching, and combinations thereof, in accordance with conventionally known techniques (e.g., JP 2006-246731 A, JP 2013-183706 A, JP 2006-166884 A).

[0023] The surface of the substrate may be formed with a fine uneven structure capable of accommodating cells together with immobilized needle-shaped structures. The fine uneven structure accommodates cells and holds them between the side walls (e.g., the inner walls of the recesses) and the bottom, restricting lateral movement of the cells (e.g., movement horizontally relative to the surface of the substrate), thereby reducing or preventing serious damage (e.g., cutting, ripping, etc.) caused by the needle-shaped structures that may occur as a result of lateral movement of the cells. In the present invention, the shape of the "fine uneven structure" is not particularly limited as long as it can achieve the above-mentioned effect, and may, for example, have a recessed shape (recess) such as a spherical crown shape, a mortar-like bowl shape (cone-like, pyramidal), or a cylindrical shape (cylindrical, prismatic, etc.). The needle-shaped structures are placed and immobilized at the bottom of this recess. The size of the recess is not particularly limited as long as it can achieve the above-mentioned effect, and may, for example, have an opening diameter or diagonal length of approximately 1 to 500 μm and a depth of approximately 1 to 500 μm. Each well may contain one cell or multiple cells.

[0024] In this specification, needle-shaped structures (needle-shaped particles) "immobilized on a substrate" may be referred to as "composite substrate for cell treatment."

[0025] In the present invention, an "intracellular introduction factor" is immobilized on the needle-shaped structure.

[0026] In one embodiment of the present invention, the "intracellular delivery factor" may be a conjugate capable of binding to an intracellular molecule. The "conjugate capable of binding to an intracellular molecule" may be any compound capable of binding, preferably selectively, and more preferably specifically, to a target intracellular molecule in the cell into which it is introduced to form a complex. It can be appropriately selected depending on the target intracellular molecule. Examples of such conjugates include, but are not limited to, nucleic acids (DNA, RNA, DNA-RNA hybrids, etc.), proteins (antibodies, antigens, enzymes, substrates, coenzymes, ligands, receptors, complex subunits, or fragments thereof), peptides, and low-molecular-weight compounds (e.g., Y-27632, Z-VAD-FMK, etc.). In the present invention, the "conjugate capable of binding to an intracellular molecule" is preferably one that does not directly affect the expression of any gene. Here, "does not directly affect" means that the introduction of the conjugate does not directly activate any intracellular signaling pathway. Preferably, the "conjugate capable of binding to an intracellular molecule" in the present invention is an antibody or a fragment thereof that can bind, preferably selectively, and more preferably specifically, to a target intracellular molecule. Antibody "fragments" include Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, sc(Fv)2, etc., and multimers of these fragments (e.g., dimers, trimers, tetramers, polymers) can also be used in the present invention.

[0027] In another aspect of the present invention, the "intracellularly introduced factor" may be a factor that exerts or is predicted to exert a desired function in the cell into which it is introduced. Examples of such factors include physiologically active substances. Examples of the "physiologically active substance" include, but are not limited to, one or more substances selected from the group consisting of nucleic acids (DNA, RNA, DNA-RNA hybrids, etc.), plasmids, virus particles, chromosomes, etc.; proteins, amino acids, oligopeptides, polypeptides, multisubunit proteins; saccharides, polysaccharides, fatty acids, cholesterol, lipids, signal transduction substances, ligand substances, hormone substances, cytokines, ions, metal particles, magnetic particles, inorganic compounds, quantum dots, organic compounds, drugs, etc.

[0028] In the present invention, the method for binding (immobilizing) the "needle-shaped structure" to the "intracellular introduction factor" can be appropriately selected depending on the type of "intracellular introduction factor" used. For example, when the "intracellular introduction factor" used is the above-mentioned "conjugate capable of binding to an intracellular molecule," the binding (immobilization) between the needle-shaped structure and the conjugate can be carried out using a method well known to those skilled in the art, as long as the strength of the bond is such that the bond is maintained even when the needle-shaped portion is inserted into a cell and then withdrawn. Examples of the binding method include physical adsorption, covalent bonding, and ionic bonding, with covalent bonding being preferred. For example, when the conjugate is a protein or peptide, the covalent bonding method can be carried out by chemically reacting a functional group (e.g., a hydroxy group, an amino group, an N-hydroxysuccinimidyl group, a sulfhydryl group, an epoxy group, a vinyl group, etc.) on the surface of the needle-shaped portion with the carboxyl terminus of the protein or peptide to form an ester bond, an amide bond, or the like. If necessary, the surface of the needle-shaped portion can be treated to introduce functional groups, for example, using polylysine, polyethyleneimine vinyl trialkoxysilane, 3-mercaptopropyl trialkoxysilane, aminoalkyl trialkoxysilane, epoxy-containing alkyl trialkoxysilane, etc.

