Mammalian frozen egg culture device and mammalian frozen egg culture method

Abstract

To provide a frozen egg cultivation apparatus that enables even an unskilled person to thaw, wash, and culture a frozen egg, and a method for cultivating frozen eggs.SOLUTION: The present invention relates to: a frozen egg cultivation apparatus comprising an accommodation part for accommodating contained frozen eggs, a liquid injection unit, a liquid discharge unit, the contained frozen eggs, and an egg outflow prevention unit positioned between the liquid injection unit and the liquid discharge unit; and a method for cultivating frozen eggs in which the frozen egg cultivation apparatus is used.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a frozen egg culturing device and a frozen egg culturing method for culturing frozen eggs (frozen fertilized eggs or frozen unfertilized eggs). [Background technology]

[0002] Conventionally, the thawing, washing, and culturing of frozen mammalian eggs such as mice are performed in an open system as shown in Figure 1. Specifically, frozen eggs placed in a container such as a cryotube are stored in liquid nitrogen. Stored frozen eggs are thawed and washed by adding a first melting solution to the container such as a cryotube, then pipetting the entire amount into a culture dish. Subsequently, the eggs (fertilized or unfertilized) are washed by transferring them sequentially with a capillary into three second melting solutions separately placed on the culture dish. The washed eggs are then transferred to a culture solution and cultured. This process, which involves handling small eggs measuring approximately 100 μm, can only be performed by a skilled technician and is not something that anyone can do.

[0003] The techniques for thawing, washing, and culturing frozen eggs are used in a variety of fields, including infertility clinics, laboratory animal facilities, and livestock farming. Therefore, if a technique could be developed that would allow even non-expert technicians to easily and reliably thaw, wash, and culture frozen eggs, it could greatly contribute to the development of these fields.

[0004] A method for cryopreserving germ cells has been proposed in which germ cells are encapsulated in hollow fibers and frozen (see, for example, Patent Document 1). According to this proposal, germ cells can be cryopreserved easily. However, the proposed device is designed to cryopreserve germ cells, but does not thaw, wash, or culture germ cells in the same device.

[0005] Furthermore, as a technology that does not impede heat conduction when thawing frozen cells and that allows for easy recovery of valuable biological cells that have leaked from a freezing container, a package that covers a freezing container containing frozen cells and that is provided with an exhaust port for discharging air from inside the package has been proposed (see, for example, Patent Document 2). However, the proposed package is a bag for thawing frozen cells, and is not intended for washing and culturing thawed cells in the same container.

[0006] Furthermore, a kit for preparing antigen-specific cytotoxic T cells has been proposed, which includes a sealed culture vessel with multiple ports as a component (see, for example, Patent Document 3). According to this proposal, it is possible to prepare antigen-specific cytotoxic T cells with a simple procedure. However, this proposal is for preparing antigen-specific cytotoxic T cells, and does not relate to techniques for thawing, washing, and culturing frozen eggs.

[0007] Therefore, at present, there is no technology available that allows even non-expert technicians to thaw, wash, and culture frozen eggs.

[0008] Furthermore, elucidating the phenomena of life in space and the possibility of reproduction have been topics of great interest since the early days of rocket development, and in recent years attention has been focused on the early life stages of mammals in the space environment. However, due to the weightlessness of space, it is thought that embryo manipulation like that performed on Earth would be impossible. In addition, there are conditional constraints such as the inability to use liquid nitrogen, and technical constraints such as the difficulty for astronauts to perform tasks such as thawing, washing, and culturing frozen eggs, which require highly advanced techniques. As a result, experiments to culture fertilized eggs in space have not yet been realized.

[0009] Therefore, from the perspective of enabling space experiments, there is a strong demand for the development of technology that allows even non-expert technicians to thaw, wash, and culture frozen eggs. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-148218 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-070402 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-130999 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to meet these demands, break through the current situation, solve the above-mentioned problems, and achieve the following objectives: The present invention aims to provide a frozen egg culture device and a frozen egg culture method that allow even non-expert technicians to thaw, wash, and culture frozen eggs. [Means for solving the problem]

[0012] As a result of extensive research into achieving the above-mentioned object, the inventors have found that by providing a frozen egg culture device comprising a storage section for storing containerized frozen eggs, a liquid injection section, a liquid discharge section, the containerized frozen eggs, and an egg outflow prevention section located between the liquid injection section and the liquid discharge section, it is possible to thaw, wash, and culture frozen eggs even in a sealed container (a closed system (a system isolated from the outside air)) without touching the frozen eggs, and therefore, even a beginner who has never handled fertilized or unfertilized eggs can thaw, wash, and culture frozen eggs without any training.

[0013] The present invention is based on the above findings of the present inventors, and the means for solving the above problems are as follows: <1> a storage unit for storing the containerized frozen eggs; A liquid injection unit; A liquid discharge portion; The frozen egg culture device is characterized by comprising the container-containing frozen eggs and an egg outflow prevention unit located between the liquid injection unit and the liquid discharge unit. <2> The aforementioned <1> The present invention relates to a method for culturing frozen eggs, characterized by using the frozen egg culturing device described above. [Effects of the Invention]

[0014] According to the present invention, it is possible to solve the above-mentioned problems in the prior art, achieve the above-mentioned object, and provide a frozen egg culture device and a frozen egg culture method that allow even non-expert technicians to thaw, wash, and culture frozen eggs. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram illustrating a conventional method for thawing and washing frozen eggs. [Figure 2A] FIG. 2A is a schematic diagram of one side of an example of a frozen egg culture device. [Figure 2B] FIG. 2B is a schematic diagram of the other side of the frozen egg culture device of FIG. 2A. [Figure 2C] FIG. 2C is a photograph of the frozen egg culture device of FIG. 2A. [Figure 3] FIG. 3 is a photograph of an example of a frozen egg storage container. [Figure 4] FIG. 4 is a diagram showing an example of a state in which a syringe and the like are connected to the frozen egg culturing device. [Figure 5] FIG. 5 shows an example of the results of culturing frozen fertilized eggs using the frozen egg culturing device of the present invention. [Figure 6] FIG. 6 is a diagram showing an example of a frozen egg storage container. [Figure 7A] FIG. 7A is a diagram showing an example of the results of culturing frozen fertilized eggs using the frozen egg culturing device of Example 2-1 that does not have a means for preventing adhesion (many of the embryos that developed were deformed). [Figure 7B]FIG. 7B is a diagram showing an example of the results of culturing frozen fertilized eggs using the frozen egg culturing device having the adhesion prevention means of Example 2-2 (many of the developed embryos maintained their round shape). [Figure 8A] FIG. 8A is a schematic diagram of a cross section of the storage section when a culture solution or the like is discharged in a frozen egg culture device that does not have a means for preventing adhesion. [Figure 8B] FIG. 8B is a schematic diagram of a cross section of the storage section that is assumed to occur when the culture medium or the like is discharged when using a frozen egg culture device having a means for preventing adhesion. DETAILED DESCRIPTION OF THE INVENTION

[0016] (Frozen egg culture device) The frozen egg culture device of the present invention has at least a storage section for storing frozen eggs in containers, a liquid injection section, a liquid discharge section, and an egg (fertilized eggs and unfertilized eggs) outflow prevention section, and may further have other components as necessary.

