Method for producing microcapsules and method for producing core beads

Abstract

The purpose of the present invention is to provide a method for producing uniformly sized microcapsules by efficiently and noninvasively enclosing various objects without requiring a microchannel device when enclosing the objects.　This method for producing microcapsules that hold an object in a hollow aqueous phase comprises the following steps 1-7 in this order.　The step 1 is a step for mixing, at a temperature equal to or lower than the dissolution temperature of a core material which dissolves or gels only by temperature changes, an aqueous phase containing a particulate core material and an object with an oil phase for forming droplets to obtain a first water droplet-in-oil suspension in which the droplets containing the particulate core material and the object are dispersed in the oil phase. The step 2 is a step for heating the first water droplet-in-oil suspension at a temperature equal to or lower than the dissolution temperature of the core material, and then cooling the same at a temperature equal to or lower than the gelling temperature of the core material to obtain core beads enclosing the object. The step 3 is a step for extracting the core beads enclosing the object into the aqueous phase. The step 4 is a step for mixing an aqueous phase containing the core beads enclosing the object and a dissolved shell raw material with an oil phase for forming droplets under the conditions in which the core beads do not dissolve, to obtain a second water droplet-in-oil suspension. The step 5 is a step for insolubilizing the shell raw material in the second water droplet-in-oil suspension under the conditions in which the core beads do not dissolve, to obtain core-shell particles having core beads enclosing the object and a shell. The step 6 is a step for extracting the core-shell particles into the aqueous phase. The step 7 is a step for providing, to the core-shell particles, the conditions in which a core material dissolves by temperature changes but a shell mate

Description

Method for producing microcapsules and method for producing core beads

[0001] The present invention relates to a method for producing microcapsules and a method for producing core beads.

[0002] Microcapsules can control the form, reactivity, influence of external pressure, etc. of the substance encapsulated therein, and thus are widely used in pharmaceuticals, cosmetics, biochemical research, cell transplantation research, etc. Many of these have an outer shell (shell) covering the contents and stably hold the contents inside the shell (core). Compared with the case where the shell is an insoluble polymer, microcapsules with a shell of a low molecular permeability hydrogel and an aqueous core can grow microorganisms and cells inside the shell by encapsulating microorganisms and cells in the core. In addition, when cells are encapsulated in hollow hydrogel microcapsules for cell transplantation, it is expected to improve the fixation rate of transplanted cells because it prevents attacks by immune system cells while allowing oxygen and nutrients to permeate. Currently, in many methods, due to the requirement of making the diameter uniform, a microchannel device is used to form a hollow shell structure, so the operation is complicated, it is difficult to fabricate in a short time, and because a microchannel device is used, there is a problem that expensive equipment is required. The invention described in Patent Document 1 provides a manufacturing technique for hollow hydrogel microcapsules that can be stably stored while suppressing damage and contamination to the object, and is a method for manufacturing a hydrogel capsule having a core containing an ionically bonded polymer and a shell containing a thermally dependent polymer. A suspension in which a sol (aqueous phase) of the thermally dependent polymer containing a core at least the surface of which is formed from the gel of the ionically bonded polymer is suspended in an oil phase is cooled to gel the thermally dependent polymer to form a shell. The oil contained in the oil phase has a hydrophobicity such that the octanol / water partition coefficient is 3 or more, and a capsule manufacturing method in which the specific gravity and viscosity satisfy a specific formula is described. The invention described in Non-Patent Document 1 generates uniform hydrogel beads using a microchannel and at the same time embeds cells inside the beads.

[0003] International Publication No. 2020 / 184680

[0004] Tan & Takeuchi, Advanced Materials, vol. 19, 2696-2701, 2007

[0005] The method described in Patent Document 1 has an uneven particle size in the core, making it difficult to obtain capsules of the desired size. Furthermore, although alginate gel is used as the core, acetic acid or calcium carbonate is used for gelation, which may damage cells when encapsulating them. The method described in Non-Patent Document 1 also has the potential to damage cells, similar to the invention in Patent Document 1, and requires the use of a microfluidic device each time microcapsules are produced. The present invention aims to provide a method for efficiently and non-invasively producing core beads and microcapsules of uniform size for various objects without requiring a microfluidic device when encapsulating the target object.

[0006] As a result of diligent research, the inventors have found that the above problems can be solved by manufacturing core beads and microcapsules using a specific method with a core material that dissolves or gels solely by temperature changes. The present invention includes the following embodiments.

[0007] <1> A method for manufacturing microcapsules that hold an object in the aqueous phase of a hollow part, comprising the following steps 1 to 7 in this order. Step 1: Mix an aqueous phase containing particulate core material and target object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil suspension in which droplets containing particulate core material and target object are dispersed in the oil phase. Step 2: Heat the first water-in-oil suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing the target object. Step 3: Remove the core beads containing the target object into the aqueous phase. Step 4: Mix the aqueous phase containing the core beads containing the target object and dissolved shell raw material with an oil phase for droplet formation under conditions in which the core beads do not dissolve to obtain a second water-in-oil suspension. Step 5: In the second water-in-oil suspension, insolubilize the shell raw material under conditions in which the core beads do not dissolve to obtain core-shell particles having core beads containing the target object and a shell. Step 6: Step of removing the core-shell particles into an aqueous phase, and Step 7: Step of dissolving the core beads by applying conditions to the core-shell particles such that the core material dissolves due to a change in temperature while the shell material does not dissolve, thereby holding the target object in the hollow aqueous phase and obtaining a microcapsule having a shell. <2> A method for producing a microcapsule according to <1>, wherein the core material comprises at least one selected from the group consisting of gelatin, collagen, agarose, and Matrigel, and the shell raw material comprises at least one selected from the group consisting of compounds that dissolve or gel with a change in temperature, compounds that become insoluble by crosslinking, and compounds that become insoluble by polymerization. <3> A method for producing a microcapsule according to <1> or <2>, wherein the target object is a biological or chemical material selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, proteins, and organic compounds. <4> A method for producing a microcapsule according to any one of <1> to <3>, wherein the target object is a cell.<5> A method for producing a microcapsule according to any one of <1> to <4>, wherein in step 1, the droplet is selected from the group consisting of a single cell, a single microorganism, a single virus particle, a single nucleic acid molecule, a single peptide molecule, a single protein molecule, and a single organic compound molecule. <6> A method for producing a microcapsule according to any one of <1> to <5>, wherein in step 1, the object includes at least one selected from the group consisting of a cell, a microorganism, a virus, nucleic acid, a peptide, an organic compound, and at least one of these being attached, modified, or bound to a solid or gel support. <7> A method for producing a microcapsule according to any one of <1> to <6>, wherein the average particle diameter of the particulate core material is 1 μm or more and 500 μm or less. <8> A method for producing a microcapsule according to any one of <1> to <7>, wherein the average particle diameter of the microcapsule is 1 μm or more and 700 μm or less. <9> A method for growing cells in a microcapsule, comprising the steps of: producing a microcapsule that holds cells in a hollow aqueous phase by the method described in <4>, and growing cells in the hollow in the presence of a culture medium. <10> A method for producing core beads containing an object, comprising the following steps 1 to 3 in this order: Step 1: Mix an aqueous phase containing particulate core material and an object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil type suspension in which droplets containing particulate core material and an object are dispersed in the oil phase; Step 2: Heat the first water-in-oil type suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing an object; Step 3: Remove the core beads containing the object into the aqueous phase. <11> A kit for forming core beads, comprising particulate core material that dissolves or gels by temperature change alone, and an oily composition for droplet formation.

