Method for manufacturing gel particles, and method for manufacturing a kit for embryologist skill training.

Abstract

The present invention provides a method for producing solid gel particles having a first surface and a coating gel layer having a second surface, wherein the interface defining the gel particles having the first surface is visible. [Solution] A method for producing gel particles and its application, comprising: performing water-in-oil emulsification using an aqueous phase A containing an ion-crosslinkable polymer and an oil phase A to form droplets, then crosslinking to obtain liquid A containing first particles; replacing the solvent in liquid A with another solvent to obtain liquid B containing first particles; mixing the first particles with the same polymer as the ion-crosslinkable polymer in aqueous phase A to obtain aqueous phase C with a concentration of the ion-crosslinkable polymer different from that of the ion-crosslinkable polymer in aqueous phase A; and performing water-in-oil emulsification using aqueous phase C and oil phase C to form droplets, then crosslinking to obtain gel particles having a double structure with a gel particle having a first surface and a coating gel layer having a second surface, and in which the first surface is visible under optical microscope observation.

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing gel particles and a method for producing a kit for training embryologists. [Background Art]

[0002] Regarding cell mimetics, techniques applying the gelation reaction of raw materials are known. For example, Patent Document 1 discloses that a gel capsule containing cells is prepared using two types of gelable substances, and the cells are cultured in the capsule to form a spherical tissue of animal cells (for example, a tissue composed of stem cells or cancer cells). [Prior Art Literature] [Patent Literature]

[0003] [Patent Document 1] Japanese Patent No. 5850416 [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] By the way, there is a demand for simulated particles that artificially mimic actual cells. As an example, in the field of reproductive medicine, there is a demand for simulated particles that substitute for human egg cells (that is, egg cell simulated particles). Gel particles are useful as a means for realizing cell simulation, but actual cells are solid and have a multilayer structure. Therefore, in order to approximate gel particles that simulate cells (cell simulated particles) to the morphology and physical properties of actual cells, solid gel particles that are solid and have a multilayer structure (more specifically, a solid gel particle having a double structure including a gel particle having a first surface and a coating gel layer having a second surface) are required. In addition, depending on the use of the gel particles such as application to egg cell simulated particles, it is also required that the interface defining the gel particle having the first surface can be visually recognized under an optical microscope observation.

[0005] This disclosure is made in view of the above circumstances, and one embodiment of this disclosure is a solid gel particle having a gel particle having a first surface and a coating gel layer having a second surface, and the object is to provide a method for producing a gel particle having visibility of the interface defining the gel particle having the first surface. Another embodiment of this disclosure aims to provide a method for manufacturing a kit for training embryologists' techniques. [Means for solving the problem]

[0006] This disclosure includes the following aspects:

[0007] <1> Step A involves performing water-in-oil emulsification using an aqueous phase A containing a first polyvalent cationic crosslinkable polymer and an oil phase A to form droplets with an average particle size of 50 μm to 190 μm, and then crosslinking the first polyvalent cationic crosslinkable polymer to obtain liquid A containing first particles containing a polymer gel. Step B involves replacing the solvent in liquid A with another solvent to obtain liquid B containing the first particle, Step C involves mixing the first particles contained in liquid B with a second polyvalent cation-crosslinkable polymer, which is the same as the first polyvalent cation-crosslinkable polymer contained in aqueous phase A, to obtain an aqueous phase C in which the concentration of the second polyvalent cation-crosslinkable polymer is different from the concentration of the first polyvalent cation-crosslinkable polymer in aqueous phase A. Step D involves performing water-in-oil emulsification using an aqueous phase C and an oil phase C to form droplets with an average particle size of 60 μm to 230 μm, and then crosslinking a second polyvalent cationic crosslinkable polymer to obtain gel particles having a double structure consisting of a gel particle having a first surface and a coating gel layer having a second surface, wherein the first surface is visible under optical microscope observation. including, A method for manufacturing gel particles. <2> The aqueous phase C further contains particles X with a particle size of 2 μm to 30 μm. <1> A method for producing gel particles as described above. <3> The mixing of particles X into the aqueous phase C is performed by in-line mixing of a liquid containing particles X into the aqueous phase C, which contains a second polyvalent cation crosslinkable polymer and the first particles. <2> Gel particles as described above. <4> Particle X is a magnetic particle, and after step D, the process includes selectively recovering the gel particles containing the magnetic particle using a magnet. <2> or <2> Gel particles described in any one of the following: <5> <1> A step of manufacturing gel particles by the gel particle manufacturing method described above, A method for manufacturing a kit for embryologists' skill training, comprising the step of heating and sterilizing gel particles at 80°C or higher. [Effects of the Invention]

[0008] According to one embodiment of the present disclosure, a method for producing solid gel particles having a first surface and a coating gel layer having a second surface is provided, wherein the interface defining the gel particles having the first surface is visible. According to another embodiment of the present disclosure, a method for manufacturing a kit for embryologist skill training can be provided. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a reference photograph illustrating the conditions corresponding to evaluation values ​​1 to 3 in the evaluation of interface visibility. [Figure 2] Figure 2 is a photograph illustrating the average thickness and coefficient of variation of the coating gel layer. [Figure 3] Figure 3 is a schematic perspective view showing a microfluidic device according to one embodiment of the present disclosure. [Figure 4] Figure 4 is a schematic cross-sectional view along the line II-II shown in Figure 3. [Figure 5] Figure 5 is a schematic exploded perspective view of the microfluidic device shown in Figure 3. [Figure 6] Figure 6 is a schematic plan view of the microfluidic device shown in Figure 3. [Figure 7] Figure 7 is a schematic plan view showing the flow path of a microfluidic device according to one embodiment of the present disclosure. [Figure 8] Figure 8 is a schematic plan view showing the flow path of a microfluidic device according to one embodiment of the present disclosure. [Figure 9] FIG. 9 is a schematic plan view showing a flow state of a fluid in a flow channel of a microchannel device according to an embodiment of the present disclosure. [Figure 10] FIG. 10 is a schematic plan view showing the microchannel device used in Examples. [Figure 11] FIG. 11 is a schematic plan view showing the microchannel device used in Examples. [Figure 12] FIG. 12 is a schematic plan view showing the microchannel device used in Examples. DESCRIPTION OF EMBODIMENTS

[0010] Hereinafter, the content according to the present disclosure will be described in detail. Descriptions of components described below are given based on representative embodiments of the present disclosure, but the present disclosure is not limited to such embodiments. In the present disclosure, a numerical range indicated using "~" means a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in the present disclosure, the upper limit or lower limit described for a certain numerical range may be replaced with the upper limit or lower limit of a numerical range described in another step. Further, in the numerical ranges described in the present disclosure, the upper limit or lower limit described for a certain numerical range may be replaced with a value shown in the examples. In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0011] In the present disclosure, when a plurality of substances corresponding to each component are present in the composition, the amount of each component in the composition means the total amount of the plurality of substances present in the composition, unless otherwise specified. In the present disclosure, the term "step" includes not only an independent step, but also a case that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. The total solid content in the present disclosure refers to the total amount of components excluding volatile components such as solvents. In this disclosure, ordinal numbers (e.g., "the first" and "the second") are terms used to distinguish between multiple components and do not limit the number of components or their relative importance.

[0012] (Gel particle manufacturing method) The manufacturing method relating to this disclosure is: Step A involves performing water-in-oil emulsification using an aqueous phase A containing a first polyvalent cationic crosslinkable polymer and an oil phase A to form droplets with an average particle size of 50 μm to 190 μm, and then crosslinking the first polyvalent cationic crosslinkable polymer to obtain liquid A containing first particles containing a polymer gel. Step B involves replacing the solvent in liquid A with another solvent to obtain liquid B containing the first particle, Step C involves mixing the first particles contained in liquid B with a second polyvalent cation-crosslinkable polymer to obtain an aqueous phase C in which the concentration of the second polyvalent cation-crosslinkable polymer is different from the concentration of the first polyvalent cation-crosslinkable polymer in aqueous phase A. Step D involves performing water-in-oil emulsification using an aqueous phase C and an oil phase C to form droplets with an average particle size of 60 μm to 230 μm, and then crosslinking a polyvalent cationic crosslinkable polymer to obtain gel particles having a double structure consisting of gel particles with a first surface and a coating gel layer with a second surface, wherein the first surface is visible under optical microscope observation. Includes.

[0013] The details of each step included in the manufacturing method relating to this disclosure are described below.

[0014] <Process A> Step A is a step in which water-in-oil emulsification is performed using an aqueous phase A containing a first polyvalent cationic crosslinkable polymer and an oil phase A to form droplets with an average particle size of 40 μm to 150 μm, and then the polyvalent cationic crosslinkable polymer is crosslinked to obtain liquid A containing first particles containing polymer gel.

[0015] <<Aqueous phase A>> Aqueous phase A contains a first polyvalent cationic crosslinkable polymer (hereinafter also simply referred to as "ionic crosslinkable polymer 1"). Aqueous phase A preferably contains a solvent, polyvalent cations, and a chelating agent, and may optionally further contain other components (for example, compounds that react with sperm proteins to directly or indirectly cause a visual change, or dyes, poorly water-soluble metal salts, preservatives, etc.). The solvent can be any aqueous solvent, such as water or an alcohol compound, but water is preferred.

[0016] [Preparation of ion-crosslinkable polymer 1] As the ion-crosslinkable polymer 1 contained in aqueous phase A, a known ion-crosslinkable polymer that can be crosslinked with polyvalent cations can be used. Examples of ionically crosslinkable polymers include pectin or its derivatives, alginic acid or its salts, gellan gum, carrageenan, polygalacturonic acid, and mixtures thereof.

[0017] Among these, the ion crosslinkable polymer 1 is preferably at least one compound selected from the group consisting of alginate, carrageenan, polygalacturonic acid, and pectin, more preferably at least one compound selected from the group consisting of alginate and carrageenan, and even more preferably alginate.

[0018] Examples of alginates include salts of alginic acid with magnesium, alkali metals, or alkaline earth metals. Among alginates, calcium alginate, magnesium alginate, and sodium alginate are preferred, with sodium alginate being more preferred, from the viewpoint of being readily available and easy to control the degree of crosslinking. Furthermore, κ-carrageenan and ι-carrageenan are preferred as carrageenan. Ion-crosslinkable polymers may be used individually or in combination of two or more types.

[0019] From the viewpoint of productivity, the viscosity range of the ionically crosslinkable polymer 1 at 25°C when 1 g of the ionically crosslinkable polymer is dissolved in 100 mL of water (1 w / vol%) is preferably 10 Cp (0.01 Pa·s) to 10,000 Cp (10 Pa·s), more preferably 10 Cp (0.01 Pa·s) to 1,000 Cp (1 Pa·s), and even more preferably 10 Cp (0.01 Pa·s) to 500 Cp (0.5 Pa·s). The viscosity of a solution containing an ionic crosslinkable polymer can be determined by the method described in JIS Z 8803 (2011) Method for Measuring the Viscosity of Liquids.

[0020] From the viewpoint of handling during manufacturing, it is preferable that the ion-crosslinkable polymer 1 be used as an ion-crosslinkable polymer mixture 1 obtained by mixing the ion-crosslinkable polymer 1 with a solvent. Suitable solvents include water and alcohol compounds, but water is preferred. The content of the ion-crosslinkable polymer 1 in the ion-crosslinkable polymer mixture 1 is preferably 0.1% to 10% by mass, and more preferably 0.3% to 5% by mass, based on the total mass of the ion-crosslinkable polymer mixture 1.