[0017] <Frozen eggs> In the present invention, "frozen eggs" refers to frozen fertilized eggs or frozen unfertilized eggs. Fertilized eggs include not only those immediately after fertilization but also early embryos. "Culturing frozen eggs" refers to thawing (melting) frozen eggs and culturing them. The species of the frozen eggs is not particularly limited and can be appropriately selected depending on the purpose, but is preferably a mammal. The mammal is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include humans, mice, rats, rabbits, dogs, cats, horses, cows, pigs, goats, sheep, and marine animals. The method for preparing the frozen eggs is not particularly limited, and any known method can be appropriately selected.

[0018] <Storage section for storing containerized frozen eggs> The storage section is a section for storing containerized frozen eggs. The shape, structure and size of the storage section are not particularly limited and can be appropriately selected depending on the purpose.

[0019] -Containerized frozen eggs- The frozen eggs are stored in a container for storing frozen eggs (hereinafter, sometimes referred to as a "frozen egg storage container"). The frozen egg storage container is not particularly limited, and any known container for frozen egg storage can be appropriately selected, such as a cryotube, a hollow straw, or a modified version thereof. These may be used alone or in combination of two or more.

[0020] The shape, structure, and size of the frozen egg storage container are not particularly limited and can be selected appropriately depending on the size of the storage section and the number of container-containing frozen eggs to be stored in the storage section.

[0021] The depth of the frozen egg storage container is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 5 mm to 15 mm in order to facilitate removal of the freezing solution and thawing solution when thawing and washing the frozen eggs. The depth refers to the length of the deepest part.

[0022] When a cryotube is used as the frozen egg storage container, examples of its shape include those shown in Figure 6. In Figure 6, "a" denotes the shape of the cryotube itself, "b" denotes the shape of a cryotube with the top cut off, "c" denotes a shape in which the bottom of the cryotube, when viewed from the front, is composed of a V-shaped portion and a skirt portion around it (sometimes referred to as an "M-shape"), "d" denotes a shape in which the skirt portion of the "M-shape" has been removed (sometimes referred to as a "top-shape"), and "e" denotes a shape in which the bottom of the cryotube, when viewed from the front, is composed of only the V-shaped portion (sometimes referred to as a "V-shape"). Among these, the V-shape is preferred because it facilitates the removal of freezing and thawing solutions during thawing and washing of frozen eggs and can increase the recovery rate of blastocysts. The shape when viewed from the front refers to the cross-sectional shape when the frozen egg storage container is placed on a flat surface with the open part facing upward and cut vertically.

[0023] The material of the frozen egg storage container is not particularly limited as long as it does not impair the effects of the present invention, and can be appropriately selected depending on the purpose. Examples include polypropylene and polyethylene.

[0024] The method for producing the frozen egg storage container is not particularly limited, and any known method can be appropriately selected for production.

[0025] <Liquid injection part> The liquid injecting section is a section used when injecting liquid into the container section. The location of the liquid injection unit is not particularly limited and can be appropriately selected depending on the purpose. For example, it is preferable to provide it at the end of the frozen egg culture device. The number of the liquid injection parts may be one or two or more, but one is preferable in terms of ease of operation.

[0026] <Liquid discharge section> The liquid discharge portion is a portion used when discharging the liquid inside the container portion. The location of the liquid discharge section is not particularly limited and can be selected appropriately depending on the purpose, but it is preferable to provide it at the same end as the end of the frozen egg culture device where the liquid injection section is located. The number of the liquid discharge parts may be one or two or more, but one is preferable in terms of ease of operation.

[0027] The distance between the liquid inlet and outlet is not particularly limited and can be appropriately selected depending on the purpose.

[0028] Furthermore, the shapes of the liquid injection section and the liquid discharge section are not particularly limited and can be selected appropriately depending on the purpose, but shapes such as a luer lock type, a needle puncture port, or a membrane tube that can be connected to the liquid injection means and liquid discharge means described below, respectively, are preferred. The ends of the liquid inlet and outlet may be provided with a member such as a cap to prevent contact with the outside.

[0029] <Egg spill prevention department> The egg outflow prevention portion is a portion used to prevent eggs (fertilized eggs and unfertilized eggs) from outflowing.

[0030] The location of the egg outflow prevention unit is not particularly limited as long as it is located between the container-held frozen eggs, the liquid injection unit, and the liquid discharge unit, and can be selected appropriately depending on the purpose.

[0031] Specific embodiments of the arrangement of the egg outflow prevention unit include, for example, an arrangement in which the unit is arranged so as to cover the openings (entrance / exit) of the liquid injection unit and the liquid discharge unit on the storage unit side; an arrangement in which the unit is arranged in the shape of a bag to accommodate the container-containing frozen eggs and is arranged in the storage unit; an arrangement in which the unit is arranged in the storage unit and separates the side of the storage unit where the container-containing frozen eggs are arranged from the side of the liquid injection unit and the liquid discharge unit (an arrangement in the shape of a wall to separate the side of the storage unit where the container-containing frozen eggs are arranged from the end side having the liquid injection unit and the liquid discharge unit (see, for example, Figures 2A and 2B)).

[0032] The wall-like arrangement can be achieved by folding the member forming the egg outflow prevention portion in half and arranging the opening of the folded portion so that it is positioned on the side where the container-containing frozen eggs are placed.

[0033] The size of the egg outflow prevention part is not particularly limited, and can be appropriately selected depending on the size of the container that contains the frozen eggs, the size of the storage part, and the like. When the egg outflow prevention part is arranged in the form of a wall, the egg outflow prevention part is arranged from one end to the other end of the storage part.

[0034] The material of the egg outflow prevention part is not particularly limited as long as it has low toxicity to eggs and can be appropriately selected depending on the purpose, and examples thereof include nylon, etc. These may be used alone or in combination of two or more types.

[0035] The structure of the egg outflow prevention part is not particularly limited as long as it does not allow eggs to pass through but allows liquids such as freezing liquid, melting liquid, and cleaning liquid to pass through, and can be appropriately selected depending on the purpose, for example, a mesh structure. The opening size of the mesh is not particularly limited as long as it does not allow eggs to pass through but allows liquids such as freezing solutions to pass through, and can be appropriately selected depending on the size of the eggs, etc. For example, for mouse eggs, the opening size may be 40 μm, etc. The egg outflow prevention part may be a single layer or a multi-layer structure consisting of two or more layers.

