Method for producing capsule and kit for producing capsule

Abstract

[Problem] To provide: a method for producing a capsule in which a to-be-embedded object vulnerable to acidic conditions can be embedded; and a kit for producing the capsule.　[Solution] The present invention pertains to a method for producing a capsule that has a core derived from an ion-binding polymer capable of making reversible sol-gel transition by means of ions, the method comprising: a step for dispersing, in a first oil phase, an aqueous phase (first aqueous phase) containing a sol of the ion-binding polymer; and a step for supplying ions to the dispersed first aqueous phase, and forming gel beads of the ion-binding polymer, at least the surfaces of the gel beads having been gelled. In the step for forming gel beads, in order to supply ions, a carboxylic acid is added to the first oil phase so as to achieve a final concentration of 0.005-0.0125 w / w%.

Description

Method for producing capsules and kit for producing capsules

[0001] The present invention relates to a method for producing a capsule and a kit for producing a capsule.

[0002] Alginate, a polymer extracted from seaweed, easily gels with divalent ions such as calcium and is therefore used as gel beads for cell embedding (see, for example, Non-Patent Document 1). Alginate gel beads have the following advantages: 1) high biodegradability, 2) the ability to protect cells from immune system factors such as cytotoxic T cells, and 3) the ability to exchange nutrients in the culture medium and gases necessary for respiration. For this reason, various studies have been conducted with the aim of cell transplantation therapy using cells embedded in alginate gel beads. Furthermore, because embedding cells in alginate gel beads can reduce external physical stimuli such as shear force, alginate gel beads are also useful for the production of physiologically active substances by liquid culture of Chinese hamster ovary (CHO) cells.

[0003] In addition, alginate gel beads, in which a polymer shell such as poly-L-lysine or polyvinylpyrrolidone is formed on the surface of the gel, have the effect of increasing the mechanical strength of the beads and preventing breakage during long-term culture (e.g., Patent Documents 1 and 2).

[0004] In contrast, hydrogel microcapsules consisting of a gel shell and a liquid core can be prepared by forming a hydrogel such as agarose on the surface of alginate gel beads and then solubilizing the alginate gel (see, for example, Patent Document 3 and Non-Patent Document 2). Although the center of hydrogel microcapsules is liquid, they are surrounded by a thick gel shell, providing sufficient physical strength for handling and long-term culture. Furthermore, the liquid core is closer to the in vivo state than gels, allowing cells to freely expand, which is expected to improve cell culture yields.

[0005] To prepare hydrogel microcapsules for embedding cells, first, alginate, the cells to be embedded, and calcium carbonate (CaCO ３ ) and dispersed in oil to form droplets. After the droplets are formed, acetic acid (CH ３COOH) is added. This releases calcium ions (Ca ２＋ ) to gel alginate, producing alginate gel beads with cells embedded inside.

[0006] Next, the alginate gel beads are mixed with agarose and dispersed in oil. This is cooled to gel the agarose and form a hydrogel shell. Finally, calcium is removed from the alginate gel using a chelating agent, turning the core into a sol.

[0007] However, even in microorganisms that are relatively tolerant to acid, growth inhibition can occur during the formation of alginate gel beads due to changes in the metabolic system caused by the influx of acetic acid into the cells (Non-Patent Document 4). For example, in the case of Escherichia coli, the growth rate is reduced by approximately half when acetic acid is added to the medium at a final concentration equivalent to 0.2 v / v% acetic acid (34 mmol / L) commonly used in the formation of alginate gel beads (Non-Patent Document 5). Furthermore, once growth inhibition occurs, the effect persists even if the influx of acetic acid into the cells is prevented.

[0008] In the aforementioned Non-Patent Document 4, if the decrease in the growth rate of microorganisms is to be suppressed to within 10% under conditions of pH 6.4, the acetic acid concentration needs to be set to a final molar concentration of approximately 5 mmol / L or less (see Fig. 3 in Non-Patent Document 4).

[0009] Furthermore, in animal cells, the pH within the body is maintained at approximately 7.4, and changes in extracellular pH have various effects on the cells. For example, mouse embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) experience a decrease in cell activity, cell viability, and undifferentiated potential when the culture medium pH becomes more acidic or alkaline than the range of 7 to 7.5 (see, for example, Non-Patent Document 3). For this reason, culture media and buffer solutions adjusted to a pH of 7 to 7.5 are used for cell culture.

[0010] Even when embedding cells in an alginate liquid core, maintaining a pH of 7 to 7.5 before and after gelation is necessary to maintain high cell viability without impairing cell function. However, as mentioned above, gelation requires the use of acid, making it difficult to gel alginate under such neutral conditions, making it difficult to embed objects that are sensitive to acidic conditions, including cells. On the other hand, acidification of the liquid by acetic acid significantly reduces the viability of the cells to be embedded.

[0011] For these reasons, there has been a demand for a new method for forming hydrogel microcapsules such as alginate gel beads that can promote the growth of microorganisms, improve the survival rate of animal cells, and even embed objects other than cells that are sensitive to acidic conditions.

[0012] JP 07-298870 JP 03-119990 WO 2020 / 184680

[0013] Rabanel JM, et. al. Biotechnol Prog vol. 25, 946-963 (2009) Aoki H, et. al. Sci Rep vol 12, 17014 (2022) Gupta P, et. al. Biochem Eng J vol 118, 25-33 (2017) Pinhal S, et. al. J Bacteriol vol 201, 10.1128 / jb. 00147-19 (2019) Poncelet D, Ann NY Acad Sci vol 944, 74-82 (2001)

[0014] Therefore, an object of the present invention is to provide a method for producing capsules capable of embedding an object to be embedded that is sensitive to acidic conditions, and a kit for producing such capsules.