[0021] [Preparation of polyvalent cations] The polyvalent cations contained in aqueous phase A are preferably divalent or trivalent cations, and more preferably divalent metal cations. Examples of divalent metal cations include calcium ions, barium ions, iron ions, zinc ions, and copper ions. From the viewpoint of excellent crosslinking with ionic crosslinkable polymer 1, among these, calcium ions are preferred as the divalent metal cation.

[0022] The polyvalent cation may be used as a salt of the polyvalent cation, or as a compound in which the polyvalent cation is coordinated to a metal chelating agent.

[0023] The polyvalent cation is preferably used as an aqueous solution of a polyvalent cation salt, which is obtained by mixing a salt of a polyvalent cation (preferably a divalent metal cation, more preferably a calcium ion) with water. The polyvalent cation salt content in the polyvalent cation salt aqueous solution is preferably 0.01% to 10% by mass, and more preferably 0.05% to 5% by mass, relative to the total mass of the polyvalent cation salt aqueous solution.

[0024] [Preparation of chelating agents] As a chelating agent contained in aqueous phase A, it can function as a gelation reaction retarder. Examples of chelating agents include edible compounds that have chelating properties. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrates, pyrophosphates, metaphosphates, and polyphosphates. From the standpoint of being readily available and inexpensive, citrates are preferable. Examples of citrates include sodium citrate (e.g., trisodium citrate).

[0025] The chelating agent is preferably used as an aqueous solution of the chelating agent (preferably citrate) mixed with water. The chelating agent content in the aqueous solution of the chelating agent is preferably 0.1% to 10% by mass, and more preferably 0.2% to 5% by mass, relative to the total mass of the aqueous solution of the chelating agent.

[0026] Aqueous phase A is preferably prepared by mixing an ion-crosslinkable polymer mixture 1, a polyvalent cation salt aqueous solution, and a chelating agent aqueous solution. In aqueous phase A, the preferred mixing ratio (a:b:c) of the ion-crosslinkable polymer mixture 1 (a), the polyvalent cation salt aqueous solution (b), and the chelating agent aqueous solution (c) is 0.1 to 5:0.1 to 5:0.2 to 5 by mass.

[0027] The concentration of the ionically crosslinkable polymer 1 in aqueous phase A is preferably 0.1% to 10% by mass, and more preferably 0.2% to 5% by mass.

[0028] - pH of aqueous phase A - The pH of aqueous phase A is preferably 7 to 9, and more preferably 7.2 to 8.0. As a pH adjusting agent, known pH adjusting agents can be used to adjust the pH. The pH of the aqueous phase is the value measured at 25°C using a pH meter, for example, using a benchtop pH meter (product name: pH METER D-51, manufactured by Horiba, Ltd.).

[0029] <<Oil phase A>> The oil phase A used in step A preferably contains oil and fat. There are no particular restrictions on the oils and fats used; they may be natural oils or synthetic oils, or mixtures thereof. Natural oils and fats may be animal oils or vegetable oils, but vegetable oils are preferred.

[0030] From the viewpoint of mimicking fat cells, the oil is preferably a saturated fatty acid or an unsaturated fatty acid, more preferably a saturated fatty acid having 12 to 30 carbon atoms or an unsaturated fatty acid having 12 to 30 carbon atoms, and even more preferably an unsaturated fatty acid having 16 to 24 carbon atoms. The number of unsaturated double bonds in an unsaturated fatty acid is preferably 1 to 3 per molecule, and more preferably 2 or 3. Examples of unsaturated fatty acids include medium-chain triglycerides (medium-chain fatty acid triglycerides) with 6 to 12 carbon atoms, such as caproic acid, caprylic acid, capric acid, and lauric acid; vegetable oils such as coconut oil, sesame oil, olive oil, corn oil, rapeseed oil, safflower oil, soybean oil, sunflower oil, nut oil, grapeseed oil, and linseed oil; and vitamin E. In one embodiment, corn oil is preferred as the oil or fat because it has low viscosity, is readily available, and is inexpensive. The oils and fats used in oil phase A may be one type used alone, or two or more types may be used in combination.

[0031] Oil phase A may contain other components besides oils and fats. Other ingredients include, for example, surfactants, etc. Nonionic surfactants are preferably used as surfactants. Examples of nonionic surfactants include glycerin fatty acid esters and sorbitan fatty acid esters, with glycerin fatty acid esters being preferred. The glycerol fatty acid ester may be a synthetic product or a commercially available product. Examples of commercially available glycerin fatty acid esters include Nikko Chemicals Co., Ltd.'s "NIKKOL® DGMS" (diglyceryl monostearate, HLB value: 5.0), "NIKKOL® MGM" (glyceryl monomyristate, HLB value: 3.5), and "NIKKOL® MGS-F50V" (glyceryl monostearate, HLB value: 3.5), and Sakamoto Pharmaceutical Co., Ltd.'s "SY Glister PS-3S" (tetraglyceryl pentastearate, HLB value: 2.6), "SY Glister PS-5S" (hexaglyceryl pentastearate, HLB value: 4.5), and "SY Glister CRS-75" (polyglyceryl-6 polyricinoleate, HLB value: 3.3). Surfactants may be used individually or in combination of two or more types.

[0032] When a surfactant is used, the surfactant content is preferably 0.05% to 3% by mass, more preferably 0.1% to 2% by mass, and even more preferably 0.5% to 1.5% by mass, based on the total mass of the oil and fat contained in oil phase A.

[0033] In step A, water-in-oil emulsification is performed using aqueous phase A and oil phase A to form droplets (WO droplets) with an average particle size of 40 μm to 150 μm. WO droplets are also called water-in-oil droplets. Methods for water-in-oil emulsification may include, for example, dispersing using conventional dispersion or emulsification devices that utilize shear action, such as stirrers, impeller-type agitators, cylindrical mills, and homomixers, or using membrane emulsification devices. However, from the viewpoint of droplet uniformity, it is preferable to use a membrane emulsification device or to mix using a microfluidic device. Details of the microfluidic device will be described later.

[0034] The mixing ratio of aqueous phase A to oil phase A can be set as appropriate. The mixing ratio of aqueous phase A to oil phase A is preferably 1:100 to 1:1 by mass, and more preferably 1:50 to 1:2.

[0035] The average particle size of the droplets formed by the water-in-oil emulsification of aqueous phase A and oil phase A is 50 μm to 190 μm. More preferably, the average particle size of the droplets is 55 μm to 185 μm, and even more preferably 60 μm to 180 μm.

[0036] The average particle size of a droplet is determined by photographing the droplet using a transmission optical microscope at a 5x objective magnification. The diameter (μm) of the droplet is then measured using measurement software (Zeiss AxioVision) in the obtained image. The measurement is performed on 10 randomly selected gel particles, and the average of the diameters obtained from these 10 points is calculated. The average particle size of the droplet is then rounded to the first decimal place. Furthermore, the average particle size of the droplets can be controlled by the flow rate ratio of the aqueous phase A and the oil phase A, the channel size of the microchannel used, and the pore size of the membrane emulsion.

[0037] Next, in step A, a polyvalent cationic crosslinkable polymer is crosslinked to obtain liquid A containing first particles containing a polymer gel. The first particle containing the polymer gel corresponds to a gel particle having a first surface (hereinafter also referred to as "gel particle X") in the gel particles obtained by the manufacturing method according to this disclosure.

[0038] Crosslinking of polyvalent cationic crosslinkable polymers is preferably carried out by contact between droplets formed by water-in-oil emulsification (WO droplets) and oils and fats containing a pH lowering agent.

[0039] It is hypothesized that when a liquid droplet (WO droplet) comes into contact with an oil containing a pH-lowering agent, the pH of the aqueous phase A constituting the droplet is lowered by the pH-lowering agent, releasing polyvalent metal ions. The ion-crosslinkable polymer then crosslinks via these released polyvalent metal ions, forming a first particle containing a polymer gel.

[0040] The pH-lowering agent used in edible oils and fats containing a pH-lowering agent (hereinafter also referred to as "hydrogenated oil") is not particularly limited as long as it is a compound that can lower the pH of the aqueous phase component of a droplet (WO droplet) and is miscible with oil. The aqueous phase component of the droplet (WO droplet) refers to the component of aqueous phase A used to form the droplet.

[0041] As the pH lowering agent, oxoacids are preferred, oxoacids having 2 to 4 carbon atoms are more preferred, oxoacids having 2 or 3 carbon atoms are even more preferred, and acetic acid is particularly preferred. Oxoacids are compounds in which a hydroxyl group (-OH) and an oxo group (=O) are bonded to the same atom, and the hydroxyl group provides an acidic proton. The pH lowering agent content is preferably 0.05% to 15% by mass, and more preferably 0.5% to 10% by mass, relative to the total mass of the hardened oil. pH lowering agents may be used individually or in combination of two or more types.

[0042] Examples of oils and fats in the hardened oil include those described in the description of oil phase A, and the preferred embodiment is the same. Furthermore, the oils and fats in the hardened oil may be the same as those used in oil phase A, or they may be different, but it is preferable that they be the same as those used in oil phase A. The oil and fat content in the hardened oil is preferably 85% to 99.95% by mass, and more preferably 90% to 99.5% by mass, relative to the total mass of the hardened oil.

[0043] Contact between the droplet (WO droplet) and the hardened oil (oil containing a pH lowering agent) may be achieved, for example, by adding the droplet to the hardened oil, by using a conventional dispersion or emulsification device that utilizes shear action such as a stirrer, impeller-type agitator, or homomixer, or by using a microfluidic device. Contact between the liquid droplet (WO droplet) and the hardened oil (oil containing a pH lowering agent) may be performed by in-line mixing. Details of the microfluidic device will be described later.

[0044] As a result of the above, liquid A containing the first particle is obtained in step A. Liquid A is a dispersion containing the first particle.

[0045] <Process B> Step B is a step in which the solvent contained in liquid A obtained in step A is replaced with another solvent to obtain liquid B containing the first particle. The solvent contained in liquid A and the other solvents may have different compositions, and components contained in one solvent may be present in the other solvent in different amounts. Other preferred solvents include water and alcohol, with water being more preferred.

[0046] Substitution with another solvent can be carried out by adding another solvent (preferably water) to solution A, mixing lightly, allowing it to stand, and then removing the supernatant (oil-water mixture) from the standing solution, repeating this process. The standing time can range from 1 minute to 5 days. In step B, the above operation is repeated to obtain liquid B containing the first particle.

[0047] <Process C> Step C is a step in which the first particles contained in liquid B are mixed with the second polyvalent cationic crosslinkable polymer (hereinafter also referred to as "ionic crosslinkable polymer 2") which is the same as the first polyvalent cationic crosslinkable polymer (ionic crosslinkable polymer 1) contained in aqueous phase A, in order to obtain aqueous phase C in which the concentration of ionic crosslinkable polymer 2 is different from the concentration of ionic crosslinkable polymer 1 in aqueous phase A.

[0048] Aqueous phase C is a dispersion containing the first particle.

[0049] The mixing of the first particles contained in liquid B and the ion-crosslinkable polymer can be done by mixing liquid B containing the first particles with the ion-crosslinkable polymer 2, or by mixing the first particles recovered from liquid B with the ion-crosslinkable polymer 2. From the viewpoint of easy adjustment of polymer concentration, it is preferable to use the first particles recovered from liquid B. The first particles can be recovered from liquid B by filtering them out using a filter.