[0036] <Other configurations> The other configurations are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose. Examples include a means for preventing adhesion, a cold storage container, and the like.

[0037] <<Means to prevent adhesion>> The adhesion prevention means is a means for preventing the upper and lower surfaces of the storage unit from coming into contact with each other when the liquid in the storage unit is discharged. By providing the adhesion prevention means in the storage unit, it is possible to form (maintain) a space between the upper and lower surfaces of the storage unit when the liquid in the storage unit is discharged, thereby improving the embryo recovery rate and suppressing embryo deformation. Therefore, it is preferable that the frozen egg culture device has the adhesion prevention means in the area of ​​the storage unit where the container-held frozen eggs are placed. The adhesion prevention means is also sometimes referred to as a space maintaining means or a crush prevention means.

[0038] The space between the upper and lower surfaces of the storage section is not particularly limited and can be selected appropriately depending on the purpose, but it is preferable that the length (thickness) between the upper and lower surfaces when discharging the liquid in the storage section is approximately 2 to 7 mm.

[0039] The adhesion prevention means may be one or more. The shape, structure, and size of the adhesion prevention means are not particularly limited and can be appropriately selected taking into consideration the thickness between the upper surface and the lower surface.

[0040] The location of the adhesion prevention means is not particularly limited and can be appropriately selected depending on the purpose. For example, when multiple adhesion prevention means are used, they can be arranged at the four corners, the four corners and the center of the area in the storage section where the container-contained frozen eggs are placed. The multiple adhesion prevention means may be arranged at equal intervals or at unequal intervals.

[0041] The adhesion prevention means may or may not be fixed in place.

[0042] When the adhesion prevention means is fixed, it can be provided in advance as a partition in the region of the storage part where the container-contained frozen eggs are placed.

[0043] When the adhesion prevention means is not fixed, the shape of the adhesion prevention means is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a planar shape such as a plate, and a three-dimensional shape such as a curved plate, a sphere, a tube, etc. Among these, a three-dimensional shape is preferred, and a curved plate or a sphere is more preferred, in terms of improving the embryo recovery rate and being excellent in the effect of suppressing embryo deformation.

[0044] When the adhesion prevention means is not fixed, the size of the adhesion prevention means is not particularly limited and can be selected appropriately depending on the purpose, but in order to prevent damage to the eggs, it is preferable that the size be greater than 1 mm wide x 1 mm long, and preferably about 3 mm wide x 10 mm long.

[0045] When the adhesion prevention means is not fixed, it is preferable that the adhesion prevention means be present in a uniformly dispersed manner in the area of ​​the storage section where the container-containing frozen eggs are placed.

[0046] The area of ​​the storage part of the adhesion prevention means that occupies the region where the container-contained frozen eggs are placed is not particularly limited and can be appropriately selected depending on the purpose, for example, about 30 to 50%, etc. The area of ​​the storage part of the adhesion prevention means that occupies the region where the container-contained frozen eggs are placed refers to the area when the frozen egg culture device is placed on a flat surface and viewed from above.

[0047] The adhesion prevention means is preferably chamfered or smoothed to prevent damage to the embryo or the device.

[0048] The material of the adhesion prevention means is not particularly limited as long as it is not toxic to the embryo, and can be appropriately selected depending on the purpose.

[0049] <<Cooler container>> By using the cold storage container, it is possible to prevent a sudden change in temperature until the melting liquid is poured into the frozen eggs in the frozen egg incubation device, thereby increasing the survival rate of the frozen eggs. The size of the cold storage container is not particularly limited and can be appropriately selected depending on the size of the frozen egg culture device. The shape of the cold storage container is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a bag shape, etc. By using a bag shape large enough to accommodate the entire frozen egg culture device, sudden temperature changes can be more effectively prevented. The material of the cold storage container is not particularly limited as long as it is a material that can be used under liquid nitrogen, and can be appropriately selected depending on the purpose. Examples include nylon mesh and small-grain aluminum. The structure of the cold storage container is not particularly limited as long as it is a structure that can be used under liquid nitrogen, and can be appropriately selected depending on the purpose. For example, it may be a structure in which small aluminum particles are contained in a mesh material such as a bag-shaped nylon mesh.

[0050] <Manufacturing method of frozen egg culture device> The method for manufacturing the frozen egg culture device is not particularly limited, and it can be manufactured by appropriately selecting known materials and using known means to achieve the above configuration. For example, it can be manufactured by attaching an egg outflow prevention part to a container capable of cryopreservation, such as a Froze Bag (Nipro Corporation).

[0051] The shape and size of the frozen egg culture device are not particularly limited and can be appropriately selected depending on the purpose.

[0052] The structure of the frozen egg culture device is not particularly limited as long as it does not have an adverse effect on the eggs contained therein, and can be appropriately selected depending on the purpose. For example, the device may be gas-permeable or non-gas-permeable.

[0053] When the frozen egg culture device is gas permeable, the frozen eggs can be cultured using a known embryo culture incubator such as a CO2 incubator.

[0054] If the frozen egg culture device is not gas permeable, it is a sealed container (closed system), so as long as the temperature is controlled, the cells can be cultured anywhere. Note that the term "sealed" means that the cells are sealed during culture. In the present invention, not having gas permeability means that the oxygen gas permeability of the outer member constituting the frozen egg culture device (for example, when the frozen egg culture device is bag-shaped, the material constituting the bag) is 5 mL / m 2 / day / MPa or less. In the present invention, the oxygen gas permeability is a value measured at a temperature of 25°C and a humidity of 80% RH in accordance with the gas permeability test method for plastic films and sheets in accordance with JIS K 7126-2.

[0055] The gas-impermeable material is not particularly limited as long as it does not impair the effects of the present invention, and any known material can be appropriately selected depending on the purpose. However, cold-resistant synthetic resins that can withstand low temperatures are preferred, and examples thereof include ultra-high molecular weight polyethylene, ethylene-vinyl acetate copolymer, fluororesin, and polyimide.

[0056] According to the frozen egg culture device of the present invention, it is possible to store the frozen eggs on dry ice instead of liquid nitrogen, and then thaw, wash, and culture them, which also makes it easier to handle, such as transporting, the frozen eggs.

[0057] (Frozen egg culture method) The method for culturing frozen eggs of the present invention is not particularly limited as long as it uses the frozen egg culturing device of the present invention described above, and can be selected appropriately depending on the purpose. However, it is preferable that the method includes at least a thawing step, a washing step, and a culturing step, and if necessary, other steps such as a fertilization step, a freezing step, a recovery step, and a refreezing step for freezing the embryos after culturing.

[0058] <Melting process> The thawing step is a step of thawing the frozen eggs in the frozen egg culturing device. The melting liquid used in the melting step is not particularly limited, and any melting liquid used in a known method for melting frozen eggs can be appropriately selected. For example, the frozen eggs can be melted using a first melting liquid with high osmotic pressure and a second melting liquid with low osmotic pressure.