[0029] The needle-shaped particle and the binder capable of binding to an intracellular molecule may be directly bonded to each other (immobilized), or may be indirectly bonded via a linker.

[0030] Furthermore, when the "factor to be introduced into cells" used is the aforementioned "biologically active substance," a bond that maintains the bond (immobilization) between the two at least until the needle-shaped part is inserted into the cell, but that dissociates / cleaves inside the cell, allowing the factor to be released into the cell, is preferred. Examples of such bonding methods include, but are not limited to, electrostatic bonding; bonding via hydrophobic interaction; chelate bonding; covalent bonds that can be cleaved inside the cell, such as disulfide bonds (SS bonds); bonding via a photocleavable linker; and bonding via a linker (enzyme-cleavable linker) having an enzyme recognition sequence, such as esterase, and can be performed using methods well known to those skilled in the art (e.g., JP 2006-166884 A).

[0031] The intracellular introduction factors immobilized on one needle-shaped structure may all be the same, or multiple different types of intracellular introduction factors may be immobilized.Furthermore, the same intracellular introduction factor may be immobilized on all multiple needle-shaped structures, or different intracellular introduction factors may be immobilized on each needle-shaped structure.

[0032] In the present invention, a predetermined intracellular introduction factor may be immobilized on the needle-shaped body in advance, or the desired intracellular introduction factor may be immobilized on the needle-shaped body as described above before use (i.e., the needle-shaped body does not need to have a predetermined intracellular introduction factor immobilized on it when provided).

[0033] The needle-shaped structure on which the intracellular transfer factor is immobilized can be used to control intracellular reactions and can be used in a method for producing modified cells in which intracellular reactions are controlled. Unless otherwise specified, the term "needle-shaped structure" below refers to multiple needle-shaped structures.

[0034] The present manufacturing method comprises: contacting the needle-shaped structure on which the intracellular transfection factor is immobilized with the cell to which the weight is attached; This allows a portion of the needle-shaped structure to be inserted into the cell, thereby introducing the intracellular introduction factor into the cell; and then extracting a portion of the needle-shaped structure from the cell; Includes.

[0035] The cells to be treated in the present invention are not particularly limited and may be either adherent cells or suspension cells, and examples of the cells include, but are not limited to, pluripotent stem cells or their differentiation-induced cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, lymphocytes, neurons, glial cells, ganglion cells, pancreatic islet cells, adrenal medulla cells, cardiomyocytes, hepatocytes, fibroblasts, epithelial cells, endothelial cells, myoblasts, retinal epithelial cells, corneal stem cells, osteoblasts, osteoclasts, and hepatocytes. The origin of the cells is not particularly limited, and mammalian cells such as those from humans, mice, rats, monkeys, dogs, pigs, cows, guinea pigs, and hamsters can be preferably used.

[0036] In the present invention, "pluripotent stem cells" refer to embryonic stem cells (ES cells) and cells with similar pluripotency, i.e., the potential to differentiate into various tissues in the body (endoderm, mesoderm, and ectoderm). Examples of cells with similar pluripotency to ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells"). In the present invention, the pluripotent stem cells are preferably human pluripotent stem cells. "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells through the introduction of specific factors (nuclear reprogramming factors), such as Oct3 / 4, Sox2, Klf4, and c-Myc. Furthermore, the "differentiation-induced cells" of the above-mentioned pluripotent stem cells refer to cells characterized by the expression of a specific phenotype or marker obtained by inducing differentiation of pluripotent stem cells. "Marker" refers to a cell antigen or its gene that is specifically expressed in a specific cell type, such as a "marker protein" or a "marker gene."

[0037] In the present invention, the cells may be cells collected from a living body, cultured cells, or frozen-thawed cells. The cells used are preferably in a dissociated or dispersed state.