[0050] In step C, the ion-crosslinkable polymer 2 used in aqueous phase C is the same ion-crosslinkable polymer 1 contained in aqueous phase A. It is preferable that the ion-crosslinkable polymers 1 and 2 used in aqueous phase A and aqueous phase C are both alginates. Aqueous phase A preferably contains a solvent, polyvalent cations, and a chelating agent, and may further contain other components (e.g., preservatives) as needed. The solvent can be any aqueous solvent, such as water or an alcohol compound, but water is preferred.

[0051] It is preferable to prepare the aqueous phase C as a mixture by mixing the ion-crosslinkable polymer mixture 2, the polyvalent cation salt aqueous solution, the chelating agent aqueous solution, and the first particles. The ion-crosslinkable polymer mixture 2, the polyvalent cation salt aqueous solution, and the chelating agent aqueous solution can be prepared in the same manner as the ion-crosslinkable polymer mixture 1, the polyvalent cation salt aqueous solution, and the chelating agent aqueous solution used in aqueous phase A.

[0052] The concentration of the first particles in aqueous phase C is preferably 0.1% to 50% by mass, and more preferably 1% to 30% by mass. Because the concentration of the first particles in the aqueous phase C is within the above range, the gel particles obtained in step D, described later, readily form a double structure having a first surface gel particle (gel particle X) derived from the first particle and a coating gel layer (hereinafter also referred to as "coating gel layer Y") having a second surface.

[0053] The concentration of ion-crosslinkable polymer 2 in aqueous phase C is different from the concentration of ion-crosslinkable polymer 1 in aqueous phase A, and is preferably 0.01% to 10% by mass, and more preferably 0.05% to 5% by mass. Having different concentrations of ion-crosslinkable polymer 2 in aqueous phase C and aqueous phase A allows for different refractive indices in the resulting gel particles in step D, described later, where the refractive index of the gel particle having the first surface (gel particle X) and the refractive index of the coating gel layer having the second surface (coating gel layer B) are different. Having different refractive indices between gel particle X and coating gel layer Y is preferable because it results in gel particles with excellent visibility of the interface defining gel particle X under optical microscope observation.

[0054] -Particle X- In one embodiment, the aqueous phase C preferably further contains particles X having a particle size of 2 μm to 30 μm.

[0055] The inclusion of particle X is preferable, for example, when the gel particles obtained by the manufacturing method according to this disclosure are used as egg cell imitation particles, because they can mimic the polar bodies of egg cells. Training aimed at improving the skill level of embryologists includes mastering the technique of sperm injection while avoiding the polar bodies of egg cells. This is because, since spindle fibers are present around the polar bodies of egg cells, sperm injection must be performed while avoiding the area around the polar bodies. The inclusion of particle X in the gel particles according to this disclosure can serve as a marker for sperm injection while avoiding the polar bodies in training using egg cell imitation particles.

[0056] The particle size of particle X is preferably 2 μm to 30 μm, and more preferably 3 μm to 25 μm, from the viewpoint of simulating polar bodies in egg cell-like particles.

[0057] Particle X is not particularly limited as long as it is visible in the gel particles obtained by the manufacturing method according to this disclosure, and may be organic particles, inorganic particles, or organic-inorganic composite particles. Particle X may be magnetic or non-magnetic, but from the viewpoint that the gel particles containing particle X can be easily separated using a magnet during the manufacturing of the gel particles, it is preferable that particle X is magnetic. Examples of particle X include silica particles, ferromagnetic particles, polymer particles, and metal particles. In one embodiment, particle X is preferably a silica particle. In another embodiment, particle X is preferably a ferromagnetic particle.

[0058] The number of particles X contained in a gel particle may be one or two or more. In one embodiment, from the viewpoint of simulating a morphology that more closely resembles an actual cell, it is preferable that the number of particles X contained in one gel particle is one.

[0059] The mixing of particles X into the aqueous phase C can be achieved by further mixing particles X into the aqueous phase C, which contains the first particles and the second ion-crosslinkable polymer (ion-crosslinkable polymer 2). In one embodiment, the mixing of particles X into the aqueous phase C is preferably carried out by in-line mixing a liquid containing particles X into the aqueous phase C, which contains an ionic crosslinkable polymer 2 and first particles.

[0060] -pH of aqueous phase C- The pH of aqueous phase C is preferably 7 to 9, and more preferably 7.2 to 8.0. As a pH adjusting agent, known pH adjusting agents can be used to adjust the pH. The pH of the aqueous phase is the value measured at 25°C using a pH meter, for example, using a benchtop pH meter (product name: pH METER D-51, manufactured by Horiba, Ltd.).

[0061] <Process D> Step D is a step in which water-in-oil emulsification is performed using aqueous phase C and oil phase C to form droplets with an average particle size of 50 μm to 200 μm, and then a polyvalent cationic crosslinkable polymer is crosslinked to obtain gel particles having a double structure consisting of gel particles having a first surface and a coating gel layer having a second surface, and in which the first surface is visible under optical microscope observation.

[0062] The aqueous phase C used in process D is the aqueous phase C obtained in process C.

[0063] The oil phase C used in process D can be the same oil phase as the oil phase A used in process A, and the preferred embodiment is also the same. Oil phase C and oil phase A may be the same oil phase.

[0064] In step D, water-in-oil emulsification is performed using aqueous phase C and oil phase C to form droplets (WO droplets) with an average particle size of 50 μm to 200 μm. Methods for water-in-oil emulsification may include, for example, dispersing using conventional dispersion or emulsification devices that utilize shear action, such as stirrers, impeller-type agitators, cylindrical mills, and homomixers, or using membrane emulsification devices. However, from the viewpoint of easily achieving uniform droplets, it is preferable to use a membrane emulsification device or to mix using a microfluidic device. Details of the microfluidic device will be described later.

[0065] The mixing ratio of the aqueous phase C to the oil phase C can be set as appropriate. The mixing ratio of the aqueous phase C to the oil phase C is preferably 1:100 to 1:1 by mass, and more preferably 1:50 to 1:2.

[0066] The average particle size of the droplets formed by water-in-oil emulsification of the aqueous phase C and the oil phase C is 60 μm to 230 μm. The droplet particle size is preferably 65 μm to 220 μm, and more preferably 70 μm to 220 μm.

[0067] The average particle size of the droplets can be confirmed by the method described in the explanation of step A, which is the method for measuring the average particle size of the droplets. Furthermore, the average particle size of the droplets can be controlled by factors such as the flow rate ratio of the aqueous phase C and the oil phase C, the channel size of the microchannel used, and the pore size of the membrane emulsion.

[0068] Next, in step D, a polyvalent cationic crosslinkable polymer is crosslinked to obtain gel particles having a double structure consisting of a gel particle having a first surface (gel particle X) and a coated gel layer having a second surface (coated gel layer Y), and in which the first surface is visible under optical microscope observation.

[0069] Crosslinking of polyvalent cationic crosslinkable polymers is preferably carried out by contact between droplets formed by water-in-oil emulsification (WO droplets) and oils and fats containing a pH lowering agent (hydrogenated oil). Contact between the droplet (WO droplet) and the hardened oil can be carried out in the same manner as the contact between the droplet (WO droplet) and the hardened oil in step A. The term "hardened oil" is synonymous with the hardened oil used in process A, and the preferred range is also the same.

[0070] Step D yields gel particles having a double structure consisting of a gel particle with a first surface and a coating gel layer with a second surface, and in which the first surface is visible under optical microscope observation. Details of the obtained gel particles will be described later.

[0071] <Separation process> The process may further include a step of separating the obtained gel particles after step D (hereinafter also referred to as the "separation step"). The separation process is not particularly limited as long as it allows for the separation of gel particles from a liquid containing gel particles. Separation methods include decantation, filtration, and extraction.

[0072] One aspect of the separation process is a step in which, when the gel particles obtained through step D contain magnetic particles as particle X, a magnet is used to selectively recover the gel particles containing magnetic particles as particle X.

[0073] Selective recovery of gel particles containing magnetic particles (particle X) involves applying magnetism to the liquid containing the gel particles using a magnet, causing only the gel particles containing the magnetic particles to aggregate and separate. Since gel particles that do not contain magnetic particles are dispersed in the liquid without agglomerating, by removing the liquid containing the gel particles that do not contain magnetic particles, only the gel particles containing magnetic particles can be efficiently recovered as particle X.

[0074] Various methods can be used to selectively collect gel particles containing magnetic particles as particle X, such as using a magnetic stand, installing a high-speed camera and a channel switching mechanism (such as a valve that switches the channel with an electromagnet) in the collection path of a microfluidic device to selectively collect only the gel particles containing the second particle using images, or installing a ferromagnetic magnet on one side of a microfluidic device and then connecting it to a bifurcated channel to increase the collection efficiency of gel particles containing particle X.

[0075] <Other processes> The manufacturing method relating to this disclosure may include steps A, B, C, D, and other steps besides the separation step.

[0076] Another aspect of the process is a step of heat sterilizing the gel particles obtained through step D (also referred to as the heat sterilization step). Including the heat sterilization step is preferable when using the gel particles according to the disclosure as egg cell simulated particles to produce an embryologist training kit. That is, the heat sterilization step may be included as one step in the method for producing an embryologist training kit. The heat sterilization process may be performed on the embryologist training kit containing gel particles and physiological saline solution. Heating methods include constant temperature baths and steam baths maintained at a heat sterilization temperature. The heating temperature is preferably 80°C or higher. The upper limit of the heating temperature is, for example, 140°C. The heating time is preferably between 1 minute and 2 hours.

[0077] In the manufacturing method according to this disclosure, it is preferable to use a microfluidic device in at least steps A, C, and D. By adjusting the channel dimensions and flow rates (e.g., the flow rates of aqueous phase A, aqueous phase C, and curing oil) in a microfluidic device, the size of gel particles X and the coating gel layer B in the gel particles can be adjusted. The use of microfluidic devices can also contribute to miniaturization of manufacturing equipment and improvement of manufacturing precision.

[0078] The following describes the details of the microfluidic device used in manufacturing method A (hereinafter also simply referred to as the "microfluidic device"). The microfluidic devices described below are just examples, and are not limited to these.

[0079] <<Microfluidic Devices>> A microfluidic device according to one embodiment of the present disclosure includes a base containing silicone, having a defining surface that defines a channel. In the present disclosure, the base or the material forming the base may be referred to as the "first base". In the present disclosure, the term "base" refers to the base (i.e., the first base) unless otherwise specified.

[0080] <<Base (First Base)>> A microfluidic device according to one embodiment of the present disclosure includes a defining surface for defining a channel and a base containing silicone.

[0081] The type of silicone is not limited. Silicones in this disclosure include known silicones. Examples of constituent units of silicone include dimethylsiloxane units (-Si(CH3)2-O-), diphenylsiloxane units (-Si(C5H6)2-O-), and methylhydrogensiloxane units (-SiH(CH3)-O-). Silicones may also contain constituent units other than those described above. Silicones may be homopolymers or copolymers. Examples of silicones include polydimethylsiloxane (PDMS), polymethylhydrogensiloxane, and silicones containing dimethylsiloxane units and methylhydrogensiloxane units. From the viewpoint of transparency and surface properties, the silicone is preferably a silicone containing at least one selected from the group consisting of dimethylsiloxane units, diphenylsiloxane units, and methylhydrogensiloxane units, and more preferably polydimethylsiloxane. The base may contain two or more types of silicone.

[0082] The silicone content in the base is not limited. From the viewpoint of the transparency of the base and the surface properties of the base (e.g., hydrophobicity and hydrophilicity improved by surface modification), the silicone content in the base is preferably 85% to 100% by mass, preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, based on the total mass of the base. The silicone content in the base may also be less than 100% by mass, based on the total mass of the base.