[0015] (1) A capsule manufacturing method according to one embodiment for achieving the above object is a method for manufacturing a capsule having a core derived from an ion-binding polymer capable of reversibly undergoing sol-gel transition in the presence of ions, the method comprising the steps of: dispersing a sol (first aqueous phase) of the ion-binding polymer in a first oil phase; and supplying the ions to the dispersed first aqueous phase to form gel beads of the ion-binding polymer, at least the surfaces of which are gelled. In the step of forming the gel beads, a carboxylic acid is added to the first oil phase to provide the ions, so as to have a final concentration of 0.005 to 0.0125 w / w %. (2) A capsule manufacturing method according to another embodiment may further comprise, after the step of forming the gel beads, the steps of: mixing a sol (second aqueous phase) of a thermo-dependent polymer capable of reversibly undergoing sol-gel transition in response to a temperature change with the gel beads, and dispersing the gel beads in a second oil phase; and cooling the dispersed second aqueous phase to form a shell in which the second aqueous phase is gelled. (3) In another embodiment, the method for producing a capsule may further include, after the shell-forming step, removing the ions contained in the gel beads and soling the gel beads to form a liquid core. (4) In another embodiment, the ion-binding polymer may be at least one selected from alginic acid, deacylated gellan gum, polyacrylic acid, and carboxymethyl cellulose. (5) In another embodiment, the first aqueous phase may further include an ion source for supplying the ions. (6) In another embodiment, the ion source may be an alkaline earth metal salt. (7) In another embodiment, the first aqueous phase may further include an object to be embedded. (8) In another embodiment, the object to be embedded may be an animal cell. (9) In another embodiment, the object to be embedded may be a microorganism.(10) In another embodiment of the method for producing a capsule, the carboxylic acid may preferably be acetic acid. (11) In another embodiment of the method for producing a capsule, the method may preferably further include a step of treating the gel beads with an enzyme that degrades the ion-binding polymer to degrade the ion-binding polymer that has not gelled. (12) In another embodiment of the method for producing a capsule, the osmotic pressure of the first aqueous phase may preferably be 260 to 320 mOsm / L. (13) In another embodiment of the method for producing a capsule, the first aqueous phase may preferably further include a cell adhesion polymer that can be used as a scaffold in the culture of adherent cells. (14) In another embodiment of the method for producing a capsule, the first aqueous phase preferably further includes a cell adhesion polymer that can be used as a scaffold in the culture of adherent cells, and the method may further include a step of adsorbing the cell adhesion polymer to the surface of the gel beads prior to the shell formation step. (15) A kit according to one embodiment for achieving the above object is a kit for producing capsules having a core derived from an ion-binding polymer capable of reversible sol-gel transition in the presence of ions, comprising at least: a material containing an aqueous phase (first aqueous phase) containing a sol of the ion-binding polymer; a material containing a first oil phase for dispersing the first aqueous phase; and a carboxylic acid for supplying the ions to the first aqueous phase and forming gel beads of the ion-binding polymer, at least the surface of which is gelled; the kit is separately filled in each container; an ion source for supplying the ions to the first aqueous phase is separately filled in a container separate from the containers and / or is contained in the material for the first aqueous phase; and the acetic acid is separately filled in advance so that the final concentration of the acetic acid during the formation of the gel beads is 0.005 to 0.0125 w / w%. (16) In another embodiment of the kit, preferably, the carboxylic acid may be acetic acid. (17) In another embodiment of the kit, preferably, the ion source may be an alkaline earth metal salt.

[0016] The present invention provides a method for producing capsules capable of embedding an object to be embedded that is sensitive to acidic conditions, and a kit for producing the capsules.

[0017] FIG. 1 shows a flow diagram of a method for producing capsules according to a first embodiment. FIG. 2 shows a schematic diagram illustrating the gelation principle of alginate, an example of an ion-binding polymer. FIG. 3 shows a flow diagram of a method for producing capsules according to a second embodiment. FIG. 4 shows phase-contrast microscope images of alginate gel beads produced by adding different final concentrations (w / w%) of acetic acid to oil. FIG. 5 shows a graph of the results of measuring the pH of the aqueous phase after adding different final concentrations (w / w%) of acetic acid to oil. FIG. 6 shows a graph of the results of measuring cell viability after adding different final concentrations (w / w%) of acetic acid to oil. FIG. 7 shows a graph of the results of measuring microbial growth rate after adding different final concentrations (w / w%) of acetic acid to oil.

[0018] Next, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below do not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiments are necessarily essential to the solution of the present invention.

[0019] [Method for Manufacturing Capsules] In this application, the term "capsule" refers to a microcontainer having a core derived from an ion-binding polymer capable of reversible sol-gel transition in the presence of ions, and an outer shell containing a hydrogel permeable to low molecular weight molecules. Specifically, the term includes gel beads with the hydrogel as the shell, and hydrogel microcapsules containing a liquid core inside a shell formed from the hydrogel. The term "hydrogel" refers to a gel containing water or an aqueous solution. The capsule shell is preferably made of hydrogel. Furthermore, the term "core derived from an ion-binding polymer" includes not only a core containing an ion-binding polymer, but also a core containing a degradation product of the ion-binding polymer degraded with a degrading enzyme. The shape and structure of the capsule are not particularly limited, but are preferably mononuclear spherical.

[0020] (First embodiment) Fig. 1 shows a flow diagram of a capsule manufacturing method according to the first embodiment. Fig. 2 shows a schematic diagram illustrating the principle of gelation of alginic acid, which is an example of an ion-binding polymer.

[0021] The capsule manufacturing method according to this embodiment is a method for manufacturing capsules having a core derived from an ion-binding polymer capable of undergoing reversible sol-gel transition in the presence of ions. The method includes the steps of dispersing an aqueous phase containing a sol of the ion-binding polymer (hereinafter referred to as the first aqueous phase) in a first oil phase (hereinafter referred to as the first aqueous phase dispersion step), supplying ions to the dispersed first aqueous phase to form gel beads of the ion-binding polymer, at least the surfaces of which are gelled (hereinafter referred to as the gel bead formation step), and recovering the gel beads from the first oil phase (hereinafter referred to as the gel bead recovery step). Furthermore, as an optional step, the method may include, after the gel bead formation step, a step of treating the gel beads with an enzyme capable of degrading the ion-binding polymer to degrade any ungelled ion-binding polymer (hereinafter referred to as the decomposition step). Each step is described in detail below.

[0022] (1) First Aqueous Phase Dispersion Step (S100) In this embodiment, the first aqueous phase dispersion step is a step of emulsifying and dispersing the first aqueous phase in the first oil phase to form microdroplets of the first aqueous phase. The first aqueous phase is an aqueous phase containing an ion-binding polymer sol, and preferably contains, in addition to the ion-binding polymer sol, an ion source for supplying ions to the ion-binding polymer and / or an embedding target. Other components depending on the embedding target (e.g., if the embedding target is cells, a buffer solution capable of maintaining the pH of the first aqueous phase within a certain range, a culture medium suitable for the growth of the cells, a salt for adjusting osmotic pressure, a cell adhesion polymer (described below), etc.) may also be included. The microdroplets of the first aqueous phase are dispersed in the first oil phase, each of the above components contained therein.

