A method for controlling intracellular reactions using needle-shaped structures in combination with hypotonic solutions

Abstract

To provide a new technique and method for controlling intracellular reaction, enabling introduction of a predetermined factor into multiple cells by simple and efficient operation.SOLUTION: A method for controlling intracellular reaction includes the steps of: bringing a spicule having an intracellular introduction factor fixed thereto into contact with a cell placed in a low osmotic pressure liquid, inserting a part of the spicule into the cell, and introducing the intracellular introduction factor into the cell; and extracting the part of the spicule 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 component in which a large number of nanoneedles are arranged on a support, formed using photolithography and dry 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 simultaneously inserted 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, etc., the positioning and up-and-down movements can be complicated, placing a heavy burden on the operator. Also, when the needle-shaped material or nanoneedle is pressed against the cell membrane, the cell membrane may sink too far inside and not penetrate, making it impossible to insert the needle-shaped material or nanoneedle into the cell (Non-Patent Document 1), which can reduce the success rate of the operation. [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 [Non-patent literature]

[0007] [Non-Patent Document 1] Trine Berthing et al. Nanotechnology.2012 Oct 19;23(41):415102. Summary of the Invention [Problem to be solved by the invention]

[0008] 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]

[0009] As a result of intensive research to solve the above problems, the present inventors have found that the volume of cells can be increased by placing them in a hypotonic solution, and that when the increased-volume cells are brought into contact with a needle-shaped body, the needle-shaped portion of the needle-shaped body is inserted into the cells more efficiently than when cells whose volume has not been increased are brought into contact.The present inventors have also found that by immobilizing an intracellular introduction factor on a needle-shaped body and bringing it into contact with the increased-volume cells, the introduction factor can be introduced into a large number of cells simply and efficiently, and that intracellular reactions can be controlled and analyzed.

[0010] The present invention is based on these new findings and includes the following inventions. [1] a. A step of contacting a needle-shaped structure on which an intracellular introduction factor is immobilized with a cell, and inserting a portion of the needle-shaped structure into the cell to introduce the intracellular introduction factor into the cell; and b. A step of extracting a portion of the needle-shaped structure from the cell; A method for producing modified cells, comprising: The method comprises placing the cells in a hypotonic solution before the start of step a and / or during step a. [2] The method according to [1], wherein the hypotonic solution comprises a 90% to 5% diluted medium. [3] The method of [1] or [2], wherein the osmotic pressure of the hypotonic solution is 264 mOsm / kg H2O or less and more than 20 mOsm / kg H2O. [4] Any of the methods [1] to [3], wherein one or more external forces selected from the group consisting of centrifugal force, magnetic force, water flow, water pressure, and electrostatic interaction are used to insert a portion of the needle-shaped structure into a cell and / or extract it from the cell. [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 structure from the cell and extracting the intracellular molecule bound to the conjugate within the cell together with the needle-shaped structure. [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] The method according to any one of [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 retaining 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 hypotonic solution for use in any of the methods [1] to

[19] .

[21] 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] .

[22] The method according to

[21] , 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.

[23] a. A step of contacting a needle-shaped structure on which an intracellular introduction factor is immobilized with a cell, and inserting a portion of the needle-shaped structure into the cell to introduce the intracellular introduction factor into the cell; and b. A step of extracting a portion of the needle-shaped structure from the cell; A method for modifying a cell, comprising: The method comprises placing the cells in a hypotonic solution before the start of step a and / or during step a.