[0083] The defining surfaces of the base define the channels of the microfluidic device. That is, the defining surfaces of the base are the surfaces of the base facing the channels. The number of defining surfaces of the base is not limited. There may be one or more defining surfaces of the base. The shape of the defining surfaces of the base is not limited. The defining surfaces of the base may be, for example, planar or curved.

[0084] The shape of the base is not limited. From the viewpoint of ease of manufacturing, the shape of the base is preferably flat.

[0085] The thickness of the base is not limited. The thickness of the base may be determined, for example, within the range of 1 mm to 10 mm. Preferably, the thickness of the base is within the range of 1 mm to 10 mm, more preferably within the range of 1 mm to 5 mm, and particularly preferably within the range of 1.5 mm to 4 mm.

[0086] <<Opponent base (second base)>> A microfluidic device according to one embodiment of the present disclosure preferably further includes a counter base that contacts the base. That is, a microfluidic device according to one embodiment of the present disclosure preferably includes a base (i.e., a first base) and a counter base that contacts the base. The counter base has a defining surface that defines a flow path together with the defining surface of the base. The flow path defined by the defining surface of the counter base is the same as the flow path defined by the defining surface of the base. That is, the flow path in a microfluidic device including a base and a counter base is formed between the base and the counter base. In the present disclosure, the counter base or the material forming the counter base may be referred to as the "second base".

[0087] The composition of the opposing base is not limited. Examples of opposing base components include silicone, glass, and stainless steel.

[0088] In one embodiment, the opposing base preferably contains silicone. The presence of silicone in the opposing base improves the adhesion between the opposing base and the base. Examples of silicone include the silicone described in the section "Base (First Base)" above. The silicone is preferably polydimethylsiloxane. The opposing base may contain one or more types of silicone.

[0089] The silicone content in the opposing base is not limited. In one embodiment, from the viewpoint of adhesion to the base and surface properties of the opposing base (e.g., ease of surface modification to improve hydrophilicity), the silicone content in the opposing base is preferably 85% to 100% by mass, preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, based on the total mass of the opposing base. The silicone content in the opposing base may also be less than 100% by mass, based on the total mass of the opposing base.

[0090] In one embodiment, the opposing base preferably includes at least one selected from the group consisting of glass and stainless steel. The opposing base may include, for example, glass, stainless steel, or both glass and stainless steel.

[0091] In one embodiment, the opposing base preferably contains glass. The type of glass is not limited. The glass in this disclosure includes known glasses. Examples of glass components include Al2O3, B2O3, CaO, Na2O, and SiO2.

[0092] In one embodiment, the opposing base is preferably made of stainless steel. The type of stainless steel is not limited. Stainless steel in this disclosure includes known stainless steels. Examples of stainless steels include SUS304 and SUS316. The surface of the opposing base, which is made of stainless steel, may be coated with a glass-like coating using a silica sol gel coating agent.

[0093] The content of at least one material selected from the group consisting of glass and stainless steel in the opposing base is not limited. In one embodiment, from the viewpoint of flatness and strength, the content of at least one material selected from the group consisting of glass and stainless steel in the opposing base is preferably 85% to 100% by mass, preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, based on the total mass of the opposing base. The content of at least one material selected from the group consisting of glass and stainless steel in the opposing base may be less than 100% by mass, based on the total mass of the opposing base.

[0094] The glass content in the opposing base is not limited. In one embodiment, from the viewpoint of transparency, the glass content in the opposing base is preferably 85% to 100% by mass, preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, based on the total mass of the opposing base. The glass content in the opposing base may also be less than 100% by mass, based on the total mass of the opposing base.

[0095] The stainless steel content in the opposing base is not limited. In one embodiment, from the viewpoint of flatness and strength, the stainless steel content in the opposing base is preferably 85% to 100% by mass, preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, based on the total mass of the opposing base. The stainless steel content in the opposing base may also be less than 100% by mass, based on the total mass of the opposing base.

[0096] The defining surfaces of the opposing base define the flow channels of the microfluidic device. That is, the defining surfaces of the opposing base are the surfaces of the opposing base that face the flow channels. The number of defining surfaces of the opposing base is not limited. There may be one or more defining surfaces. The shape of the defining surfaces of the opposing base is not limited. The defining surfaces of the opposing base may be, for example, planar or curved.

[0097] The shape of the opposing base is not limited. From the viewpoint of ease of manufacturing, the shape of the opposing base is preferably flat.

[0098] The thickness of the opposing base is not limited. The thickness of the opposing base may be determined, for example, within the range of 1 mm to 20 mm.

[0099] Hereinafter, a microfluidic device according to one embodiment of this disclosure will be described with reference to Figures 3, 4, 5, and 6. Figure 3 is a schematic perspective view showing a microfluidic device according to one embodiment of this disclosure. Figure 4 is a schematic cross-sectional view along the line II-II shown in Figure 3. Figure 5 is a schematic exploded perspective view of the microfluidic device shown in Figure 3. Figure 6 is a schematic plan view of the microfluidic device shown in Figure 1.

[0100] The microfluidic device 100 shown in Figure 3 includes a base (first base) 10 and a counter base (second base) 20. The counter base 20 is positioned on top of the base 10 and is in contact with the base 10.

[0101] As shown in Figures 4 and 5, the channel 30 of the microfluidic device 100 is formed between the base 10 and the opposing base 20. As shown in Figure 4, the channel 30 is a space enclosed by the wall surface of a groove (i.e., a recess) formed on the surface of the base 10 and the surface of the opposing base 20. The surface surrounding the channel 30 is the defining surface in this disclosure. The shape of the channel 30 corresponds to the shape of the groove formed on the surface of the base 10. As shown in Figure 6, the channel 30 branches at confluence point 30A and confluence point 30B.

[0102] As shown in Figures 5 and 6, the microfluidic device 100 includes four openings that penetrate the base 10. The four openings include opening 40, opening 41, opening 42, and opening 43. Each opening is connected to the flow path 30. For example, three openings are used as fluid inlets, and the remaining opening is used as a fluid outlet. As shown in Figure 4, the shape of each opening in plan view is circular. However, the shape of the openings in plan view is not limited to circular. The dimensions of each opening are determined by considering, for example, the dimensions of the flow path (e.g., the width of the flow path).

[0103] <<Flow path>> In one embodiment of the present disclosure, the flow path of a microfluidic device is defined by at least the defining surface of the base. The flow path of a microfluidic device including a base and an opposing base is defined by the defining surface of the base and the defining surface of the opposing base.

[0104] The shape of the channel is not restricted. Examples of cross-sectional shapes of the channel include square, circular, and semicircular shapes. For example, a channel with a square cross-section is defined by being surrounded by four planar defining surfaces. For example, a channel with a circular cross-section is defined by being surrounded by one cylindrical defining surface or two curved defining surfaces. For example, a channel with a semicircular cross-section is defined by being surrounded by one curved defining surface and one planar defining surface. However, the above specific examples do not restrict the relationship between the shape of the channel and the characteristics of the defining surfaces (e.g., the number and shape of the defining surfaces).

[0105] The width of the channel is not limited. The width of the channel may be determined, for example, within the range of 1 μm to 2,000 μm. Preferably, the width of the channel is within the range of 5 μm to 1,000 μm, more preferably within the range of 10 μm to 500 μm, and particularly preferably within the range of 20 μm to 400 μm.

[0106] Preferably, the flow path includes a main flow path and at least one branch flow path branching off from the main flow path. A flow path having the above structure can merge at least two types of fluids at the junction of the main flow path and the branch flow path. For example, the second fluid can be merged with the first fluid by introducing a first fluid into the main flow path and then introducing a second fluid into a first branch flow path branching off from the main flow path. For example, the second and third fluids can be sequentially merged with the first fluid by introducing a first fluid into the main flow path, introducing a second fluid into a first branch flow path branching off from the main flow path, and then introducing a third fluid into a second branch flow path branching off from the main flow path downstream of the junction of the main flow path and the first branch flow path. The number of branch flow paths branching off from the main flow path may be one or more. The number of junctions between the main flow path and the branch flow paths may be one or more. The shape of the junction between the main channel and the branch channel is not limited. The shape of the junction between the main channel and the branch channel may be determined, for example, according to the number of branch channels and the position where the branch channels merge with the main channel. Examples of shapes for the junction between the main channel and the branch channel include T-shapes, Y-shapes, and cross shapes.

[0107] The configuration of the flow channels will be described below with reference to Figures 5 and 6. Figure 7 is a schematic plan view showing the flow channels of a microfluidic device according to one embodiment of this disclosure. Figure 8 is a schematic plan view showing the flow channels of a microfluidic device according to one embodiment of this disclosure. However, the configuration of the flow channels is not limited to the configurations shown below.

[0108] In one embodiment, the flow path preferably includes a first flow path section, a second flow path section that merges with the first flow path section, and a third flow path section connected to the confluence point of the first and second flow path sections. In the above embodiment, the flow path may include, for example, four or more flow path sections and two or more confluence points.

[0109] In an example of a flow path including the components described above, the flow path 31 shown in Figure 7 includes a first flow path section 31a, a second flow path section 31b that merges with the first flow path section 31a, and a third flow path section 31c that connects to the confluence point 31A of the first flow path section 31a and the second flow path section 31b. The shape of the confluence point 31A is T-shaped.

[0110] In the flow path 31 shown in Figure 7, for example, the first fluid introduced into the first flow path section 31a merges with the second fluid introduced into the second flow path section 31b at the confluence point 31A. The first and second fluids that merge at the confluence point 31A then flow through the third flow path section 31c.

[0111] In one embodiment, the flow path preferably includes a first flow path section, a second flow path section that merges with the first flow path section, a third flow path section connected to the confluence point of the first and second flow path sections, a fourth flow path section that merges with the third flow path section, and a fifth flow path section connected to the confluence point of the third and fourth flow path sections. In the above embodiment, the flow path may include, for example, six or more flow path sections and three or more confluence points.

[0112] In an example of a flow path including the components described above, the flow path 32 shown in Figure 8 includes a first flow path section 32a, a second flow path section 32b that merges with the first flow path section 32a, a third flow path section 32c that connects to the confluence point 32A of the first flow path section 32a and the second flow path section 32b, a fourth flow path section 32d that merges with the third flow path section 32c, and a fifth flow path section 32e that connects to the confluence point 32B of the third flow path section 32c and the fourth flow path section 32d. The shape of the confluence point 32A is T-shaped. The shape of the confluence point 32B is T-shaped.

[0113] In the flow path 32 shown in Figure 8, for example, the first fluid introduced into the first flow path section 32a merges with the second fluid introduced into the second flow path section 32b at the confluence point 32A. The first and second fluids that merge at the confluence point 32A pass through the third flow path section 32c and merge with the third fluid introduced into the fifth flow path section 32e at the confluence point 32B. The first, second, and third fluids that merge at the confluence point 32B flow through the fourth flow path section 32d.

[0114] One method for delivering liquid into the flow path of a microfluidic device is to use a liquid delivery pump. The type of liquid delivery pump is not limited. The type of liquid delivery pump may be determined, for example, according to the type of liquid and the amount of liquid to be delivered. Examples of liquid delivery pumps include syringe pumps, plunger pumps, mono pumps, diaphragm pumps, tubing pumps, and pneumatic pumps. From the viewpoint of low pulsation during liquid delivery, syringe pumps, pneumatic pumps, multi-plunger pumps, and mono pumps are preferred.

[0115] One method for combining an oil-containing liquid and a water-containing liquid is to combine them in a flow path that includes a main flow path and at least one branch flow path branching off from the main flow path.