[0038] In the present invention, a weight is attached to a cell. Attaching the weight increases the weight of the cell, increasing the impact upon contact between the cell and the needle-shaped structure, and facilitating insertion of the needle-shaped portion of the needle-shaped structure into the cell (i.e., increasing the probability of insertion). The "weight" can be one or more selected from the group consisting of resin, metal, magnetic substance, and combinations thereof. The shape of the weight is not particularly limited, but it can be, for example, bead-shaped. For example, in the present invention, magnetic beads such as Dynabeads (registered trademark, Veritas Corporation) can be suitably used as the "weight" for the cell. Binding of the cell to the weight can be performed using methods well known to those skilled in the art, such as one or more of antibodies or fragments thereof, avidin-biotin, streptavidin-biotin, etc.

[0039] The needle-shaped portion of the needle-shaped body can be inserted into cells by contacting the cells with the needle-shaped body. This contact between the cells and the needle-shaped body can be achieved by adding the needle-shaped body to the cells (or by adding the cells to the needle-shaped body), or by adding the cells to a needle-shaped body immobilized on a substrate (a composite substrate for cell treatment). If necessary, an external force may be applied to bring the cells into contact with the needle-shaped body. Here, "external force" refers to a force applied to the cells and / or the needle-shaped body, and includes centrifugal force, magnetic force, electrostatic interaction, water flow (water pressure), etc., and one or more selected from these can be used. Centrifugation can be performed under any condition that does not damage the cells, for example, at 100 to 30,000 g for 30 to 600 seconds. The water flow (water pressure) can be generated by pipetting. The use of an external force increases the impact when the cells come into contact with the needle-shaped body, making it easier for the needle-shaped portion of the needle-shaped body to be inserted into the cells (i.e., increasing the insertion probability).

[0040] The insertion of the needle-shaped part of the acicular particle into the cell may be carried out in a culture medium, preferably in a cell culture environment, for example, while maintaining culture conditions of 37°C and 5% CO. If the insertion time is short, it can also be carried out at room temperature in an atmospheric environment.

[0041] If the intracellular introduction factor immobilized on the needle-shaped body is a "conjugate capable of binding to an intracellular molecule," the insertion of the needle-shaped part of the needle-shaped body into the cell may be maintained for a time sufficient for the conjugate immobilized on the inserted needle-shaped body to bind to the target intracellular molecule within the cell. This time can be appropriately set by a person skilled in the art depending on the type of conjugate and the target intracellular molecule, but can be, for example, 1 to 120 minutes, preferably 5 to 60 minutes, and more preferably about 30 minutes.

[0042] The needle-shaped portion can then be removed from the cell together with the target intracellular molecule bound to the conjugate, thereby reducing or eliminating the amount of the target intracellular molecule in the cell, thereby obtaining a modified cell in which the function of the intracellular molecule has been removed or reduced.

[0043] Furthermore, when the intracellular introduction factor immobilized on the needle-shaped body is the aforementioned "physiologically active substance," the insertion of the needle-shaped part of the needle-shaped body into the cell may be maintained for a time sufficient for the physiologically active substance immobilized on the needle-shaped body to be released from the needle-shaped body within the cell (and, if necessary, a bond severing process may be performed). This time can be appropriately set by those skilled in the art depending on the type of physiologically active substance and the type of bond, but can be, for example, 1 to 120 minutes, preferably 5 to 60 minutes, and more preferably about 30 minutes.

[0044] The needle-shaped portion is then removed from the cell, and the physiologically active substance is left inside the cell, thereby increasing the amount of the physiologically active substance within the cell, thereby obtaining modified cells in which the function of the physiologically active substance has been introduced or enhanced.

[0045] The needle-shaped portion can be removed from the cell by applying an external force to separate the contacting cell and the needle-shaped structure. Here, the "external force" can be as defined above.

[0046] In this method, the steps of inserting the needle-shaped portion of a needle-shaped object into a cell and removing the needle-shaped portion from the cell can be performed once or multiple times on the same cell. Here, "multiple times" means two or more, three or more, four or more, or five or more times, and there is no particular upper limit, but from the viewpoint of reducing the burden on the cell and maintaining its viability and physiological state in good condition, 15 or fewer times or 10 or fewer times is preferred. Furthermore, "performed multiple times" here means not only when the step is performed multiple times consecutively, but also when the step is performed multiple times at regular time intervals.

