JP6843343B2 - Mammalian embryo freezer - Google Patents

Description

The present invention relates to a mammalian embryo freezing device having a cooling treatment unit and the like. More specifically, it has a cooling means and a cooling processing unit, and the cooling processing unit is an elongated one capable of inserting a refrigerant tank containing a cooling medium and a straw tube containing a fertilized egg into a hollow. A tubular member is provided, and a plurality of tubular members penetrate the wall surface of the refrigerant tank and are formed so as to project into the tank. Therefore, the outer peripheral side of each tubular member is in contact with the cooling medium, and the hollow portion is in contact with the cooling medium. It relates to a mammalian embryo freezing device isolated from a cooling medium.

Embryo transfer (or fertilized egg transfer, Embryo Transfer; ET) is a breeding technique in which a fertilized egg (embryo) collected from a female or an embryo produced by in vitro fertilization is transferred to a separate female. Embryo transfer is widely used as a means of artificial breeding along with artificial insemination because it is useful for efficient use of eggs of excellent females and increase of production of specific varieties and strains.

Generally, embryo transfer is performed by the following procedure. First, the ovulatory female is overovulated by hormone administration. The female is then artificially inseminated, and after a few days, the fertilized egg is collected from the female's reproductive organs and cryopreserved. Next, after thawing the frozen embryo, the embryo is transplanted to a separate female (egg-bearing female) to become pregnant and give birth.

In addition, eggs are collected from females, fertilized in vitro, and after in vitro fertilization, the fertilized eggs are transplanted to separate females to produce pregnant embryos and give birth, and cloned embryos are produced by micromanipulation and transplanted to females. Research on techniques for pregnancy and childbirth is also progressing, and some have become practical techniques. In addition, a technique for sex selection by transplanting sex-determined embryos has already been put into practical use.

Techniques for cryopreserving embryos are needed to make embryo transfer widely available. By cryopreserving the embryo, long-term storage of the embryo becomes possible, which is advantageous for transportation and trading, and the transplantation time can be adjusted.

For example, in the case of the slow freezing method, which is the most widely used method for cryopreserving embryos at present, the embryos are stored in a straw tube, and the straw tube is extremely slow at 0.3 to 0.5 ° C / min up to around -30 ° C. After cooling at a rate, place in liquid nitrogen and store frozen.

At that time, for example, it is possible to manually adjust the cooling rate while pouring a small piece of dry ice into the alcohol tank, but a computer-controlled program freezer is usually used.

As a cooling method for the program freezer used for embryo freezing, a method using a refrigerant tank, a method using a Peltier element, a method using a Stirling type cooler, and the like have been proposed or put into practical use.

For example, Patent Document 1 is a program freezer that injects liquid nitrogen in a mist form to lower the temperature inside a chamber (refrigerant tank), and Patent Document 2 is a 1-liquid 2-tank type that uses an alcohol solution as a refrigerant. Patent Document 3 includes a straw tube freezing device for cooling by a cooling means using a Perche element, and Patent Document 4 includes a Sterling type cooler and a local cooling means using a Perche element. The freezing devices for the cells are described respectively.
JP-A-58-47975 Japanese Unexamined Patent Publication No. 2003-621 Japanese Patent Application Laid-Open No. 2007-97872 Japanese Unexamined Patent Publication No. 2012-34667

When the mammalian embryo is frozen using a program freezer or the like, for example, when cooling is performed in a refrigerant tank, when the straw tube is immersed in the refrigerant for cooling, the mammalian embryo housed in the straw tube is used. May be contaminated. On the other hand, for example, when cooling by a cooling means using a Peltier element, it is difficult to increase the number of processing lines (the number of straw tubes that can be cooled at the same time in one operation). Further, when cooling is performed by a Stirling type cooler, the degree of cooling may differ for each straw pipe or for each part in the straw pipe, and temperature unevenness is likely to occur. If temperature unevenness occurs when the mammalian embryo is frozen, the quality of the embryo is likely to deteriorate or deteriorate.