[24] A method for inserting a portion of a needle-shaped structure into a cell, comprising: A method comprising placing the cell in a hypotonic solution before and / or during the step of inserting a portion of a needle-shaped body into the cell. [Effects of the Invention]

[0011] 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]

[0012] [Figure 1] FIG. 1 is a graph showing the results of measuring the diameter / volume (black circles) and viability (white squares) of cells treated with solutions having various medium ratios (osmolality). [Figure 2] FIG. 2 is a schematic diagram showing a method for producing a composite substrate for cell treatment on which acicular particles are immobilized. [Figure 3] Figure 3 is a graph showing the residual rate of GAPDH in cells obtained by inserting and removing the needle-shaped part of a Panatetra device coated with anti-glyceraldehyde-3-phosphate dehydrogenase (hereinafter simply referred to as "GAPDH") antibody into and from cells placed in solutions with various medium ratios (osmolality). DETAILED DESCRIPTION OF THE INVENTION

[0013] In the present invention, the term "needle-shaped structure" means a fine structure having at least one needle-shaped portion.

[0014] 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.

[0015] 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.

[0016] 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 shape of a needle-shaped particle or one immobilized on a substrate.

[0017] 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 a single acicular particle varies depending on the length and number of the needle-shaped portions, but can be large enough to fit within a sphere with a radius of 0.5 μm to 100 μm.

[0018] In this specification, the numerical values ​​relating to the sizes of the "acicular bodies," "acicular particles," and "acicular portions" are expressed as average values.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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).

[0024] 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.

[0025] In this specification, needle-shaped structures (needle-shaped particles) "immobilized on a substrate" may be referred to as "composite substrate for cell treatment."

[0026] In the present invention, an "intracellular introduction factor" is immobilized on the needle-shaped structure.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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).

[0032] 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.

[0033] 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).

[0034] 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.

[0035] The present manufacturing method comprises: a. Bringing a needle-shaped structure on which an intracellular introduction factor has been immobilized into a cell into contact with the cell, thereby inserting a portion of the needle-shaped structure into the cell and introducing the intracellular introduction factor into the cell; and then b. Extracting a portion of the needle-shaped structure from the cell; Includes: This production method is characterized in that the cells are placed in a hypotonic solution before the start of step a or during step a.

[0036] 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.

[0037] 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."

[0038] 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.

[0039] In the present invention, a weight may be attached to the cell. Attaching a 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.

[0040] The needle-shaped portion of the needle-shaped structure can be inserted into cells by contacting the cells with the needle-shaped structure. This contact between the cells and the needle-shaped structure can be achieved by adding the needle-shaped structure to the cells (or by adding the cells to the needle-shaped structure), or by adding the cells to a needle-shaped structure 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 structure. Here, "external force" refers to a force applied to the cells and / or the needle-shaped structure, 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 structure, making it easier for the needle-shaped portion of the needle-shaped structure to be inserted into the cells (i.e., increasing the insertion rate). The application of an external force can be performed one or more times.

[0041] In the present invention, the insertion of the needle-shaped part of the needle-shaped particle into the cell is carried out in a hypotonic solution.

[0042] In the present invention, "hypotonic" refers to an osmotic pressure lower than that of intracellular fluid, and "hypotonic fluid" refers to a culture medium having an osmotic pressure lower than that of intracellular fluid. As used herein, "culture medium" refers to a liquid capable of maintaining cell growth and / or survival, and examples of such media include, but are not limited to, culture media, buffer solutions, and solutions containing one or more selected from the group consisting of dimethyl sulfoxide, salts, and sugars (e.g., physiological saline, glucose solution, cell cryopreservation solution, etc.).

[0043] By placing cells in a hypotonic solution, extracellular water flows into the cells, increasing the cell volume, which makes it easier for the needle-shaped portion of the needle-shaped structure to be inserted into the cells (i.e., increasing the insertion probability). This is thought to be because increasing the cell volume increases the tension of the cell membrane, which prevents the cell membrane from sinking too deeply inward when the needle-shaped portion of the needle-shaped structure comes into contact with the cell membrane, making it easier for the needle-shaped portion of the needle-shaped structure to penetrate the cell membrane. That is, in one aspect, the present invention provides a method for efficiently inserting the needle-shaped portion of a needle-shaped structure into cells (with a high insertion probability).