[0116] The formation of droplets will be explained with reference to Figure 9. Figure 9 is a schematic plan view showing the fluid flow in a channel of a microfluidic device according to one embodiment of the present disclosure. As shown in Figure 9, for example, droplet D1 is formed when two liquids merge at a T-shaped confluence point in the channel.

[0117] The following describes in detail each step of the manufacturing method for microfluidic devices.

[0118] [Process (1)] In step (1), a first base having a grooved surface and containing silicone is prepared. Hereinafter, the grooved surface on the first base may be referred to as the "specific surface of the first base."

[0119] Examples of silicones include the silicones described in the "Base (First Base)" section above. The silicone is preferably polydimethylsiloxane.

[0120] The specific surface of the first base includes a region that contacts the second base in step (5) described later and a region that defines the flow path formed in step (5) described later. The specific surface of the first base is subjected to plasma treatment in step (2) described later and then contacts the second base in step (5) described later. The shape of the groove is not limited. The shape of the groove is determined, for example, according to the shape of the desired flow path. Examples of cross-sectional shapes of the groove include a square and a semicircle. The method of forming the groove is not limited. An example of a method of forming the groove is a method using a mold made by photolithography. For example, many of the flow paths of known microfluidic devices are formed using molds made by photolithography. For example, a first base having grooves can be obtained by contacting silicone or a composition containing silicone material with a mold made by photolithography and curing the composition. For example, the shape of the convex portion of the mold corresponds to the shape of the groove (i.e., concave portion) of the first base.

[0121] The shape of the first base is not limited. From the viewpoint of ease of manufacture, the shape of the first base is preferably flat. For example, a flat first base has a first main surface and a second main surface on the opposite side of the first main surface. In a flat first base, the specific surface of the first base may be the first main surface or the second main surface.

[0122] [Process (2)] In step (2), a specific surface of the first base is subjected to plasma treatment in the presence of a gas containing at least one selected from the group consisting of oxygen, argon, and nitrogen. In step (2), if necessary, plasma treatment may also be performed on the portion that comes into contact with the composition containing the surfactant described later. For the plasma treatment, for example, a known plasma treatment apparatus may be used.

[0123] The gas comprises at least one selected from the group consisting of oxygen, argon, and nitrogen. In some embodiments, the gas preferably contains oxygen. In some embodiments, the gas preferably contains oxygen and argon. Plasma treatment in the presence of a gas containing at least oxygen can improve the adsorption of the surfactant to the defining surface of the first base, and can also improve the adhesion between the first base and the second base.

[0124] The oxygen flow rate in plasma processing is preferably 5 sccm to 300 sccm, more preferably 10 sccm to 200 sccm, and particularly preferably 20 sccm to 100 sccm.

[0125] The flow rate of argon in plasma processing is preferably 0 sccm to 300 sccm, more preferably 0 sccm to 250 sccm, and particularly preferably 0 sccm to 200 sccm. When the gas used in plasma processing contains argon, the lower limit of the argon flow rate is preferably 1 sccm, more preferably 5 sccm, and particularly preferably 20 sccm.

[0126] The pressure in plasma processing is preferably 10 Pa to 300 Pa, more preferably 15 Pa to 200 Pa, and particularly preferably 20 Pa to 150 Pa.

[0127] The processing time in plasma treatment is preferably 5 to 120 seconds, more preferably 10 to 100 seconds, and particularly preferably 15 to 80 seconds.

[0128] The output power in plasma processing is preferably 5W to 300W, more preferably 10W to 250W, and particularly preferably 15W to 200W. The output power is RF (Radio Frequency) output.

[0129] [Process (3)] In step (3), a second base is prepared, which has a surface for contacting the first base. Hereinafter, the surface of the second base that contacts the first base may be referred to as the "specific surface of the second base."

[0130] The components of the second base are not limited. Preferred components of the second base are the same as preferred components of the opposing base described in the section "Opposite Base (Second Base)" above.

[0131] A specific surface of the second base is subjected to plasma treatment in step (4) described later, and then comes into contact with the first base in step (5) described later. A groove may be formed on the specific surface of the second base. The shape of the groove is not limited. The shape of the groove is determined, for example, according to the shape of the desired flow path. Examples of groove cross-sectional shapes include a square and a semicircle. As a method for forming the groove, for example, a method using a mold made by photolithography as described in the section "Step (1)" above can be used.

[0132] The shape of the second base is not limited. From the viewpoint of ease of manufacture, the shape of the second base is preferably flat. For example, a flat second base has a first main surface and a second main surface on the opposite side of the first main surface. In a flat second base, the specific surface of the second base may be the first main surface or the second main surface.

[0133] [Process (4)] In step (4), a specific surface of the second base is subjected to plasma treatment in the presence of a gas containing at least one selected from the group consisting of oxygen, argon, and nitrogen. In step (4), if necessary, plasma treatment may also be performed on the portion that comes into contact with the composition containing the surfactant described later. The preferred conditions for plasma treatment are the same as the preferred conditions for plasma treatment described in the section "Step (2)" above. It is preferable to perform step (4) simultaneously with step (2). That is, in steps (2) and (4), it is preferable to perform plasma treatment simultaneously on the specific surface of the first base and the specific surface of the second base in the presence of a gas containing at least one selected from the group consisting of oxygen, argon, and nitrogen. Performing steps (2) and (4) simultaneously shortens the time required from the end of steps (2) and (4) to the start of step (5), and improves the adhesion between the first base and the second base in step (5), which will be described later. Steps (2) and (4) may be performed simultaneously, for example, in a single plasma processing apparatus.

[0134] [Process (5)] In step (5), the specific surface of the second base that has undergone the plasma treatment is brought into contact with the specific surface of the first base that has undergone the plasma treatment, thereby forming a flow path defined by the first base and the second base. After the plasma treatment, the second base is brought into contact with the first base, causing the second base to adhere to the first base. In step (5), the groove of the first base is covered by the second base, thereby forming a space (i.e., a flow path) surrounded by the surface of the first base and the surface of the second base.

[0135] In step (5), after bringing a specific surface of the second base into contact with a specific surface of the first base, a load may be applied to the first base and the second base. Applying a load to the first base and the second base improves the adhesion between the first base and the second base. The load may be, for example, 50 g / cm². 2 ~500g / cm 2It may be determined within the range of [this range]. From the viewpoint of improving the adhesion between the first base and the second base, it is more preferable in step (5) to bring a specific surface of the second base into contact with a specific surface of the first base under the temperature conditions described later, and then apply a load to the first base and the second base.

[0136] In step (5), after bringing a specific surface of the second base into contact with a specific surface of the first base, the first base and the second base may be heated. Heating the first base and the second base improves the adhesion between the first base and the second base. The temperature may be determined, for example, in the range of 100°C to 300°C.

[0137] (Gel particles) The gel particles obtained from the manufacturing method described herein will be explained below.

[0138] The gel particles obtained by the manufacturing method according to this disclosure are solid gel particles having a particle diameter of 50 μm to 200 μm, and the solid gel particles have a double structure having a gel particle having a first surface and a coating gel layer having a second surface, and the first surface is visible under optical microscope observation and contains a polymer gel which is a crosslinked product of a polyvalent cationic crosslinkable polymer, the particle diameter of the gel particle having the first surface is 40 μm to 150 μm, and the average thickness of the coating gel layer is 5 μm to 100 μm.

[0139] The gel particles are solid gel particles having a first surface and a coating gel layer having a second surface, and the interface defining the gel particles having the first surface is visible.

[0140] In some embodiments, gel particles are preferably used as egg cell-mimicking particles. The reasons for this are as follows: As the need for artificial insemination (i.e., intracytoplasmic sperm injection) as a fertility treatment expands, universities and other academic institutions use egg cells from non-human organisms such as mice and cows during training aimed at improving the skill level of embryologists performing assisted reproductive technology. Clinics and other medical institutions often use egg cells that are scheduled to be discarded. However, cells from other organisms are not the same as human egg cells, and the number of human egg cells that are discarded is limited, so they are often not available when needed. In addition, there are constraints regarding human egg cells from a bioethical standpoint and regarding storage period. The gel particles relating to this disclosure can be constructed to closely resemble the form and physical properties of actual cells. Therefore, it can contribute to improving the skill level of embryologists while avoiding the limitations that have been present when using egg cells from other organisms or discarded human egg cells.

[0141] The individual components of gel particles are described below.

[0142] The gel particle is a solid gel particle having a double structure consisting of a gel particle having a first surface and a coating gel layer having a second surface.

[0143] In this disclosure, a solid gel particle means a gel particle having a multilayer structure in which a gel particle further contains another gel particle. Specifically, in the double structure of the gel particle according to this disclosure, both the gel particle having the first surface and the coating gel layer having the second surface contain gel.

[0144] Being a solid gel particle offers many advantages over hollow gel particles when used as an egg cell imitation particle. Actual mammalian egg cells consist of an egg cell and a surrounding glycoprotein matrix membrane (called the zona pellucida). If the gel particles are hollow, it is not possible to reproduce the physical properties of two gel-like bodies with different elastic moduli (the inner egg cell and the outer zona pellucida), as is the case with actual egg cells and zona pellucida. This makes it difficult to properly reproduce the handling of simulated particles (i.e., particle adsorption at the pipette tip, particle rotation, and sperm injection) during embryologist training. Furthermore, during actual sperm injection into egg cells, it is necessary to position the manipulator perpendicular to the zona pellucida, perform a two-stage puncture from the zona pellucida to the egg cell, and reliably inject the sperm into the center of the egg cell. However, if the gel particles are hollow, depending on the thickness of the gel layer, the gel particles may deform significantly, potentially making puncture easier than intended. Considering such a situation, hollow gel particles are not suitable as simulated particles for embryologist training.

[0145] On the other hand, since the gel particles obtained by the manufacturing method according to this disclosure are solid gel particles, when used as egg cell imitation particles, the gel particles having the first surface can be configured to mimic the portion corresponding to an actual egg cell, and the covering gel layer having the second surface can be configured to mimic the transparent body that surrounds an actual egg cell. Furthermore, because the particles are solid gel particles, their hardness can be controlled from hard to soft, making it possible to approximate the deformation of actual egg cells or to construct even more complex simulated particles. Furthermore, in one embodiment, the gel particles may include particles X having a predetermined particle size, and when the gel particles are used as egg cell imitation particles, these particles X will mimic the polar body or spindle. Confirmation and control of the injection position using the polar body as a marker is extremely important during sperm injection into egg cells, and in order to position the particles X that mimic the polar body at the predetermined location, it is advantageous that the particles are solid gel particles with a double structure.

[0146] The particle size of the gel particles is 50 μm to 200 μm from the viewpoint of applicability to cell-mimicking particles. Preferably, the particle size of the gel particles is 55 μm to 195 μm, and more preferably 60 μm to 190 μm.

[0147] The particle size of the gel particles is measured according to the following measurement method and conditions. The gel particles relating to this disclosure are photographed using a transmission optical microscope at an objective magnification of 5x to obtain an image. The diameter (μm) of the gel particles is measured using measurement software (Zeiss, AxioVision) in the obtained image. The measurement is performed on 10 randomly selected gel particles, and the average value of the diameters obtained from these 10 points is calculated and rounded to the first decimal place to determine the particle diameter of the gel particles. For measuring the particle size of gel particles, a transmission optical microscope known as an inverted microscope can be used. In this disclosure, images and photographs are obtained using the following image acquisition environment. Inverted microscope: Axio Obserber Z1 (manufactured by Zeiss) Objective lens: ×5 Camera: AxioCamMR3 Light source: Power Supply 232 (Zeiss, halogen)

[0148] The gel particles obtained by the manufacturing method according to this disclosure are solid gel particles, and these solid gel particles have a double structure comprising a gel particle having a first surface (gel particle X) and a coated gel layer having a second surface (coated gel layer Y), and the first surface is visible under optical microscope observation.