[0023] (Ionic-binding polymer) An ionic-binding polymer is a water-soluble polymer that can crosslink a portion of its main chain via ionic bonds formed by counter ions. An ionic-binding polymer can undergo a reversible sol-gel transition in the presence of ions, and is independent of the temperature of a system containing the ionic-binding polymer. This makes it preferable in that even when blended into a thermodependent polymer (described below), the gelled state can be maintained regardless of temperature changes.

[0024] The ion-binding polymer is not particularly limited as long as it has the above-mentioned properties, but is preferably at least one selected from polysaccharides having cationic or anionic functional groups in the molecule, such as alginic acid, deacylated gellan gum, polyacrylic acid, and carboxymethylcellulose, and more preferably alginic acid. In this application, "alginic acid" is not limited to alginic acid itself, but also includes salts soluble in aqueous solvents, such as sodium alginate, potassium alginate, and ammonium alginate.

[0025] (First aqueous phase) The first aqueous phase is an aqueous phase containing a sol of an ion-binding polymer in which an ion-binding polymer is dissolved in an aqueous solvent, and gelation occurs due to crosslinking of the main chains when ions are supplied by some means. In addition to the sol of the ion-binding polymer, the first aqueous phase preferably contains an ion source and an object to be embedded, as described below. The pH of the first aqueous phase during this step is 6.5 to 7.5, from the viewpoint of preventing immediate gelation by the ion source, as described below. When the object to be embedded is a cell, the pH is preferably 7 to 7.5, more preferably 7.1 to 7.5, and even more preferably 7.2 to 7.5.

[0026] The aqueous solvent for dissolving the ion-binding polymer is not particularly limited and may be purified water, physiological saline, or a buffer solution such as phosphate buffer, Tris buffer, or HEPES buffer, but is preferably a buffer solution from the viewpoint of being able to be appropriately selected depending on the embedding target. When the embedding target is a cell, the aqueous solvent is not particularly limited as long as it can maintain the pH of the first aqueous phase within the range of 7 to 7.5, and various known aqueous solvents can be used. Among these, HEPES buffer is preferred because it does not form precipitates with calcium ions and has low cytotoxicity.

[0027] (Ion Source) The ion source is a substance that releases counter ions for gelling the first aqueous phase. The ion source is preferably an alkaline earth metal salt such as calcium, magnesium, strontium, barium, or zinc, and more preferably a calcium salt. Examples of calcium salts include calcium carbonate, calcium citrate, and calcium phosphate. Among these, calcium carbonate is preferred because it is sparingly soluble under neutral conditions and does not dissolve immediately. When calcium carbonate is added to a first aqueous phase adjusted to neutral conditions (pH approximately 6.5 to 7.5) and emulsified and dispersed in the first oil phase, the resulting mixture can be uniformly dispersed as fine droplets without forming gel beads.

[0028] (Embedding Target) The embedding target is not particularly limited, but is preferably a microorganism such as bacteria, virus, or yeast; a cell such as an animal cell or a plant cell; a synthetic polymer such as a microbead or polymer immobilized with synthetic DNA; a proton pump inhibitor; a cytokine; an antibody drug; or other pharmaceutical product, and more preferably an animal cell or a microorganism. When the embedding target is a cell, the cell may be dispersed or may be a collection of multiple cells (e.g., a tissue fragment or a cell mass). The cell may be a biological sample, a clinical specimen, or a cultured cell. The cell may also be adult stem cells, embryonic stem cells, induced pluripotent stem cells, or differentiated cells of each. The capsules produced by the manufacturing method according to this embodiment prevent microbial growth inhibition and are particularly suitable for embedding an embedding target that is unstable under acidic conditions.

[0029] (Osmotic Pressure of the First Aqueous Phase) The osmotic pressure of the first aqueous phase may be adjusted appropriately depending on the embedding target. Specifically, when the embedding target is a cell, the osmotic pressure of the first aqueous phase is preferably 260 to 320 mOsm / L, more preferably 260 to 300 mOsm / L. If the osmotic pressure of the first aqueous phase is within the above range, it is close to the osmotic pressure of extracellular fluid in vivo, thereby maintaining a high viability of the embedded animal cells. To adjust the osmotic pressure, various water-soluble salts that do not participate in the gelation of the ion-binding polymer, such as sodium chloride or potassium chloride, or water-soluble sugars such as glucose, maltose, trehalose, and sucrose, or sugar alcohols such as glycerol, may be added to the first aqueous phase. The osmotic pressure can be measured by the method described in "2.47 Osmotic Pressure Measurement (Osmolality Measurement)" of the "General Test Methods" section of the 18th Edition of the Japanese Pharmacopoeia, or by various other known methods.

[0030] (Cell Adhesion Polymer) In this embodiment, the term "cell adhesion polymer" refers to any polymer that can be used as a scaffold for cells when adhesive cells are used as the embedding target during the culture of the adherent cells. Specific examples include extracellular matrices such as laminin, collagen, fibronectin, vitronectin, fibrinogen, and elastin, fragments thereof (e.g., laminin 511E8 fragment), proteins such as gelatin, and polymers such as poly-L-lysine, polyornithine, chitosan, poly(2-methoxyethyl acrylate) (PMEA), alginic acid bound to the cell adhesive arginine-glycine-aspartic acid (RGD) peptide, dextran, and polyethylene glycol (PEG). The cell adhesion polymer may be added to the first aqueous phase together with the embedding target and ion source, if necessary.

[0031] (Other) In addition to the components described above, the first aqueous phase may contain various substances appropriate for the embedding target. For example, if the embedding target is animal cells, the first aqueous phase may contain a buffer solution, a culture medium, salts for adjusting osmotic pressure, and the like. The culture medium may be appropriately selected from various known media with a composition suitable for culturing each cell, and examples include MEM medium, MEMα medium, Dulbecco's Modified Eagle Medium (DMEM medium), RPMI 1640 medium, and Ham's F12 medium. The culture medium may also contain additives commonly used in cell culture, such as FBS (fetal bovine serum). Furthermore, if the embedding target is a microorganism, the first aqueous phase may contain a medium suitable for the growth of the microorganism and antibiotics that inhibit the growth of microorganisms other than the embedding target. These media and antibiotics can be appropriately selected from various known media, and examples of such media include LB medium, SOC medium, M9 medium, 2xYT medium, and TB medium. Examples of such antibiotics include, but are not limited to, ampicillin, kanamycin, tetracycline, streptomycin, and chloramphenicol.

[0032] (First Oil Phase) The first oil phase is an oil phase for emulsifying and dispersing the first aqueous phase to form fine droplets, and is a continuous phase. The first oil phase contains an oil capable of emulsifying and dispersing the first aqueous phase, and preferably further contains a surfactant.