[0059] <Cleaning process> The washing step is a step of washing the thawed eggs (fertilized eggs or unfertilized eggs) after the thawing step. The washing solution used in the washing step is not particularly limited, and any washing solution used in a known egg washing method can be appropriately selected. Examples include a culture medium used in the culture step described below and a fertilization medium used in the fertilization step.

[0060] The type of each of the culture media is not particularly limited, and any known medium that can be used for culturing fertilized eggs or fertilizing unfertilized eggs can be appropriately selected depending on the purpose, such as CZB medium.

[0061] When the frozen egg culture device is gas permeable, the culture medium can be used in a commonly used manner.

[0062] When the frozen egg culture device does not have gas permeability, the culture medium to be used is one in which the CO2 concentration in the culture medium is optimized. The CO2 concentration in the culture medium with the optimized CO2 concentration is not particularly limited as long as it allows eggs to be cultured, and can be appropriately selected depending on the purpose, but is preferably 3 to 6%, more preferably 4 to 5%. A concentration in the preferred range is advantageous in that it increases the development rate.

[0063] The method for optimizing the CO2 concentration in each of the culture media is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include a method of leaving the media in a CO2 incubator at 37°C for a certain period of time. The certain period of time is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include 24 hours. Alternatively, for example, the CO2 concentration in each of the culture media can be adjusted to 4-5% by exposing the media to a carbon dioxide gas generating agent called an anero-pouch for about 4 hours. The timing for optimizing the CO2 concentration is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, 1 to 2 days before the cleaning step.

[0064] The methods for the thawing step and the washing step are not particularly limited as long as the frozen egg culture device of the present invention described above is used, and can be appropriately selected depending on the purpose.

[0065] For example, in order to inject the thawing liquid and the washing liquid through the liquid injecting portion of the frozen egg culture device, the thawing liquid and the washing liquid are sucked into an injecting means such as a syringe, respectively. From the viewpoint of simple, error-free and rapid operation, the injection means is preferably attached to a connector of a three-way stopcock having a member for connecting to the liquid injection part, such as a tube with a needle.

[0066] In addition, in order to discharge the liquid in the storage section through the liquid discharge section of the frozen egg culture device, a member for connecting the liquid discharge section to the discharge means, such as a tube with a needle, is attached to the discharge means, such as a syringe.

[0067] Next, the injection means is connected to the liquid injection section of the stored frozen egg culture device, and the liquid discharge means is connected to the liquid discharge section. If the frozen egg culture device has been stored in the cold storage container, at least the liquid inlet and outlet parts of the frozen egg culture device are removed from the cold storage container, and the liquid outlet means is connected to each of the liquid outlet parts.

[0068] Thereafter, the melted liquid is injected into the container through the liquid injection section. Next, the melted liquid is discharged from the container through the liquid discharge portion. When a plurality of types of melts are used, the above procedure is carried out for each of the melts.

[0069] The melt may be poured all at once, or may be poured in a number of times (by repeating the pouring and discharging). When the first melting liquid having a high osmotic pressure and the second melting liquid having a low osmotic pressure are used as the melting liquids, it is preferable to add the entire amount of the first melting liquid at once and add half of the entire amount of the second melting liquid in two separate additions, as this increases the development rate and the recovery rate of blastocysts.

[0070] The amount of the melting liquid used is not particularly limited and can be selected appropriately depending on the amount of frozen eggs (amount including freezing agent, hereinafter sometimes referred to as "contents"), and it is preferable to use, for example, about three times the amount of the contents.

[0071] Thereafter, the cleaning liquid is injected into the container through the liquid injection section. Next, the cleaning liquid is discharged from the container through the liquid discharge portion.

[0072] The washing solution may be injected all at once, or may be divided into several portions and injected and discharged repeatedly. However, it is preferable to divide the total amount into two portions and add half of each portion, as this increases the development rate and blastocyst recovery rate.

[0073] It is preferable to perform the washing step at least twice or more, as this increases the development rate and the recovery rate of blastocysts. In the present invention, performing the washing step at least twice or more means performing the washing step once and then performing the washing step again after a certain period of time has elapsed. The certain period of time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 30 minutes to 3 hours, and more preferably 1 to 2 hours. If it is within the preferred range, more of the freezing solution can be removed, which is advantageous in that the development rate and the recovery rate of blastocysts can be further increased. The conditions such as temperature during the certain period of time are not particularly limited and can be appropriately selected depending on the purpose, and examples include a temperature of 20 to 40° C. under light-shielded conditions.

[0074] The amount of the washing solution used is not particularly limited, and can be selected appropriately depending on the amount of frozen eggs (amount including the freezing agent, hereinafter sometimes referred to as "contents").

[0075] The time and temperature for performing each treatment in the thawing step and the washing step are not particularly limited, and can be appropriately selected depending on the development rate, blastocyst recovery rate, and the like.

[0076] <Fertilization process> When frozen unfertilized eggs are used as the frozen eggs, a fertilization step is carried out after the washing step and before the culturing step described below. The fertilization step is a step of placing sperm into the frozen egg culture device containing the unfertilized eggs and fertilizing them.

[0077] The method for introducing sperm into the frozen egg culture device is not particularly limited and can be selected appropriately depending on the purpose. For example, a method can be used in which a liquid containing the sperm is injected through the liquid injection section of the frozen egg culture device in the same manner as in the melting process and washing process described above.

[0078] The sperm to be used may be unfrozen sperm (fresh sperm), or frozen sperm that have been thawed may be used. The amount of sperm used is not particularly limited and can be appropriately selected depending on the amount of the unfertilized eggs, etc.

[0079] The temperature and time for the fertilization step are not particularly limited and can be appropriately selected depending on the purpose, for example, at 37°C for 4 to 6 hours.

[0080] By providing the fertilization step, the frozen unfertilized eggs can be thawed, washed, fertilized, and cultured using the frozen egg culture device.

[0081] <Culture process> The culturing step is a step of culturing the fertilized eggs washed in the washing step or the fertilized eggs fertilized in the fertilization step.

[0082] The temperature for the culture is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include about 37°C. The culture period is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, about 3 to 4 days. In the present invention, when the frozen egg culture device is not gas permeable, the culture medium is one in which the CO2 concentration is optimized. In this case, embryos can be cultured in a completely sealed container, and as long as the temperature can be controlled, the embryos can be cultured anywhere.

[0083] After the culture, the cultured product can be collected by opening the frozen egg culture device.

[0084] The frozen egg culture device of the present invention allows the processes from thawing to culturing to be carried out in the same device, and blastocysts can be obtained from frozen eggs. It has also been confirmed that the blastocysts obtained by the present invention can produce liveborn offspring when transplanted.