[0008] According to the present invention, a method is provided for efficiently and non-invasively manufacturing microbeads and microcapsules of uniform size for various objects, without requiring a microfluidic device to encapsulate the object.

[0009] Figure 1 shows images of microcapsules obtained in the example (using U937 cells, which are suspension cells) on day 0 and day 3. Figure 2 shows images of microcapsules obtained in the example (using HEK293T cells, which are adherent cells) on day 0 and day 3. Figure 3 shows an image of cup-shaped gelatin particles (gelatin cups) obtained in the example. Figure 4 shows an image of core beads containing cells obtained using cup-shaped gelatin particles (gelatin cups) in the aqueous phase.

[0010] The present invention will be described in detail below using exemplary embodiments as examples, but the present invention is not limited to the embodiments described below. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in the practice of the present invention. Furthermore, all publications and patents cited herein in connection with the invention described herein constitute part of this specification, for example, as indicating methods, materials, and other things that can be used in the present invention.

[0011] In this specification, the numerical range indicated by "A to B" means a numerical range that includes the endpoints A and B. The same applies to "A to B". In this specification, "microcapsule" means a minute container structure with a size (diameter) generally ranging from a few micrometers to several thousand micrometers. In this specification, "shell" means the outer shell structure of a microcapsule that can cover its contents. In this specification, the shell is preferably composed of a hydrogel. In this specification, "core bead" means a granular structure that can be located in the core of a core-shell particle. In this specification, "core material" means the material that constitutes the core bead. Furthermore, "single cell" means one cell, i.e., a single cell, and when simply referred to as "cell," it includes any case of multiple types of cells, one or more identical cells. Furthermore, "identical cells" means genetically identical cells. The same applies to "single microorganism".

[0012] [Method for manufacturing microcapsules] The method for manufacturing microcapsules that hold the object in the hollow aqueous phase of this embodiment (hereinafter also simply referred to as "microcapsule manufacturing method") comprises the following steps 1 to 7 in this order. Step 1: Mix an aqueous phase containing particulate core material and target object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil suspension in which droplets containing particulate core material and target object are dispersed in the oil phase. Step 2: Heat the first water-in-oil suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing the target object. Step 3: Remove the core beads containing the target object into the aqueous phase. Step 4: Mix the aqueous phase containing the core beads containing the target object and dissolved shell raw material with an oil phase for droplet formation under conditions in which the core beads do not dissolve to obtain a second water-in-oil suspension. Step 5: In the second water-in-oil suspension, insolubilize the shell raw material under conditions in which the core beads do not dissolve to obtain core-shell particles having core beads containing the target object and a shell. Step 6: Step of extracting the core-shell particles into an aqueous phase, and Step 7: Step of dissolving the core beads by applying conditions under which the core material dissolves due to temperature changes while the shell material does not, thereby holding the target object in the hollow aqueous phase and obtaining a microcapsule having a shell. According to the present invention, a method is provided for efficiently and non-invasively producing microcapsules of uniform size for various targets without requiring a microfluidic device when encapsulating the target object. In this embodiment, particulate core material is used, and by using a material that dissolves or gels only with temperature changes as the core material, the target object can be encapsulated in the core beads by temperature changes alone. Furthermore, by using particulate core material with a uniform particle size beforehand, the particle size of the core beads encapsulating the target object can also be made uniform. In addition, it is possible to encapsulate the target object in the core beads without using acids, bases, metal salts, etc., and as a result, damage to the target object is suppressed, and the target object can be encapsulated in the core beads non-invasively. Steps 1 to 7 will be described below.

[0013] <Step 1> Step 1 is a process in which an aqueous phase containing particulate core material and target object is mixed with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels with temperature changes alone, thereby obtaining a first water-in-oil suspension in which droplets containing particulate core material and target object are dispersed in the oil phase. The materials used in Step 1 will be described below.

[0014] [Particulate Core Material] The particulate core material used in step 1 melts or gels with temperature changes alone. The core material melts or gels with temperature changes alone, and is preferably a hydrogel. The core material preferably contains at least one selected from the group consisting of gelatin, collagen, agarose, and Matrigel, more preferably contains at least one selected from the group consisting of gelatin, collagen, and agarose, even more preferably contains gelatin, and even more preferably is gelatin.

[0015] When the core material is gelatin, from the viewpoint of reducing the impact on the target object, the temperature at which the material is melted by raising the temperature is preferably 30°C or higher, more preferably 35°C or higher, and preferably 60°C or lower, more preferably 50°C or lower, and even more preferably 40°C or lower. When gelation is performed by lowering the temperature, the temperature is preferably 16°C or lower, more preferably 10°C or lower, even more preferably 4°C or lower, and preferably above 0°C, more preferably 1°C or higher, and even more preferably 3°C or higher. When the core material is other than gelatin, the temperature should be appropriately selected according to the core material.

[0016] The core material is particulate. The particulate core material is not limited to a spherical shape, but can be any shape that can form droplets containing the particulate core material and the target object. Examples of particulate core material shapes include spherical, flattened, hemispherical, cup-shaped (a shape in which a part of the spherical core material is hollowed out to form a depression), and porous (a shape with a large number of holes or irregularities on the surface or inside). Among these, a spherical or cup-shaped core material is preferred from the viewpoint of ease of manufacturing. The shape of the particulate core material can be appropriately selected depending on the type and size of the target object and the amount of target object to be held in the hollow part of the resulting microcapsule. For example, if the target object is a cell, a spherical core material is selected when holding a single cell in a microcapsule, and a cup-shaped core material is selected when holding multiple cells in a microcapsule. Because the particulate core material is cup-shaped, droplets containing a large amount of target object can be easily formed in step 1.

[0017] The average particle size of the particulate core material is not particularly limited and can be appropriately determined considering ease of manufacturing, ease of encapsulation of the target object, etc., but is preferably 1 μm or more, more preferably 5 μm or more, even more preferably 10 μm or more, even more preferably 20 μm or more, even more preferably 30 μm or more, and preferably 500 μm or less, more preferably 300 μm or less, even more preferably 200 μm or less, and even more preferably 100 μm or less. The average particle size may be appropriately changed depending on the target object to be encapsulated by the microcapsule. When the particulate core material is a cup-shaped core material, from the viewpoint of obtaining core beads that encapsulate multiple cells, etc., as described later, and from the viewpoint of ease of manufacturing the cup-shaped core material, the average particle size is preferably 10 μm or more, more preferably 20 μm or more, even more preferably 30 μm or more, even more preferably 50 μm or more, and preferably 300 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. The average particle size of the particulate core material is measured by the method described in the examples.

[0018] (Method for manufacturing particulate core material) The method for manufacturing particulate core material is not particularly limited, and it is sufficient to obtain particulate core material having a desired particle size. However, it is preferable to manufacture it by forming droplets of uniform size using a microfluidic device and then gelling them. More specifically, an aqueous solution of the core material and a droplet-generating oil are delivered to a microfluidic device to generate an emulsion containing the core material, and the particulate core material can be obtained by keeping this emulsion below a certain temperature.

