JP7780065B2 - Mammalian frozen egg culture device and mammalian frozen egg culture method - Google Patents

Description

The present invention relates to a frozen egg incubation device and a frozen egg incubation method for culturing frozen eggs (frozen fertilized eggs or frozen unfertilized eggs).

Conventionally, 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. After that, the eggs (fertilized or unfertilized) are washed while being transferred sequentially with a capillary into three second melting solutions separately placed on the culture dish. After washing, the eggs are then transferred to 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.

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 allows even non-expert technicians to easily and reliably thaw, wash, and culture frozen eggs, it could greatly contribute to the development of these fields.

A method for cryopreserving germ cells has been proposed to date, 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 easily cryopreserved. However, the proposed device is designed to cryopreserve germ cells, and does not thaw, wash, or culture the germ cells in the same device.

Furthermore, as a technology that does not impede heat conduction when thawing frozen cells and 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 is equipped with an exhaust port for venting 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.

A kit for preparing antigen-specific cytotoxic T cells has also been proposed, which includes a sealed culture vessel with multiple ports as a component (see, for example, Patent Document 3). According to this proposal, antigen-specific cytotoxic T cells can be prepared with a simple procedure. However, this proposal is intended for preparing antigen-specific cytotoxic T cells, and does not relate to techniques for thawing, washing, and culturing frozen eggs.

As a result, there is currently no technology available that allows unskilled technicians to thaw, wash, and culture frozen eggs.

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 not be possible. 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 cultivate fertilized eggs in space have not yet been realized.

Therefore, from the perspective of making it possible to conduct 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.

JP 2009-148218 A Japanese Patent Application Laid-Open No. 2001-070402 JP 2010-130999 A

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

As a result of extensive research into achieving the above-mentioned objectives, the inventors discovered that by creating a frozen egg incubation device that includes 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 beginners who have never handled fertilized or unfertilized eggs can thaw, wash, and culture frozen eggs without any training.

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 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> A method for culturing frozen eggs, characterized by using the frozen egg culturing device described in <1>.

The present invention solves the above-mentioned problems of the prior art and achieves the above-mentioned objectives, providing a frozen egg incubation device and a frozen egg incubation method that allow even non-expert technicians to thaw, wash, and culture frozen eggs.

FIG. 1 is a diagram illustrating a conventional method for thawing and washing frozen eggs. FIG. 2A is a schematic diagram of one side of an example of a frozen egg culturing device. FIG. 2B is a schematic diagram of the other side of the frozen egg culturing apparatus of FIG. 2A. FIG. 2C is a photograph of the frozen egg culturing apparatus of FIG. 2A. FIG. 3 is a photograph of an example of a frozen egg storage container. 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. FIG. 5 is a diagram showing an example of the results of culturing frozen fertilized eggs using the frozen egg culturing device of the present invention. FIG. 6 is a diagram showing an example of a frozen egg storage container. FIG. 7A shows 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 shows an example of the results of culturing frozen fertilized eggs using the frozen egg culturing device with the adhesion prevention means of Example 2-2 (many of the embryos that developed maintained their round shape). FIG. 8A is a schematic diagram of a cross section of the storage section when a culture medium or the like is discharged in a frozen egg culture device that does not have a means for preventing adhesion. 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.

(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 also have other components as necessary.

<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.

<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.

- 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 processed version thereof. These may be used alone or in combination of two or more.

There are no particular restrictions on the shape, structure, or size of the frozen egg storage container, and they 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.

There are no particular restrictions on the depth of the frozen egg storage container and it can be selected appropriately depending on the purpose, but a depth of 5 mm to 15 mm is preferred in order to facilitate the removal of freezing and thawing liquids when thawing and washing the frozen eggs. The depth refers to the length of the deepest part.

When a cryotube is used as the frozen egg storage container, examples of the shape include those shown in Fig. 6. In Fig. 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, consists 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, consists 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.

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 selected appropriately depending on the purpose. Examples include polypropylene and polyethylene.

There are no particular limitations on the method for manufacturing the frozen egg storage container, and it can be manufactured by appropriately selecting any known method.

<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 preferred in terms of ease of operation.

<Liquid discharge part>
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 portions may be one or two or more, but one is preferred in terms of ease of operation.

There are no particular restrictions on the spacing between the liquid inlet and outlet, and it can be selected appropriately depending on the purpose.

