A method for controlling intracellular reactions using needle-shaped particles

Abstract

To provide a new technique and method for controlling an intracellular reaction that enable a large number of cells to be treated through a simple operation and prevent the introduced factors from remaining in the treated cells.SOLUTION: Provided are a needle-shaped particle immobilized with a binding body boundable to an intracellular molecule, and a composite base material for cell treatment comprising the same, and means for controlling intracellular reaction utilizing the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to acicular particles used for controlling intracellular reactions, on which binders capable of binding to intracellular molecules are immobilized, a cell treatment composite substrate comprising the acicular particles, and a method for controlling intracellular reactions using them. [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. There are also concerns that factors introduced into cells through genetic recombination or genome editing may themselves induce some kind of intracellular reaction, or that such factors may remain in the cells. For this reason, new technologies and methods for controlling intracellular reactions are still desperately needed 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, etc., the positioning and up-and-down movements can be complicated, and substances such as antibodies immobilized on the needle-shaped materials or nanoneedles can also bind to parts other than the needle-shaped materials or nanoneedles (on the support), resulting in loss (waste) of these substances. [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] The present invention aims to provide new technologies and methods for controlling intracellular reactions that enable the processing of a large number of cells with simple manipulations and that do not allow introduced factors to remain in the cells after manipulation. [Means for solving the problem]

[0008] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that by using acicular particles having immobilized thereon binders capable of binding to intracellular molecules, and a composite substrate for cell treatment comprising such acicular particles, it is possible to insert the acicular portion into a cell and extract the target intracellular molecule, thereby making it possible to treat a large number of cells with simple manipulations, and to control and analyze intracellular reactions without leaving any introduced factors remaining in the cells after manipulation.

[0009] The present invention is based on these new findings and includes the following inventions. [1] A needle-shaped particle onto which a binder capable of binding to an intracellular molecule is immobilized. [2] The acicular particles according to [1], wherein the length of the acicular part of the acicular particles is 1 to 50 μm. [3] The acicular particles of [1] or [2], wherein the acicular particles are zinc oxide. [4] The needle-shaped particle according to any one of [1] to [3], wherein the conjugate is a nucleic acid, a protein, a peptide, or a low-molecular-weight compound. [5] The needle-shaped particle according to [4], wherein the conjugate is an antibody or a fragment thereof.

[0010] [6] A composite substrate for cell treatment, in which acicular particles are immobilized on a substrate. [7] The composite substrate for cell treatment according to [6], wherein the length of the needle-shaped part of the needle-shaped particles is 1 to 50 μm. [8] The composite substrate for cell treatment according to [6] or [7], wherein the acicular particles are zinc oxide. [9] The composite substrate for cell treatment according to any one of [6] to [8], wherein the substrate is made of a resin.

[10] The composite substrate for cell treatment according to any one of [6] to [9], wherein the substrate has a binder on its surface, and a portion of the acicular particles is immobilized to the substrate by the binder.

[11] The composite substrate for cell treatment according to

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

[12] The composite substrate for cell treatment according to any one of [6] to

[11] , wherein a fine uneven structure capable of accommodating cells is formed on the surface of the substrate on which the acicular particles are immobilized.

[13] The composite substrate for cell treatment according to any one of [6] to

[12] , wherein a binder capable of binding to an intracellular molecule is immobilized on the acicular particles.

[14] The composite substrate for cell treatment according to

[13] , wherein the conjugate is a nucleic acid, a protein, a peptide, or a low molecular weight compound.

[15] The composite substrate for cell treatment according to

[14] , wherein the conjugate is an antibody or a fragment thereof.

[0011]

[16] A step of inserting the acicular particles of any one of [1] to [5] or a part of the acicular particles of any one of

[13] to

[15] in the cell treatment composite substrate into a cell; A step of binding an intracellular molecule to the conjugate immobilized on the needle-shaped particle; and extracting the acicular particles from the cells together with the intracellular molecules bound to them via the conjugate; Including, A method for producing modified cells in which the function of the intracellular molecule is eliminated or reduced.