[0033] (Oil for Dispersing the First Aqueous Phase) The oil is not particularly limited as long as it can emulsify and disperse the first aqueous phase to form microdroplets as described above. Examples of oils include saturated or unsaturated higher fatty acids, alkyl esters of such higher fatty acids, polyglycerol esters, glycerides, saturated or unsaturated higher aliphatic alcohols, and aliphatic hydrocarbons. Among these, chemically synthesized oils are preferred because they are less likely to be contaminated by microorganisms, and because they are highly safe, easy to remove by centrifugation or washing, and easy to dissolve carboxylic acids such as acetic acid used for gelation. Examples of higher aliphatic alcohols include capryl alcohol, lauryl alcohol, oleyl alcohol, stearyl alcohol, and isostearyl alcohol, with isostearyl alcohol being preferred.

[0034] (Surfactant) From the viewpoint of emulsifying and dispersing droplets of the first aqueous phase in the first oil phase, the surfactant is preferably a w / o emulsion-forming surfactant. The surfactant may be cationic, anionic, amphoteric, or nonionic, as long as it is capable of forming a w / o emulsion. Furthermore, the surfactant may be a naturally occurring surfactant or a synthetic surfactant, and in the case of a naturally occurring surfactant, lecithin is preferred. Adding a surfactant to the first oil phase improves the dispersibility of the first aqueous phase and suppresses fusion of the microdroplets. The concentration of the surfactant in the first oil phase is not particularly limited, but is preferably 0.5 to 5 w / w%.

[0035] The stirring method for dispersing the first aqueous phase is not particularly limited and can be appropriately designed depending on the scale of preparation of the first aqueous phase droplets, and examples thereof include stirring with a stirrer.

[0036] (2) Gel Bead Formation Step (S200) In this embodiment, the gel bead formation step involves adding a carboxylic acid to the first oil phase to dissolve an ion source, and then supplying ions to the dispersed first aqueous phase to form gel beads of ion-binding polymers, at least the surfaces of which are gelled. Here, "at least the surfaces are gelled" refers to a state in which the ion-binding polymer that constituted the outer periphery of the droplets during dispersion has gelled, and the embedded object inside the gel beads is isolated from the external environment by the gelled ion-binding polymer. Gel beads do not necessarily have to be gelled all the way to the inside; preferably, they have a core containing ungelled ion-binding polymer. When the embedded object is a cell, the gel beads are preferably gelled only on the surface to avoid interfering with the extension of the internal cells and to maintain a neutral core. The degree of gelation can be confirmed by known observation techniques, such as shape observation using a phase-contrast microscope.

[0037] (Gel beads) Gel beads are bead-like structures formed by gelling a first aqueous phase dispersed in an oil phase to form droplets, thereby gelling at least the ion-binding polymer on the surface of the droplets. The gel beads can be used alone as capsules for embedding an object to be embedded therein, or can be used as the core of a capsule further formed with a shell on the outside, as in the second embodiment described below.

[0038] (Carboxylic Acid) The carboxylic acid in this embodiment is not particularly limited as long as it is miscible with water and an organic solvent and does not impair the effects of the present invention, and examples of monocarboxylic acids include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, examples of dicarboxylic acids include succinic acid, glutaric acid, tartaric acid, malic acid, and maleic acid, and examples of tricarboxylic acids include citric acid. Among these, monocarboxylic acids are preferred from the viewpoint of not forming a chelate bond with a counter ion, and acetic acid is more preferred from the viewpoint of being inexpensive and not having reducing properties or an unpleasant odor.

[0039] (Final Concentration of Carboxylic Acid) The carboxylic acid is added to the first oil phase so that the final concentration in the first oil phase is 0.005 to 0.0125 w / w%. This final concentration is preferably 0.005 to 0.01 w / w%. When the embedding target is a microorganism, if acetic acid is used as the carboxylic acid and the final concentration is 0.005 to 0.01 w / w%, this final concentration corresponds to a final acetic acid concentration of 0.7 to 1.4 mmol / L in molar terms, thereby suppressing microbial growth inhibition to 10% or less (see Non-Patent Document 4).

[0040] Furthermore, by keeping the final concentration within this range, the pH of the first aqueous phase can be maintained at 7 to 7.5, allowing the gelation reaction of the ion-binding polymer to proceed under neutral conditions. This allows the capsules produced in this embodiment to embed even objects that are sensitive to acidic conditions, and can improve cell viability in the case of animal cells. Furthermore, the ion-binding polymer gel beads can maintain sufficient strength and prevent fusion, deformation, and breakage of the gel beads. When the object to be embedded is a cell, the pH inside the gel beads after the addition of carboxylic acid is preferably 7 to 7.5, more preferably 7.1 to 7.4, and even more preferably 7.2 to 7.4, from the viewpoint of improving cell viability.

[0041] The principle of the gel bead formation process will be explained in more detail below with reference to Figure 2. In the system shown in Figure 2, alginic acid is used as the ion-binding polymer, calcium carbonate as the ion source, and acetic acid as the carboxylic acid. While specific compound names will be used in the following explanations in Figure 2 and this paragraph, the present invention is not limited to these. When acetic acid is added to oil, the acetic acid migrates into the alginic acid suspension droplets (Reaction Formula 1). Part of the migrated acetic acid dissociates and becomes hydrogen ions (H ＋ ) (Reaction Formula 2). The released hydrogen ions dissolve calcium carbonate and release calcium ions (Ca ２＋ ) (Reaction Scheme 3). The released calcium ions crosslink and gel alginate to form gel beads (Reaction Scheme 4). Some acetate ions (CH ３ COO － ) is a hydroxide ion (OH －) and neutralizes the hydrogen ions produced in Reaction 2. Therefore, if excess acetic acid is added and all of the calcium carbonate is dissolved, gelation can occur to the center of the core, but the acidic conditions will result in cell death. Reducing the amount of acetic acid will gel only the surface of the core, maintaining a neutral condition in the ungelled center, allowing cells to survive. However, if the amount of acetic acid is insufficient, the alginate gel beads will not maintain sufficient strength, resulting in fusion of the beads due to ungelled alginate, and deformation or destruction due to insufficient gel strength. Therefore, it is important to add an optimal amount of acetic acid. By optimizing the amount of acetic acid added, it is possible to form gel beads that gel a portion of the outer periphery of the droplet at the surface of the core, while leaving a sol portion inside the core, as shown in Figure 2.