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.

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

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

Specific examples of the arrangement of the egg outflow prevention unit include an arrangement in which it is arranged so as to cover the openings (gateways) of the liquid inlet and outlet on the storage unit side of the liquid inlet and outlet, an arrangement in which it is arranged in the storage unit in the shape of a bag to accommodate the container-containing frozen eggs, and an arrangement in which it is arranged in the storage unit and separates the side of the storage unit where the container-containing frozen eggs are placed from the side of the liquid inlet and liquid outlet (an arrangement in the shape of a wall that separates the side of the storage unit where the container-containing frozen eggs are placed from the end side having the liquid inlet and liquid outlet (see, for example, Figures 2A and 2B)).

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

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.

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

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 liquid 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 consisting of two or more layers.

<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.

<<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.

There are no particular restrictions on the space between the upper and lower surfaces of the storage section and it 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 inside the storage section is approximately 2 to 7 mm.

The adhesion prevention means may be one or more. There are no particular restrictions on the shape, structure, or size of the adhesion prevention means, and they can be selected appropriately taking into account the thickness between the upper and lower surfaces.

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

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

If the adhesion prevention means is fixed, it can also be installed in advance as a partition in the area of the storage section where the containerized frozen eggs are placed.

When the adhesion prevention means is not fixed, the shape of the adhesion prevention means is not particularly limited and can be selected appropriately depending on the purpose. Examples include planar shapes such as a plate, and three-dimensional shapes such as a curved plate, sphere, or tube. Among these, three-dimensional shapes are preferred, with curved plate and sphere shapes being more preferred, as they are more effective in improving the embryo recovery rate and suppressing embryo deformation.

When the adhesion prevention means is not fixed, there are no particular restrictions on the size of the adhesion prevention means and it can be selected appropriately depending on the purpose. However, in order to prevent damage to the eggs, it is preferable for the size to be greater than 1 mm wide x 1 mm long, and preferably approximately 3 mm wide x 10 mm long.

When the adhesion prevention means is not fixed, it is preferable that the adhesion prevention means be uniformly dispersed in the area of the storage unit where the container-containing frozen eggs are placed.

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

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

There are no particular restrictions on the material of the adhesion prevention means, as long as it is not toxic to the embryo, and it can be selected appropriately depending on the purpose.

<<Cold storage 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.

<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).

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

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

When the frozen egg culture device is gas permeable, the cells can be cultured using a known embryo culture incubator such as a CO ₂ incubator.

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 culture can be carried out anywhere. Note that the sealed container means that the container is sealed during the 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/ ^m2 /day/MPa or less. Note that 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.

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

The frozen egg incubation device of the present invention allows frozen eggs to be stored on dry ice instead of liquid nitrogen, and then thawed, washed, and incubated, making it easier to transport and handle the frozen eggs.

(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.

<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.

<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.

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 selected appropriately depending on the purpose, such as CZB medium.

If the frozen egg culture device is gas permeable, the culture media can be used in the same manner as commonly used methods.

When the frozen egg culture device does not have gas permeability, the culture medium to be used is one in which the CO ₂ 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%. The preferred range is advantageous in that it results in a higher development rate.

The method for optimizing the _CO2 concentration in each of the culture solutions is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include a method in which the culture solution is left standing for a certain period of time in a _CO2 incubator at 37°C. 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 solutions can be adjusted to 4 to 5% by exposure to a carbon dioxide gas generating agent called an anero-pouch for about 4 hours.
The timing for optimizing the CO ₂ 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.

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

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.

In addition, in order to discharge the liquid from the storage unit through the liquid discharge unit of the frozen egg culture device, a member for connecting the liquid discharge unit to the discharge means, such as a needle-equipped tube, is attached to the discharge means, such as a syringe.

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.

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.

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.

There are no particular restrictions on the amount of melting liquid used, and it can be selected appropriately depending on the amount of frozen eggs (including the amount of freezing agent, hereinafter sometimes referred to as "contents"). For example, it is preferable to use an amount approximately three times the amount of the contents.

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.

The washing solution can be injected all at once, or the entire amount can be divided into multiple times (by repeatedly injecting and discharging), but it is preferable to divide the entire amount into two parts and add half of each part, as this increases the development rate and blastocyst recovery rate.