[0019] Furthermore, by introducing an aqueous solution of a water-soluble compound that phase-separates from the core material (hereinafter, "water-soluble compound that phase-separates from the core material" is also referred to as "compound A") into a microfluidic device, in addition to an aqueous solution of the core material and a droplet-forming oil, or by introducing an aqueous solution containing the core material and compound A, and a droplet-forming oil into a microfluidic device, an emulsion containing the core material and compound A is generated. After lowering this emulsion to a certain temperature or below, compound A is dissolved in water to obtain a particulate core material with a cup-shaped form in which the compound A portion is missing. Compound A is selected as a compound that is water-soluble even below the dissolution temperature of the core material and phase-separates from the core material. Specifically, when the core material is gelatin, collagen, agarose, or Matrigel, examples of compound A include polyethylene glycol, dextran, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), Ficol, cellulose derivatives (carboxymethylcellulose, hydroxyethylcellulose, etc.), starch derivatives, and hyaluronic acid.

[0020] The particle size of the particulate core material can be adjusted by appropriately changing the liquid delivery rate, liquid delivery volume, flow path diameter, pressure, etc. Alternatively, the obtained particulate core material may be classified using known methods to obtain particulate core material with uniform particle size (narrow particle size distribution).

[0021] [Subject Matter] In this embodiment, the subject matter is the object to be held in the aqueous phase of the hollow part of the microcapsule. Preferably, it is a biological or chemical material selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, proteins, and organic compounds, and more preferably, it is a cell. In step 1, it is also preferable that the subject matter contained in the droplet is one selected from the group consisting of a single cell, a single microorganism, one virus particle, one molecule of nucleic acid, one molecule of peptide, one molecule of protein, and one molecule of organic compound. Among these, it is more preferable that the subject matter contained in the droplet in step 1 is a single cell or a single microorganism. For example, if the droplet contains a single cell, cloning can be performed by the proliferation of one cell in the aqueous phase of the hollow part of the microcapsule obtained by the manufacturing method of this embodiment. In step 2, it is also preferable that the subject matter contained in the droplet is multiple cells of the same species. By culturing cells in the hollow part of the microcapsule obtained by the manufacturing method of this embodiment, organoids or spheroids can be formed. Differentiation induction or maturation may occur during cultivation. These organoids and spheroids are expected to be used, for example, to evaluate drug responsiveness.

[0022] Furthermore, the object may include at least one selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, organic compounds, and at least one of these attached, modified, or bound to a solid or gel-phase support. That is, it is also preferable that the object is one in which at least one selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, and organic compounds attached, modified, or bound to a solid or gel-phase support. For example, if the object is an organic compound attached, modified, or bound to a solid or gel-phase support, the organic compound may be one type or multiple types. Also, multiple units of one type of organic compound may be attached, modified, or bound to the support. Furthermore, for example, cells attached, modified, or bound to a support may be used in combination with organic compounds attached, modified, or bound to a support, and the cells and organic compounds may be attached, modified, or bound to the same support or to separate supports. Moreover, cells and organic compounds attached, modified, or bound to a support may be used in combination. Among these, a preferred embodiment is one in which one selected from the group consisting of the same type of cell, the same type of microorganism, the same type of virus, the same type of nucleic acid, the same type of peptide, and the same type of organic compound is adhered, modified, or bound to a solid-phase or gel-phase support. Objects that may pass through the shell are preferably adhered, modified, or bound to the support and supported on the support. The support is not particularly limited, but it is preferably particulate (bead-shaped). Specific examples of supports include polystyrene beads, latex beads, agarose, polyacrylamide, and PEG. Examples of methods for adhesion, modification, or binding to the support include avidin-biotin interaction, antigen-antibody interaction, cell adhesion proteins, and cell adhesion by ionic bonding.Specifically, for a method of synthesizing nucleic acid-encoded compounds (DNA-encoded libraries) on solid-phase beads (TentaGel Beads), refer to Andrew B. MacConnell, et al., “DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis”, ACS Comb. Sci., 2015, Vol 17, Issue 9, pp. 518-534. Additionally, Wesley G. Cochrane, et al., “Activity-Based DNA-Encoded Library Screening”, ACS Comb. Sci., Vol 12, Issue 5, pp. 425-435 describes a method for performing activity-based screening using beads synthesized in this way. Furthermore, for a method of synthesizing a combinatorial peptide library of "one-bead-one compound" (OBOC) compounds bound to solid-phase beads (TentaGel) and a screening method using this library, refer to Kit S Lam, et al., “Synthesis and Screening of “One-Bead One-Compound” Combinatorial Peptide Libraries”, Methods Enzymol., 2003, Vol. 369, pp. 298-322. Also, for a method of immobilizing cells onto solid-phase beads, refer to Doyeon Koo, et al., “Defining T cell receptor repertoires using nanovial-based binding and functional screening”, Proc Natl Acad Sci USA., 2024, Vol. 121, No. 14, e2320442121.

[0023] Furthermore, the object may include multiple types of objects as described above. For example, if the object is a cell and an organic compound (which may be bound to a support), it is possible to evaluate the effect of the organic compound on the cell. By using cells into which various genes have been introduced as the object, and having the microcapsule contain these cells, it is possible to evaluate the effect of the compound on cells with many types of genes.

[0024] [Oil phase for droplet formation] Various oil phases known as droplet oils are used as the oil phase for droplet formation, and are oily compositions containing, for example, fluorine oils such as 2-(trifluoromethyl)-3-ethoxidedecafluorohexane and surfactants.

[0025] The mixing temperature of the aqueous phase and the oil phase in step 1 is below the melting temperature of the core material. When gelatin is used as the core material, it is preferably 30°C or lower, more preferably 20°C or lower, and even more preferably 10°C or lower. Furthermore, from the viewpoint of suppressing the freezing of the aqueous phase, the mixing temperature is preferably above 0°C, more preferably 1°C or higher, and even more preferably 3°C or higher. If the core material is other than gelatin, the temperature can be appropriately selected according to the core material. It is preferable to keep the mixing temperature within the above range because it suppresses the melting of particulate core material, prevents the freezing of the aqueous phase, and reduces damage to the object.

[0026] A water-in-oil suspension is prepared by mixing particulate core material and target material in an aqueous phase and adding an oil phase. Alternatively, the aqueous phase may be centrifuged before adding the oil phase to obtain pellets containing particulate core material and target material, to which the oil phase may be added. From the viewpoint of obtaining a stable water-in-oil suspension, the amount of oil phase for droplet formation per 100 volumes of aqueous phase (or pellets containing particulate core material and target material) is preferably 100 volumes or more, more preferably 200 volumes or more, even more preferably 500 volumes or more, and preferably 2000 volumes or less, more preferably 1500 volumes or less, and even more preferably 1000 volumes or less.

[0027] The mixing of the aqueous phase containing particulate core material and target object with the oil phase for droplet formation is not particularly limited as long as an oil-in-water droplet suspension is obtained in which droplets containing particulate core material and target object are dispersed in the oil phase. Examples of mixing methods include mixing with a vortex mixer, mixing with a magnetic stirrer, and mixing by pipetting. Among these, mixing with a vortex mixer is preferred because it is easy to operate. It is also preferable to control the temperature so that the temperature in the system does not rise during mixing.

[0028] <Step 2> Step 2 is a step in which the water-in-oil suspension obtained in Step 1 is heated to a temperature above the melting temperature of the core material, and then cooled to a temperature below the gelation temperature of the core material to obtain core beads (hereinafter also simply referred to as "core beads") that contain the target object. The heating temperature is above the melting temperature of the core material, and when the core material is gelatin and the target object is cells, it is preferably 30°C or higher and 40°C or lower. When the core material is other than gelatin or when the target object is different, the temperature can be appropriately selected according to the core material and the target object. The heating time is not particularly limited as long as it is sufficient time for the core material to dissolve, for example, preferably 1 minute or more, more preferably 3 minutes or more, and from the viewpoint of suppressing the coalescence of water droplets in oil, it is preferably 30 minutes or less, more preferably 15 minutes or less, and even more preferably 10 minutes or less.

