Gel particles and embryologist training kits

Abstract

To provide solid gel particles having a multilayer structure, wherein the surface of the core region of the multilayer structure is visible under optical microscope observation, and which are useful as cell-like particles, and to provide such gel particles and their applications. [Solution] A solid gel particle having a particle diameter of 50 μm to 200 μm, wherein the solid gel particle has a double structure comprising 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 being 40 μm to 150 μm, and the average thickness of the coating gel having the second surface being 5 μm to 100 μm, and the gel particle and its application.

Description

Technical Field

[0001] The present disclosure relates to gel particles and kits for training embryologists.

Background Art

[0002] Regarding cell mimetics, a technique applying the gelation reaction of raw materials is known. For example, Patent Document 1 discloses that by producing a gel capsule encapsulating cells using two types of gelable substances and culturing the cells in the capsule, a spherical tissue of animal cells (for example, a tissue composed of stem cells or cancer cells) is formed.

Prior Art Literature

Patent Literature

[0003]

Patent Document 1

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 the morphology and physical properties of actual cells for gel particles simulating cells (cell simulated particles), they are required to be solid gel particles having a solid and multilayer structure (more specifically, solid gel particles having a double structure including a gel particle having a first surface and a coating gel layer having a second surface). In addition, depending on the usage 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 is visible under observation with an optical microscope.

[0005] This disclosure is made in view of the above circumstances, and one embodiment of this disclosure aims to provide a solid gel particle having a gel particle having a first surface and a coating gel layer having a second surface, wherein the interface defining the gel particle having the first surface is visible. Another embodiment of this disclosure aims to provide an embryologist training kit comprising the gel particles described above. [Means for solving the problem]

[0006] This disclosure includes the following aspects:

[0007] <1> These are solid gel particles with a particle size of 50 μm to 200 μm. The solid gel particle has a double structure comprising 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. It contains a polymer gel which is a crosslinked product of a polyvalent cationic crosslinkable polymer. The particle size of the gel particles having the first surface is 40 μm to 150 μm. The average thickness of the coated gel layer is 5 μm to 100 μm. Gel particles. <2> The gel particles having the first surface and the coating gel layer have different refractive indices. <1> Gel particles as described above. <3> Furthermore, the first surface is located outside the second surface, and the second surface is located inside the first surface, containing particles X with a particle diameter of 2 μm to 30 μm. <1> or <2> Gel particles as described above. <4> The gel particles having the first surface contain compounds or dyes that react with sperm proteins to produce a visual change, either directly or indirectly. <1> ~ <3> Gel particles described in any one of the following: <5> These are egg cell-like particles, <1> ~ <4> Gel particles as described in one of the following: <5> <5> A training kit for embryologists containing the gel particles described above. [Effects of the Invention]

[0008] According to one embodiment of the present disclosure, it is possible to provide a gel particle having a first surface and a solid gel particle having a coating gel layer having a second surface and the gel particle having a first surface, wherein the interface defining the gel particle having the first surface is visible. According to another embodiment of the present disclosure, a kit for embryologist skill training, comprising the gel particles described above, 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] Figure 9 is a schematic plan view showing the fluid flow patterns in the flow channels of a microfluidic device according to one embodiment of the present disclosure. [Figure 10] Figure 10 is a schematic plan view showing the microfluidic device used in the embodiment. [Modes for carrying out the invention]

[0010] Hereinafter, the content according to the present disclosure will be described in detail. Description of constituent elements described below may be made based on exemplary embodiments according to the present disclosure, but the present disclosure is not limited to such embodiments. In the present disclosure, a numerical range indicated by 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 in a certain numerical range may be replaced with the upper limit or lower limit of another stepwise described numerical range. Further, in the numerical ranges described in the present disclosure, the upper limit or lower limit described in a certain numerical range may be replaced with the values 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 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 where the intended purpose of the step is achieved even if it cannot be clearly distinguished from other steps. The total solid content in the present disclosure refers to the total amount of components excluding volatile components such as solvents. In the present disclosure, ordinal numbers (for example, "first" and "second") are terms used to distinguish a plurality of constituent elements, and do not limit the number of constituent elements or the superiority or inferiority of constituent elements.