[0149] Details of the gel particles having a first surface (gel particle X) and the coated gel layer having a second surface (coated gel layer Y) will be described later.

[0150] The method for determining whether or not the first surface of a gel particle X is visible under optical microscope observation is as follows: Gel particles are photographed using a transmission optical microscope at a 5x objective magnification to obtain images. It is then determined whether the boundary defining the outer edge of gel particle X is visible in the obtained images. As a transmission optical microscope, a transmission optical microscope known as an inverted microscope can be used. In this disclosure, images are obtained using the following image acquisition environment. Inverted microscope: Axio Obserber Z1 (manufactured by Zeiss) Objective lens: ×5 Camera: AxioCamMR3 Light source: Power Supply 232 (Zeiss, halogen)

[0151] The gel particles contain polymer gels, which are crosslinked products of polyvalent cationic crosslinkable polymers. The details regarding polyvalent cationic crosslinkable polymers and crosslinking have been previously described and will not be explained here.

[0152] <Polyvalent cations> The polyvalent cations used for crosslinking the ionic crosslinkable polymer are not particularly limited. The details regarding polyvalent cations have already been described, and will not be explained here.

[0153] In gel particles, it is preferable that the refractive index of the gel particle having the first surface (gel particle X) and the coating gel layer (coating gel layer Y) are different. This is preferable because the different refractive indices of the gel particle X and the coating gel layer Y result in gel particles with excellent visibility of the interface defining the gel particle X under optical microscope observation.

[0154] Methods for adjusting the refractive index of the gel particles X and the coating gel layer Y to be different include adjusting the gel concentration, mixing different gels or adjusting the mixing ratio, and adjusting the degree of gel crosslinking by adjusting the salt concentration of polyvalent cations.

[0155] <Gel particle having a first surface (gel particle X)> The gel particles relating to this disclosure have a gel particle (gel particle X) having a first surface. In the gel particle X according to this disclosure, the gel particle X is located in the core portion, and a coating gel layer (coating gel layer Y) having a second surface is located outside the gel particle X.

[0156] The gel particles X are particles containing at least a gel, and preferably contain a polymer gel which is a crosslinked product of a polyvalent cationic crosslinkable polymer. In gel particles X, the polyvalent cation-crosslinkable polymer that forms the polymer gel, and the polyvalent cation used for crosslinking the polyvalent cation-crosslinkable polymer are used in aqueous phase A. Since the terms '<Ionic Crosslinkable Polymer>' and '<Polyvalent Cation>' are synonymous, and the preferred embodiments are also the same, a detailed explanation is omitted here.

[0157] In the gel particles X, the polymer gel content is preferably 0.01% to 10% by mass, and more preferably 0.05% to 5% by mass, relative to the total solid content of the components constituting the gel particles X.

[0158] In one embodiment, the gel particles X preferably contain a compound or dye that reacts with sperm proteins to produce a visual change, either directly or indirectly. Compounds or dyes that react with sperm proteins to produce a visual change, either directly or indirectly, are preferably included in the aqueous phase A in step A.

[0159] It is preferable, for example, when the gel particles X are used as egg cell imitation particles, that the gel particles X contain compounds or dyes that react with sperm proteins to produce a visual change directly or indirectly. Training aimed at improving the skill level of embryologists is to acquire the accuracy of injecting sperm into egg cells. It is advantageous for acquiring the accurate technique of sperm injection in training using egg cell imitation particles if the gel particles X contain compounds or dyes that react with sperm proteins to produce a visual change directly or indirectly.

[0160] During the formation of sperm, a type of germ cell, the nuclear proteins are replaced from histones to protamines. Protamines form higher-order structures through disulfide bonds. As a result, unlike somatic cell nuclei, the chromatin structure in sperm nuclei is highly condensed. The compounds or dyes that react with sperm proteins to produce visual changes directly or indirectly are preferably compounds that specifically adsorb to or react with the disulfide bonds abundant in protamine to produce color, discoloration, or luminescence. The gel particle X contains a compound or dye that reacts with sperm proteins to produce a visual change, either directly or indirectly. When embryologists use the gel particle according to this disclosure as an egg cell imitation particle for sperm injection technique training, a change in the color and luminescence of the gel particle occurs only when sperm are successfully injected into the gel particle, allowing for visual determination of successful injection. Examples of compounds or dyes that react with sperm proteins to produce visual changes directly or indirectly include dithiobis(2-nitrobenzoic acid) (DTNB), Ellman's reagent, monobromobiman (mBBr), and 4-dithiopyridine (4-DPS).

[0161] The gel particles X may contain other components besides those described above, to the extent that they do not impair the effects relating to this disclosure. The other components of gel particle X are the same as the other components used in aqueous phase A, and their preferred ranges are also the same; therefore, we will omit their explanation here.

[0162] The particle size of gel particle X is 40 μm to 150 μm, from the viewpoint of approximating the actual cell diameter. Preferably, the particle size of gel particle X is 45 μm to 145 μm, and more preferably 50 μm to 140 μm.

[0163] In the gel particles obtained by the manufacturing method described herein, the particle size of the gel particle X is measured by the following measurement method and measurement conditions. The gel particles relating to this disclosure are photographed using a transmission optical microscope at an objective magnification of 5x to obtain an image. The diameter (μm) of the gel particles X is measured using measurement software (Zeiss, AxioVision) in the obtained image. The measurement is performed on 10 randomly selected gel particles X, and the average value of the diameters obtained from these 10 points is calculated and rounded to the first decimal place to obtain the particle diameter of the gel particles X. In this disclosure, the image acquisition environment used for measuring the particle size of gel particles X is the same as that used for measuring the particle size of gel particles according to this disclosure.

[0164] From the viewpoint of providing gel particles with excellent uniformity, the coefficient of variation (CV value) of the particle size of the gel particles X is preferably 10% or less, more preferably 8.5% or less, and even more preferably 7% or less. There are no specific restrictions on the lower limit of the coefficient of variation, but it may be 0% or greater. The coefficient of variation is calculated using the following formula with respect to the gel particle X. Coefficient of variation (%) of gel particle X = [(Standard deviation of diameters at 10 points) / (Average value of diameters at 10 points)] × 100

[0165] <Coating gel layer having a second surface> The gel particles obtained by the manufacturing method according to this disclosure have a coated gel layer (coated gel layer Y) having a second surface. The covering gel layer Y is a layer located on the outside of the gel particles X. The second surface of the coating gel layer Y refers to the surface of the coating gel layer Y that is furthest from the gel particle X, out of the two opposing surfaces of the coating gel layer Y.

[0166] The coating gel layer Y is a layer containing at least a gel, and preferably contains a polymer gel which is a crosslinked product of a polyvalent cationic crosslinkable polymer. In the coated gel layer Y, the polyvalent cationic crosslinkable polymer that forms the polymer gel, and the polyvalent cation used for crosslinking the polyvalent cationic crosslinkable polymer, are the same as the <ion crosslinkable polymer 2> and <polyvalent cation> used in the aqueous phase C, and the preferred embodiment is also the same, so the explanation is omitted here. Examples similar to the ion-crosslinkable polymers and polyvalent cations described above can be cited.

[0167] In the coating gel layer Y, the polymer gel content is preferably 0.01% to 10% by mass, and more preferably 0.05% to 5% by mass, relative to the total solid content of the components constituting the coating gel layer Y.

[0168] The coating gel layer Y may contain other components besides the polymer gel, as long as they do not impair the effects relating to this disclosure. The other components contained in the coating gel layer Y are the same as the other components used in the aqueous phase C, and the preferred embodiments are also the same; therefore, a detailed explanation is omitted here.

[0169] The average thickness of the coating gel layer Y is 5 μm to 100 μm, from the viewpoint of closely resembling the appearance of actual cells and zona pellucida. The average thickness of the coating gel layer Y is preferably 7 μm to 95 μm, and more preferably 10 μm to 90 μm.

[0170] In the gel particles relating to this disclosure, the average thickness of the coating gel layer Y is measured by the following measurement method and measurement conditions. The gel particles relating to this disclosure are photographed using a transmission optical microscope at an objective magnification of 5x to obtain an image photograph. In the obtained image, three radiation lines are drawn randomly at 120° intervals from the center of the gel particle X. At each of the three radiation lines, the length from the boundary between the gel particle X and the coating gel layer Y to the outermost surface of the coating gel layer (i.e., the second surface) is measured using measurement software (Zeiss, AxioVision). The average value of the three obtained length measurements is calculated. Here, we will supplement the above measurement with reference to the image shown in Figure 2. In Figure 2, the three dashed lines indicate radiation drawn randomly at 120° intervals from the center of the gel particle X. The length indicated by the white double arrows represents the length from the boundary between the gel particle X and the coating gel layer Y to the outermost surface (second surface) of the coating gel layer. The photograph shown in Figure 2 is used solely to explain the measurement of the average thickness of the coating gel layer Y. The same measurements as described above are performed on 10 randomly selected gel particles. The average values ​​obtained from these 10 points are averaged and rounded to the first decimal place to obtain the average thickness of the coating gel layer Y. In this disclosure, the image acquisition environment used to measure the average thickness of the coated gel layer Y is the same as the image acquisition environment used to measure the particle size of the gel particles related to this disclosure.

[0171] From the viewpoint of obtaining gel particles with excellent uniformity, the coating gel layer Y preferably has a coefficient of variation (CV value) of particle size of 10% or less, more preferably 8.5% or less, and even more preferably 7% or less. There are no specific restrictions on the lower limit of the coefficient of variation, but it may be 0% or greater. The coefficient of variation is calculated using the following formula with respect to the gel particle X. Coefficient of variation (%) of coating gel layer Y = [(Standard deviation of coating thickness at 10 points) / (Average value of coating thickness at 10 points)] × 100

[0172] (Applications of gel particles) The gel particles obtained by the manufacturing method described herein are preferably used as egg cell imitation particles, and are particularly preferably used as human egg cell imitation particles.

[0173] (Embryologist training kit and method for manufacturing the same) The embryologist training kit relating to this disclosure includes the gel particles relating to this disclosure. An embryologist is a medical technician whose job is to perform assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) under the guidance of a physician.

[0174] The embryologist training kit described herein, containing the gel particles described herein, can contribute to improving the skill level of embryologists without using egg cells from organisms other than humans, or without using discarded human egg cells.

[0175] An embryologist training kit can be prepared, for example, by adding the gel particles according to this disclosure to physiological saline and sealing it in a storage bag such as an infusion bag. Physiological saline refers to an inorganic salt solution that has been adjusted to be isotonic with the human body, and may also have buffering properties. Examples of physiological saline include saline containing 0.9 w / v% (mass / volume%) sodium chloride, phosphate-buffered saline (PBS), and Tris-buffered saline.

[0176] A method for manufacturing a kit for embryologists' training techniques includes a step of manufacturing gel particles using the manufacturing method described herein, and a step of heating and sterilizing the gel particles at 80°C or higher (heat sterilization step). The details of the conditions (heating method, heating temperature, and heating time) applied to the heat sterilization process are as previously described. [Examples]

[0177] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to the following examples unless it exceeds the spirit of the disclosure. Unless otherwise specified, "parts" and "%" are based on mass.