Therefore, the present invention provides a novel means that is less likely to cause contamination, can maintain the quality of mammalian embryos at a certain level or higher even after freezing, and can freeze mammalian embryos with relatively high efficiency. With the goal.

In the present invention, it is a mammalian embryo freezing device having a cooling means and a cooling processing unit, in which the cooling processing unit has a refrigerant tank containing a cooling medium and a straw tube containing the mammalian embryo in a hollow cavity. An elongated tubular member that can be inserted is provided, and a plurality of the tubular members penetrate the wall surface of the refrigerant tank and are formed so as to protrude into the tank, whereby the outer periphery of each tubular member is formed. Provided is a mammalian embryo freezing device or the like in which each side is in contact with the cooling medium and the inside of the hollow is isolated from the cooling medium.

The endothermic portion of the cooling means is brought into contact with the outer surface of the refrigerant tank to cool the cooling medium contained in the refrigerant tank. Then, the cooling medium in the refrigerant tank cools the tubular member from the outer peripheral side, so that the straw pipe inserted in the hollow of the tubular member is cooled.

In this device, the tubular member penetrates the wall surface of the refrigerant tank and is formed so as to protrude into the tank, and the hollow portion of the tubular member is completely isolated from the cooling medium in the refrigerant tank. Therefore, the cooling medium in the refrigerant tank is in contact with the outer peripheral side of the tubular member, but cannot enter the hollow. Therefore, since the straw tube inserted in the hollow of the tubular member does not come into contact with the cooling medium at all, it is possible to prevent the occurrence of contamination due to the cooling medium invading the straw tube, resulting in poor quality of the mammalian embryo. Deterioration and deterioration can be suppressed.

Further, by forming a plurality of tubular members so as to project into the refrigerant tank, the straw tubes can be cooled simultaneously by the number of tubular members in one operation, so that the number of simultaneous treatments can be increased and the mammalian embryo can be increased. It is possible to improve the efficiency of the freezing process.

Further, in the present invention, the cooling means is not brought into direct contact with the straw pipe, but the tubular member is cooled from the outer peripheral side by a cooling medium to cool the straw pipe in the hollow, so that the tubular member is tubular. The member can be cooled substantially uniformly over its entire length and from its entire circumference. Therefore, by inserting the straw tube in which the mammalian embryo is housed into the hollow of the tubular member, temperature unevenness is less likely to occur during the freezing treatment of the mammalian embryo. As a result, at the time of embryo transfer, the frequency of embryo quality deterioration / deterioration due to freezing / thawing of mammalian embryos can be reduced, and the embryo quality can be maintained at a certain level or higher. Therefore, it becomes possible to succeed in embryo transfer more reliably and stably.

In addition, since the tubular member can be cooled substantially uniformly over its entire length and from its entire circumference, its heat can be transferred substantially uniformly to the straw tube in which the mammalian embryo is housed. As a result, the solution around the mammalian embryo in the straw tube is cooled first, so that ice nuclei are planted and ice crystal formation is induced, so that ice can be easily planted without any additional operation. , Thereby, the occurrence of intracellular freezing (cell damage) of mammalian embryos can be suppressed.

In addition, in the present invention, since the straw pipe is not taken in and out of the cooling medium, and thereby, the refrigerant tank can be formed substantially closed and the cooling medium can be stored in the cooling medium. Is difficult to lose weight. Therefore, the present invention has an advantage that the amount of the cooling medium used can be significantly reduced, and the frequency and labor of replenishing the cooling medium can also be significantly reduced.

According to the present invention, contamination can be prevented from occurring, the quality of mammalian embryos can be maintained at a certain level or higher, and mammalian embryos can be frozen with relatively high efficiency.

The present invention is a mammalian embryo freezing device having a cooling means and a cooling processing unit, wherein the cooling processing unit has a refrigerant tank containing a cooling medium and a straw tube containing the mammalian embryo in a hollow cavity. An elongated tubular member that can be inserted is provided, and a plurality of the tubular members penetrate the wall surface of the refrigerant tank and are formed so as to protrude into the tank, whereby the outer periphery of each tubular member is formed. Each side is in contact with the cooling medium, and the inside of the hollow includes all the mammalian embryo freezing devices isolated from the cooling medium.