[0012] (Gel Particles) The gel particles according to the present disclosure are solid gel particles having a particle diameter of 50 µm to 200 µm. The solid gel particles have a double structure including gel particles having a first surface and a coated gel layer having a second surface, the first surface is visible under observation with an optical microscope, and the solid gel particles comprise a polymer gel that is a crosslinked product of a polyvalent cation crosslinkable polymer. The particle diameter of the gel particles having the first surface is 40 µm to 150 µm, and the average thickness of the coated gel layer is 5 µm to 100 µm.

[0013] The gel particles according to this disclosure are 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.

[0014] In one embodiment, the gel particles relating to this disclosure 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 it is often difficult to obtain egg cells 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.

[0015] The constituent elements of the gel particles relating to this disclosure are described below.

[0016] The gel particles relating to this disclosure are solid gel particles having a double structure comprising a gel particle having a first surface and a coating gel layer having a second surface.

[0017] 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.

[0018] Being a solid gel particle offers many advantages compared to hollow gel particles when the gel particles according to this disclosure are used as egg cell-mimicking particles. 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 (i.e., 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.

[0019] On the other hand, since the gel particles relating 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 according to this disclosure may include particles X having a predetermined particle size. 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. In order to position the particles X that mimic the polar body at a predetermined location, it is advantageous that the particles X have a double-layered solid gel particle structure.

[0020] The particle size of the gel particles relating to this disclosure is 50 μm to 200 μm from the viewpoint of applicability to cell-mimicking particles. The particle size of the gel particles is preferably 55 μm to 195 μm, and more preferably 60 μm to 190 μm.

[0021] The particle size of the gel particles relating to this disclosure shall be 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 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)

[0022] The gel particles relating to this disclosure are solid gel particles, and these solid gel particles have a double structure comprising a gel particle having a first surface (hereinafter also referred to as "gel particle X") and a coating gel layer having a second surface (hereinafter also referred to as "coating gel layer Y"), and the first surface is visible under optical microscope observation.

[0023] 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.

[0024] The method for determining whether or not the first surface of the gel particle X is visible under optical microscope observation in the gel particle relating to this disclosure is as follows. The gel particles relating to this disclosure are photographed using a transmission optical microscope at an objective magnification of 5x to obtain an image. It is determined whether the boundary defining the outer edge of the gel particle X is visible in the obtained image. 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)

[0025] The gel particles relating to this disclosure include polymer gels which are crosslinked products of polyvalent cationic crosslinkable polymers.

[0026] <Polyvalent cationic crosslinkable polymer> The polymer gel contained in the gel particles relating to this disclosure is a crosslinked product of a polyvalent cationic crosslinkable polymer. As the polyvalent cationic crosslinkable polymer that forms the crosslinked product of the polyvalent cationic crosslinkable polymer (hereinafter also simply referred to as "ionic crosslinkable polymer"), known ionic crosslinkable polymers 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.

[0027] Among these, the ion-crosslinkable polymer 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.

[0028] 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.

[0029] From the viewpoint of productivity, the viscosity range of the ionically crosslinkable polymer 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.

[0030] <Polyvalent cations> The polyvalent cations used for crosslinking the ionic crosslinkable polymer are not particularly limited. The polyvalent cation is preferably a divalent or trivalent cation, and more preferably a divalent metal cation. 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 polymers, among these, calcium ions are preferred as the divalent metal cations.

[0031] 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.

[0032] In the gel particles according to this disclosure, it is preferable that the refractive indices 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.

[0033] 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.

[0034] The refractive indices of gel particles X and the coating gel layer Y can be measured, for example, by repeatedly collecting gel particles X, sputtering them from the surface with an ion beam, and performing mass spectrometry on the secondary ions to analyze the composition in the depth direction, and then converting the refractive indices of gel particles X and the coating gel layer Y from the difference in composition before and after the thickness of the coating gel layer Y.

[0035] The absolute value of the refractive index difference between the gel particles X and the coating gel layer Y is preferably 0.001 or greater, and more preferably 0.005 or greater. The refractive indices of the gel particles X and the coating gel layer Y may be such that the refractive index of the gel particles X is higher than that of the coating gel layer Y, or the refractive index of the coating gel layer Y is higher than that of the gel particles.