[0178] [Example 1] (1) Fabrication of microfluidic devices We fabricated a microfluidic device to serve as a gel particle formation site. Fabrication method: Microfluidic devices were fabricated according to the following procedure. The microfluidic device includes a base (first base) and a counter base (second base) that contacts the first base.

[0179] (Step 1: Prepare the photomask) A photomask (System Advance Co., Ltd.) was prepared, containing a blue glass plate and a patterned chromium thin film. One side of the blue glass plate (hereinafter referred to as the "first side") is covered with the patterned chromium thin film. The area of ​​the first side of the blue glass plate covered with the patterned chromium thin film forms the light-shielding portion of the photomask. The area of ​​the first side of the blue glass plate not covered with the patterned chromium thin film forms the light-transmitting portion of the photomask. The photomask includes a T-shaped light-transmitting portion. The line width of the T-shaped light-transmitting portion is 100 μm.

[0180] (Step 2: Mold making) A 4-inch (1 inch = 25.40 mm) silicon wafer (Electronics End Materials Corporation) was cleaned using acetone and ethanol. The cleaned silicon wafer was dried at 100°C for 10 minutes using a hot plate (HP-1SA, AS ONE Corporation). The dried silicon wafer was placed in a spin coater (MS-A150, Mikasa Corporation) by suction. 5 mL of "SU-8 3050" (KAYAKU Advanced Materials) was dropped onto the silicon wafer. "SU-8 3050" is a negative-type photoresist. After removing air bubbles from the "SU-8 3050" dropped onto the silicon wafer, the silicon wafer was rotated at a rotation speed of 1,300 rpm (revolutions per minute) for 30 seconds. A silicon wafer covered with a thin film of "SU-8 3050" was pre-baked at 65°C for 5 minutes and then at 95°C for 40 minutes, after which the silicon wafer was cooled to room temperature. Next, the silicon wafer was placed in the mask aligner by suction. The chromium thin film of the photomask was brought into contact with the thin film of "SU-8 3050" formed on the silicon wafer, and 8.0 mW / cm² of condensation was applied to the thin film of "SU-8 3050". 2The silicon wafer was irradiated with ultraviolet light (wavelength: 365 nm) for 25 seconds. The silicon wafer was baked at 65°C for 1 minute and then at 95°C for 15 minutes, after which it was cooled to room temperature. The silicon wafer and 10 mL of "SU-8 developer" (KAYAKU Advanced Materials) were placed in a 120 mm diameter glass petri dish, and then developed using a shaker (NR-10, Taitec Co., Ltd.) for 10 minutes. The "SU-8 3050" and "SU-8 developer" remaining on the silicon wafer were washed with isopropyl alcohol (Fujifilm Wako Pure Chemical Industries, Ltd.). The silicon wafer was hard baked at 150°C for 20 minutes using a hot plate. A mold was obtained by the above procedure. The mold includes the silicon wafer and a pattern formed on the silicon wafer using photoresist.

[0181] (Step 3: Fabrication of the first base) The mold was cleaned with acetone and ethanol, and then dried on a hot plate at 100°C for 10 minutes. A composition of "Sylgard 184" (DuPont-Toray Specialty Materials Co., Ltd.) main component and hardener mixed in a 10:1 (mass ratio) was placed on the mold, which was placed in a glass petri dish. After degassing, the composition was cured by heating it on a hot plate at 90°C for 1 hour. By peeling the cured material from the mold, a first base containing polydimethylsiloxane (PDMS) was obtained. Grooves were formed on the surface of the first base exposed by peeling. The shape of the grooves formed on the first base corresponds to the shape of the pattern on the mold. The groove depth, measured using a laser optical microscope (VK8550, Keyence Corporation), was in the range of 100 μm to 105 μm. Three openings (i.e., holes) were formed in the first base of the microfluidic device using a 1.5 mm diameter biopsy trephine (Kai Corporation). Each opening is connected to the end of the T-shaped channel in step 5 (bonding), which will be described later.

[0182] (Step 4: Plasma treatment) A plate made of polydimethylsiloxane (PDMS) was prepared as the second base. The first and second bases were placed in the chamber of a tabletop etcher (14-147, U-Tech Co., Ltd.). The pressure inside the chamber was adjusted to 50 Pa, 100 sccm of Ar (argon gas) and 20 sccm of O2 (oxygen gas) were introduced into the chamber, and plasma treatment was performed for 20 seconds at a 20 W RF (Radio Frequency) output. The surface to be treated of the first base was the surface in which the groove was formed. The surface to be treated of the second base was one side of the second base (i.e., the surface of the second base that will come into contact with the first base in step 5 described later).

[0183] (Step 5: Bonding) Immediately after plasma treatment, the plasma-treated surface of the second base was brought into contact with the plasma-treated surface of the first base (i.e., the surface of the first base where the groove was formed), thereby bonding the first and second bases together. Specifically, the bonding of the first and second bases was performed within 30 minutes. In the microfluidic device obtained by bonding the first and second bases, a T-shaped channel is formed between the first and second bases, as shown in Figure 10. The width of the channel is 100 μm. The openings 50, 51, and 52 shown in the microfluidic device 200 in Figure 10 are used as inlet 1, inlet 2, and outlet 1, respectively.

[0184] After plasma treatment (step 4) and bonding (step 5), the microchannels were left to stand at room temperature for at least one day until the microchannel surface returned to hydrophobicity before liquid delivery began.

[0185] (2) Formation of gel particles having a first surface (gel particle X) (steps A and B) Each of the following solutions with the composition shown below was prepared. Solution A, Solution B, and Solution C were each stirred with a stirrer for at least one hour until their components were completely dissolved in water. Both liquids E and F were stirred with a stirrer for at least one hour each until their components were completely dissolved in the corn oil.

[0186] Solution A: Sodium alginate (FFWK Co., Ltd., sodium alginate 80-120) 2% by mass, pure water 98% by mass Solution B: Trisodium citrate (manufactured by FFWK, trisodium citrate) 5.88% by mass, pure water 94.12% by mass Solution C: Calcium chloride (FFWK Co., Ltd., calcium chloride) 1.11% by mass, pure water 98.8% by mass D liquid: A liquid 25% by mass, B liquid 25% by mass, C liquid 50% by mass (aqueous phase A) Liquid E: Polyglyceryl-6 polyricinoleate (manufactured by Sakamoto Pharmaceutical Co., Ltd., SY Glister CRS-75) 1% by mass, corn oil (manufactured by FFWK, corn oil) 99% by mass (oil phase A) Liquid F: Polyglyceryl-6 polyricinoleate (SY Glister CRS-75, manufactured by Sakamoto Pharmaceutical Co., Ltd.) 1% by mass, Acetic acid (Acetic acid, manufactured by FFWK) 2% by mass, Corn oil (Corn oil, manufactured by FFWK) 97% by mass (Hydrogenated oil)

[0187] Solutions E and D were each filled into 5 ml glass syringes (HARVARD) and then placed in a syringe pump (LEGATO200, kdScientific). A glass syringe filled with solution E was connected to the opening 50 of the microfluidic device 200, and a glass syringe filled with solution D was connected to the opening 51 of the microfluidic device 200, both via PFA tubing (outer diameter 1 / 16 inch mm, inner diameter 1 mm, manufactured by AS ONE).

[0188] Next, solution E was introduced at a flow rate of 2 ml / hr from inlet 1 (opening 50) and solution D at a flow rate of 0.1 ml / hr from inlet 2 (opening 52). The formed WO droplets (water droplets in oil) were collected for 1 hour from outlet 1 (opening 56) via a PFA tube (outer diameter 1 / 16 inch mm, inner diameter 1 mm, manufactured by AS ONE) into a glass vial (20 ml) filled with 5 ml of solution F. The collected solution (solution A) was then allowed to stand for 2 hours.

[0189] To 7.1 ml of this recovered liquid, 10 ml of pure water was added and gently mixed. After standing for 30 minutes, 15 ml of the supernatant (oil-water mixture) was removed with a dropper. Furthermore, pure water was added to bring the total volume to 17.1 ml, and after light mixing, it was allowed to stand for 30 minutes. Then, 15 ml of the supernatant (oil-water mixture) was removed with a dropper. This procedure was repeated until the pH of the liquid after removing the supernatant reached 6 or higher. The liquid was then filtered through a SUS mesh filter (NR0597-058, manufactured by Flon Chemical Co., Ltd.) to recover the gel particles (gel particles X) having the first surface.

[0190] (3) Formation of a coating gel layer having a second surface (steps C and D) Each of the following solutions with the composition shown below was prepared. Solution A, Solution B, and Solution C were each stirred with a stirrer for at least one hour until their components were completely dissolved in water. Both liquids E and F were stirred with a stirrer for at least one hour each until their components were completely dissolved in the corn oil.

[0191] Solution A: Sodium alginate (FFWK Co., Ltd., sodium alginate 80-120) 2% by mass, pure water 98% by mass Solution B: Trisodium citrate (manufactured by FFWK, trisodium citrate) 5.88% by mass, pure water 94.12% by mass Solution C: Calcium chloride (FFWK Co., Ltd., calcium chloride) 1.11% by mass, pure water 98.8% by mass Solution D': Solution A 12.5% ​​by mass, Solution B 12.5% ​​by mass, Solution C 25% by mass, recovered gel particles A 2% by mass, pure water 23% by mass (aqueous phase C) Liquid E: Polyglyceryl-6 polyricinoleate (SY Glister CRS-75, manufactured by Sakamoto Pharmaceutical Co., Ltd.) 1% by mass, corn oil (corn oil, manufactured by FFWK) 99% by mass (oil phase C) Liquid F: Polyglyceryl-6 polyricinoleate (SY Glister CRS-75, manufactured by Sakamoto Pharmaceutical Co., Ltd.) 1% by mass, Acetic acid (Acetic acid, manufactured by FFWK) 2% by mass, Corn oil (Corn oil, manufactured by FFWK) 97% by mass (Hydrogenated oil)

[0192] Solutions E and D' were each filled into 5 ml glass syringes (HARVARD) and then placed in a syringe pump (LEGATO200, kdScientific). A glass syringe filled with solution E was connected to the opening 50 of a microfluidic device 200 having the configuration shown in Figure 10 via a PFA tube (outer diameter 1 / 16 inch mm, inner diameter 1 mm, manufactured by AS ONE), and a glass syringe filled with solution D' was connected to the opening 51 of a microfluidic device 1 via a PFA tube (outer diameter 1 / 16 inch mm, inner diameter 1 mm, manufactured by AS ONE).

[0193] Next, solution E was introduced at a flow rate of 2.5 ml / hr from inlet 1 (opening 50) and solution D' at a flow rate of 0.12 ml / hr from inlet 2 (opening 51). The formed WO droplets (water droplets in oil) were collected for 1 hour from outlet 1 (opening 52) via a PFA tube (outer diameter 1 / 16 inch mm, inner diameter 1 mm, manufactured by AS ONE) into a glass vial (20 ml) filled with 5 ml of solution F. The collected solution was then allowed to stand for 2 hours.

[0194] To this recovered liquid (7.62 ml), 10 ml of pure water was added and gently mixed. After standing for 30 minutes, 15 ml of the supernatant (oil-water mixture) was removed using a dropper. Furthermore, pure water was added to bring the total volume to 17 ml, and after lightly mixing, it was allowed to stand for 30 minutes. Then, 15 ml of the supernatant (oil-water mixture) was removed with a dropper. This operation was repeated until the pH of the liquid after removing the supernatant reached 6 or higher, thereby obtaining Gel Particle 1 of Example 1 (a solid gel particle produced by the manufacturing method according to this disclosure) having a double structure consisting of a gel particle having a first surface (gel particle X) and a coated gel layer having a second surface (coated gel layer B).