[0029] In step 2, after heating as described above, the core material is cooled to a temperature below its gelation temperature. The cooling temperature is below the gelation temperature of the core material, and if the core material is gelatin, it is preferably 16°C or lower, more preferably 10°C or lower, and even more preferably 4°C or lower. The mixing temperature is preferably above 0°C, more preferably 1°C or higher, and even more preferably 3°C or higher, from the viewpoint of suppressing freezing of the aqueous phase. If the core material is not gelatin, the temperature can be appropriately selected according to the core material. The cooling time is not particularly limited as long as it is sufficient for the core material to gel, for example, preferably 1 minute or more, more preferably 3 minutes or more, and even more preferably 5 minutes or more, and from the viewpoint of shortening the manufacturing time and reducing the impact on the object, it is preferably 60 minutes or less, more preferably 30 minutes or less.

[0030] <Step 3> Step 3 is the process of extracting the beads containing the target substance obtained in Step 2 into the aqueous phase. In Step 2, the beads containing the target substance are contained in the oil phase for droplet formation. In addition to the beads containing the target substance, the oil phase contains small droplets. These small droplets are thought to originate from the aqueous phase that does not contain particulate core material when the aqueous phase and oil phase are mixed in Step 1. In Step 3, it is preferable to remove the small droplets before extracting the beads containing the target substance into the aqueous phase. Since the small droplets are contained in the turbid oil phase below the emulsion phase, it is preferable to gently mix the suspension, let it stand, remove the turbid oil phase, and repeat the operation of adding a new oil phase for droplet formation until the turbid oil phase is gone. After the above operation, the beads containing the target substance can be extracted into the aqueous phase by adding a fluorinated liquid containing perfluorooctanol and the aqueous phase (e.g., liquid culture medium). By removing the oil phase or isolating the aqueous phase, an aqueous phase containing beads encapsulating the target substance can be obtained.

[0031] <Steps 4 and 5> Step 4 is a step of mixing an aqueous phase containing core beads encapsulating the target substance and dissolved shell raw material with an oil phase for droplet formation under conditions in which the core beads do not dissolve to obtain a second water-in-oil type suspension. Step 5 is a step of insolubilizing the shell raw material in the second water-in-oil type suspension under conditions in which the core beads do not dissolve to obtain core-shell particles having core beads encapsulating the target substance and a shell.

[0032] [Shell Material] The shell material satisfies the following requirements: (i) it can be dissolved in the aqueous phase at a temperature at which the core beads do not dissolve; (ii) it can be insoluble under conditions at which the core beads do not dissolve; and (iii) the insoluble shell material (hereinafter, the insoluble shell material, i.e., the material constituting the shell, is also called the "shell material") does not dissolve under conditions at which the core material of the core beads dissolves. Preferably, the shell material is at least one selected from the group consisting of compounds that dissolve or gel with temperature changes, compounds that become insoluble by crosslinking, and compounds that become insoluble by polymerization.

[0033] (Compounds that dissolve or gel with temperature changes) When the shell material is a compound that dissolves (including melting) or gels with temperature changes, the shell material used is one that can maintain a dissolved state in the aqueous phase at a temperature lower than the melting temperature of the core beads, and does not dissolve at the melting temperature of the core beads. Examples of shell materials include gelatin, collagen, agarose, and Matrigel, and among these, it is preferable that the core material is gelatin and the shell material is agarose. When the core material is gelatin, the remelting temperature of the core beads is about 30°C, and when the shell material is agarose, the melting temperature of low-melting-point agarose is about 50°C and the gelation temperature is about 8°C.

[0034] (Compounds that become insoluble by crosslinking) When the shell material is a compound that becomes insoluble by crosslinking, the form of crosslinking is not particularly limited, but ionic crosslinking, primary amino group (-NH ２ Examples include the formation of amide bonds between a hydroxysuccinimide ester and an N-hydroxysuccinimide ester, the formation of thioether bonds between a sulfhydryl group (-SH) and a maleimide group, the crosslinking of diacetone acrylamide and adipic acid dihydrazide, and the formation of amide bonds between a carboxyl group and a primary amino group using a carbodiimide compound. These insolubilization processes must be carried out in an aqueous phase, and the crosslinking reaction must proceed below the dissolution temperature of the core beads.

[0035] When the shell material is a compound that becomes insoluble through ionic crosslinking, an example is the crosslinking of sodium alginate with calcium ions. Sodium alginate is water-soluble, and by adding divalent or higher metal ions such as calcium ions to an aqueous solution of sodium alginate, ionic crosslinking is formed and gelation occurs. The gel formed by this crosslinking is a hydrogel having a three-dimensional network structure, and for long-term cultivation of the target object, it is preferable that this three-dimensional network structure can pass through the culture medium and waste products from the target object.

[0036] When the shell raw material is sodium alginate, for example, in step 4, it is preferable to use an aqueous phase containing sodium alginate, a metal compound containing a divalent or higher metal, and a metal chelating agent as the aqueous phase containing the shell raw material, and in step 5, to crosslink the alginic acid with metal by adding acid. However, it is not limited to this, and sodium alginate and a metal compound containing a divalent or higher metal that does not dissolve in neutral but dissolves under weak acidity may also be used. As the divalent or higher metal, alkaline earth metals such as calcium, magnesium, strontium, barium, and zinc are preferred, and calcium is more preferred from the viewpoint of suppressing crosslinking reactivity and the effect on the target material. When ethylenediaminetetraacetic acid is used as the metal chelating agent, under acidic conditions, protons take lone pairs of electrons, so the coordination bond becomes weaker than under basic conditions, and the ability to form a complex decreases. Therefore, by adding acid in step 5, calcium ions are released, and alginic acid is crosslinked with calcium ions to form a gel. As a metal compound containing a divalent or higher metal that does not dissolve in neutral but dissolves under weak acidity, calcium carbonate is a preferred example. Among these, in step 4, it is preferable to use an aqueous phase containing sodium alginate, a metal compound containing a divalent or higher metal, and a metal chelating agent as the aqueous phase containing the shell raw material.

[0037] Furthermore, as a crosslinking agent, a primary amino group (-NH ２ When using the formation of an amide bond with N-hydroxysuccinimide ester, for example, by using TAPEG (terminal amino group modified tetrapolyethylene glycol) and TNPEG (terminal N-hydroxysuccinimide ester modified tetrapolyethylene glycol) represented by the following formula, an amide bond is formed, a cross-linked structure is formed, and the material becomes insoluble.

[0038]

[0039] In step 4, TAPEG may be used as a shell raw material in the aqueous phase, and insolubilization may be achieved by adding TNPEG in step 5. Alternatively, TNPEG may be used as a shell raw material in the aqueous phase in step 4, and insolubilization may be achieved by adding TAPEG in step 5. There is no particular limitation.

[0040] When using crosslinking by forming a thioether bond between a sulfhydryl group (—SH) and a maleimide group, for example, by using dithiothreitol (DTT) and TPEG-MAL (terminal maleimide group-modified tetraethylene glycol) represented by the following formula, a thioether bond is formed and insolubilization occurs by crosslinking.

[0041]

[0042] It is preferable to use TPEG-MAL as a shell raw material in the aqueous phase in step 4 and insolubilize by adding DTT in step 5.