[17] The method of

[16] , wherein the modified cells are used to analyze the function of the intracellular molecule.

[18] The method of

[16] or

[17] , wherein the step of inserting a portion of the acicular particle 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.

[19] The method according to any one of

[16] to

[18] , wherein a weight is attached to the cell.

[0012]

[20] A step of inserting the acicular particles of any one of [1] to [5] or a part of the acicular particles of any one of

[13] to

[15] in the cell treatment composite substrate into a cell; A step of binding an intracellular molecule to the conjugate immobilized on the needle-shaped particle; extracting the acicular particles from the cells together with the intracellular molecules bound to them via the conjugate; and analyzing the state of the cells; Including, A method for analyzing the function of the intracellular molecule.

[21] The method according to

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

[22] The method of

[20] or

[21] , wherein the step of inserting a portion of the acicular particle 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.

[23] The method according to any one of

[20] to

[22] , wherein a weight is attached to the cell. [Effects of the Invention]

[0013] According to the present invention, it is possible to treat a large number of cells with simple manipulation, and it is possible to provide new technologies and methods for controlling intracellular reactions, which do not allow introduced factors to remain in the cells after manipulation. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing a method for producing a composite substrate for cell treatment. [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

[0015] 1.Acicular particles In the present invention, the term "acicular particles" refers to fine particles having at least one needle-shaped portion.

[0016] 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.

[0017] The shape of the acicular particles is formed by one or more needle-shaped portions arranged / connected at the center, and may be, for example, rod-shaped, L-shaped, V-shaped, T-shaped, Y-shaped, or radial (e.g., tripod-shaped, quatropod-shaped, tetrapod-shaped, etc.), but is not limited to these. When multiple needle-shaped portions are provided, the needle-shaped portions 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 particles may vary depending on the length and number of the needle-shaped portions provided, but can be large enough to fit into a sphere with a radius of 0.5 μm to 100 μm.

[0018] The above numerical values ​​regarding the sizes of the acicular particles and acicular portions are expressed as average values.

[0019] The acicular particles are made of a material that has little or no toxicity to cells, and preferably no toxicity at all. 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 crystals, zirconium, etc.), glass, plastics (e.g., polyethylene, polypropylene, cyclic polyolefins, cyclic olefin copolymers, polyester, polystyrene, polymethyl acrylate, polylactic acid, polyether ether ketone, fluororesin, etc.), silicon nitride, silicon, etc., and acicular solid materials can be suitably used.

[0020] 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.

[0021] In the present invention, the needle-shaped particles to which a binder capable of binding to an intracellular molecule is immobilized can be used.

[0022] 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 to form a complex, and 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 signal transduction pathway. Preferably, in the present invention, the "conjugate capable of binding to an intracellular molecule" 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.

[0023] The binding (immobilization) of the needle-shaped particle to a binder capable of binding to an intracellular molecule can be performed using methods well known to those skilled in the art, as long as the binding is strong enough to maintain the bond even when the needle-shaped portion is inserted into a cell and then withdrawn. Examples of such binding methods include physical adsorption, covalent bonding, and ionic bonding, with covalent bonding being preferred. For example, when the binder is a protein or peptide, covalent bonding can be performed by chemically reacting functional groups (e.g., hydroxyl groups, amino groups, N-hydroxysuccinimidyl groups, sulfhydryl groups, epoxy groups, vinyl groups, etc.) on the surface of the needle-shaped portion with the carboxyl terminus of the protein or peptide to form an ester bond or an amide bond. If necessary, the surface of the needle-shaped portion can be treated to introduce functional groups, which can be achieved using, for example, polylysine, polyethyleneimine vinyl trialkoxysilane, 3-mercaptopropyl trialkoxysilane, aminoalkyl trialkoxysilane, or epoxy-containing alkyl trialkoxysilane.