[0042] (3) Gel Bead Recovery Step (S300) In this embodiment, the gel bead recovery step is a step for recovering the gel beads from the first oil phase. Specifically, washing with a buffer solution containing a surfactant may be performed. In addition, it is preferable to add a salt such as sodium chloride to the buffer solution to adjust the osmotic pressure.

[0043] (4) Degradation Step The capsule manufacturing method of this embodiment may optionally include, after the above-described gel bead production, a step of treating the ungelled ion-binding polymer inside the gel beads (i.e., in a sol state) with a degrading enzyme. Examples of ion-binding polymer degrading enzymes include alginate lyase, κ-carrageenase, and gellan gum degrading enzyme. This allows the outer shells of the gel beads to remain intact, while the interior of the gel beads becomes a liquid core containing degraded ion-binding polymers. This allows, for example, cells to be embedded and cultured under conditions closer to those in vivo.

[0044] The above steps allow for the production of microcapsules containing ion-binding polymer gels that can embed acid-sensitive embedding targets under neutral conditions. By appropriately selecting the embedding target, the capsules can be used for efficient cell and microorganism culture and transplantation, single-cell analysis, and as delivery agents in drug delivery.

[0045] Second Embodiment FIG. 3 shows a flow diagram of a capsule manufacturing method according to a second embodiment. The second embodiment is a method for manufacturing a capsule having a core made of gel beads of an ion-binding polymer obtained in the first embodiment and a shell made of a thermo-dependent polymer. Specifically, the method further includes a step of dispersing an aqueous phase (hereinafter referred to as the second aqueous phase) containing the ion-binding polymer gel beads obtained in the first embodiment and a sol of a thermo-dependent polymer capable of reversible sol-gel transition upon temperature change into a second oil phase (hereinafter referred to as the second aqueous phase dispersion step), and a step of cooling the dispersed second aqueous phase to form a shell by gelling the second aqueous phase (hereinafter referred to as the shell formation step). Furthermore, after the shell formation step, the method further includes a step of removing ions contained in the gel beads and solizing the gel beads to form a liquid core (hereinafter referred to as the liquid core formation step). The capsule manufacturing method according to this embodiment is similar to the first embodiment up to the gel bead recovery step. Hereinafter, explanations of portions overlapping with the first embodiment will be omitted.

[0046] (5) Second aqueous phase dispersion step (S400) The second aqueous phase dispersion step is a step of adding the above-mentioned ion-binding polymer gel beads to a thermo-dependent polymer sol, and emulsifying and dispersing the second aqueous phase containing the gel beads and the thermo-dependent polymer sol in a second oil phase to form microdroplets.

[0047] (Thermo-dependent polymer) The thermo-dependent polymer is a polymer that can undergo reversible sol-gel transition at a certain temperature, regardless of whether or not ions are present in a system containing the thermo-dependent polymer. Furthermore, from the viewpoint of forming a shell, which will be described later, the thermo-dependent polymer is preferably a polymer that is transparent under visible light and hydrophilic. Examples of such polymers include agarose, agar, gelatin, carrageenan (preferably kappa-carrageenan), and native gellan gum, and preferably agarose, agar, or gelatin, and more preferably agarose.

[0048] (Second Oil Phase) The second oil phase is an oil phase for emulsifying and dispersing the second aqueous phase to form fine droplets, and is a continuous phase. The second oil phase contains an oil capable of emulsifying and dispersing the second aqueous phase, and preferably further contains a surfactant.

[0049] (Oil for dispersing the second aqueous phase) Any known oil can be used for the second oil phase as long as it can prevent the thermodependent polymer gel from settling and agglomerating after the shell formation step described below. Specific examples of such oils include the oils disclosed in Japanese Patent No. 7018685 (Patent Document 3).

[0050] (Surfactant) From the viewpoint of emulsifying and dispersing droplets of the second aqueous phase in the second oil phase, the surfactant is preferably a w / o type emulsion-forming surfactant. The surfactant may be the same as or different from the surfactant added to the first oil phase. Among the surfactants, synthetic surfactants such as sorbitan monolaurate (Span (registered trademark) 20), sorbitan monopalmitate (Span (registered trademark) 40), sorbitan monostearate (Span (registered trademark) 60), sorbitan tristearate (Span (registered trademark) 65), sorbitan monooleate (Span (registered trademark) 80), sorbitan trioleate (Span (registered trademark) 85), sorbitan isostearate (Span (registered trademark) 120), polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 20), polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 20), polyoxyethylene sorbitan tristearate (Span (registered trademark) 65), polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 85), polyoxyethylene sorbitan tristearate (Span (registered trademark) 65), polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 20 ... It is preferable to use ethylene sorbitan monolaurate (Tween® 21), polyoxyethylene sorbitan monopalmitate (Tween® 40), polyoxyethylene sorbitan monostearate (Tween® 60), polyoxyethylene sorbitan monostearate (Tween® 61), polyoxyethylene sorbitan tristearate (Tween® 65), polyoxyethylene sorbitan monooleate (Tween® 80), Pluronic® type nonionic surfactants, and the like.

[0051] (6) Shell Formation Step (S500) The shell formation step is a step of cooling the dispersed second aqueous phase droplets to form a shell containing a thermodependent polymer gel around the gel beads of the ion-binding polymer. The cooling method is not particularly limited, and the container containing the second aqueous phase droplets may be cooled with ice or the like. Furthermore, to rapidly form the shell, the second aqueous phase may be dispersed in the second oil phase at room temperature, and then mixed with the cooled second oil phase to form a gel. Using a cooled second oil phase allows the second aqueous phase droplets to be directly cooled, thereby contributing to a reduction in the time required for shell preparation. When the second oil phase is cooled, it is preferably cooled to a temperature within the range of 4 to 10°C, from the viewpoint of maintaining the temperature lower than the gelation temperature of the thermodependent polymer.

[0052] (7) Shell Recovery Step (S600) The shell recovery step is a step of recovering the capsules after shell formation from the second oil phase. The capsules after shell formation have an outer shell containing a thermodependent polymer gel and inner gel beads made of an ion-binding polymer with at least the surface gelled. The gelation of both polymers prevents the capsules from fusing or bonding to each other. Therefore, similar to the gel bead recovery step described above, the capsules can be suitably recovered from the second oil phase by washing with a buffer solution or the like.

[0053] (8) Liquid Core Formation Step (S700) The liquid core formation step is a step of removing ions contained in gel beads of ion-binding polymer in the capsule to convert the gelled portion of the ion-binding polymer into a sol, thereby forming a liquid core. More specifically, this step is a step of chelating counter ions such as alkaline earth metal ions that crosslink the gel beads by ionic bonds with a chelating agent, and dissolving them outside the shell.