[0043] (Compounds that become insoluble by polymerization) When the compound becomes insoluble by polymerization, it may be a radical polymerization reaction, a cationic polymerization reaction, or anionic polymerization, but a radical polymerization reaction is preferred from the viewpoint that the monomer, which is the shell raw material, is water-soluble and that the polymerization reaction proceeds at a low temperature in an aqueous system. In a radical polymerization reaction, the radical polymerizable compound is a compound having an unsaturated double bond, and addition polymerization is preferred, and as the radical polymerizable compound, compounds having a vinyl group, a (meth)acryloyl group, or a (meth)acrylamide group are preferred. In step 5, when the compound becomes insoluble by polymerization, it is preferable that in step 4 the aqueous phase contains the radical polymerizable compound and a radical polymerization initiator, or that in step 4 the aqueous phase contains the radical polymerizable compound and in step 5 the radical polymerization initiator is added. The radical polymerization initiator may be a thermal polymerization initiator or a photopolymerization initiator, but a photopolymerization initiator is preferred from the viewpoint of suppressing the melting of the core beads. The radical polymerization initiator is preferably water-soluble. Examples of water-soluble photopolymerization initiators include 2-hydroxy-2-methylpropiophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate. Examples of water-soluble thermal polymerization initiators include 2,2'-Azobis(2-methylpropionamidine) dihydrochloride and 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride.

[0044] Examples of the water-soluble radically polymerizable compound include polyethylene glycol diacrylate, acrylamide, allyl (meth)acrylate, trimethylolpropane tri(meth)acrylate, triallylamine, etc. Among these, polyethylene glycol diacrylate and acrylamide are more preferable.

[0045] In Step 5, it is preferable to initiate polymerization by ultraviolet irradiation to polymerize the shell raw material. During the ultraviolet irradiation, it is preferably carried out under cooling so that the core beads do not dissolve. Also, from the viewpoint of carrying out under the condition that the core beads do not dissolve, when using a compound other than those that dissolve or gelate due to temperature change as the shell raw material, it is preferably carried out under cooling.

[0046] <Step 6> Step 6 is a step of taking out the core-shell particles into the aqueous phase. Step 6 can be carried out in the same manner as Step 3.

[0047] <Step 7> Step 7 is a step of providing conditions to the core-shell particles such that the core material dissolves due to temperature change while the shell material does not dissolve, dissolving the core beads, holding the object in the hollow part aqueous phase, and obtaining microcapsules having a shell. The core-shell particles taken out into the aqueous phase are heated under the condition that the core beads dissolve and the shell does not dissolve, so that the core material is removed into the aqueous phase through the shell. Thereby, microcapsules in which the object is held in the hollow part aqueous phase inside the shell are obtained.

[0048] When the core material is gelatin and the material constituting the shell is agarose, it is preferable to heat under the condition that the core beads dissolve and the shell does not dissolve. Specifically, it is preferably 30°C or higher, more preferably 35°C or higher, and preferably 50°C or lower, more preferably 40°C or lower.

[0049] The average particle size of the obtained microcapsules is controlled by the particle size of the particulate core material, the thickness of the shell, etc. From the viewpoint of ease of manufacturing and appropriate size according to the target object, the average particle size of the microcapsules is preferably 1 μm or more, more preferably 5 μm or more, even more preferably 15 μm or more, even more preferably 30 μm or more, even more preferably 50 μm or more, and preferably 700 μm or less, more preferably 500 μm or less, even more preferably 300 μm or less, and even more preferably 150 μm or less. Note that the average particle size may be appropriately changed depending on the target object to be encapsulated. The average particle size of the obtained microcapsules is measured by the method described in the examples.

[0050] [Method for Manufacturing Core Beads] The present invention also relates to a method for manufacturing core beads containing an object, comprising the following steps 1 to 3 in this order. Step 1: Mix an aqueous phase containing particulate core material and an object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil suspension in which droplets containing particulate core material and an object are dispersed in the oil phase. Step 2: Heat the first water-in-oil suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing an object. Step 3: Remove the core beads containing the object into the aqueous phase. Steps 1 to 3 are the same as steps 1 to 3 in the above-described method for manufacturing microcapsules, and preferred embodiments are also the same.

[0051] [Core bead formation kit] The present invention also relates to a core bead formation kit, which includes a particulate core material that dissolves or gels with temperature changes alone, and an oily composition for droplet formation. The particulate core material that dissolves or gels with temperature changes alone is the same as the particulate core material described in the above-mentioned [Method for producing microcapsules], and the preferred range is also the same. The oily composition for droplet formation is the same as the oily phase for droplet formation described above.

[0052] [Applications of Core Beads and Microcapsules] The core beads containing the target object of this embodiment can be used for functional analysis of individual cells by avoiding physical contact with other cells through compartmentalization with hydrogel. Furthermore, it is possible to proliferate cells inside the core beads. In addition, cells encapsulated in the gel can be densely aligned in the microchannel by utilizing the elasticity and uniformity of size of the gel, thereby enabling highly reliable gel and cell encapsulation in droplets, which does not follow the Poisson distribution.

[0053] The microcapsules of this embodiment can be used for various applications. One embodiment illustrates a method for growing cells within a microcapsule, which includes the steps of: manufacturing a microcapsule that holds cells in a hollow aqueous phase; and growing cells within the hollow phase in the presence of a culture medium. In step 1, by adjusting the concentration of the target cell so that one cell is contained in the droplet, a microcapsule containing cells grown from one cell in the hollow aqueous phase is obtained, and cloning can be efficiently performed within the microcapsule. By contacting the microcapsule containing the cloned cells with a substrate or drug according to the purpose and evaluating it, it is possible to screen for desired cells.

[0054] Furthermore, this technology can also be applied to large-scale screening using microcapsules. By simultaneously applying some kind of perturbation to the contents of a large number of microcapsules, large-scale screening can be performed. For example, a population of cells can be encapsulated inside the capsule, and substances smaller than the pore size of the microcapsule, such as viruses or small molecule compounds, can be introduced from the outside. Moreover, if a support containing a cell bound to a single compound is enclosed in each of the large number of microcapsules, and the compound is detached from the support by light irradiation, the compound will act on the cells inside the capsule. Subsequently, the capsules can be fed into a cell sorter, and the compound's action on the cells can be read by optical measurement. By selecting the capsules based on their action on cells, it is thought that compound screening based on the effect on cells can be performed.

[0055] The present invention will be described in more detail by the following examples, but these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0056] The materials and cells used in the following examples are as follows: • Complete medium: Prepared by adding 10% FBS, 1% Pen / Strep, and a final concentration of 20 mM L-Alanyl-L-Glutamine (L-Ala / L-Glu) to RPMI medium. Here, RPMI was #183-02165 from Fujifilm Wako Pure Chemical Industries, Ltd., and Pen / Strep was #15140-122 from Gibco, added to a 1 / 100 dilution (1%). L-Alanyl-L-Glutamine was #016-21841 from Fujifilm Wako Pure Chemical Industries, Ltd. • Cells: U937 cells (JCRB Cell Bank, #9021) • EDTA: Ethylenediaminetetraacetic acid