[0085] <Other processes> The other steps are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose. Examples of such steps include a freezing step and a fixation step.

[0086] <<Freezing process>> The freezing step is a step of freezing fertilized eggs or unfertilized eggs in the frozen egg storage container. The method for freezing the fertilized eggs or unfertilized eggs is not particularly limited, and any known method can be appropriately selected. For example, a method can be used in which the fertilized eggs or unfertilized eggs are immersed in a low osmotic pressure freezing solution, and then the fertilized eggs or unfertilized eggs are transferred to the frozen egg storage container containing a high osmotic pressure freezing solution, and then rapidly frozen in liquid nitrogen.

[0087] The frozen eggs are placed in the storage section together with the frozen egg storage container through the open section of the frozen egg culture device, and then the open section is sealed by heat sealing or the like to create a frozen egg culture device in which the storage container containing the frozen eggs is placed in the storage section.

[0088] The frozen egg culture device may be stored as is, or may be stored in the cooling container. The storage temperature is not particularly limited and can be appropriately selected depending on the type of freezing liquid, for example, between liquid nitrogen temperature and -30°C.

[0089] <<Fixation process>> The fixation step is a step of chemically fixing the cultured embryo. The fixation step is not particularly necessary in the fields of infertility treatment and livestock farming. On the other hand, in the field of basic biology, embryo fixation is necessary for detailed observations in space experiments, where long-term storage is required.

[0090] The fixation method is not particularly limited, and any known method can be appropriately selected depending on the purpose. However, in consideration of space experiments, fixation with less than 1% paraformaldehyde is preferable.

[0091] The fixation can be carried out in the frozen egg culture device in the same manner as the thawing and washing steps. After the fixation step, a washing treatment can be carried out as necessary, and the eggs can be stored for a long period of time in a refrigerator or freezer. Furthermore, while conventional methods of chemical fixation use a mouthpiece or the like, which poses the risk of ingesting a toxic fixative, the frozen egg culture device of the present invention eliminates such a risk and is therefore highly safe. [Example]

[0092] The present invention will be described in detail below with reference to the following Production Examples and Examples, but the present invention is not limited to these Production Examples and Examples.

[0093] (Manufacturing Example 1: Manufacturing of frozen egg culture device) A frozen egg culture device was manufactured by modifying a Froze Bag F-050 (volume 250 mL, Nipro Corporation). Schematic diagrams of the frozen egg culture device (symbol: 1) are shown in Figures 2A and 2B.

[0094] Figure 2A is a schematic diagram of one side of the frozen egg culture device, and Figure 2B is a schematic diagram of the other side of the frozen egg culture device. In Figures 2A and 2B, reference numeral 2 denotes a storage section for storing container-contained frozen eggs, reference numeral 3 denotes a liquid injection section, reference numeral 4 denotes a liquid discharge section, and reference numeral 5 denotes an egg outflow prevention section.

[0095] The liquid inlet and outlet are ports provided at the ends of the Froze Bag F-050.

[0096] The egg outflow prevention section was made by folding a 40 mm long, 50 mm wide nylon mesh (330 mesh, 40 μm mesh size, AS ONE Corporation) in half at a point 25 mm long, and securing the horizontal end to the F-050 using a heat sealer (P-300, Fuji Impulse Co., Ltd.). The nylon mesh was secured to the F-050 in a wall-like manner, 25 to 50 mm long from the end of the F-050 opposite the end where the port was provided, and 5 mm wide from the horizontal end of the F-050. The nylon mesh was positioned so that the opening of the folded portion was located opposite the liquid inlet and outlet. The nylon mesh does not allow fertilized or unfertilized eggs to pass through, but allows liquids such as freezing solution, thawing solution, and culture medium to pass through.

[0097] (Manufacturing Example 2: Manufacturing of frozen egg storage containers) The top of a cryotube (Ceramic Tube, Sumitomo Bakelite Co., Ltd., MS-4501, 1 mL) was cut to produce a frozen egg storage container consisting of only the bottom, which has a V-shaped shape when viewed from the front. A photograph of the frozen egg storage container is shown in Figure 3.

[0098] (Production Example 3: Production of adhesion prevention means) Using a cryotube as a raw material, an adhesion prevention means was manufactured as follows. The cryotube was cut into approximately 5 equal parts in the vertical direction (depth direction), and the side portion of the cryotube was cut off. The cut pieces were then cut in half again to obtain curved adhesion prevention means measuring approximately 3 to 5 mm in width and approximately 10 mm in length. The adhesion prevention means was chamfered.

[0099] Example 1: Freezing, thawing, washing, and culturing of fertilized eggs In this experiment, the freezing and thawing of mouse fertilized eggs was carried out with reference to the hyperosmotic vitrification method.

[0100] <Preparation of mouse fertilized eggs> In this experiment, 2-cell stage oviduct-perfused embryos collected by oviduct perfusion as described below were used as fertilized mouse eggs.

[0101] -Egg collection by fallopian tube irrigation- The mice used were male and female ICR or B6D2F1 strain mice. Female mice were superovulated by intraperitoneally injecting 7.5 IU of PMSG (serotropin, Asuka Animal Health Co., Ltd.) using a 1 mL syringe (Terumo Corporation) and a 26-gauge needle (Terumo Corporation), followed approximately 48 hours later by injecting 7.5 IU of hCG (gonadotropin, Asuka Animal Health Co., Ltd.) using the same syringe. The female mice that had been superovulated by hormone injection were housed overnight in the same cage as the male mice. The next morning, they were checked for vaginal plugs, and female mice with confirmed plugs were used the following day. For perfusion, oviducts were collected from mice euthanized by cervical dislocation, and a 30-gauge syringe needle was inserted into the fimbria (the entrance to the oviduct on the ovarian side) and approximately 0.1 mL of Hepes-CZB medium (see below, hereafter sometimes referred to as "H-CZB medium") was injected. The fertilized eggs collected by perfusion were cultured in a chamber filled with CZB medium (see below) and covered with mineral oil (M8410, Sigma). Culture was carried out in an incubator (5% CO2, 37°C, 100% humidity).