[0054] (Chelating Agent) The chelating agent is not particularly limited. Examples of chelating agents that can be used include ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), trans-1,2-cyclohexanediaminetetraacetic acid (CyDTA), and nitrilotriacetic acid (NTA). In addition, to prevent the addition of the chelating agent from causing the system containing the capsules to lose its neutral condition, a buffer solution having chelating ability, such as a citrate buffer solution, may be added together.

[0055] (9) Adsorption Step: The capsule manufacturing method according to this embodiment may further include, as an optional step, a step of adsorbing a cell adhesion polymer onto the surface of gel beads prior to the shell formation step. Specifically, when the ion-binding polymer is alginate, the gel beads have a negative charge on their surfaces. After electrostatically adsorbing a cationic polymer such as poly-L-lysine onto the gel beads, a cell adhesion polymer, such as laminin or fibronectin, which has a negative charge under neutral conditions, is then electrostatically adsorbed. Alternatively, poly-L-lysine to which isothiocyanobenzyl NTA has been covalently introduced is adsorbed onto the gel beads, and an expressed cell adhesion protein fused with a histidine tag that binds to NTA is immobilized onto the gel beads. Adding the immobilized cell adhesion polymer to the alginate gel beads prior to shell formation allows the cell adhesion polymer to be electrostatically adsorbed onto the gel beads, and a scaffold for cell adhesion can be formed inside the shell after the liquid core formation step. To improve the strength of the gel beads, a positively charged polymer such as poly-L-lysine, chitosan, or polyethyleneimine may be electrostatically adsorbed. Furthermore, a negatively charged polymer such as alginic acid, polyacrylic acid, or collagen may be adsorbed onto the adsorbed positively charged polymer. Furthermore, the adsorption of a positively charged polymer and a negatively charged polymer may be alternately repeated one or more times.

[0056] This allows the production of hollow hydrogel microcapsules that can embed acid-sensitive or acid-sensitive objects under neutral conditions. By selecting the appropriate object, the capsules can be used for efficient cell and microorganism culture and transplantation, single-cell analysis, and as a delivery agent for drug delivery.

[0057] [Kit for Producing Capsules] The present invention further includes a kit for producing capsules using the capsule production method described above. This kit is for producing capsules having a core derived from an ion-binding polymer capable of reversible sol-gel transition in the presence of ions. Specifically, the kit includes at least a material for an aqueous phase (first aqueous phase) containing a sol of the ion-binding polymer, a material containing a first oil phase for dispersing the first aqueous phase, and a carboxylic acid for supplying ions to the first aqueous phase to form gel beads of the ion-binding polymer with at least the surface gelled, all of which are packed separately in respective containers. Furthermore, an ion source for supplying ions to the first aqueous phase is packed separately in a container separate from the respective containers and / or is included in the material for the first aqueous phase.

[0058] (Materials for the First Aqueous Phase) The materials for the first aqueous phase included in the kit are packed separately in a single container independent of the other containers, and include at least the ion-binding polymer described above, and preferably an ion source. In the materials for the first aqueous phase, the ion-binding polymer may be included in the form of a raw material powder, or in the form of a sol in which it has been dissolved in an aqueous solvent in advance. In addition to the ion source, any constituent component described below may also be included. Components other than the ion-binding polymer may be packed separately in a container separate from the sol. Furthermore, components other than the ion-binding polymer and the ion source do not need to be included in the kit.

[0059] The user of the kit can prepare a desired object to be embedded and suspend the object in the ion-binding polymer sol by the method described above. Furthermore, when the object to be embedded is a cell, various components, such as a buffer solution capable of maintaining the pH of the first aqueous phase within the range of 7 to 7.5, a medium suitable for growing cells, and a cell adhesive polymer that serves as a scaffold for adherent cells, may be included in the materials for the first aqueous phase depending on the intended commercial use and use, and / or may be prepared and added separately by the user of the kit.

[0060] (Materials for the First Oil Phase) The materials for the first oil phase included in the kit are packed separately in a container independent of the other containers, and include the oil for dispersing the first aqueous phase described above, and preferably a surfactant. The surfactant may be packed separately from the materials for the first oil phase in a container separate from the container and attached to the kit, or may not be included in the kit.

[0061] (Carboxylic Acid) The carboxylic acid included in the kit is separately filled in a single container independent of the other containers. Furthermore, the carboxylic acid is separately filled in a state in which its concentration is appropriately adjusted so that, when the predetermined procedure described in the manual or the like attached to the kit is performed, the final concentration in the first oil phase during gel bead formation is 0.005 to 0.0125 w / w%. The final concentration is preferably adjusted to 0.005 to 0.01 w / w%. The various types of carboxylic acid mentioned above can be used, but acetic acid is preferred.

[0062] (Ion Source) The ion source included in the kit may be contained in the container for the material of the first aqueous phase as described above, or may be packed separately in a container separate from the material of the first aqueous phase and attached, but is preferably contained in the container for the material of the first aqueous phase. The various ion sources described above can be used, and preferably are alkaline earth metal salts, and more preferably are calcium carbonate.

[0063] (Other) In addition to the above-mentioned components, the kit may include various substances depending on the desired use and function. For example, raw material powder of the above-mentioned thermodependent polymer may be included as a material for the second aqueous phase, or the oil contained in the second oil phase and, if necessary, a surfactant may be included as a material for the second oil phase. Furthermore, in the case of a kit for embedding cells, buffer solutions, culture media, salts for adjusting osmotic pressure, etc. may be included separately and / or contained in each of the above-mentioned containers so that the cells can be embedded alive.

[0064] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0065] The reagents used in each test example described below are as follows: Sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd.) HEPES (manufactured by Tokyo Chemical Industry Co., Ltd.) Calcium carbonate (CaCO ３ , manufactured by Shiraishi Kogyosho) MEM medium (manufactured by Sigma) Sodium chloride (NaCl, manufactured by Wako Pure Chemical Industries) Lecithin (manufactured by Wako Pure Chemical Industries) Isostearyl alcohol (manufactured by Kokyu Alcohol Kogyosho) Acetic acid (manufactured by Wako Pure Chemical Industries)

[0066] In each of the following test examples, concentrations are shown as final concentrations unless otherwise specified.