[0057] [Production of Particulate Core Material - 1 (Production of Spherical Gelatin)] Spherical gelatin was produced as a particulate core material. The spherical gelatin was prepared by forming a gelatin solution into uniformly sized droplets using a microfluidic device, and then gelling it. Specifically, spherical gelatin with an average particle size of 39.5 μm was prepared using the following procedure: A 10 w / w% gelatin solution (#077-03155, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared and filtered using a 0.22 μm syringe filter (#SLGVJ13SL, manufactured by Merck, Burlington, MA, USA). This gelatin solution was transferred to a 2 mL tube, and using FLPG plus silent (#FLPG005J, Fluigent, Le Kremlin-Bicetre, France) and Flow EZ 2000 mbar (# LU-FEZ-2000, Fluigent), a pressure of 1200 mBar was applied to the tube, and the solution was delivered to a microfluidic device through PEEK tubing (0.25 mm inner diameter, #NPK-007, Nirei Kogyo Co., Ltd.). The droplet-generating oil (#1864112, Bio-Rad, CA, USA) was filled into a 5 mL syringe (#SS-05LZ, Terumo Corporation) and delivered to the microfluidic device at a rate of 150 μL / min through PEEK tubing (0.5 mm inner diameter, #NPK-008, Nirei Kogyo Co., Ltd.) using a syringe pump (70-4505 Elite Pump, Harvard Apparatus, Holliston, MA, USA). To prevent the gelatin solution from cooling and gelling during delivery, the 2 mL tube containing the gelatin solution was heated to 37°C using a heat block. The microfluidic device was also placed on a thermoplate (#TPi-CKX53X, Tokai Hit Co., Ltd.) and heated to 37°C during delivery. The obtained droplets were collected in a 15 mL tube through a PEEK tubing (0.5 mm inner diameter, #NPK-008, manufactured by Nirei Kogyo Co., Ltd.) via an outlet. The collection tube was then cooled at 4°C for 15 minutes to gel the gelatin.Next, HFE-7200 (3M, Saint Paul, MN, USA), containing phosphote-buffered saline (PBS) and 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol (#324-90642, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), was added, and spherical gelatin was recovered from the emulsion into the PBS. The obtained spherical gelatin was stored at 4°C until use.

[0058] The obtained spherical gelatin with an average particle diameter of 39.5 μm exhibited high uniformity in particle size, and the CV value (Coefficient of Variation) shown below was 6.2%. CV (%) = (σ / D) × 100, where σ is the standard deviation and D is the average particle diameter. The average particle diameter of the spherical gelatin is the number-average particle diameter measured from magnified images acquired with a phase-contrast microscope. Furthermore, by changing the microfluidic device and the liquid delivery speed, spherical gelatin with an average particle diameter of 57.5 μm (CV value = 6.4%) and an average particle diameter of 79.4 μm (CV value = 3.0%) was produced using the same method. In the case of the cup-shaped gelatin described later, the number-average particle diameter of the largest diameter in the magnified image acquired with a phase-contrast microscope was used as the average particle diameter.

[0059] [Production of Particulate Core Material - 2 (Production of Cup-Shaped Gelatin (Gelatin Cups))] Cup-shaped gelatin (gelatin cups) was produced as a particulate core material. Gelatin cups were produced by dropletizing a mixed solution containing gelatin and polyethylene glycol (PEG) using a microfluidic device, followed by phase separation and gelation. Specifically, gelatin cups with an average particle size of approximately 75 μm were produced by the following procedure. First, a 16 w / w% gelatin solution (#077-03155, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in purified water. Next, a 3.4 w / w% polyethylene glycol solution (average molecular weight 20,000, #168-11285, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in purified water. Equal volumes of both solutions were mixed to obtain a mixed solution containing a final concentration of 8 w / w% gelatin and 1.7 w / w% polyethylene glycol. This mixed solution was transferred to a 2 mL tube, and using FLPG plus silent (#FLPG005J, Fluigent, Le Kremlin-Bicetre, France) and Flow EZ 2000 mbar (# LU-FEZ-2000, Fluigent), a pressure of 500 mBar was applied to the tube, allowing the solution to be delivered to a microfluidic device through PEEK tubing (0.25 mm inner diameter, #NPK-007, Nirei Kogyo Co., Ltd.). The droplet-generating oil (#1864112, Bio-Rad, CA, USA) was filled into a 5 mL syringe (#SS-05LZ, Terumo Corporation) and delivered to the microfluidic device at a rate of 140 μL / min through PEEK tubing (0.5 mm inner diameter, #NPK-008, Nirei Kogyo Co., Ltd.) using a syringe pump (70-4505 Elite Pump, Harvard Apparatus, Holliston, MA, USA). During delivery, to prevent the gelatin solution in the mixed solution from cooling and gelling, the 2 mL tube containing the mixed solution was heated to 65°C using a heat block. The microfluidic device was also placed on a thermoplate (#TPi-CKX53X, Tokai Hit Co., Ltd.) and heated to 37°C during delivery.The obtained droplets were collected in a 15 mL tube through PEEK tubing (0.5 mm inner diameter, #NPK-008, manufactured by Nirei Kogyo Co., Ltd.) from the outlet. The obtained droplets were collected through the outlet tube and incubated for 1 hour in a thermomix set to 37°C to promote phase separation of gelatin and polyethylene glycol. Subsequently, the mixture was cooled on ice for 15 minutes to gel the gelatin phase. Next, HFE-7200 (manufactured by 3M, Saint Paul, MN, USA) containing phosphote-buffered saline (PBS) and 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol (#324-90642, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the particulate gelatin cups were collected from the emulsion into the PBS. The obtained particulate gelatin cups were stored at 4°C until use. The obtained gelatin cups are shown in Figure 3. As shown in Figure 3, gelatin particles with a hollowed-out (cup-shaped) form were obtained from spherical gelatin particles.

[0060] [Production of Hollow Agarose Microcapsules by Two-Step Particle-Templated Emulsification - 1] <Step 1 (Process of Encapsulating Cells and Particle Gelatin Emulsion)> Cells and spherical gelatin were encapsulated in the same emulsion by particle-templated emulsification. The following operations using spherical gelatin were performed on ice to prevent dissolution. The required amount of spherical gelatin was transferred to a new 15 mL tube, centrifuged at 200 × g for 5 minutes, the supernatant was removed, and it was made into a pellet. 500 μL of the spherical gelatin pellet and 3 × 10 ６The cells were mixed in a chilled 50 mL tube. The cell count was calculated using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific). To this mixture of spherical gelatin and cells, 4 mL of droplet-forming oil (#1864112, Bio-Rad) was added, and the mixture was vortexed at 2,700 rpm for 30 seconds (Vortex-Genie 2, Scientific Industries, Bohemia, NY, USA). This generated an emulsion containing cells and gelatin beads.

[0061] <Step 2 (Process of encapsulating cells in gelatin beads by dissolving and gelling particulate gelatin)> In the emulsion state, spherical gelatin was dissolved and then gelled again to encapsulate the cells in the gelatin beads. Specifically, the spherical gelatin was first dissolved by incubating the emulsion at 37°C for 5 minutes while stirring at 300 rpm. Next, the gelatin was gelled again by incubating at 4°C for 15 minutes.

[0062] <Step 3 (Removal of cell-encapsulated gelatin beads from the oil phase to the aqueous phase)> To remove small-sized droplets (satellite droplets) from the emulsion, the following procedure was performed: The tube was inverted and mixed, and allowed to stand for 1 minute. The cloudy oil phase below the emulsion phase was discarded, and 1 mL of new droplet-generating oil (#1864112, Bio-Rad) was added. This was repeated several times until the cloudiness below the emulsion phase disappeared. After the removal of satellite droplets was complete, 10 mL of complete medium and 2 mL of HFE-7200 (3M) containing 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol (PFO, #324-90642, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the cell-encapsulated gelatin beads were recovered from the emulsion into the complete medium. Adding PFO causes the emulsion to disemulsify, and the gelatin beads containing cells, which were encapsulated within the emulsion, move into the aqueous phase. Since the gelatin beads containing cells dissolve very easily, care was taken to ensure that the procedure was always performed on ice.