[0024] 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.

[0025] The binders immobilized on one acicular particle may all be the same, or a plurality of different binders may be immobilized on the same acicular particle.Furthermore, the same binder may be immobilized on all of the acicular particles, or a different binder may be immobilized on each acicular particle.

[0026] The acicular particles may be provided in a form in which the predetermined binder is immobilized in advance, or may be used after the desired binder is immobilized to the acicular particles as described above (i.e., the acicular particles may not have the predetermined binder immobilized in advance when provided).

[0027] 2. Composite substrate for cell treatment In the present invention, the term "composite substrate for cell treatment" refers to a composite substrate in which a plurality of the acicular particles are immobilized on the surface of a substrate. Hereinafter, unless otherwise specified, the acicular particles immobilized on the surface of a substrate refer to a plurality of acicular particles.

[0028] The "substrate" may be any material capable of immobilizing the acicular particles, and is not particularly limited. Examples include 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 acicular particles, 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, but are not limited to, polyethylene film, polyethylene plate, magnetic beads, etc.

[0029] The acicular particles can be immobilized on the surface of a substrate by any method known to those skilled in the art, as long as it allows the needle-shaped portions of the particles to be inserted into cells and then withdrawn. Examples of such immobilization methods include, but are not limited to, physical adsorption, covalent bonding, ionic bonding, embedding in a substrate, and use of a binder (adhesive).

[0030] The "binder" usable in the present invention may be any material capable of immobilizing the acicular particles on the surface of a substrate, and is not particularly limited. Examples include polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PolyHEMA), polyvinylpyrrolidone, vinyl acetate resin, vinyl chloride resin, epoxy resin, urethane 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 may be used. The binder is applied to a predetermined area on the surface of the substrate, and upon solidification, the acicular particles, which are partially in contact with, or preferably partially embedded in, the binder, adhere and immobilize them on the surface of the substrate. Preferably, the binder has low binding or adsorption properties to the conjugate. Examples of such binders include PVA and PolyHEMA. By using such binders, when attempting to immobilize the conjugate to acicular particles immobilized on a substrate, binding or adsorption of the conjugate to portions other than the acicular particles (e.g., the substrate) can be suppressed, thereby suppressing loss of the conjugate that is not immobilized on the acicular particles. Furthermore, the binder can contain a molecule that interacts 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. By using such molecules, deterioration of the cell condition due to changes from the normal culture environment during cell treatment can be suppressed.

[0031] The surface of the substrate may be formed with a microrelief structure capable of accommodating cells together with immobilized acicular particles. The microrelief structure accommodates cells and holds them between the side walls (e.g., 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, tearing, etc.) caused by the acicular particles that may occur as a result of lateral movement of the cells. In the present invention, the shape of the "microrelief 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 acicular particles are arranged 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 about 1 to 500 μm and a depth of about 1 to 500 μm. Each well may contain one cell or multiple cells.

[0032] The "composite substrate for cell treatment" of the present invention may be provided in a form in which the predetermined binder is immobilized in advance on the acicular particles immobilized on the substrate, or may be used after the desired binder is immobilized on the acicular particles immobilized on the substrate as described above (i.e., the predetermined binder does not have to be immobilized in advance on the acicular particles when provided).

[0033] In the "composite substrate for cell treatment" of the present invention, the conjugates immobilized on one acicular particle may all be the same, or a plurality of different conjugates may be immobilized on the substrate. Furthermore, the same conjugate may be immobilized on all of the multiple acicular particles immobilized on the substrate, or a different conjugate may be immobilized on each acicular particle.

[0034] 3. Production and analysis of engineered cells The acicular particles having immobilized thereon a binder capable of binding to an intracellular molecule, and the cell treatment composite substrate comprising the acicular particles having immobilized thereon a binder capable of binding to an intracellular molecule, can be used in a method for producing modified cells. Hereinafter, unless otherwise specified, any acicular particle refers to a plurality of acicular particles.