[0036] <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.

[0037] 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. The polyvalent cationic crosslinkable polymers that form polymer gels, and the polyvalent cations used for crosslinking the polyvalent cationic crosslinkable polymers, are the same as those described in the sections on <Polyvalent Cationic Crosslinkable Polymers> and <Polyvalent Cations>.

[0038] The ion-crosslinkable polymer that forms the polymer gel contained in the gel particles X 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. 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, due to their easy availability and ease of controlling the degree of crosslinking.

[0039] In gel particles X, the polyvalent cations used for crosslinking the ionically crosslinkable polymer are not particularly limited. The polyvalent cations used for crosslinking the ionically crosslinkable polymer 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 polymers, among these, calcium ions are preferred as the divalent metal cations.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 firmly 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).

[0044] 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. Other components contained in gel particles X include, for example, chelating agents, poorly water-soluble metal salts, and so on. As a chelating agent, it can function as a gel 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. Citrates are preferred because they are readily available and inexpensive. Examples of citrates include sodium citrate (e.g., trisodium citrate). Poorly water-soluble metal salts can also function as gelation reaction retarders. Examples of poorly water-soluble metal salts include edible compounds that contain metal salts with low water solubility. Examples of poorly water-soluble metal salts include calcium sulfate, calcium gluconate, calcium benzoate, calcium oxalate, and tricalcium phosphate. From the viewpoint of water solubility, which allows for an appropriate gel reaction delay effect, calcium sulfate is preferred. Other components may be used individually or in combination of two or more.

[0045] 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.

[0046] In the gel particles relating to this disclosure, 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.

[0047] 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

[0048] <Coating gel layer having a second surface> The gel particles relating 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.

[0049] 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. The polyvalent cationic crosslinkable polymers that form polymer gels, and the polyvalent cations used for crosslinking the polyvalent cationic crosslinkable polymers, are the same as those described in the sections on <Polyvalent Cationic Crosslinkable Polymers> and <Polyvalent Cations>.

[0050] The ion-crosslinkable polymer that forms the polymer gel contained in the gel particles X is more preferably an alginate. 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, due to their easy availability and ease of controlling the degree of crosslinking. The polyvalent cations used for crosslinking the ionic crosslinkable polymer are not particularly limited.

[0051] In the coated gel layer Y, the polyvalent cations used for crosslinking the ionically crosslinkable polymer are not particularly limited. The polyvalent cations used for crosslinking the ionically crosslinkable polymer 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 polymers, among these, calcium ions are preferred as the divalent metal cations.

[0052] 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.

[0053] 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. Other components of the coating gel layer Y include the same components as those of the gel particles X, and the preferred embodiments are also the same.

[0054] 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.

[0055] 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.

[0056] 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

[0057] <Particle X> In one embodiment, the gel particles according to this disclosure preferably include particles X having a particle diameter of 2 μm to 30 μm, located outside the first surface of the gel particle X and inside the second surface of the coating gel layer Y.

[0058] The inclusion of particle X in the gel particles relating to this disclosure is preferable, for example, because when the gel particles relating to this disclosure are used as egg cell imitation particles, they can be used as imitation bodies of the polar bodies of egg cells. Training aimed at improving the skill level of embryologists includes mastering the technique of injecting sperm 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 relating to this disclosure can serve as a marker for injecting sperm while avoiding the polar bodies in training using egg cell imitation particles.

[0059] 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.

[0060] Particle X is not particularly limited as long as it is visible within the gel particles, and may be an organic particle, an inorganic particle, or an organic-inorganic composite particle. 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.

[0061] The number of particles X contained in the gel particles according to this disclosure 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.

[0062] Particle X can be introduced when forming the coating gel layer in the manufacturing of the gel particles according to this disclosure.

[0063] (Applications of gel particles) The gel particles relating to this disclosure are preferably used as egg cell imitation particles, and are particularly preferably used as human egg cell imitation particles.

[0064] (Embryologist Training Kit) 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.

[0065] 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.

[0066] 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.

[0067] It is preferable that the gel particles sealed in the containment bag be heat-sterilized. Details of the heat sterilization process can be described later as the heat sterilization process.