[0063] <Step 4 (Step to obtain an emulsion containing cell-encapsulating core beads and dissolved agarose)> Agarose (Ultra-low Gelling Temperature, #45030-1G, manufactured by Sigma) was dissolved in PBS (phosphate buffered saline) at 95°C to a concentration of 1 w / w%, and filtered using a 0.22 μm syringe filter (#SLGVJ13SL, manufactured by Merck). The cell-encapsulating gelatin beads were centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the mixture was formed into a pellet. 500 μL of the gelatin bead pellet and 300 μL of the above agarose solution were mixed in a 50 mL tube at 23°C. 4 mL of droplet-generating oil (#1864112, manufactured by Bio-Rad) was added to this mixture, and the mixture was vortexed at maximum speed for 5 to 15 seconds (Vortex-Genie 2, manufactured by Scientific Industries). As a result, the gelatin beads containing cells were encapsulated in agarose, forming an emulsion.

[0064] <Step 5 (Step to insolubilize agarose and obtain core-shell particles having a core bead containing cells and a shell)> After vortexing, the agarose was immediately left to stand in ice for 15 minutes, which insolubilized (gelled) the agarose, and core-shell particles having a core bead containing cells and an agarose shell were obtained.

[0065] <Step 6: Step to extract core-shell particles into the aqueous phase> To remove satellite droplets from the emulsion containing the core-shell particles, the following procedure was performed: The tube was inverted and mixed, and allowed to stand for 1 minute. The cloudy oil phase below the emulsion phase was discarded, and 1 mL of new droplet-forming oil (#1864112, Bio-Rad) was added. This was repeated several times until the cloudiness below the emulsion phase disappeared. After the removal of satellite droplets was complete, 10 mL of complete medium and 2 mL of HFE-7200 (3M) containing 20 v / v% 1H,1H,2H,2H-perfluoro-1-octanol (#324-90642, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the agarose-encapsulated gelatin beads were recovered from the emulsion into the complete medium.

[0066] <Step 7: Dissolving core beads to hold cells in the hollow aqueous phase and obtain microcapsules with a shell> The core-shell particles recovered in the complete medium were incubated at 37°C for 5 minutes to dissolve the inner gelatin and obtain a hollow agarose microcapsule structure. The obtained microcapsules held cells in the hollow aqueous phase and had a shell made of agarose. The average particle size of the obtained microcapsules was 63.8 μm for the outer diameter (CV value = 10.2%) and 52.2 μm for the inner diameter (CV value = 10.8%). The outer and inner diameters of the microcapsules are number-average particle sizes obtained by analyzing magnified images acquired with a phase-contrast microscope.

[0067] Images of the obtained microcapsules on day 0 and day 3 are shown in Figure 1. The microcapsules shown in Figure 1 were obtained by adding FITC (fluorescein isothiocyanate isomer I) to the agarose used as the shell and by performing nuclear staining with Hoechst 33342. Microcapsules were obtained in the same manner except that adherent cells HEK293T cells were used instead of suspension cells U937 cells. Images of the obtained microcapsules on day 0 and day 3 are shown in Figure 2. In Figure 2, as in Figure 1, agarose with FITC added was used as the shell and nuclear staining was performed with Hoechst 33342. From Figures 1 and 2, it can be seen that whether suspension cells U937 cells or adherent cells HEK293T cells were used, microcapsules with cells held in the aqueous phase of the hollow portion were obtained in the same way, and cell proliferation was observed in the aqueous phase of the hollow portion of the microcapsule after 3 days of culture.

[0068] Microcapsules holding U937 cells in the hollow aqueous phase were fabricated using the same method as above, except that a particulate core material (particulate gelatin) with an average particle size of 57.5 μm was used. The inner diameter of the obtained microcapsules was 73.1 μm (CV value = 3.2%) and the outer diameter was 83.6 μm (CV value = 4.1%). Microcapsules holding U937 cells in the hollow aqueous phase were also fabricated using the same method as above, except that a particulate core material (particulate gelatin) with an average particle size of 79.4 μm was used. The inner diameter of the obtained microcapsules was 101.8 μm (CV value = 2.5%) and the outer diameter was 111.3 μm (CV value = 2.8%).

[0069] [Manufacturing of Hollow Agarose Microcapsules by Two-Stage Particle-Templated Emulsification - 2] <Step 1 (Step of Encapsulating Cells and Particulate Gelatin in an Emulsion)> Except for encapsulating cells and cup-shaped gelatin (gelatin cups) in the same emulsion by particle-templated emulsification, an emulsion containing cells and gelatin beads was produced in the same manner as in [Manufacturing of Hollow Agarose Microcapsules by Two-Stage Particle-Templated Emulsification - 1] above.

[0070] <Steps 2 to 7> Hollow agarose microcapsules were manufactured in the same manner as described in [Production of Hollow Agarose Microcapsules by Two-Step Particle Templated Emulsification - 1] above. Figure 4 shows the core beads containing the target substance in the aqueous phase, obtained in Step 3. As shown in Figure 4, when gelatin cups were used as the particulate core material, core beads containing multiple cells were obtained.

[0071] [Production of hollow polyacrylamide microcapsules by two-step particle template emulsification] <Steps 1 to 3> Gelatin beads containing cells were extracted into the aqueous phase using the same method as in steps 1 to 3 of the above [Production of hollow agarose microcapsules by two-step particle template emulsification].

[0072] <Step 4 (Step to obtain an emulsion containing cell-encapsulated core beads and acrylamide)> The acrylamide premixture (7.5 w / v% acrylamide (#1610156, Bio-Rad) and 0.1 × Tris-buffered saline EDTA (TBSET: 10 mM Tris-HCl pH 8.0, 137 mM NaCl, 20 mM EDTA, 1.4 mM KCl, 0.1 v / v% Triton-X100)) was filtered using a 0.22 μm syringe filter (#SLGVJ13SL, Merck). The gelatin beads containing cells were centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the mixture was formed into a pellet. 500 μL of the gelatin bead pellet and 300 μL of the acrylamide premixture were mixed in a chilled 50 mL tube. To this mixture, 4 mL of droplet-forming oil (#1864112, Bio-Rad) containing 0.2 v / v% 2-Hydroxy-2-methylpropiophenone (#H0991, Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was vortexed at 2,700 rpm for 5 seconds (Vortex-Genie 2, Scientific Industries). This encapsulated the cell-containing gelatin beads in the acrylamide premixture, forming an emulsion.

[0073] <Step 5 (Step to insolubilize acrylamide and obtain core-shell particles having a core bead containing cells and a shell)> To gel the acrylamide, light with a wavelength of 365 nm is applied at 40 J / cm ２ The tube was irradiated for 5 to 10 minutes. The LED lamp was placed 1 to 5 cm away from the tube.

[0074] <Step 6 (Step of extracting core-shell particles into the aqueous phase) and Step 7 (Step of dissolving core beads to hold cells in the hollow aqueous phase and obtain microcapsules having a shell)> By the same method as in Steps 6 and 7 of the above [Production of hollow agarose microcapsules by two-step particle template emulsification], a hollow polyacrylamide microcapsule structure that holds cells in the hollow aqueous phase was obtained.