Hereinafter, examples of embodiments of the present invention will be described with reference to FIGS. 1 to 3. The present invention is not narrowly limited to the embodiments illustrated below.

FIG. 1 is a schematic partial external perspective view showing an example of a mammalian embryo freezing device according to the present invention, and FIG. 2 is a schematic vertical sectional view of the same partial. It should be noted that both drawings are individual schematic views, and their respective configurations and lengths do not necessarily correspond to each other.

The mammalian embryo freezing device A illustrated in FIG. 1 or FIG. 2 has a cooling means A1 and a cooling processing unit A2, and the cooling processing unit A2 in both figures includes a refrigerant tank 1 accommodating a cooling medium M and a cooling medium tank 1. It is provided with an elongated tubular member 2 capable of inserting a straw tube containing a mammalian embryo into the hollow 21, and a heat insulating member 3 surrounding the outer surface of the refrigerant tank 1. In FIG. 2, a cushioning member 4 is interposed between the refrigerant tank 1 and the heat absorbing portion A11 of the cooling means A1, and the stirring means 5 is arranged in the refrigerant tank 1. ..

The cooling means A1 is a heat source equipment for cooling. For example, the heat absorbing portion A11 of the cooling means A1, is contacted directly or via a cushioning member 4 to the outer surface 11 of the coolant vessel 1, by moving the heat to the refrigerant tank 1, it is accommodated in the coolant vessel 1 Cool the cooling medium M.

As the cooling means A1, a wide range of known cooling means A1 capable of adjusting the amount of heat absorption and thereby cooling the cooling medium M in the refrigerant tank 1 from at least 0 ° C. to about -30 ° C. Can be adopted. For example, when the cooling means A1 is a Stirling cooler, the entire mammalian embryo freezing device A can be made lighter and smaller, whereby the device can be easily transported and moved. At the same time, it can be installed and used in various places, so that convenience can be enhanced.

The cooling treatment unit A2 is a site for freezing the mammalian embryo. A straw tube containing a mammalian embryo is inserted into the hollow 21 of the tubular member 2 of the cooling processing unit A2, and the tubular member 2 is cooled from the outer peripheral 22 side by the cooling medium M in the refrigerant tank 1. Thereby, the mammalian embryo in the straw tube is frozen.

The refrigerant tank 1 is a portion for accommodating the cooling medium M, and the cooling medium M is stored and used in the tank 1.

The shape of the refrigerant tank 1 is not particularly limited as long as it is formed so as to accommodate and store the cooling medium M. For example, the external shape may be formed to be substantially box-shaped, substantially tubular, or the like. Further, for example, if an injection port for injecting the cooling medium M into the refrigerant tank 1 is formed, a top surface may be provided and the refrigerant tank 1 may be formed in a substantially closed state.

As the material of the refrigerant tank 1, a known material having resistance to extremely low temperatures and high thermal conductivity can be widely adopted. As the material of the refrigerant tank 1, for example, aluminum, aluminum alloy, copper, nickel, magnesium, stainless steel and the like can be adopted. They may be appropriately subjected to a known surface treatment or the like depending on the material or the like.

The endothermic portion A11 of the cooling means A1 is brought into contact with the outer surface 11 of the refrigerant tank 1, for example, a part of the bottom surface 12 or the side surface 13 of the outer surface of the refrigerant tank 1 directly or via the buffer member 4. Thus, the heat is moved into the coolant vessel 1, cooling the cooling medium M contained in the refrigerant tank 1.

The cooling medium M is a heat medium used to transfer the cold heat from the cooling means A1 to the tubular member 2 during cooling. As the cooling medium M, known ones can be widely adopted, and although not particularly limited, a liquid having a freezing point of at least -30 ° C. or lower, for example, alcohol such as methanol or ethanol can be preferably used.