[0068] (Gel particle manufacturing method) The method for producing the gel particles relating to this disclosure is not limited, but is preferably the method described below (hereinafter also referred to as Method A).

[0069] Manufacturing method A 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, 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 diameter of 60 μm to 230 μm, and then crosslinking a 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. Includes.

[0070] The following describes the details of each step included in manufacturing method A.

[0071] <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 50 μm to 190 μm, and then the first polyvalent cationic crosslinkable polymer is crosslinked to obtain liquid A containing first particles containing polymer gel.

[0072] <<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.

[0073] [Preparation of ion-crosslinkable polymer A] The ion-crosslinkable polymer 1 contained in aqueous phase A is synonymous with the ion-crosslinkable polymer previously described as the ion-crosslinkable polymer that forms the polymer gel contained in gel particle X in the description of gel particles in this disclosure, and the preferred range is also the same. 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.

[0074] [Preparation of polyvalent cations] The polyvalent cations contained in aqueous phase A are the same as the polyvalent cations previously described in the description of gel particles X in this disclosure as polyvalent cations used for crosslinking the ionically crosslinkable polymer, and the preferred range is also the same. 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.

[0075] [Preparation of chelating agents] The chelating agent contained in aqueous phase A is the same as the chelating agent described above as an example of other components in gel particle X in the description of gel particles relating to this disclosure, and the preferred range is also the same. 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.

[0076] Aqueous phase A is preferably prepared as a mixture by mixing an ion-crosslinkable polymer mixture, 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.

[0077] 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.

[0078] - 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.).

[0079] <<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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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 50 μm to 190 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 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.

[0084] 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.

[0085] 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 or less.

[0086] The average particle size of the droplets can be confirmed by the same method as described above as a method for measuring the particle size of gel particles according to this disclosure. 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 B, the channel size of the microchannel used, and the pore size of the membrane emulsion.

[0087] Next, in step A, the polyvalent cationic crosslinkable polymer 1 is crosslinked to obtain liquid A containing first particles containing polymer gel. The first particle containing the polymer gel corresponds to the gel particle (gel particle X) having a first surface in the gel particle according to this disclosure.

[0088] The crosslinking of the ionic crosslinkable polymer 1 is preferably carried out by contact between droplets formed by water-in-oil emulsification (WO droplets) and an oil containing a pH lowering agent.

[0089] 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 1 then crosslinks via these released polyvalent metal ions, forming a first particle containing a polymer gel.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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. Details of the microfluidic device will be described later.

[0094] 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.

[0095] <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.

[0096] 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.

[0097] <Process C> Step C is a process in which the first particles contained in liquid B and the second polyvalent cationic crosslinkable polymer (hereinafter also referred to as ion crosslinkable polymer 2), which is the same as the first ion crosslinkable polymer (ion crosslinkable polymer 1) contained in aqueous phase A, are mixed to obtain aqueous phase C in which the concentration of the polyvalent cationic crosslinkable polymer is different from that of aqueous phase A.

[0098] Aqueous phase C is a dispersion containing the first particle.

[0099] The mixing of the first particles contained in liquid B and the ion-crosslinkable polymer 2 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.

[0100] 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.

[0101] 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.

[0102] 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 particle 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 (coating gel layer Y) having a second surface.

[0103] 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 ion-crosslinkable polymer 1 in 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.

[0104] -Particle X- In one embodiment, the aqueous phase C preferably further contains particles X having a particle size of 2 μm to 30 μm. The details of particle X are the same as those of particle X described in the description of gel particles in this disclosure, and the preferred range is also the same.

[0105] 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 polyvalent cationic crosslinkable polymer (ionic crosslinkable polymer 2). In one embodiment, it is preferable to mix the particles X into the aqueous phase C by in-line mixing of the liquid containing particles X into the aqueous phase C, which contains the ion-crosslinkable polymer 2 and the first particles.

[0106] -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.).

[0107] <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.

[0108] The aqueous phase C used in process D is the aqueous phase C obtained in process C.

[0109] 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.

[0110] 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 60 μm to 230 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, it is preferable to use a membrane emulsification device or to mix using a microfluidic device, as this makes it easier to achieve uniform droplet size. Details of the microfluidic device will be described later.

[0111] 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.