[0067] (Test Example 1) Investigation of the final concentration of acetic acid in the preparation of alginate gel beads 2 w / v% sodium alginate, 0.5 w / v% CaCO ３, 10 v / v% MEM medium, and 10 mmol / L HEPES buffer solution (pH 7.5) with NaCl added to adjust the osmolality to within the range of 260-300 mOsm / L were prepared (hereinafter referred to as the alginate suspension). The osmolality was measured using the method described in "2.47 Osmolality Measurement Method (Osmolality Measurement Method)" of the "General Test Methods" section of the 18th Edition of the Japanese Pharmacopoeia, as described above. 9 mL of isostearyl alcohol (ISA) containing 3 wt% lecithin was prepared. While stirring the ISA with a stirrer, 1 mL of the alginate suspension was added dropwise to emulsify and disperse the alginate suspension, forming microdroplets. After the droplet formation, 0.0025, 0.005, 0.01, 0.0125, 0.025, 0.05, 0.1, or 0.2 wt% acetic acid was added, and CaCO was added. ３ The alginate droplets were dissolved in ISA. The calcium ions generated by the dissolution gelled the alginate droplets, producing alginate gel beads. The gel beads were recovered from the ISA by centrifugation (150 × g, 3 min) using a centrifuge (Beckman, Model No. 5702R). After recovery, the gel beads were washed three times with 10 mL of washing buffer consisting of 10 mmol / L HEPES buffer (pH 7.5) containing 170 mmol / L NaCl. The washed alginate gel beads were suspended in half the amount of the washing buffer, and their shape was observed using a phase-contrast inverted microscope (Olympus, Model No. IX71).

[0068] Figure 4 shows phase-contrast microscope images of alginate gel beads prepared by adding different final concentrations (w / w%) of acetic acid to ISA oil. The scale bar in the figure indicates 500 μm. In the samples to which 0.005 to 0.2 w / w% acetic acid was added, normal spherical alginate gel beads were formed, and no misshapen beads were observed. On the other hand, in the sample to which 0.0025 w / w% acetic acid was added, misshapen alginate gel beads, as indicated by the arrows, were observed. These misshapen alginate gel beads were formed by adhesion and gelation due to insufficient surface gelation, and are therefore unsuitable for use as capsules.

[0069] Therefore, it was suggested that a final concentration of acetic acid of at least 0.005 w / w% was necessary to maintain the normal shape of the alginate gel beads.

[0070] (Test Example 2) Measurement of pH in the aqueous phase with changes in the final concentration of acetic acid Next, the pH in the aqueous phase with changes in the final concentration of acetic acid was measured under the conditions of Test Example 1. However, since it is difficult to measure the pH once alginic acid has gelled, an experiment was carried out without adding alginic acid. Specifically, an aqueous phase with the same composition as Test Example 1 was prepared, except that sodium alginate was not added to the alginic acid suspension of Test Example 1. Specifically, a 0.5 w / v% CaCO solution containing 10 mmol / L HEPES buffer (pH 7.5), 65 mmol / L NaCl, and 10 v / v% MEM was used. ３ The suspension was used as the aqueous phase. Then, the aqueous phase was emulsified and dispersed in ISA under the same conditions as in Test Example 1 to form microdroplets. After the droplets were formed, 0.005, 0.01, 0.0125, 0.025, 0.05, 0.1, or 0.2 wt. % acetic acid was added, and the CaCO ３ The aqueous phase was then separated and collected by centrifugation (3,000×g, 3 min). The pH of the collected aqueous phase was measured with a pH meter (manufactured by Horiba, Ltd., model number LAQUAtwin-pH-11B).

[0071] Figure 5 shows a graph of the results of measuring the pH of the aqueous phase after adding different final concentrations (w / w%) of acetic acid to the oil. The vertical axis of the graph represents the pH of the aqueous phase, and the horizontal axis represents the final concentration of acetic acid. pH measurements were performed three times for each condition, and the average values ​​and standard deviations were plotted. As a result, the aqueous phases to which 0.005 to 0.0125 w / w% acetic acid was added exhibited a pH of 7.3 or higher and a pH of 7.5 or lower, respectively. On the other hand, the aqueous phases to which 0.025 w / w% or higher acetic acid was added exhibited a pH of less than 7.0.

[0072] The optimal pH for many cells is within the range of 7 to 7.5, and cells are damaged and their growth is inhibited outside this range. Therefore, when cells are to be embedded, it is estimated that the final concentration of acetic acid added to alginate gel beads should preferably be in the range of 0.005 to 0.0125 w / w%.

[0073] (Test Example 3) Measurement of cell viability at different final concentrations of acetic acid The cell viability was examined at each acetic acid concentration. ６ An alginate suspension was prepared with the same composition as in Test Example 1, except that MCF-7 cells (RIKEN BioResource Center, catalog number 1904) suspended in MEM medium (+10% FBS (Gibco), 1 mmol / L sodium pyruvate (Nacalai), 0.1 mmol / L MEM non-essential amino acids (Gibco), and 1 / 100 amount of penicillin-streptomycin (Nacalai)) (hereinafter also referred to as cell culture medium) were added to a concentration of 100 cells / mL. 1 mL of the alginate suspension after cell addition was emulsified and dispersed in 9 mL of ISA as in Test Example 1 to form microdroplets. After the droplet formation, 0.005, 0.01, 0.05, or 0.2 w / w% acetic acid was added to gel the alginate, producing alginate gel beads with cells embedded in them. The produced alginate gel beads were centrifuged (150 × g, 3 min) and collected from the ISA. After collection, the gel beads were washed with a washing buffer of the same composition as in Test Example 1. The washed alginate gel beads were suspended in the cell culture medium described above, and trypan blue (Wako Pure Chemical Industries, Ltd.) was added to stain the cells embedded in the alginate gel beads. The stained gel beads were observed under a phase-contrast microscope, and the number of live and dead cells within the gel beads was visually counted. The cell viability was calculated from the ratio of live cells to the total number of cells counted (number of live cells + number of dead cells). Cells not embedded in alginate gel beads were similarly stained with trypan blue, and the cell viability was calculated in the same manner as above, and used as a control without acetic acid.

[0074] Figure 6 shows a graph of the results of measuring cell viability after adding different final concentrations (w / w%) of acetic acid to oil. The vertical axis of the graph represents cell viability, and the horizontal axis represents the final concentration of acetic acid. When 0.005 to 0.05 w / w% acetic acid was added, cell viability of over 90% was observed. On the other hand, when 0.2 w / w% acetic acid was added, cell viability remained at approximately 33%.