[0044] The hypotonic solution of the present invention may be any solution that can increase the volume of cells placed in the solution (when the cell diameter is used as an index, the length is, for example, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, or 1.09 times or more, with the upper limit being preferably approximately 1.3 times or less, 1.2 times or less, 1.15 times or less, or 1.1 times or less), and the osmotic pressure and composition of the solution can be determined appropriately depending on the cells used.

[0045] A hypotonic solution capable of increasing the volume of a cell can be determined by a simple preliminary study, i.e., by placing the target cell in a hypotonic solution having a predetermined composition and / or osmolality and confirming whether the increase in volume is achieved. Once a hypotonic solution capable of increasing the volume of the target cell is determined, the preliminary study does not need to be performed every time, and the determined hypotonic solution can be used thereafter.

[0046] In one embodiment, a diluted medium can be used as the hypotonic solution of the present invention. The diluted medium refers to a medium obtained by diluting a medium typically used to maintain the growth and / or survival of target cells. For example, when the concentration of a typically used medium is taken as 100%, a medium diluted to a concentration of 90% to 5%, preferably 80% to 10%, more preferably 80% to 20%, and even more preferably 70% to 30% can be used. Ultrapure water, etc., can be used to dilute the medium, although there are no particular limitations.

[0047] Furthermore, in one embodiment, when the target cells are mammalian cells, preferably human cells, the hypotonic solution of the present invention can be a medium having an osmotic pressure selected from the range of 264 mOsm / kg HO or less, or more than 20 mOsm / kg HO, for example, 260 to 25 mOsm / kg HO, preferably 260 to 30 mOsm / kg HO, more preferably 250 to 30 mOsm / kg HO, even more preferably 200 to 30 mOsm / kg HO, and particularly preferably 196 to 84 mOsm / kg HO (e.g., 100 to 30 mOsm / kg HO).

[0048] The treatment of placing cells in a hypotonic solution can be carried out by adding the cells to the hypotonic solution or by adding a hypotonic solution to the cells. The treatment is effective when carried out before the start of the step of inserting the needle-shaped portion of the acicular particle into the cells, but it can also be effective when carried out during the step as long as the insertion can newly occur.

[0049] The insertion of the needle-shaped part of the acicular particle into the cell is preferably carried out in a hypotonic solution 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.

[0050] When the intracellular introduction factor immobilized on the needle-shaped body is the aforementioned "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, and can be carried out by leaving the cells stationary for this time.

[0051] 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.

[0052] Alternatively, when the intracellular introduction factor immobilized on the needle-shaped body is the aforementioned "biologically 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 aforementioned 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, and can be carried out by leaving the cells to stand for this time.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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).

[0058] In another embodiment, the resulting modified cells can be used to analyze the function of the introduced intracellular transfection factor.

[0059] 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 of these selected states may be quantified and analyzed.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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]

[0064] Experiment 1: Treatment of cells with hypotonic solutions (preliminary study) 1-1.Cell preparation Jurkat E6.1 (purchased from DS Pharma Biomedical) was cultured in ALyS505N-0 (Cell Science Institute) medium supplemented with 2% fetal bovine serum (Thermo Fisher).

[0065] 1-2. Preparation of culture medium RPMI1640 (osmolality 280 mOsm / kg H2O; Thermo Fisher Scientific) and ultrapure water were prepared and mixed in the specified ratios to prepare solutions with medium ratios of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%.

[0066] 1-3. Cell immersion treatment and evaluation Jurkat 5×10 5 The cells were suspended in 1 mL of the above solution containing various media ratios and allowed to stand at 4°C for 10 minutes, followed by 20 minutes at 37°C. A portion of the suspension was then taken and the cell viability and diameter were measured using a NucleoCounter NC-200 (Chemometec). Cell diameter measurements using the NucleoCounter NC-200 were calculated from the spread of fluorescence from cells stained with a fluorescent substance.

[0067] 1-4.Measurement results The results of viability and diameter measurements of cells treated with the above solutions with various media ratios are shown in FIG.