[0102] [CZB medium] 200 mL of Milli-Q was placed in a glass bottle, and the following reagents were thoroughly dissolved using a slurrier to prepare a stock solution with the following composition. The solution was left to stand overnight in a refrigerator and sterilized using a filter. - Composition of stock solution - The following composition represents the amount per 100 mL of CZB medium. NaCl 476 mg (Fujifilm Wako Pure Chemical Corporation) KCl 36 mg (Fujifilm Wako Pure Chemical Corporation) MgSO4 7H2O 29mg (Nacalai Tesque, Inc.) KH2PO4 16mg (Fujifilm Wako Pure Chemical Corporation) EDTA 2Na 4mg (Fujifilm Wako Pure Chemical Corporation) 211mg NaHCO3 (Fujifilm Wako Pure Chemical Corporation) Sodium lactate 0.53mL (60% syrup) (Sigma) D-glucose 100mg (Fujifilm Wako Pure Chemical Corporation) Penicillin G 5mg (ICN Biomedicsls Inc) Streptomycin 7mg (ICN Biomedicsls Inc) Phenol red, appropriate amount (10mg / ml soln.in saline) (Sigma)

[0103] The following reagents were added to 50 mL of the stock solution of CZB medium, and the mixture was left to stand overnight in a refrigerator. The following composition represents the amount per 100 mL of CZB medium. Sodium pyruvate 3mg (Fujifilm Wako Pure Chemical Corporation) L-Glutamine 15mg (Sigma) 25 mg CaCl2 2H2O (Fujifilm Wako Pure Chemical Corporation) BSA 500mg (Sigma, GIBCO AlbuMAX)

[0104] The solution was sterilized using a 0.22 μm filter (MILLEX GV) in a clean bench, dispensed into assist tubes, or placed in glass bottles and stored in a refrigerator.

[0105] [H-CZB medium] 200 mL of Milli-Q was placed in a glass bottle, and the following reagents were thoroughly dissolved using a slurrier to prepare a stock solution with the following composition. The solution was left to stand overnight in a refrigerator and sterilized using a filter. - Composition of stock solution - The following composition represents the amount per 100 mL of H-CZB medium. NaCl 476 mg (Fujifilm Wako Pure Chemical Corporation) KCl 36 mg (Fujifilm Wako Pure Chemical Corporation) MgSO4 7H2O 29mg (Nacalai Tesque, Inc.) KH2PO4 16mg (Fujifilm Wako Pure Chemical Corporation) EDTA 2Na 4mg (Fujifilm Wako Pure Chemical Corporation) 211mg NaHCO3 (Fujifilm Wako Pure Chemical Corporation) · Hepes·Na(basic) ··· 520mg (Sigma) Sodium lactate 0.53mL (60% syrup) (Sigma) D-glucose 100mg (Fujifilm Wako Pure Chemical Corporation) Penicillin G 5mg (ICN Biomedicsls Inc) Streptomycin 7mg (ICN Biomedicsls Inc) Phenol red, appropriate amount (10mg / ml soln.in saline) (Sigma)

[0106] The following reagents were added to 50 mL of the H-CZB medium stock solution, and the mixture was left to stand overnight in a refrigerator. The following composition represents the amount per 100 mL of H-CZB medium. Sodium pyruvate 3mg (Fujifilm Wako Pure Chemical Corporation) L-Glutamine 15mg (Sigma) 25 mg CaCl2 2H2O (Fujifilm Wako Pure Chemical Corporation) PVA 10mg (Sigma)

[0107] The solution was sterilized using a 0.22 μm filter (MILLEX GV) in a clean bench, dispensed into assist tubes, and stored in a refrigerator.

[0108] <Freezing of mouse fertilized eggs and their packaging in a frozen egg culture device> The mouse fertilized eggs in CZB medium prepared as described above were transferred together with a small amount of culture medium to 50 μL of hypotonic freezing solution 1 (see below, sometimes referred to as "EFS 20 solution") that had been brought to room temperature.

[0109] [Freezing liquid 1] -FS fluid for EFS 20- The following reagents and solutions were placed in a 50 mL centrifuge tube and mixed thoroughly to dissolve. After complete dissolution, 60.0 mg of BSA was placed on top and allowed to dissolve naturally. This was used as the FS solution for EFS 20 solution. The composition of PB1 (BSA-) is described below. PB1(BSA-) ※ 14.0mL Ficoll 6.0g (GE Healthcare) Sucrose 3.4g (Fujifilm Wako Pure Chemical Corporation)

[0110] -EFS 20 liquid- The EFS 20 liquid was prepared by mixing the FS liquid for EFS 20 with ethylene glycol in the following ratio. FS solution for EFS 20 4 (v / v) Ethylene glycol 1 (v / v) (Fujifilm Wako Pure Chemical Corporation)

[0111] After 2 minutes had passed since the fertilized eggs were transferred to freezing solution 1, the fertilized eggs were transferred to the frozen egg storage container of Example 2 containing 50 μL of hyperosmotic freezing solution 2 (see below, sometimes referred to as "EFS 42.5 cd solution") that had been returned to room temperature.

[0112] [Freezing liquid 2] -FS liquid for EFS 42.5cd- The following reagents and solutions were placed in a 50 mL centrifuge tube, mixed well to dissolve, and left to stand overnight in a refrigerator at 4°C to prepare FS solution for EFS 42.5 cd. The composition of PB1 (BSA-) is described below. PB1(BSA-) ※ 9.0mL Ficoll 6.0g (GE Healthcare) Sucrose 12.0g (Fujifilm Wako Pure Chemical Corporation)

[0113] -EFS 42.5cd liquid- The FS solution for EFS 42.5cd was mixed with ethylene glycol in the following ratio to prepare EFS 42.5cd solution. FS solution for EFS 42.5cd 57.5(v / v) Ethylene glycol 42.5 (v / v) (Fujifilm Wako Pure Chemical Corporation)

[0114] After one minute had passed since the fertilized eggs were transferred to freezing solution 2, the frozen egg storage container was picked up with tweezers and placed in a dish filled with liquid nitrogen, and was held there for 5 to 10 seconds to be rapidly frozen.

[0115] A mark was made on either the liquid injection part or the liquid discharge part of the frozen egg culture device, and a hole was made with a tube. The frozen egg culture device was cooled in the gas layer of liquid nitrogen. Then, the frozen egg storage container containing the frozen fertilized eggs was placed in the storage part of the frozen egg culture device. Next, the open part of the frozen egg culture device (the end opposite to the end having the liquid injection part and the liquid discharge part) was sealed and packed with an upper and lower heat sealer (Fuji Impulse Co., Ltd., T-130K, T230K), and cooled with liquid nitrogen.

[0116] The frozen egg culture device was stored at -80°C after being frozen with liquid nitrogen.

[0117] In addition, the compositions of PB1(BSA-) and PB1(BSA+) described above are as follows. [PB(BSA- / +)] (1) Ten PBS tablets (Takara Bio Inc.) were placed in a screw-cap bottle, 1,000 mL of Milli-Q was taken, and dissolved and sterilized by autoclaving. Using a graduated cylinder, Milli-Q was added to make it 1,000 mL, and PBS - was adjusted to 1,000 mL and returned to the screw-cap bottle.