[0112] 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. Preferably, the average particle size of the droplets is 65 μm to 220 μm, and more preferably 70 μm to 220 μm or less.

[0113] The average particle size of the droplets can be confirmed by the same method as described above for measuring the particle size of gel particles according to this disclosure. 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.

[0114] Next, in step D, the ion-crosslinkable polymer 2 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.

[0115] The crosslinking of the ionic crosslinkable polymer 2 is preferably carried out by contact between droplets (WO droplets) formed by water-in-oil emulsification and an oil or fat (hydrogenated oil) containing a pH lowering agent. 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.

[0116] The gel particles obtained by process D are the gel particles according to this disclosure. Details of the gel particles relating to this disclosure are as previously described and will not be explained here.

[0117] <Separation process> Manufacturing method A 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.

[0118] 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.

[0119] 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.

[0120] <Other processes> Manufacturing method A may include processes other than process A, process B, process C, process D, and the separation process.

[0121] 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 prepare 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.

[0122] In manufacturing method A, 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.

[0123] 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.

[0124] <<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.

[0125] <<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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] The shape of the base is not limited. From the viewpoint of ease of manufacturing, the shape of the base is preferably flat.

[0130] 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.

[0131] <<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".

[0132] The composition of the opposing base is not limited. Examples of opposing base components include silicone, glass, and stainless steel.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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).

[0148] <<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.

[0149] 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).

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The following describes in detail each step of the manufacturing method for microfluidic devices.

[0163] [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."

[0164] Examples of silicones include the silicones described in the "Base (First Base)" section above. The silicone is preferably polydimethylsiloxane.

[0165] 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.

[0166] 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.

[0167] [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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] [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."

[0175] 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.

[0176] 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.

[0177] 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.

[0178] [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.

[0179] [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.

[0180] 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.

[0181] 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. [Examples]

[0182] 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.

[0183] [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.

[0184] (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.

[0185] (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.

[0186] (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.

[0187] (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).

[0188] (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 of the microfluidic device 200 in Figure 10 are used as inlet 1, inlet 2, and outlet 1, respectively.

[0189] 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.

[0190] (2) Formation of gel particles having a first surface (gel particle X) (steps A and B) Each liquid having the following composition was prepared. Solutions A, B, and C were each stirred with a stirrer for at least one hour until their components were completely dissolved in water. Solution E and Solution F were each stirred with a stirrer for at least one hour 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 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)

[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 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).

[0193] 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.

[0194] 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. Then, the liquid (liquid B) containing the gel particles (gel particle X) was filtered through a SUS mesh filter (NR0597-058, manufactured by Flon Chemical Co., Ltd.) to recover the gel particles (gel particle X) having the first surface.

[0195] (3) Formation of a coating gel layer having a second surface (steps C and D) Each liquid having the following composition was prepared. Solutions A, B, and 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.

[0196] 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)

[0197] 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).

[0198] 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 through 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.

[0199] 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 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 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 which is the gel particle according to this disclosure), which has 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).

[0200] (4) Measurement and evaluation <Interface Visibility> The gel particle 1 obtained in Example 1 was subjected to transmission observation using an inverted microscope (Axio Obserber Z1 (Zeiss)) and the visibility of the boundary between the gel particle X (the gel particle having the first surface) and the coating gel layer B was evaluated according to the following three evaluation criteria (evaluation values ​​1 to 3).

[0201] 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.

[0202] =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.

[0203] 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.

[0204] The interface visibility of gel particle 1 in Example 1 was "evaluation value 3," indicating that it had a good interface visibility.

[0205] <Size of gel particles> For the gel particles 1 obtained in Example 1, the size of gel particles X (average diameter and coefficient of variation), the size of the coating gel layer B (average coating thickness and coefficient of variation), and the particle diameter of gel particles 1 were measured.

[0206] 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

[0207] (Examples 2-17, Comparative Examples 1-4) In the preparation of the gel particles of Example 1, gel particles 2-17 of Examples 2-18 and gel particles C1-C4 of Comparative Examples 1-3 were prepared in the same manner as in Example 1, except that some of the synthesis conditions were changed as shown in Table 1 below. The obtained gel particles were measured and evaluated in the same manner as in Example 1. The results are shown in Table 1.