[0075] Therefore, when the final concentration of acetic acid was 0.005 to 0.01 w / w%, alginate gel beads were formed in the same manner as in the conventional method, while the pH of the aqueous phase was maintained within the range of 7 to 7.5, and a high cell survival rate was achieved.

[0076] (Test Example 4) Measurement of Microbial Viability Depending on Final Acetic Acid Concentration The viability of microorganisms in alginate gel beads prepared at final acetic acid concentrations of 0.01 and 0.2 w / w% was measured. Escherichia coli, which has the same optimal pH as the animal cells described above, was used as the microorganism. Instead of MEM medium, 5.2 x 10 Escherichia coli DH5α strain (Takara Bio Inc.) suspended in sterilized water was used. ６ An alginate suspension was prepared with the same composition as in Test Example 1, except that the alginate suspension was added so that the concentration was 1 / mL. As in Test Example 1, 1 mL of the alginate suspension after the addition of E. coli was emulsified and dispersed in 9 mL of ISA to form microdroplets. After the droplets were formed, 0.01 or 0.2 w / w% acetic acid was added to gel the alginate. The resulting alginate gel beads with embedded E. coli were centrifuged (150 × g, 3 min) and then washed twice with 10 mL of a washing buffer with the same composition as in Test Example 1, and once with 10 mL of NaCl-free LB medium (hereinafter referred to as LB (NaCl-) medium) consisting of 1 w / v% Bacto tryptone and 0.5 w / v% Bacto yeast extract (both manufactured by Becton Dickinson). After washing, the alginate gel beads were suspended in LB (NaCl-) medium in an amount half that of the beads, and then dispensed in 20 μL aliquots into a 96-well plate (manufactured by Watson). 200 μL of LB (NaCl-) medium was dispensed into each well containing the alginate gel beads, and the plate was incubated overnight at 30°C. The amount of E. coli was measured using a microplate reader (Bio-Rad, Model 550) as turbidity, measuring absorbance at 595 nm. Each sample was measured three times, and the average and standard deviation were calculated. The average and standard deviation are shown in the graph in Figure 7. Additionally, 20 μL of the above-mentioned E. coli-embedded alginate gel beads prepared with 0.01 w / v% acetic acid and 200 μL of LB (NaCl-) medium were dispensed into wells, and the turbidity was measured once without incubation, which served as a control.

[0077] Figure 7 shows a graph of the results of culturing microorganism-embedded alginate gel beads gelled with 0.01 or 0.2 w / v% acetic acid. The vertical axis of the graph represents turbidity, and the horizontal axis represents the concentration of acetic acid used in the alginate gel beads. As shown, reducing the amount of acetic acid added was effective in promoting microbial growth. Therefore, when the final concentration of acetic acid was 0.01 w / w%, an improvement in growth rate was achieved not only for animal cells but also for microorganisms with an optimal pH near neutral.

[0078] From the above results, it is clear that the capsules produced by the production method according to the present invention can be used to embed even objects that are susceptible to acidic conditions, including cells.

[0079] The capsule manufacturing method and kit according to the present invention can be used, for example, as a method for manufacturing ion-binding polymer capsules for encapsulating cells, and as a kit for manufacturing such capsules.

Claims

1. A method for producing capsules having a core derived from an ion-binding polymer capable of reversible sol-gel transition in the presence of ions, comprising the steps of: dispersing an aqueous phase (first aqueous phase) containing a sol of the ion-binding polymer in a first oil phase; and supplying the ions to the dispersed first aqueous phase to form gel beads of the ion-binding polymer, at least the surfaces of which are gelled; and in the step of forming the gel beads, adding a carboxylic acid to the first oil phase to provide the ions, so as to give a final concentration of 0.005 to 0.0125 w / w%.

2. The method for producing capsules according to claim 1, further comprising the steps of: after the step of forming the gel beads, dispersing an aqueous phase (second aqueous phase) prepared by mixing the gel beads with a sol of a thermodependent polymer capable of reversibly undergoing sol-gel transition upon temperature change into a second oil phase; and cooling the dispersed second aqueous phase to form a shell in which the second aqueous phase is gelled.

3. The method for producing a capsule according to claim 2, further comprising the steps of: removing the ions contained in the gel beads and converting the gel beads into a sol to form a liquid core after the step of forming the shell.

4. The method for producing capsules according to claim 1, wherein the ion-binding polymer is at least one selected from the group consisting of alginic acid, deacylated gellan gum, polyacrylic acid, and carboxymethyl cellulose.

5. The method for producing a capsule according to claim 1, wherein the first aqueous phase further comprises an ion source for supplying the ions.

6. The method for producing capsules according to claim 5, wherein the ion source is an alkaline earth metal salt.

7. The method for producing capsules according to claim 1, wherein the first aqueous phase further contains an object to be embedded.

8. The method for producing capsules according to claim 7, wherein the object to be embedded is an animal cell.

9. The method for producing capsules according to claim 7, wherein the object to be embedded is a microorganism.

10. The method for producing capsules according to claim 1, wherein the carboxylic acid is acetic acid.

11. The method for producing capsules according to claim 1, further comprising the step of treating the gel beads with an enzyme that degrades the ionic polymer to degrade the ionic polymer that has not formed a gel.

12. The method for producing capsules according to claim 1, wherein the osmotic pressure of the first aqueous phase is 260 to 320 mOsm / L.

13. The method for producing capsules according to claim 1, wherein the first aqueous phase further contains a cell adhesion polymer that can be used as a scaffold for culturing adherent cells.

14. The method for producing capsules according to claim 3, wherein the first aqueous phase further contains a cell adhesion polymer that can be used as a scaffold for culturing adherent cells, and the method further comprises a step of adsorbing the cell adhesion polymer onto the surface of the gel beads prior to the step of forming the shell.

15. A kit for producing capsules having a core derived from an ion-binding polymer capable of reversible sol-gel transition in the presence of ions, comprising at least the following materials, packed separately in respective containers: a material for an aqueous phase (first aqueous phase) containing a sol of the ion-binding polymer; a material containing a first oil phase for dispersing the first aqueous phase; and a carboxylic acid for supplying the ions to the first aqueous phase and forming gel beads of the ion-binding polymer, at least the surface of which is gelled; wherein an ion source for supplying the ions to the first aqueous phase is packed separately in a container separate from the respective containers and / or is contained in the material for the first aqueous phase; and the kit is packed separately in advance so that the final concentration of the carboxylic acid when the gel beads are formed is 0.005 to 0.0125 w / w%.

16. The kit of claim 15, wherein the carboxylic acid is acetic acid.

17. The kit of claim 15, wherein the ion source is an alkaline earth metal salt.

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