[0035] The manufacturing method includes the following steps: inserting into a cell the needle-shaped portion of a needle-shaped particle to which a binder capable of binding to an intracellular molecule is immobilized, or the needle-shaped portion of a needle-shaped particle in a cell treatment composite substrate comprising needle-shaped particles to which a binder capable of binding to an intracellular molecule is immobilized; binding a target intracellular molecule to the conjugate immobilized on the needle-shaped particle in the cell; and and withdrawing the needle-shaped portion from the cell together with the intracellular molecule bound thereto via the conjugate.

[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] The needle-shaped portions of acicular particles can be inserted into cells by contacting the cells with the acicular particles, or the cells with acicular particles immobilized on the cell treatment composite substrate. This contact between the cells and each acicular particle can be achieved by adding the acicular particles to the cells (or by adding the cells to the acicular particles), or by adding the cells to the acicular particles in the cell treatment composite substrate. If necessary, an external force may be applied to bring the cells into contact with the acicular particles. Here, "external force" refers to a force applied to the cells and / or acicular particles, and includes centrifugal force, magnetic force, electrostatic interaction, water flow (water pressure), and the like, and one or more selected from these can be used. Centrifugation can be performed under conditions that do not damage the cells, for example, at 100 to 30,000 g for 30 to 600 seconds. The water flow (water pressure) can be achieved by pipetting. By using an external force, it is possible to increase the impact when the cell comes into contact with the acicular particle, thereby facilitating the insertion of the needle-shaped portion of the acicular particle into the cell (ie, increasing the insertion probability).

[0040] If necessary, a weight may be attached to the cells. Attaching a weight increases the weight of the cells, increasing the impact upon contact between the cells and the acicular particles, and facilitating the insertion of the needle-shaped portion of the acicular particles into the cells (i.e., increasing the probability of insertion). The "weight" may be a bead-shaped material made of resin, metal, magnetic material, or the like, but is not particularly limited thereto as long as it can achieve the above-mentioned effect. For example, in the present invention, magnetic beads such as Dynabeads (registered trademark, Veritas Corporation) can be suitably used as the "weight" for the cells. Binding of the cells to the weight can be performed using methods well known to those skilled in the art, such as one or more of an antibody or its fragment, avidin-biotin, streptavidin-biotin, etc.

[0041] 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.

[0042] The insertion of the needle-shaped part of the acicular particle into the cell may be maintained for a time sufficient for the conjugate immobilized on the inserted needle-shaped part inside the cell 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.

[0043] 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.

[0044] The needle-shaped part can be removed from the cell by applying an external force to separate the contacting cell and needle-shaped particle. Here, the "external force" can be as defined above.

[0045] In this method, the steps of inserting the needle-shaped portion of the acicular particle into the 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.

[0046] The resulting modified cells vary depending on the intracellular function of the intracellular molecule of interest. For example, if the intracellular molecule is a factor that suppresses or inhibits 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)), activated modified cells can be obtained by removing or reducing the function of the factor. Furthermore, if the intracellular molecule of interest is a factor that suppresses or inhibits cell differentiation induction, differentiation-induced modified cells can be obtained by removing or reducing the function of the factor (examples of modified cells are not limited to these).

[0047] Alternatively, the resulting modified cells can be used to analyze the function of intracellular molecules of interest.

[0048] The function of a target intracellular molecule can be analyzed by analyzing the state of modified cells obtained by the above-mentioned method, in which the function of the intracellular molecule has been eliminated or reduced. The state of the modified cells to be analyzed includes, 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 can be quantified and analyzed.