It is preferable to perform the washing step at least twice or more, since 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 hour to 2 hours. If it is within the preferred range, the freezing solution can be removed more effectively, which is advantageous in that the development rate and the recovery rate of blastocysts are 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 thereof include a temperature of 20 to 40° C. under light-shielded conditions.

There are no particular restrictions on the amount of cleaning solution used, and it can be selected appropriately depending on the amount of frozen eggs (including the amount of freezing agent, hereinafter sometimes referred to as "contents").

There are no particular limitations on the time and temperature for each treatment in the thawing and washing steps, and these can be selected appropriately depending on factors such as the development rate and blastocyst recovery rate.

<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.

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 may be used in which a liquid containing the sperm is injected through the liquid injection section of the frozen egg culture device, similar to the thawing and washing processes described above.

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.

There are no particular restrictions on the temperature and time for the fertilization step, and these can be selected appropriately depending on the purpose. Examples include 4 to 6 hours at 37°C.

By including the fertilization process, the frozen unfertilized eggs can be thawed, washed, fertilized, and cultured using the frozen egg culture device.

<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.

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 period of the culture 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, the fertilized eggs can be cultured in a completely sealed container, and the fertilized eggs can be cultured anywhere as long as the temperature can be controlled.

After the incubation, the cultured product can be collected by opening the frozen egg incubation device.

The frozen egg culture device of the present invention allows processes from thawing to culture to be carried out within the same device, making it possible to obtain blastocysts from frozen eggs. It has also been confirmed that blastocysts obtained using the present invention can produce live offspring when transplanted.

<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.

<<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.

The frozen eggs are placed in the storage section together with the frozen egg storage container through the open section of the frozen egg incubation device, and the open section is then sealed by heat sealing or the like, thereby creating a frozen egg incubation device in which the storage container containing the frozen eggs is placed in the storage section.

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 the liquid nitrogen temperature and -30°C.

<<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.

There are no particular limitations on the fixation method, and any known method can be selected appropriately depending on the purpose, but considering space experiments, fixation with less than 1% paraformaldehyde is preferable.

The fixation can be carried out in the frozen egg culture device in the same manner as the thawing and washing processes. After the fixation process, washing can be performed as necessary, and the eggs can be stored for long periods of time in a refrigerator or freezer. Furthermore, while conventional methods involve chemical fixation using a mouthpiece or the like, which poses the risk of ingesting toxic fixatives, the frozen egg culture device of the present invention eliminates this risk and is extremely safe.

The following describes in detail the manufacturing examples and working examples of the present invention, but the present invention is not limited to the following manufacturing examples and working examples.

(Production Example 1: Production 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.

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 represents a storage section for storing containerized frozen eggs, reference numeral 3 represents a liquid injection section, reference numeral 4 represents a liquid discharge section, and reference numeral 5 represents an egg outflow prevention section.

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

The egg outflow prevention section was made by folding a 40 mm long, 50 mm wide nylon mesh (330 mesh, 40 μm opening, AS ONE Corporation) in half at a point 25 mm long, and securing the horizontal edge to the Froze Bag F-050 using a heat sealer (P-300, Fuji Impulse Co., Ltd.). The nylon mesh was secured to the Froze Bag F-050 vertically, 25 to 50 mm from the end opposite the port-equipped end, and horizontally, 5 mm from the horizontal edge of the Froze Bag F-050, forming a wall. The nylon mesh was positioned so that the opening of the folded portion was located opposite the liquid inlet and outlet sections. Fertilized and unfertilized eggs cannot pass through the nylon mesh, but liquids such as freezing solution, thawing solution, and culture medium can pass through.

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

(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.

(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.

<Preparation of mouse fertilized eggs>
In this experiment, oviduct-perfused embryos at the two-cell stage, collected by oviduct perfusion as described below, were used as fertilized mouse eggs.

- 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 a similar 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 syringe needle (30 gauge) 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% CO ₂ , 37°C, 100% humidity).