[0118] (2) 10 mL of MgCl2·6H2O (FUJIFILM Wako Pure Chemical Corporation) adjusted to 100-fold concentration was dissolved in 1,000 mL of the PBS - adjusted in (1). After completely dissolving, 10 mL of CaCl2·2H2O (FUJIFILM Wako Pure Chemical Corporation) adjusted to​​​​​​​​​​​​​​​​ (Fujifilm Wako Pure Chemical Corporation) Penicillin G 5.1 mg (ICN Biomedicsls Inc) D-glucose 80mg (Fujifilm Wako Pure Chemical Corporation)

[0120] (4) 400 mL of the PB1 (BSA-) prepared in (3) above was dispensed into a 500 mL storage bottle (Corning), 1,200 mg of BSA (AlbuMAX) was placed on the water surface, and the bottle was left to stand in a refrigerator until it dissolved naturally. It was then sterilized using a filter to prepare PB1 (BSA+).

[0121] <Thawing, washing, and culturing frozen fertilized eggs> -Preparation- Three 30 mL syringes were prepared, and 30 mL of melting solution 1 (high osmolality melting solution, see below), melting solution 2 (low osmolality melting solution, see below), and culture solution (the above-mentioned CZB medium) that had been returned to room temperature were drawn into each syringe. The culture medium was placed in a gas-permeable nematode bag and left to stand in an incubator (5% CO2, 37°C, 100% humidity) for 24 hours, and the CO2 concentration in the culture medium was optimized (4-5%).

[0122] [Melted liquid 1] (1) 179.7 g of sucrose was dissolved in the PB1 (BSA-) in a 1,000 mL storage bottle (Corning) with shaking, and the total volume was adjusted to the 700 mL mark on the storage bottle. (2) 2,100 mg of BSA was placed on the surface of 700 mL of the 0.75 M sucrose-PB1 prepared in (1) above, and left to stand in a refrigerator until it dissolved naturally. The solution was then sterilized using a filter to prepare Melting Solution 1 (hereinafter sometimes referred to as "0.75 M sucrose-PB1 (BSA+)").

[0123] [Melted liquid 2] 400 mL of the PB1 (BSA+) was mixed with 200 mL of the 0.75 M sucrose-PB1 (BSA+), which was then sterilized using a filter to obtain thawed solution 2 (hereinafter, sometimes referred to as "0.25 M sucrose-PB1 (BSA+)").

[0124] A tube with a needle was attached to the waste liquid syringe (100 mL) for connecting to the liquid discharge part of the frozen egg culture device.

[0125] A three-way stopcock was prepared and a tube with a needle attached was attached to connect it to the liquid injection part of the frozen egg culture device. From the side opposite the side where the needle tube was attached to the three-way stopcock, a syringe containing thawing solution 1, a syringe containing thawing solution 2, and a syringe containing culture solution were attached in this order.

[0126] - Melting and cleaning - (1) The frozen egg culture device stored at -80°C was removed from the freezer. A tube connected to the waste syringe was attached to the liquid outlet of the frozen egg culture device, and a tube connected to the three-way stopcock was attached to the liquid inlet (see FIG. 4). The cock of the tube connected to the waste liquid syringe was opened, and the air was removed using the syringe, and then the cock was closed. This step (1) was carried out for 1 minute and 30 seconds at room temperature.

[0127] (2) The entire amount (30 mL) of the melting solution 1 was poured into the container of the frozen egg culture device, the stopcock was closed, and the frozen egg culture device was shaken five times. This step (2) was carried out for 4 minutes and 30 seconds at room temperature.

[0128] (3) The cock of the tube connected to the waste syringe was opened, and the injected melting liquid 1 was completely removed, and then the cock was closed. 15 mL of the melting solution 2 was placed in the container of the frozen egg culture device, the stopcock was closed, and the frozen egg culture device was shaken five times. The cock of the tube connected to the waste syringe was opened, and the injected melted liquid 2 was completely removed, and then the cock was closed. Next, 15 mL of the melting solution 2 was placed in the container of the frozen egg culture device, the stopcock was closed, and the frozen egg culture device was shaken five times. This step (3) was carried out for 3 minutes at room temperature.

[0129] (4) The cock of the tube connected to the waste syringe was opened, and the injected melted liquid 2 was completely removed, and then the cock was closed. 15 mL of the culture medium was placed in the container of the frozen egg culture device, the stopcock was closed, and the frozen egg culture device was shaken five times. The cock of the tube connected to the waste syringe was opened, and after the culture medium was completely removed, the cock was closed. 15 mL of the culture medium was placed in the container of the frozen egg culture device, the stopcock was closed, and the frozen egg culture device was shaken five times.

[0130] (5) After carrying out the step (4), the frozen egg culture device was left to stand at room temperature (25°C) or 37°C (hereinafter, sometimes referred to as the "temperature until the second medium change") for 0.5 hours, 1 hour, or 2 hours (hereinafter, sometimes referred to as the "time until the second medium change"). During this time, the frozen egg culture device was covered with aluminum foil. Then, the step (4) was repeated once more.

[0131] -culture- After the thawing and washing operations, the tube connected to the three-way stopcock was slowly removed while holding the hard part of the port of the liquid injection part, and a rubber stopper was placed on the liquid injection part. Similarly, the tube connected to the liquid outlet was slowly removed, and the liquid outlet was capped with a rubber stopper. Next, the frozen egg culture device was placed in an incubator at 37°C and cultured for 3 days.

[0132] After the incubation, the frozen egg incubation device was opened and the contents were transferred to a 10 cm dish. The percentage of blastocysts recovered was calculated. The results are shown in Table 1 below. An example of the results of microscopic observation is shown in Figure 5. The recovery rate, survival rate, and blastocyst rate were calculated using the following formulas. Recovery rate (%) = (number of recovered embryos / number of eggs tested) x 100 Viability (%) = (number of surviving embryos / number of recovered embryos) x 100 Blastocyst rate (%) = (number of blastocysts / number of retrieved embryos) x 100

[0133] [Table 1]

[0134] As shown in Table 1 and FIG. 5, it was confirmed that frozen eggs could be cultured to the blastocyst stage by using the frozen egg culture device of the present invention.

[0135] Furthermore, when ICR mice were used as recipients and the obtained blastocyst-stage embryos were transplanted into the uterus of pseudopregnant mice on day 2.5, liveborn offspring were obtained (see Table 2), confirming that the blastocysts obtained by culturing in the frozen egg culture device of the present invention are of such quality that they can be used to produce liveborn offspring.

[0136] [Table 2]

[0137] In the fields of infertility treatment and livestock farming, there is no need to fix the embryos after the culture. However, when conducting experiments on the space station, it is necessary to fix the embryos after the culture for preservation reasons. Furthermore, from the viewpoint of handling, it is preferable to use less than 1% paraformaldehyde (hereinafter sometimes referred to as "PFA") on the space station. After 4 days of incubation, 0.99% PFA was added to the frozen egg culture device, followed by fixation for 1 hour and washing with PBS containing 0.1% polyvinyl alcohol. The injection and discharge of the fixative and the injection of the washing solution were carried out in the same manner as in the thawing and washing procedures. The frozen egg culture device was opened and the blastocysts were collected. The recovered blastocysts were immunostained using standard methods, and the inner cell mass (ICM) and trophectoderm (TE) were clearly stained. Furthermore, gene expression was confirmed using a next-generation sequencer, and the expression of Pttg1, a gene required for the M phase of the cell cycle, was also confirmed.