[0068] Compared to the 100% medium solution, the diameter of cells treated with solutions containing 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10% medium was larger (black circles). More specifically, the diameter of cells treated with the 100% medium solution was 13.4 μm, while the diameter of cells treated with the 30% medium solution was 14.5 μm.

[0069] It was confirmed that the diameter of cells treated with solutions with a medium ratio lower than 30% (20%, 10%) significantly increased, but the viability of these cells (open squares) significantly decreased.

[0070] The diameter of cells treated with a solution with a 0% medium ratio (ultrapure water) was similar to that of cells treated with a solution with a 100% medium ratio, but it was confirmed that the cell viability was significantly reduced.

[0071] From the above results, it was confirmed that by placing cells in a hypotonic solution with a medium ratio of approximately 30%, the cell diameter, i.e., volume, can be increased without significantly reducing the cell viability.

[0072] Experiment 2: Cell modification by treatment of cell treatment composite matrix with hypotonic solution 2-1. 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 2. A linear low-density polyethylene (LLDPE) film (masking) with a 4 mm diameter hole was placed on top of the LLDPE film, 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 with a silicone spatula.

[0073] A 5% ethanol dispersion of acicular particles (Panatetra (registered trademark) WZ-0501L (Amtec); average needle length 20 μm) was dropped onto the substrate, and the substrate was coated with Panatetra using a bar coater (No. 20).

[0074] 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.

[0075] 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.

[0076] 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 Panatetra, part of which was embedded and fixed in the water-soluble photosensitive resin, was observed.

[0077] 2-2. Coating of antibodies onto cell treatment composite substrates To the cell treatment composite substrate obtained in 2-1 above, 25 μL of anti-GAPDH antibody (Biolegend) at a concentration of 50 μg / mL was added and left to stand for 2 hours at 37°C to coat the Panatetra with the antibody. The obtained antibody-coated cell treatment composite substrate was washed once with 60 μL of PBS (Nacalai Tesque) and then used for the following cell treatment.

[0078] 2-3.Cell Preparation 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).

[0079] 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.

[0080] 2-4. Cell treatment and evaluation using cell treatment composite substrate The anti-GAPDH antibody-coated cell treatment composite substrate obtained in 2-2 above was treated with the beads-bound cells in 2-3 above at a concentration of 1.0 × 10 cells / well together with RPMI 1640 medium (osmolality: 280 mOsm / kg HO; Thermo Fisher Scientific; Comparative Example 1) or solutions diluted with ultrapure water to a medium ratio of 70%, 50%, or 30% (osmolality: 196, 140, and 84 mOsm / kg HO, respectively; Examples 1, 2, and 3). 5 The cells were added and centrifuged (2000 g, 4°C, 3 minutes), and the needle-shaped part of a Panatetra that had been coated with anti-GAPDH antibody was inserted into the cells, which were then left to stand at 37°C for 30 minutes.

[0081] The cells were lifted by pipetting, detached from the cell treatment composite substrate, fixed with Fixation Buffer (R&D Systems), stained with Alexa488®-labeled anti-GAPDH antibody (Biolegend), and analyzed for fluorescence intensity using a flow cytometer, CytoFLES S (Beckman Coulter). Two tubes containing cells without the Panatetra anti-GAPDH antibody coating were prepared for each solution. During flow cytometer analysis, one tube was stained with anti-GAPDH antibody to serve as a positive control (Posi-con), and the other was treated with Alexa488®-labeled isotype antibody (Biolegend) to serve as a negative control (Nega-con).

[0082] To analyze the residual rate of GAPDH, a population of cells with normal morphology was sorted, and the median values ​​of FSC and GAPDH fluorescence intensity were calculated from the sorted population, and the relative residual amount of GAPDH was evaluated using the calculation method described below.