[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...476mg
(Fujifilm Wako Pure Chemical Corporation)
・KCl...36mg
(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)
・_NaHCO3 ...211mg
(Fujifilm Wako Pure Chemical Corporation)
・ Sodium lactate... 0.53 mL
(60% syrup) (Sigma)
・D-glucose... 100mg
(Fujifilm Wako Pure Chemical Corporation)
・Penicillin G・・・5mg
(ICN Biomedics Inc.)
・Streptomycin... 7mg
(ICN Biomedics Inc.)
Phenol red (10 mg/ml soln.in saline) (Sigma)

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)
・_CaCl2.2H2O ... _25mg
(Fujifilm Wako Pure Chemical Corporation)
・BSA...500mg
(Sigma, GIBCO AlbuMAX)

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 the refrigerator.

[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...476mg
(Fujifilm Wako Pure Chemical Corporation)
・KCl...36mg
(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)
・_NaHCO3 ...211mg
(Fujifilm Wako Pure Chemical Corporation)
・Hepes・Na(basic)...520mg
(Sigma)
・ Sodium lactate... 0.53 mL
(60% syrup) (Sigma)
・D-glucose... 100mg
(Fujifilm Wako Pure Chemical Corporation)
・Penicillin G・・・5mg
(ICN Biomedics Inc.)
・Streptomycin... 7mg
(ICN Biomedics Inc.)
Phenol red (10 mg/ml soln.in saline) (Sigma)

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)
・_CaCl2.2H2O ... _25mg
(Fujifilm Wako Pure Chemical Corporation)
・PVA...10mg
(Sigma)

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.

<Freezing of mouse fertilized eggs and their placement in a frozen egg culture device>
The mouse fertilized eggs in the 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 returned to room temperature.

[Freezing liquid 1]
-FS liquid 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)

-EFS 20 liquid-
The FS liquid for EFS 20 was mixed with ethylene glycol in the following ratio to prepare EFS 20 liquid.
FS solution for EFS 20: 4 (v/v)
Ethylene glycol... 1 (v/v)
(Fujifilm Wako Pure Chemical Corporation)

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 Production Example 2 containing 50 μL of hyperosmotic Freezing Solution 2 (see below, sometimes referred to as "EFS 42.5c-d solution") that had been returned to room temperature.

[Freezing solution 2]
-FS liquid for EFS 42.5c-d-
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 42.5c-d. 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)

-EFS 42.5c-d liquid-
Ethylene glycol was mixed with the FS solution for EFS 42.5c-d in the following ratio to prepare EFS 42.5c-d solutions.
FS solution for EFS 42.5c-d: 57.5 (v/v)
Ethylene glycol... 42.5 (v/v)
(Fujifilm Wako Pure Chemical Corporation)

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 held there for 5 to 10 seconds to rapidly freeze them.

A mark was made on either the liquid inlet or outlet of the frozen egg culture device, and a hole was made with a tube. The frozen egg culture device was cooled in a gas layer of liquid nitrogen. The frozen egg storage container containing the frozen fertilized eggs was then placed in the storage section of the frozen egg culture device. Next, the open section of the frozen egg culture device (the end opposite the end having the liquid inlet and outlet sections) was sealed and packed with a top-bottom heat sealer (Fuji Impulse Co., Ltd., T-130K, T230K) and cooled with liquid nitrogen.

The frozen egg culture device was frozen in liquid nitrogen and then stored at -80°C.

The compositions of the above-mentioned PB1 (BSA-) and PB1 (BSA+) are as follows:
[PB1 (BSA-/+)]
(1) Ten PBS tablets (Takara Bio Inc.) were placed in a screw-cap bottle, and 1,000 mL of Milli-Q was added. This was dissolved and sterilized in an autoclave. Milli-Q was added to the bottle using a measuring cylinder to bring the total volume to 1,000 mL, and 1,000 mL of ^PBS was added. The solution was then returned to the screw-cap bottle.

(2) 10 mL of 100x concentrated _MgCl2 · _6H2O (FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved in 1,000 mL ^of the PBS- prepared in (1) above. Once completely dissolved, 10 mL of 100x concentrated _CaCl2 · _2H2O (FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved therein to prepare 1,020 mL of PBS ⁺ .

(3) The following reagents were thoroughly dissolved in 1,020 mL of the PBS ⁺ prepared in (2) above to prepare PB1 (BSA-).
<Composition of PB1 (BSA-) (per 80 mL)>
・PBS ⁺ ... 80mL
・ Sodium pyruvate ・・・ 2.9 mg
(Fujifilm Wako Pure Chemical Corporation)
Penicillin G: 5.1 mg
(ICN Biomedics Inc.)
・D-glucose... 80mg
(Fujifilm Wako Pure Chemical Corporation)

(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+).