[0195] (4) Measurement and evaluation <Interface Visibility> The obtained gel particles were observed using an inverted microscope (Axio Obserber Z1 (Zeiss)) and the visibility of the boundary between gel particle X (gel particle with the first surface) and the coating gel layer B was evaluated according to the following three evaluation criteria (evaluation values ​​1-3).

[0196] The inverted microscope used for evaluation is a standard optical microscope commonly used in the work of embryologists. The fact that the boundary between gel particle X and the covering gel layer B is visible means that, under optical microscopy, gel particle X has a primary surface.

[0197] =Evaluation Criteria= Evaluation score 1: The boundary is completely invisible. Evaluation score 2: The boundary is visible, but somewhat unclear. Rating 3: The boundary is clearly visible.

[0198] The states corresponding to evaluation values ​​1 to 3 can be visually observed, for example, as shown in the reference photographs in Figure 1, which are presented as evaluation example 1 and evaluation example 2.

[0199] The interface visibility of gel particle 1 in Example 1 was "evaluation value 3," indicating that it had a good interface visibility.

[0200] <Size of gel particles> For the obtained gel particle 1, the size of gel particle X (average diameter and coefficient of variation) and the size of the coating gel layer B (average coating thickness and coefficient of variation) were measured.

[0201] The results were as follows: Average diameter of gel particle X: 101 μm, coefficient of variation: 4.2% Average coating thickness of coating gel layer B: 23 μm, coefficient of variation: 5.6% Particle size of gel particle 1: 124 μm

[0202] (Example 2) Gel particles 2 of Example 2 were prepared in the same manner as in Example 1, except that the following three points were changed in the preparation of the gel particles of Example 1. (1) The microfluidic device 300 was fabricated in the same manner as the microfluidic device 200 shown in Figure 10, except that an inlet 3 (opening 62) was added to the microfluidic device 300 as shown in Figure 11. The openings 60, 61, 62, and 63 of the microfluidic device 300 in Figure 11 are used as inlet 1, inlet 2, inlet 3, and outlet 1, respectively. (2) In the formation of gel particles having the first surface (gel particle X), a microfluidic device 300 was used instead of the microfluidic device 200. Furthermore, liquid F was flowed through the inlet 3 (opening 62) at a flow rate of 5 ml / hr, collected in an empty glass vial (20 ml) for 1 hour, and then allowed to stand for 2 hours. (3) In forming a coating gel layer having a second surface, a microfluidic device 300 was used instead of the microfluidic device 200. Furthermore, liquid F was flowed through inlet 3 at a flow rate of 5 ml / hr, collected in an empty glass vial (20 ml) for 1 hour, and then allowed to stand for 2 hours.

[0203] <Size of gel particles> For the obtained gel particles 2, the size of gel particle X (average diameter and coefficient of variation) and the size of the coating gel layer B (average coating thickness and coefficient of variation) were measured.

[0204] The results were as follows: Average diameter of gel particle X: 99 μm, coefficient of variation: 4.0% Average coating thickness of coating gel layer B: 25 m, coefficient of variation: 4.8% Particle size of gel particle 2: 125 μm

[0205] The interface visibility of gel particle 2 in Example 2 was "evaluation value 3," indicating that it had a good interface visibility.

[0206] (Example 3) Gel particle 3 of Example 3 was prepared in the same manner as in Example 2, except that the following two points were changed in the preparation of gel particle 2 of Example 2. (1) As shown in Fig. 12, a microchannel device 400 was produced in the same manner as the microchannel device 300 shown in Fig. 11, except that the microchannel device 400 additionally had an inlet 4 (opening 72). The opening 70, opening 71, opening 72, opening 73 and opening 74 of the microchannel device 400 in Fig. 12 are used as inlet 1, inlet 2, inlet 4, inlet 3 and outlet 1, respectively. (2)(3) In forming a coating gel layer having a second surface, solution D'-2 and solution D'-3 shown below were newly prepared, the microchannel device 400 was used instead of the microchannel device 200, solution D'-2 was flowed into inlet 2 (opening 71) at a flow rate of 92.4 μl / hr, and solution D'-3 was flowed into inlet 4 (opening 72) at a flow rate of 27.6 μl / hr, respectively. ·Solution D'-2: 12.5 mass% of solution A, 12.5 mass% of solution B, 25 mass% of solution C, 2 mass% of gel particles recovered in step (2) ·Solution D'-3: 0.1 mass% of silica particles (manufactured by Micromod, sicastar, particle diameter: 10 μm, 50 mg / ml), 22.9 mass% of pure water

[0207] <Size of Gel Particles> For the obtained gel particles 3, the size (average diameter and coefficient of variation) of gel particles X and the size (average coating thickness and coefficient of variation) of coating gel layer B were measured.

[0208] The results were as follows. Average diameter of gel particles X: 103 μm, coefficient of variation: 4.0% Average coating thickness of coating gel layer B: 27 μm, coefficient of variation: 6.3% Particle diameter of gel particles 3: 132 μm

[0209] The interface visibility of the gel particles 3 of Example 3 was "Evaluation value 3", and the gel particles had an interface with good visibility.

[0210] Furthermore, the added silica particles were positioned outside the gel particles (gel particle X) having the first surface, and inside the second surface (outermost surface) of the covering gel layer B having the second surface, resulting in gel particle 3 having an appearance similar to the polar body in the oocyte-zona pellucida.

[0211] (Example 4) Except for the following two changes made in the preparation of gel particle 3 in Example 3, gel particle 4 of Example 4 was prepared in the same manner as in Example 3. (1)(3) In forming a coating gel layer having a second surface, the D'-2 solution and D'-4 solution were newly prepared, and the microfluidic device 400 was used instead of the microfluidic device 200. The D'-2 solution was flowed through inlet 2 (opening 71) at a flow rate of 92.4 μl / hr, and the D'-3 solution was flowed through inlet 4 (opening 72) at a flow rate of 27.6 μl / hr. • D'-2 solution: 12.5% ​​by mass of solution A, 12.5% ​​by mass of solution B, 25% by mass of solution C, and 2% by mass of gel particles recovered in step (2). ·D'-4 liquid: Magnetic particles (manufactured by Spherotech, Carboxyl Ferromagnetic Particles CFM-80-5, particle size: 8.0μm~8.9μm, 0.5% by mass), pure water 22.9% by mass

[0212] (2)(3) In forming the coating gel layer having a second surface, when collecting the recovered liquid from outlet 1, a 1.5 mL Eppendorf tube was placed on a magnetic stand (Tamagawa TAB4899N12) instead of a 20 mL glass vial, and the liquid was collected there for 10 mins, after which it was left to stand for 1 hour. From this 1.27 ml of recovered liquid, the magnetic separation section was avoided and the remaining liquid was removed using a 1 ml Eppendorf pipette. 1 ml of pure water was added to the remaining liquid, and after removing it from the magnetic stand, the Eppendorf tube was gently agitated and the liquid was transferred to a 20 ml vial. 16 ml of pure water was then added, mixed lightly, and allowed to stand for 30 minutes. 15 ml of the supernatant (oil-water mixture) was then removed using a dropper. Furthermore, pure water was added to bring the total volume to 17 ml, and after lightly mixing, it was allowed to stand for 30 minutes. Then, 15 ml of the supernatant (oil-water mixture) was removed with a dropper. This process was repeated until the pH of the liquid after removing the supernatant reached 6 or higher, thereby producing solid gel particles having a double structure consisting of gel particles with a first surface and a coating gel layer with a second surface.

[0213] <Size of gel particles> For the obtained gel particles 3, the size of the gel particles X (average diameter and coefficient of variation) and the size of the coating gel layer B (average coating thickness and coefficient of variation) were measured.

[0214] The results were as follows: Average diameter of gel particle X: 103 μm, coefficient of variation: 4.0% Average coating thickness of coating gel layer B: 24 m, coefficient of variation: 6.0% Particle size of gel particle 3: 127 μm

[0215] The interface visibility of gel particle 4 in Example 4 was "evaluation value 3," indicating that it had a good interface visibility. Furthermore, the added magnetic particles were positioned outside the gel particles (gel particle X) having the first surface, and inside the second surface (outermost surface) of the coating gel layer having the second surface, resulting in gel particle 4 having an appearance similar to the polar body in the oocyte-zona pellucida. Compared to gel particle 3 of Example 3, almost no gel particles that did not contain particle X were observed among the obtained gel particles.

[0216] (Example 5) The gel particles 1 prepared in Example 1 were added to physiological saline (FFWK 0.01 mol / L phosphate-buffered physiological saline) at a concentration of 0.001% by mass, and this was placed in an infusion bag, pouched, and sealed. This bag was immersed in a constant temperature bath heated to 90°C for 5 minutes to simulate heat sterilization. Subsequently, the gel particles were removed from the infusion bag and observed under an inverted microscope. It was confirmed that the gel particles did not rupture or deform, and that gel particle 1 had a double structure consisting of a gel particle X having a first surface and a covering gel layer B having a second surface, with sufficient visibility of the interface. [Explanation of symbols]

[0217] 10: Base (First base) 20: Opposing base (second base) 30, 31, 32: Flow channels 31a, 32a: First flow channel 31b, 32b: Second flow channel section 31c, 32c: Third flow channel section 32d: Fourth flow channel 32e: Fifth flow channel 30A, 30B, 31A, 32A, 32B: Confluence 40, 41, 42, 43: Openings 100, 200, 300, 400: Microfluidic devices D1: Droplet 50, 51, 60, 61, 62, 70, 71, 72, 73: Openings for liquid inflow 52, 63, 74: Openings that serve as collection ports.

Claims

1. Step A involves performing water-in-oil emulsification using an aqueous phase A containing a first polyvalent cationic crosslinkable polymer and an oil phase A to form droplets with an average particle size of 50 μm to 190 μm, and then crosslinking the first polyvalent cationic crosslinkable polymer to obtain a liquid A containing first particles containing a polymer gel. Step B involves replacing the solvent contained in liquid A with another solvent to obtain liquid B containing the first particles, Step C involves mixing the first particles contained in the liquid B with a second polyvalent cation-crosslinkable polymer which is the same as the first polyvalent cation-crosslinkable polymer contained in the aqueous phase A, thereby obtaining an aqueous phase C in which the concentration of the second polyvalent cation-crosslinkable polymer is different from the concentration of the first polyvalent cation-crosslinkable polymer in aqueous phase A. Step D involves performing water-in-oil emulsification using an aqueous phase C and an oil phase C to form droplets with an average particle size of 60 μm to 230 μm, and then crosslinking the second polyvalent cationic crosslinkable polymer to obtain gel particles having a double structure consisting of gel particles having a first surface and a coating gel layer having a second surface, wherein the first surface is visible under optical microscope observation. including, A method for manufacturing gel particles.

2. The method for producing gel particles according to claim 1, wherein the aqueous phase C further comprises particles X having a particle size of 2 μm to 30 μm.

3. The method for producing gel particles according to claim 2, wherein the mixing of the particles X into the aqueous phase C is performed by in-line mixing of a liquid containing the particles X into the aqueous phase C containing the second polyvalent cation crosslinkable polymer and the first particles.

4. The method for producing gel particles according to claim 2 or 3, wherein the particle X is a magnetic particle, and after step D, the method includes selectively recovering the gel particles containing the magnetic particle using a magnet.

5. A step of producing gel particles by the method for producing gel particles described in claim 1, A method for manufacturing a kit for embryologists' skill training, comprising the step of heating and sterilizing the gel particles at 80°C or higher.

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