[0047] The resulting modified cells vary depending on the function of the intracellularly introduced factor. When the introduced intracellularly introduced factor is a "conjugate capable of binding to an intracellular molecule," for example, when the intracellular molecule that binds to the conjugate is a factor that functions to suppress or inhibit cell activation (e.g., factors such as CIN85, Sts-2, and Cbl in T cells (Mei Suen Kong et al., Science Signaling 05 Feb 2019: Vol. 12, Issue 567, eaav4373)), the intracellular response of the factor can be controlled by removing or reducing the function of the factor, i.e., activated modified cells can be obtained. Furthermore, when the intracellular molecule that binds to the conjugate is a factor that functions to suppress or inhibit cell differentiation induction, for example, when the function of the factor is removed or reduced, modified cells can be obtained in which the intracellular response of the factor is controlled, i.e., differentiation-induced modified cells (examples of modified cells are not limited to these examples).

[0048] If the introduced intracellular factor is the aforementioned "bioactive substance," and for example, if it is a factor that has the function of promoting cell activation, then by introducing or enhancing the function of the factor, the intracellular reaction of the factor can be controlled, i.e., modified cells with promoted activation can be obtained. Furthermore, if, for example, the bioactive substance is a factor that has the function of promoting cell differentiation induction, then by introducing or enhancing the function of the factor, the intracellular reaction of the factor can be controlled, i.e., modified cells with induced differentiation can be obtained (examples of modified cells are not limited to these).

[0049] In another embodiment, the resulting modified cells can be used to analyze the function of the introduced intracellular transfection factor.

[0050] The function of the introduced intracellular transfection factor can be analyzed by analyzing the state of the modified cells obtained by the above-mentioned method. The state of the modified cells to be analyzed may include, but is not limited to, cell proliferation rate, survival rate, metabolism, reactive oxygen species, gene expression, etc., and one or more states selected from these may be quantified and analyzed.

[0051] For example, if the intracellularly introduced factor is a "conjugate capable of binding to an intracellular molecule," and the resulting modified cells show a decrease, suppression, or disappearance in growth rate, viability, metabolism, reactive oxygen species, or gene expression compared to unmodified cells, it can be determined that the conjugate has such a function, i.e., that the intracellular molecule targeted by the conjugate has a function that contributes to the maintenance or promotion of growth rate, viability, metabolism, reactive oxygen species, or gene expression. Alternatively, if the resulting modified cells show an increase or induction in growth rate, viability, metabolism, reactive oxygen species, or gene expression compared to unmodified cells, it can be determined that the conjugate has such a function, i.e., that the intracellular molecule targeted by the conjugate has a function to suppress or inhibit growth rate, viability, metabolism, reactive oxygen species, or gene expression.

[0052] Alternatively, when the intracellularly introduced factor is a "bioactive substance," if the growth rate, viability, metabolism, reactive oxygen species, or gene expression in the resulting modified cells is reduced, suppressed, or eliminated compared to unmodified cells, it can be determined that the bioactive substance has the function of contributing to the reduction, suppression, or elimination of cellular growth rate, viability, metabolism, reactive oxygen species, or gene expression. Alternatively, for example, if the growth rate, viability, metabolism, reactive oxygen species, or gene expression in the resulting modified cells is increased or induced compared to unmodified cells, it can be determined that the bioactive substance has the function of increasing or inducing growth rate, viability, metabolism, reactive oxygen species, or gene expression.

[0053] In analyzing gene expression in modified cells, any gene and protein encoded thereby can be used as the target of analysis, but it is preferable that they are different from the target intracellular molecule, the conjugate, and the introduced physiologically active substance so that the behavior of the cell can be clarified.

[0054] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0055] Experiment 1: Cell modification by treatment of cell-treatment composite substrate 1-1. Preparation of composite substrate for cell treatment An outline of the method for preparing the cell treatment composite substrate with immobilized acicular particles used in this experiment is shown in Figure 1. A linear low-density polyethylene (LLDPE) film was covered with an LLDPE film (masking) with a 4 mm diameter hole, and a 1% aqueous solution of a water-soluble photosensitive resin (BIOSURFINE® AWP, manufactured by Toyo Gosei) was added, after which the excess liquid was scraped off using a polystyrene cylinder.

[0056] Needle-shaped particles (Panatetra (registered trademark) WZ-0501 (Amtec)) were attached to a sponge puff (107 (NBR) for emulsion pact (Shiseido)), and the surface of the substrate to which the photosensitive resin had been added was lightly tapped several times with this sponge puff to coat it with Panatetra.