[0075] [Production of hollow polyPEGDA microcapsules by two-stage particle template emulsification] <Steps 1 to 3> Gelatin beads containing cells were extracted into the aqueous phase using the same method as in steps 1 to 3 of the above [Production of hollow agarose microcapsules by two-stage particle template emulsification].

[0076] <Step 4 (Step to obtain an emulsion containing cell-encapsulated core beads and polyethylene glycol diacrylate (PEGDA))> A PEGDA premixture (10 v / v% PEGDA (#437441, Merck) and 0.1 × Tris-buffered saline EDTA (TBSET: 10 mM Tris-HCl pH 8.0, 137 mM NaCl, 20 mM EDTA, 1.4 mM KCl, 0.1 v / v% Triton-X100)) was filtered using a 0.22 μm syringe filter (#SLGVJ13SL, Merck). The gelatin beads containing cells were centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the mixture was formed into a pellet. 500 μL of the gelatin bead pellet and 300 μL of the PEGDA premixture were mixed in a chilled 50 mL tube. To this mixture, 4 mL of droplet-forming oil (#1864112, Bio-Rad) containing 0.2 v / v% 2-Hydroxy-2-methylpropiophenone (#H0991, Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was vortexed at 2,700 rpm for 5 seconds (Vortex-Genie 2, Scientific Industries). This encapsulated the cell-containing gelatin beads in the PEGDA premixture, forming an emulsion.

[0077] <Step 5 (Step to insolubilize PEGDA and obtain core-shell particles having a core bead containing cells and a shell)> To gel PEGDA, light with a wavelength of 365 nm is applied at 40 J / cm ２ The tube was irradiated for 5 to 10 minutes. The LED lamp was placed 1 to 5 cm away from the tube.

[0078] <Step 6 (Step of extracting core-shell particles into the aqueous phase) and Step 7 (Step of dissolving core beads to hold cells in the hollow aqueous phase and obtain a microcapsule having a shell)> A hollow polyPEGDA capsule structure that holds cells in the hollow aqueous phase was obtained by the same method as in Steps 6 and 7 of the above [Production of hollow agarose microcapsules by two-step particle template emulsification].

[0079] [Production of hollow alginate microcapsules by two-step particle template emulsification] <Steps 1 to 3> Gelatin beads containing cells were extracted into the aqueous phase using the same method as in steps 1 to 3 of the above [Production of hollow agarose microcapsules by two-step particle template emulsification].

[0080] <Step 4 (Step to obtain an emulsion containing core beads and alginate containing cells)> A sodium alginate-calcium-EDTA solution was prepared by mixing a 2 w / w% sodium alginate solution (IL-6, manufactured by Kimika Co., Ltd.) and a calcium-EDTA solution (50 mM CaCl2, 50 mM EDTA; pH adjusted to 7.2) in a 1:1 ratio, and filtered using a 0.22 μm syringe filter (#SLGVJ13SL, manufactured by Merck). The gelatin beads containing cells were centrifuged at 200 × g for 5 minutes, the supernatant was removed, and the mixture was formed into a pellet. 500 μL of the gelatin bead pellet and 300 μL of the sodium alginate-calcium-EDTA solution were mixed in a chilled 50 mL tube. To this mixture, 4 mL of droplet-forming oil (#1864112, Bio-Rad) was added, and the mixture was vortexed at 2,700 rpm for 5 seconds (Vortex-Genie 2, Scientific Industries). This encapsulated the cell-containing gelatin beads in a sodium alginate-calcium-EDTA solution.

[0081] <Step 5 (Step to gel sodium alginate and obtain core-shell particles having a core bead containing cells and a shell)> To gel the emulsion of sodium alginate, the oil phase below the emulsion was removed, 1 mL of droplet-forming oil (#1864112, Bio-Rad) containing 0.1 v / v% acetic acid was added, and the mixture was inverted and mixed for 30 seconds.

[0082] <Step 6 (Step of extracting core-shell particles into the aqueous phase) and Step 7 (Step of dissolving core beads to hold cells in the hollow aqueous phase and obtain a microcapsule having a shell)> A hollow alginate capsule structure that holds cells in the hollow aqueous phase was obtained by the same method as in Steps 6 and 7 of the above [Production of hollow agarose microcapsules by two-step particle template emulsification].

Claims

1. A method for manufacturing microcapsules that hold an object in the aqueous phase of a hollow portion, comprising the following steps 1 to 7 in this order. Step 1: Mix an aqueous phase containing particulate core material and target object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil suspension in which droplets containing particulate core material and target object are dispersed in the oil phase. Step 2: Heat the first water-in-oil suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing the target object. Step 3: Remove the core beads containing the target object into the aqueous phase. Step 4: Mix the aqueous phase containing the core beads containing the target object and dissolved shell raw material with an oil phase for droplet formation under conditions in which the core beads do not dissolve to obtain a second water-in-oil suspension. Step 5: In the second water-in-oil suspension, insolubilize the shell raw material under conditions in which the core beads do not dissolve to obtain core-shell particles having core beads containing the target object and a shell. Step 6: Step of extracting the core-shell particles into the aqueous phase, and Step 7: Step of applying conditions to the core-shell particles such that the core material dissolves due to temperature changes while the shell material does not dissolve, thereby dissolving the core beads, holding the target object in the hollow aqueous phase, and obtaining microcapsules having shells.

2. The method for producing microcapsules according to claim 1, wherein the core material comprises at least one selected from the group consisting of gelatin, collagen, agarose, and Matrigel, and the shell material comprises at least one selected from the group consisting of compounds that dissolve or gel with temperature changes, compounds that become insoluble by crosslinking, and compounds that become insoluble by polymerization.

3. The method for producing microcapsules according to claim 1 or 2, wherein the object is a biological or chemical material selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, proteins, and organic compounds.

4. The method for producing microcapsules according to claim 1 or 2, wherein the object is a cell.

5. The method for producing a microcapsule according to claim 1 or 2, wherein in step 1, the droplet is one selected from the group consisting of a single cell, a single microorganism, a single virus particle, a single nucleic acid molecule, a single peptide molecule, a single protein molecule, and a single organic compound molecule.

6. The method for producing microcapsules according to claim 1 or 2, wherein in step 1, the object comprises at least one selected from the group consisting of cells, microorganisms, viruses, nucleic acids, peptides, organic compounds, and at least one of these being adhered, modified, or bound to a solid or gel phase support.

7. The method for producing microcapsules according to claim 1 or 2, wherein the average particle diameter of the particulate core material is 1 μm or more and 500 μm or less.

8. A method for producing microcapsules according to claim 1 or 2, wherein the average particle size of the microcapsules is 1 μm or more and 700 μm or less.

9. A method for growing cells in a microcapsule, comprising the steps of: producing a microcapsule that holds cells in a hollow aqueous phase by the method described in claim 4; and growing cells in the hollow in the presence of a culture medium.

10. A method for manufacturing core beads containing an object, comprising the following steps 1 to 3 in this order: Step 1: Mix an aqueous phase containing particulate core material and an object with an oil phase for droplet formation at a temperature below the dissolution temperature of a core material that dissolves or gels by temperature change alone, to obtain a first water-in-oil suspension in which droplets containing particulate core material and an object are dispersed in the oil phase; Step 2: Heat the first water-in-oil suspension above the dissolution temperature of the core material, then cool it below the gelation temperature of the core material to obtain core beads containing an object; Step 3: Remove the core beads containing the object into the aqueous phase.

11. A core bead formation kit comprising a particulate core material that dissolves or gels solely by temperature changes, and an oily composition for droplet formation.

Images