The tubular member 2 is a portion into which a straw tube containing a mammalian embryo is inserted into the hollow 21.

The tubular member 2 is, for example, (1) a tubular and elongated hollow rod-shaped member, and (2) a tube so that a straw tube containing a mammalian embryo can be inserted into the hollow 21. (4) The outer peripheral 22 side thereof excludes at least one or both tip portions (reference numerals 211, or 212 and 212) so that the hollow 21 side of the shape member 2 is cut off from the inside of the refrigerant tank 1 without flowing. The entire surface is opened in the refrigerant tank 1 so as to be in contact with the cooling medium M stored in the refrigerant tank 1, and (5) at least one tip portion 211 of the hollow 21 is not blocked and is outside the refrigerant tank 1. It is formed as if it were open.

Therefore, the tubular member 2 is formed so as to penetrate the wall surface (for example, reference numeral 13) of the refrigerant tank 1 and project into the tank 1. As a method for forming the tubular member 2 so as to protrude into the refrigerant tank 1, known methods can be widely adopted and are not particularly limited. For example, as shown in FIGS. 1 and 2, the tubular member 2 may be arranged so as to penetrate the inside of the refrigerant tank 1. In this way, the tubular member 2 is allowed to penetrate the inside of the refrigerant tank 1 from one side surface 13 of the refrigerant tank 1 to the side surface 14 at the opposite position, and both tip portions 211 and 212 are located outside the refrigerant tank 1. A portion other than the above is arranged at a position in the refrigerant tank 1, and the portion (a through hole forming portion of the wall surface 13 of the refrigerant tank 1 and a tubular shape) is formed so that the cooling medium M does not leak from the penetrating portion. The tubular member 2 is also substantially sealed (the gap between the outer circumference 22 of the member 2) so that the outer peripheral 22 side of the tubular member 1 is in contact with the cooling medium M and the inside of the hollow 21 is isolated from the cooling medium M. Can be arranged.

At least one of the two tip portions 211 and 212 of the tubular member 2 is formed so as to be open to the outside of the refrigerant tank 1 so that the straw pipe can be inserted and removed. The other may be closed, or may be similarly formed so as to be open to the outside of the refrigerant tank 1.

The direction in which the tubular member 2 is projected can be appropriately determined and is not particularly limited. For example, as shown in FIGS. 1 and 2, the tubular member 2 may be arranged substantially parallel to the bottom surface of the refrigerant tank 1, that is, substantially horizontally.

The inner diameter of the tubular member 2 is such that the straw tube can be inserted into the hollow 21 of the tubular member 2, and the wall surface (inner wall surface) of the tubular member 2 on the hollow 21 side and the outer peripheral surface of the straw tube are formed at the time of insertion. Designed to be in close contact, eg, substantially identical (although only slightly larger than the outer diameter of the straw tube to be inserted).

As the material of the tubular member 2, a known material that can be formed in a substantially tubular shape, has resistance to extremely low temperatures, and has high thermal conductivity can be widely adopted. As the material of the tubular member 2, for example, aluminum, aluminum alloy, copper, nickel, magnesium, stainless steel and the like can be adopted. They may be appropriately subjected to a known surface treatment or the like depending on the material or the like.

In the present invention, a plurality of tubular members 2 are arranged. For example, as shown in FIG. 1 or 2, a plurality of tubular members 2 are formed so as to project from one side surface 13 of the refrigerant tank 1 in substantially the same direction, and are arranged substantially parallel in the vertical direction and / or the horizontal direction. The tubular member 2 may be arranged. As a result, the straw tubes can be cooled at the same time by the number of arrangements of the tubular members 2 in one operation, so that the number of simultaneous treatments can be increased and the freezing treatment of mammalian embryos can be made highly efficient. Although it depends on the dimensions of the refrigerant tank 1, for example, 50 to 250 tubular members 2 may be arranged in one refrigerant tank 1 in the arrangement as shown in FIG.