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

[Melted liquid 1]
(1) 179.7 g of sucrose was dissolved in the PB1 (BSA-) in a 1,000 mL storage bottle (Corning) while 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 obtain Melting Solution 1 (hereinafter, sometimes referred to as "0.75 M sucrose-PB1 (BSA+)").

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

A tube with a needle was attached to the waste syringe (100 mL) to connect it to the liquid outlet of the frozen egg culture device.

A three-way stopcock was prepared and a tube with a needle attached was attached to connect to the liquid injection section of the frozen egg culture device. From the side of the three-way stopcock opposite the side with the needle attached, a syringe containing melting liquid 1, a syringe containing melting liquid 2, and a syringe containing culture medium were attached in that order.

- 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 culturing 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.

(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.

(3) The cock of the tube connected to the waste syringe was opened, and the injected melt 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.

(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.

(5) After carrying out 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, step (4) was repeated once more.

-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.

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
Survival rate (%) = (number of surviving embryos/number of recovered embryos) x 100
Blastocyst rate (%) = (number of blastocysts/number of retrieved embryos) x 100

As shown in Table 1 and Figure 5, it was confirmed that frozen eggs can be cultured to the blastocyst stage using the frozen egg culture device of the present invention.

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 using the frozen egg culture device of the present invention are of such high quality that they can be used to produce liveborn offspring.

In the fields of infertility treatment and livestock farming, it is not necessary 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 incubation device for 1 hour to fix the cells, and then the cells were washed 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.
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 distinctly. 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.

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.

<Example 2-1: Frozen egg culture device without adhesion prevention means (control)>
Culturing from frozen eggs to blastocysts was carried out in the same manner as in Example 1, except that in step (5) of -thawing and washing- (time and temperature until the second medium exchange) was set to 37±1°C and 1 to 2 hours.

<Example 2-2: Frozen egg culturing 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 produced in Production 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.

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).

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 a 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 embryo (12).

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 space until now.

The present invention includes, for example, the following aspects.
<1> A storage unit for storing 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 outflow prevention unit is disposed in the storage unit,
The frozen egg culture device according to <1>, wherein the side of the storage section where the container-containing frozen eggs are placed is separated from the liquid injection section and the liquid discharge section.
<3> The frozen egg culture device according to any one of <1> and <2>, wherein the container for the frozen eggs contained in the container has a V-shape when viewed from the front.
<4> The frozen egg culturing device according to any one of <1> to <3>, wherein the frozen eggs are frozen mammalian eggs.
<5> The frozen egg culturing device according to any one of <1> to <4>, wherein the storage unit has a means for preventing adhesion.
<6> A method for culturing frozen eggs, characterized by using the frozen egg culturing device according to any one of <1> to <5>.
<7> A thawing step of thawing frozen eggs;
A washing step of washing the thawed eggs;
The method for culturing frozen eggs according to <6>, further comprising: a culturing step of culturing the washed eggs.
<8> The method for culturing frozen eggs according to <7>, wherein the washing step is carried out at least twice.
<9> The method for culturing frozen eggs according to any one of <6> to <8>, wherein a culture medium having a _CO2 concentration of 3 to 6% is used.

1 Frozen egg culture device 2 Storage section 3 Liquid injection section 4 Liquid discharge section 5 Egg outflow prevention section 6 Syringe (for melting liquid 1)
7. Syringe (for melting liquid 2)
8. Syringe (for culture medium)
9. Syringe (for waste liquid)
10 Upper surface of storage section 11 Lower surface of storage section 12 Embryo 13 Adhesion prevention means

Claims (8)

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. The mammalian frozen egg culture device according to claim 1, wherein the liquid discharge unit is located at the same end as the end of the mammalian frozen egg culture device where the liquid injection unit is located. 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 . The storage section has a sticking prevention means for maintaining 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. A method for culturing frozen mammalian eggs, comprising using the mammalian frozen egg culturing device according to any one of claims 1 to 4. a thawing step of thawing frozen mammalian eggs;
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. The method for culturing frozen mammalian eggs according to claim 6, wherein the washing step is carried out at least twice. CO ₂ The method for culturing frozen mammalian eggs according to any one of claims 5 to 7, wherein a culture medium having a concentration of 3 to 6% is used.