[0083]

number

[0084] 2-5.Results The needle-shaped part of the Panatetra, coated with anti-GAPDH antibody, is inserted into a cell and left there, allowing the anti-GAPDH antibody on the needle to bind to GAPDH inside the cell.When the needle-shaped part is then removed, the GAPDH bound to the anti-GAPDH antibody is also extracted outside the cell.It was confirmed that the fluorescence intensity of GAPDH in cells subjected to this treatment was reduced compared to that of cells not subjected to this treatment.

[0085] The cells were then subjected to the above treatment using RPMI1640 medium (osmolality: 280 mOsm / kg HO; Thermo Fisher Scientific; Comparative Example 1) or solutions diluted with ultrapure water to a medium ratio of 70%, 50%, or 30% (osmolality: 196, 140, or 84 mOsm / kg HO, respectively; Examples 1, 2, and 3). The analysis results of the residual GAPDH activity are shown in Figure 3.

[0086] It was confirmed that the residual rate of GAPDH was lower in cells treated with solutions containing reduced media ratios (70%, 50%, 30%) (Examples 1-3) than in cells treated with a solution containing 100% media ratio (Comparative Example 1), i.e., more GAPDH was removed from the cells.

[0087] The results of Experiments 1 and 2 above confirmed that placing cells in a solution with a reduced culture medium ratio (i.e., a culture medium with reduced osmotic pressure) can increase the volume of the cells, thereby increasing the efficiency of insertion of the needle-shaped part onto which the intracellular introduction factor is immobilized into the cells. It was also confirmed that the intracellular introduction factor can be introduced and acted on a large number of cells with simple procedures, enabling efficient cell modification.

Claims

1. a. Adding cells to a substrate on which a plurality of needle-shaped structures having intracellular introduction factors immobilized thereon are immobilized, bringing the needle-shaped structures into contact with the cells, and inserting a portion of the needle-shaped structures into the cells to introduce the intracellular introduction factors into the cells; b) A step of extracting a part of the needle-shaped structure from the cell; A method for producing modified cells, comprising: The method comprises placing the cells in a hypotonic solution at least during step a.

2. 2. The method of claim 1, wherein the hypotonic fluid consists of 90% to 5% diluted medium.

3. 3. The method of claim 1 or 2, wherein the osmolality of the hypotonic solution is 264 mOsm / kg H2O or less and more than 20 mOsm / kg H2O.

4. The method according to any one of claims 1 to 3, wherein one or more external forces selected from the group consisting of centrifugal force, magnetic force, water flow, water pressure, and electrostatic interaction are used to insert a portion of the needle-shaped structure into a cell and / or extract it from a cell.

5. The method according to any one of claims 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 of claim 5 , wherein the conjugate is a nucleic acid, a protein, a peptide, or a small molecule compound.

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

8. The method according to any one of claims 1 to 4, wherein the intracellular introduction factor is a physiologically active substance, and the method comprises extracting the needle-shaped structure from the cell and retaining the intracellular introduction factor within the cell.

9. The method according to claim 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 claim 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 according to claim 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 claims 1 to 11, wherein the needle-shaped bodies are needle-shaped particles.

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

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

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

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

18. The method according to claim 15, 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.

19. The method according to any one of claims 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 hypotonic solution for use in the method according to any one of claims 1 to 19.

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

22. The method of claim 21, 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.

23. a. Adding cells to a substrate on which a plurality of needle-shaped structures having intracellular introduction factors immobilized thereon are immobilized, bringing the needle-shaped structures into contact with the cells, and inserting a portion of the needle-shaped structures into the cells to introduce the intracellular introduction factors into the cells; b) A step of extracting a part of the needle-shaped structure from the cell; A method for modifying a cell, comprising: The method comprises placing the cells in a hypotonic solution at least during step a.

24. A method for inserting parts of multiple needle-shaped structures immobilized on a substrate into cells, comprising: The method comprises placing the cells in a hypotonic solution at least during the steps of adding cells to the substrate, contacting the needle-shaped body with the cells, and inserting a portion of the needle-shaped body into the cells.

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