[0057] The masking was removed, and the photosensitive resin was UV-crosslinked using a low-pressure mercury lamp (TUV15W / G15T8 (Philips)) to adhere the Pana-Tetra. The UV irradiation conditions were 12 cm distance and 30 minutes. Next, the substrate was washed with water to wash away any excess Pana-Tetra that was not sufficiently adhered.

[0058] A ring-shaped LDPE member (inner diameter 4 mm) and the Panatetra-coated area (φ4 mm) on the substrate were aligned in the center and overlapped, and heat-sealed to prepare a container-shaped composite substrate for cell treatment.

[0059] A portion of the obtained cell treatment composite substrate was removed for observation, and the bottom surface was cut out and observed with an ultra-deep multi-angle microscope (VHX-D500, manufactured by Keyence). A coating of needle-shaped particles of Pana-Tetra, partially embedded and fixed in the water-soluble photosensitive resin, was observed (Figure 2).

[0060] 1-2. Coating of antibodies onto composite substrates for cell treatment 50 μL of anti-Cbl antibody (Biolegend) at a concentration of 10 μg / mL was added to the cell treatment composite substrate obtained in 1-1 above, and the substrate was left to stand at 37°C for 2 hours to coat the Panatetra with the antibody. The obtained antibody-coated cell treatment composite substrate was washed twice with 60 μL of PBS (Nacalai Tesque) and then used for the following cell treatment.

[0061] 1-3.Cell preparation PBMCs (peripheral blood mononuclear cells, HameCare) were thawed and cultured for one day. The medium used was ALyS505N-7 (Cell Science Institute) supplemented with 10% fetal bovine serum (Thermo Fisher).

[0062] To stimulate PBMC activation, Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) in an amount equal to the number of cells was added to the cell culture and incubated at 4°C for 1 hour to stimulate the PBMCs and bind beads (weights) to the cell surface.

[0063] 1-4. Cell treatment and evaluation using cell treatment composite substrate The antibody-coated cell treatment composite substrate obtained in 1-2 above was treated with 2.5 x 10 PBMCs stimulated in 1-3 above together with the medium. 5 The cells were added and centrifuged (21880 g, 23°C, 3 minutes), and the needle-shaped part of a Panatetra that had been coated with anti-Cbl antibody was inserted into the cells, which were then allowed to stand at 23°C for 30 minutes.

[0064] The cells were lifted by pipetting, detached from the cell treatment composite substrate, and collected in a 96-well plate. 150 μL of medium was added and the cells were cultured at 37°C in a 5% CO2 environment for 1 or 3 days (Example 1).

[0065] On the other hand, as comparative examples, cells were used that were treated in the same manner as above using a cell treatment composite substrate prepared in the same manner as the above cell treatment composite substrate, except that neither the anti-Cbl antibody nor the Panatetra was coated, or a cell treatment composite substrate prepared in the same manner as the above cell treatment composite substrate, except that the Panatetra was not coated with the anti-Cbl antibody (cells treated with the former cell treatment composite substrate are referred to as "Comparative Example 1", and cells treated with the latter cell treatment composite substrate are referred to as "Comparative Example 2").

[0066] One and three days after the start of culture in a 96-well plate, each cell culture was harvested and stained with anti-CD3 antibody (fluorescence: APC-Cy7, Biolegend), anti-CD25 antibody (fluorescence: APC, Biolegend), and anti-CD137 antibody (fluorescence: Brilliant Violet 421, Biolegend). The staining intensity of each antibody was then analyzed using a flow cytometer, CytoFLES S (Beckman Coulter). For analysis, the CD3-positive population was sorted, and the positivity rates of CD25 and / or CD137 were calculated. Unstained cells were used as the standard for determining whether cells were positive or negative.

[0067] 1-5.Results In general, PBMCs are known to be activated via an intracellular signaling cascade based on CD3 stimulation, resulting in the promotion of expression of the activation markers CD25 and CD137 (Endocrine Journal 2005, 52(5), 635-641; J Immunol Methods. 2008 Nov 30; 339(1):23-37.). Meanwhile, it is known that part of this intracellular signaling cascade based on CD3 stimulation is inhibited by the ubiquitin ligase Cbl, suppressing cell activation, i.e., suppressing expression of CD25 and CD137 (Science Signaling 05 Feb 2019: Vol. 12, Issue 567, eaav4373).