[0208] [Table 1]

[0209] (Example 18) In the preparation of gel particles 1 of Example 1, the solution D' used in step (3) of forming a coated gel layer having a second surface was changed as follows, and gel particles 18 were prepared in the same manner as in Example 1, except that the inside of the syringe pump was stirred with a small stirrer during liquid feeding by the syringe pump. Solution D': 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), 0.1 mass% of magnetic particles (manufactured by Spherotech, Carboxyl Ferromagnetic Particles CFM-80-5, particle size: 8.0 to 8.9 µm, 0.5 mass%), 22.9 mass% of pure water,

[0210] The obtained gel particles 18 were measured and evaluated in the same manner as in Example 1. As a result, regarding the size of the gel particles 18 obtained in Example 18, the average diameter of the gel particles having a first surface (gel particles X) was 103 µm, and the coefficient of variation was 4.0%; the average coating thickness of the coated gel layer having a second surface (coated gel layer B) was 25 µm, and the coefficient of variation was 5.2%. The particle diameter of the gel particles 18 was 130 µm. The evaluation value of interface visibility was 3, and the gel particles had an interface with good visibility. Furthermore, the added magnetic particles were arranged outside the gel particles X and inside the surface of the coated gel layer B, and the gel particles 18 had an appearance similar to the polar body in an ovocyte-zona pellucida.

[0211] (Example 19) In the preparation of gel particles 1 in Example 1, the D' solution used in step (3) forming a coated gel layer having a second surface was changed as described below, and the contents of the syringe pump were stirred with a small agitator while the liquid was being delivered by the syringe pump. Gel particles 19 were prepared in the same manner as in Example 1. The obtained gel particles 19 were measured and evaluated in the same manner as in Example 1. Solution D': Solution A 2.5% by mass, Solution B 12.5% ​​by mass, Solution C 25% by mass, Gel particles recovered in step (2) 2% by mass, Silica particles (Micromod, sicastar, particle size: 10 μm, 50 mg / ml) 0.1% by mass, Pure water 22.9% by mass

[0212] The obtained gel particles 19 were measured and evaluated in the same manner as in Example 1. As a result, the size of the gel particles 19 obtained in Example 19 was as follows: the average diameter of the gel particles with the first surface (gel particle X) was 101 μm, with a coefficient of variation of 4.3%; the average coating thickness of the coated gel layer with the second surface (coated gel layer B) was 24 μm, with a coefficient of variation of 5.6%; and the particle diameter of the gel particles 19 was 128 μm. Furthermore, the interface visibility rating was 3, indicating that it had a highly visible interface. Furthermore, the added magnetic particles were positioned on the outside of the gel particles X and inside the second surface (outermost surface) of the coating gel layer B, resulting in the gel particles 19 having an appearance similar to the polar bodies in the egg cell-zona pellucida.

[0213] (Example 20) 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 infusion 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 gel particle 1 showed no rupture or deformation, and had a double structure consisting of a gel particle (gel particle X) with a first surface and a covering gel layer with a second surface, with sufficient visibility of the interface. [Explanation of symbols]

[0214] 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: Microfluidic devices D1: Droplet 50, 51: Openings for liquid to flow in 52: Opening which serves as the collection port

Claims

1. These are solid gel particles with a particle size of 50 μm to 200 μm. The solid gel particle has a double structure comprising 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. It contains a polymer gel which is a crosslinked product of a polyvalent cationic crosslinkable polymer. The particle size of the gel particles having the first surface is 40 μm to 150 μm. The average thickness of the coating gel layer is 5 μm to 100 μm. Gel particles.

2. The gel particle according to claim 1, wherein the gel particle having the first surface and the coated gel layer have different refractive indices.

3. Furthermore, the gel particle according to claim 1, wherein the particle X having a particle diameter of 2 μm to 30 μm is located outside the first surface and inside the second surface.

4. The gel particle according to claim 1, wherein the gel particle having the first surface contains a compound or dye that reacts with sperm proteins to produce a visual change directly or indirectly.

5. A gel particle according to any one of claims 1 to 4, which is an egg cell-like particle.

6. A kit for embryologist skill training, comprising the gel particles described in claim 5.

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