[0049] For example, if the growth rate, viability, metabolism, reactive oxygen species, or gene expression of the resulting modified cells is reduced, suppressed, or eliminated compared to unmodified cells, it can be determined that the intracellular molecule has a function that contributes to maintaining or promoting the growth rate, viability, metabolism, reactive oxygen species, or gene expression. Alternatively, if the growth rate, viability, metabolism, reactive oxygen species, or gene expression of the resulting modified cells is increased or induced compared to unmodified cells, it can be determined that the intracellular molecule has a function that suppresses or inhibits the growth rate, viability, metabolism, reactive oxygen species, or gene expression. In analyzing gene expression, any gene and its encoded protein can be used, but it is preferable that they are different from the target intracellular molecule or the conjugate so that the behavior of the cell can be clarified.

[0050] 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]

[0051] 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 used in this experiment is shown in Figure 1. 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 using a polystyrene cylinder.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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).

[0056] 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.

[0057] 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).

[0058] 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 by binding the beads to their surface.

[0059] 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.

[0060] 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).

[0061] 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").

[0062] 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.

[0063] 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).

[0064] 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)).

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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). 5 This 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] The results of Experiments 1 and 2 above confirmed that by using needle-shaped particles to which binders capable of binding to intracellular molecules are immobilized, it is possible to remove the target intracellular molecules from a large number of cells with simple operations and control intracellular reactions without allowing the introduced factors to remain in the cells even after the operation.

Claims

1. A composite substrate for cell treatment, comprising a container-shaped substrate on which acicular particles are immobilized.

2. 2. The composite substrate for cell treatment according to claim 1, wherein the length of the needle-shaped part of the acicular particles is 1 to 50 μm.

3. The composite substrate for cell treatment according to claim 1 or 2, wherein the acicular particles are zinc oxide.

4. The composite substrate for cell treatment according to any one of claims 1 to 3, wherein the substrate is made of a resin.

5. The composite substrate for cell treatment according to any one of claims 1 to 4, wherein the substrate has a binder on its surface, and a portion of the acicular particles is immobilized to the substrate by the binder.

6. The composite substrate for cell treatment according to claim 5 , wherein the binder is made of a protein non-adsorbing material.

7. The composite substrate for cell treatment according to any one of claims 1 to 6, wherein a fine uneven structure capable of accommodating cells is formed on the surface of the substrate on which the acicular particles are immobilized.

8. The composite substrate for cell treatment according to any one of claims 1 to 7, wherein a binder capable of binding to an intracellular molecule is immobilized on the acicular particles.

9. The composite substrate for cell treatment according to claim 8 , wherein the conjugate is a nucleic acid, a protein, a peptide, or a low molecular weight compound.

10. The composite substrate for cell treatment according to claim 9 , wherein the binder is an antibody or a fragment thereof.

11. A step of inserting a part of the acicular particle in the cell treatment composite substrate according to any one of claims 8 to 10 into a cell; A step of binding an intracellular molecule to the conjugate immobilized on the needle-shaped particle; and extracting the acicular particles from the cells together with the intracellular molecules bound to them via the conjugate; Including, A method for producing modified cells in which the function of the intracellular molecule is eliminated or reduced.

12. The method of claim 11 , wherein the modified cells are used to analyze the function of the intracellular molecule.

13. The method of claim 11 or 12, wherein the step of inserting a portion of the acicular particle 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.

14. The method according to any one of claims 11 to 13, wherein a weight is attached to the cell.

15. A step of inserting a part of the acicular particles in the cell treatment composite substrate according to any one of claims 8 to 10 into a cell. A step of binding an intracellular molecule to the conjugate immobilized on the needle-shaped particle; extracting the acicular particles from the cells together with the intracellular molecules bound to them via the conjugate; and analyzing the state of the cells; Including, A method for analyzing the function of the intracellular molecule.

16. The method according to claim 15, wherein the step of analyzing the state of the 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.

17. The method of claim 15 or 16, wherein the step of inserting a portion of the acicular particle 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.

18. The method of any one of claims 15 to 17, wherein the cells are attached to a weight.

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