[0068] In the case of cells inserted into and removed from the needle-shaped part of the Panatetra coated with anti-Cbl antibody (Example 1), the number of non-activated cells (CD25 - and CD137 - ) ratio decreased, and the live CD25 + and CD137 - , CD25 + and CD137 + , and CD25 + and CD137 -It was confirmed that the proportion of activated cells, which are expressed by the phenotype of , increased significantly in each case (FIG. 3). Furthermore, in all of Example 1, Comparative Example 1, and Comparative Example 2, the proportion of activated cells was higher after 3 days of culture than after 1 day, and the proportion of activated cells in Example 1 was higher than in Comparative Examples 1 and 2. This confirmed that activation progressed more quickly and was more intense in Example 1 than in Comparative Examples 1 and 2 (FIG. 3 (A) vs. (B)).

[0069] These results suggest that the needle-shaped part of the Panatetra, coated with anti-Cbl antibody, was inserted into PBMCs, allowing the anti-Cbl antibody on the needle to bind to intracellular Cbl, and then when the needle was removed, the Cbl bound to the anti-Cbl antibody was released outside the cells, thereby removing the inhibition of Cbl in the intracellular signal cascade based on CD3 stimulation and promoting cell activation. Furthermore, it was confirmed that the removal of Cbl by this treatment is not a temporary effect but can have a long-term effect lasting at least 3 days.

[0070] Experiment 2: Improving the efficiency of cell treatment using a composite substrate by attaching weights to cells 2-1. Cell preparation and binding of the spindle Jurkat E6.1 (purchased from DS Pharma Biomedical) was cultured in ALyS505N-0 (manufactured by Cell Science Institute) supplemented with 2% fetal bovine serum (manufactured by Thermo Fisher).

[0071] Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher) in an amount equal to the number of cells was added to the cell culture and reacted at 4°C for 1 hour, allowing the beads to bind to the surface of Jurkat cells to form weights.

[0072] 2-2. Efficiency of insertion of Panatetra needle by centrifugal treatment of cells The spindle-bound Jurkat cells prepared in 2-1 above were added to the composite substrate for cell treatment obtained in 1-1 above, and 2.5 × 10 cells were added together with the medium (50 μL). 5This was centrifuged (2000 g, 23°C, 1 minute), the needle of the Panatetra was inserted into the cells, the cells were detached by pipetting, and the centrifugation was repeated again. Centrifugation was performed a total of 10 times.

[0073] After the final (10th) centrifugation, the cells were suspended by pipetting and mixed with an equal volume of trypan blue solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The cells were placed on a hemocytometer and observed under a microscope. Stained cells were counted as dead cells, and unstained cells were counted as live cells. The viability was calculated from these values ​​(Example E).

[0074] On the other hand, as a comparative example, a cell viability test (Comparative Example F) was performed in which a cell treatment composite substrate prepared in the same manner as the cell treatment composite substrate described above, except that it was not coated with Panatetra, was used, and the cell viability test was performed in the same manner as in Example E described above.

[0075] In addition, the viability of cells treated in the same manner as in Example E above, except that Jurkats without weights were used (Comparative Example C), and the viability of cells treated in the same manner as in Comparative Example F above, except that Jurkats without weights were used (Comparative Example D) were used.

[0076] Furthermore, the viability of cells treated in the same manner as in Comparative Example C above, except that the number of cells added was increased fourfold (Reference Example A), and the viability of cells treated in the same manner as in Comparative Example D above, except that the number of cells added was increased fourfold (Reference Example B) were used.

[0077] 2-3.Results The survival rate of each type of cell after repeated centrifugation is shown in FIG. It was confirmed that the cell viability (Example E, 81%) when weight-bound cells were allowed to interact with a cell treatment composite substrate by centrifugation was lower than the viability (Comparative Example F, 95.9%) when the cells were allowed to interact with a cell treatment composite substrate not coated with Pana-Tetra. This decrease in viability can be attributed to the repeated insertion of the needle-shaped part of Pana-Tetra into the cells due to repeated centrifugation. In other words, the decrease in viability indicates that the needle-shaped part of Pana-Tetra was successfully inserted into the cells, and the lower the viability, the higher the efficiency of insertion of the needle-shaped part of Pana-Tetra into the cells.