[0138] Example 2 A comparison was made between a frozen egg culture device without a means for preventing adhesion and a frozen egg culture device with a means for preventing adhesion.

[0139] <Example 2-1: Frozen egg culture device without adhesion prevention means (control)> In Example 1, the frozen eggs were cultured to blastocysts in the same manner as in Example 1, except that step (5) in -thawing and washing- (time and temperature until the second medium change) was set to 37±1°C and 1 to 2 hours.

[0140] <Example 2-2: Frozen egg culture device with adhesion prevention means> In Example 1 <Freezing of mouse fertilized eggs and sealing in a frozen egg culture device>, when the frozen egg storage container was placed in the storage section of the frozen egg culture container, the 10 adhesion prevention means manufactured in Manufacturing Example 3 were also placed in the storage section, and the frozen eggs were cultured to blastocysts in the same manner as in Example 1, except that step (5) in -thawing and washing- (time and temperature until the second medium change) was set to 37±1°C and 1 to 2 hours.

[0141] The results of the recovery rate and blastocyst rate calculated in the same manner as in Example 1 are shown in Table 2 below. Examples of the results of microscopic observation are shown in Figure 7A (when a frozen egg culture device without a means for preventing adhesion was used) and Figure 7B (when a frozen egg culture device with a means for preventing adhesion was used).

[0142] [Table 2]

[0143] As shown in Table 2 and Figures 7A and 7B, it was confirmed that the use of a frozen egg culture device equipped with a means for preventing adhesion can increase the embryo recovery rate and prevent embryo deformation. This is thought to be because, as shown in the schematic diagrams of Figures 8A and 8B, the provision of the means for preventing adhesion (13) makes it possible to maintain space within the storage section when the culture medium, etc. is discharged, preventing the upper surface (10) and lower surface (11) of the storage section from coming into contact with each other, and as a result, preventing deformation of the embryos (12).

[0144] The frozen egg incubation device of the present invention allows for thawing, washing, and incubation without direct contact with the frozen eggs. Therefore, even those who have never manipulated embryos before can incubate frozen eggs without any practice. Furthermore, by using a sealed frozen egg incubation device, frozen eggs can be incubated anywhere as long as the temperature is controlled. Therefore, it can be suitably used in institutions that handle fertilized or unfertilized eggs, such as infertility clinics, animal testing facilities, and livestock farming fields, and it will also enable experiments that have not been possible to conduct in outer space until now.

[0145] The present invention includes, for example, the following aspects. <1> a storage unit for storing the containerized frozen eggs; A liquid injection unit; A liquid discharge portion; The frozen egg culture device is characterized by comprising the container-containing frozen eggs and an egg outflow prevention unit located between the liquid injection unit and the liquid discharge unit. <2> The egg spill prevention unit is disposed in the storage unit, The side of the storage unit where the container-contained frozen eggs are placed is separated from the liquid injecting unit and the liquid discharging unit side. <1> 1. The frozen egg culture device according to claim 1. <3> The container of the container-packed frozen eggs has a V-shaped container when viewed from the front. <1> from <2> 1. The frozen egg culture device according to claim 1, wherein the frozen egg culture device is a frozen egg culture device. <4> The frozen eggs are mammalian frozen eggs. <1> from <3> 1. The frozen egg culture device according to claim 1, wherein the frozen egg culture device is a frozen egg culture device. <5> The container has a means for preventing adhesion. <1> from <4> 1. The frozen egg culture device according to claim 1, wherein the frozen egg culture device is a frozen egg culture device. <6> The aforementioned <1> from <5> The present invention relates to a method for culturing frozen eggs, characterized by using the frozen egg culturing device described in any one of the above. <7> a thawing step of thawing the frozen eggs; A washing step of washing the thawed eggs; and a culturing step of culturing the washed eggs. <6> This is a method for culturing frozen eggs described in . <8> The washing step is carried out at least twice. <7> This is a method for culturing frozen eggs described in . <9> The culture medium used had a CO2 concentration of 3-6%. <6> from <8> 1. A method for culturing frozen eggs according to any one of the above. [Explanation of symbols]

[0146] 1 Frozen egg culture device 2. Storage section 3 Liquid injection part 4 Liquid drain 5 Egg spill prevention section 6 syringes (for melting liquid 1) 7 Syringe (for melting liquid 2) 8 syringes (for culture medium) 9 Syringe (for waste liquid) 10 Top of the storage section 11 Underside of storage unit 12 embryos 13. Adhesion prevention measures

Claims

1. A storage section, A liquid injection unit; A liquid discharge portion; An apparatus for freezing and culturing mammalian eggs, comprising an egg outflow prevention unit, The accommodation unit is an accommodation space of the mammalian frozen egg culture device, the liquid injection part is a part used when injecting a liquid into the storage part, and is located at an end of the mammalian egg cryopreservation device; the liquid discharge part is a part used when discharging the liquid in the storage part, and is located at an end of the mammalian egg freezing culture device, the egg outflow prevention part has a mesh structure and is arranged in the form of a wall in the storage part so as to divide the storage part into a part for storing containers containing frozen mammalian eggs and a part for not storing containers containing frozen mammalian eggs; A mammalian frozen egg culture device, characterized in that the areas where the container containing the mammalian frozen eggs is not accommodated are located on the liquid inlet side and the liquid outlet side.

2. A mammalian frozen egg culture device as described in Claim 1, wherein the liquid discharge section is located at the same end as the end of the mammalian frozen egg culture device at which the liquid injection section is located.

3. 3. The mammalian frozen egg culture device according to claim 1, wherein the container containing the mammalian frozen eggs has a V-shaped shape when viewed from the front with the opening facing up.

4. The storage section has a means for preventing adhesion to maintain a space between the upper surface and the lower surface of the storage section, 4. The mammalian frozen egg culture device according to claim 1, wherein the adhesion prevention means maintains the distance between the upper and lower surfaces of the storage section at 2 to 7 mm.

5. A method for culturing frozen mammalian eggs, characterized by using a mammalian frozen egg culturing device described in any one of claims 1 to 4.

6. A method for producing frozen mammalian eggs, comprising: A washing step of washing the thawed eggs; The method for culturing frozen mammalian eggs according to claim 5, further comprising a culturing step of culturing the washed eggs.

7. A method for culturing frozen mammalian eggs as described in claim 6, wherein the washing step is performed at least twice.

8. The method for culturing frozen mammalian eggs according to any one of claims 5 to 7, wherein a culture medium having a CO 2 concentration of 3 to 6% is used.

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