[0078] On the other hand, when cells not bound to a weight were allowed to interact with a cell treatment composite substrate by centrifugation, the cell viability (Comparative Example C, 95.7%) was lower than the viability when the cells were allowed to interact with a cell treatment composite substrate not coated with Pana-Tetra (Comparative Example D, 98.9%), but was higher than the viability when cells were used with weight-bound cells (Example E, 81%).This result suggests that by attaching a weight to the cells, the force pressing the cells against the needle-shaped part of Pana-Tetra by centrifugation was increased, making it easier for the needle-shaped part to insert into the cells.

[0079] Furthermore, it was confirmed that when the number of cells without attached weights was increased (Reference Example A, 81.1%), a survival rate comparable to that of cells with attached weights was obtained. However, when the number of cells is large, the processing efficiency may decrease in practice, which may not be desirable.

[0080] The results of Experiments 1 and 2 above confirmed that by contacting acicular particles with immobilized intracellular introduction factors with lead-bound cells, the intracellular introduction factors can be introduced and acted on a large number of cells in a simple and efficient manner, and intracellular reactions can be controlled.

Claims

1. a step of contacting a needle-shaped structure on which an intracellular introduction factor is immobilized with a cell to which a bead-shaped weight is attached, and inserting a portion of the needle-shaped structure into the cell to introduce the intracellular introduction factor into the cell; and a step of extracting a portion of the needle-shaped structure from the cell; A method for producing modified cells, comprising:

2. The method of claim 1 , wherein the weight is one or more materials selected from the group consisting of resin, metal, magnetic material, and combinations thereof.

3. The method according to claim 1 or 2, wherein the step of inserting a portion of the needle-shaped structure into a cell and / or extracting it from a cell is carried out using one or more external forces selected from the group consisting of centrifugal force, magnetic force, water flow, water pressure, and electrostatic interaction.

4. The method according to any one of claims 1 to 3, wherein the intracellular introduction factor is a conjugate capable of binding to an intracellular molecule, and the method comprises extracting the needle-shaped body from the cell and extracting the intracellular molecule bound to the conjugate within the cell together with the needle-shaped body.

5. The method of claim 4 , wherein the conjugate is a nucleic acid, a protein, a peptide, or a small molecule compound.

6. The method of claim 4 or 5, wherein the binder is an antibody or a fragment thereof.

7. The method according to any one of claims 1 to 3, wherein the intracellular introduction factor is a physiologically active substance, and the method comprises removing the needle-shaped structure from the cell and leaving the intracellular introduction factor within the cell.

8. The method according to claim 7, wherein the physiologically active substance is one or more substances selected from the group consisting of nucleic acids, peptides, proteins, sugars, polysaccharides, fatty acids, cholesterol, lipids, signal transduction substances, ligand substances, hormone substances, cytokines, ions, metal particles, magnetic particles, inorganic compounds, quantum dots, organic compounds, and drugs.

9. The method according to claim 7 or 8, wherein the bond between the physiologically active substance and the needle-shaped structure is a bond that can be separated within a cell.

10. The method according to claim 9, wherein the bond between the intracellular introduction factor and the needle-shaped structure is at least one selected from the group consisting of an electrostatic bond, a bond due to hydrophobic interaction, a chelate bond, a covalent bond that is cleaved within the cell, a bond via a photocleavable linker, and a bond via an enzyme-cleavable linker.

11. The method according to any one of claims 1 to 10, wherein the needle-shaped bodies are needle-shaped particles.

12. The method according to claim 11, wherein the length of the needle-shaped part of the acicular particles is 1 to 50 μm.

13. 13. The method of claim 11 or 12, wherein the acicular particles are zinc oxide.

14. The method according to any one of claims 1 to 13, wherein the needle-shaped structure is immobilized on a substrate.

15. The method according to claim 14, wherein the substrate has a binder on its surface, and a portion of the needle-shaped structure is fixed to the substrate by the binder.

16. The method of claim 15, wherein the binder comprises a non-protein-adsorbing material.

17. The method according to claim 14, wherein the needle-shaped structure is manufactured using one or more selected from the group consisting of photolithography, dry etching, wet etching, and combinations thereof.

18. The method according to any one of claims 14 to 17, wherein the surface of the substrate having the needle-shaped structures has a fine uneven structure capable of accommodating cells.

19. A method for analyzing the function of the intracellularly introduced factor, comprising the step of analyzing the state of modified cells produced by the method according to any one of claims 1 to 18.

20. The method of claim 19, wherein the step of analyzing the state of the modified cells analyzes one or more selected from the group consisting of cell proliferation rate, viability, metabolism, reactive oxygen species quantification, and gene expression.

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