The heat insulating material 3 is a member for preventing heat transfer. By surrounding the outer surface 11 of the refrigerant tank 1 with the heat insulating material 3 and blocking the outside air from the refrigerant tank 1 as much as possible, the temperature of the cooling medium M in the refrigerant tank 1 can be stabilized, and the cold heat generated by the cooling means A1 can be stabilized. The loss can be suppressed as much as possible, and the heat absorption by the cooling means A1 can be made highly efficient.

As for the material of the heat insulating material 3, known materials can be widely applied and are not limited. The location of the heat insulating material 3 is also not particularly limited, but may be arranged on the bottom surface 12 and the side surface 13 of the refrigerant tank 1 as shown in FIG. 1 or 2, or may be further arranged on the top surface. May be good. In addition, for example, the heat insulating material 3 can be opened and closed at the opening portion of the tubular member 2 (the portion where the straw pipe is inserted, for example, reference numeral 211), the portion where the injection port for injecting the cooling medium M into the refrigerant tank 1 is arranged, and the like. Alternatively, it may be formed so as to be removable.

The cushioning member 4 shown in FIG. 2 is a member for alleviating the vibration generated when the cooling means A1 is being driven, and is an arbitrary component. By sandwiching the buffer member 4 between the heat absorbing portion A11 of the cooling means A1 and the refrigerant tank 1, it is possible to prevent the vibration generated while driving the cooling means A1 from being transmitted to the refrigerant tank 1. This makes it possible to reduce the possibility that the mammalian embryo housed in the straw tube will be damaged or deteriorated. As the cushioning member 4, a known member capable of absorbing vibration and having high thermal conductivity can be widely adopted.

The stirring means 5 in the refrigerant tank 1 shown in FIG. 2 is a member that stirs the cooling medium M. By stirring the cooling medium M by the stirring means 5, heat can be smoothly and efficiently transferred from the endothermic portion A11 of the cooling means A1 to the refrigerant tank 1, and temperature unevenness in the cooling medium M occurs. Can be suppressed. Therefore, the cold heat from the cooling means A1 can be uniformly and uniformly transferred to the tubular member 2 via the cooling medium M. As the stirring means 5, known ones can be widely adopted.

FIG. 3 is a diagram showing an example of a system configuration in the mammalian embryo freezing device A according to the present invention. The present invention is not narrowly limited to this configuration example.

In FIG. 3, the control means B includes a temperature control unit B1, a CPU (reference numeral B2), a memory B3, an input means B4, and the like, and is connected to the cooling means A1, the heating means C, the temperature detecting means D, and the like.

The control means B controls the operation of cooling the inside of the cooling processing unit A2 (inside the cooling medium M or the hollow 21 of the tubular member 2) according to a preset cooling processing procedure. The control means B may be formed of, for example, an electronic substrate and mounted in the mammalian embryo freezing device A.

At that time, for example, the temperature detecting means D is installed in the cooling processing unit A2 (inside the cooling medium M or the hollow 21 of the tubular member 2), and the control means B adjusts the cooling or heat treatment based on the temperature information. It may be set to.

For example, the temperature control unit B1 compares the assumed temperature at a specific timing in the preset cooling process procedure with the actual temperature detected by the temperature detecting means D, and the actual temperature is assumed at that timing. When it is determined that the temperature is higher than the threshold value, the temperature control unit B1 instructs the cooling means A1 to promote the drive to increase the amount of heat absorption, or to instruct the degree of the drive. The cooling means A1 receiving the command from the temperature control unit B1 promotes the drive so as to increase the amount of heat absorption, whereby the inside of the cooling processing unit A2 (inside the cooling medium M or the hollow 21 of the tubular member 2) is moved. Be cooled.

Further, for example, in the temperature control unit B1, the assumed temperature at a specific timing in the preset cooling process procedure is compared with the actual temperature detected by the temperature detecting means D, and both temperatures are within the threshold range. When it is determined that the temperature is inside, the temperature control unit B1 orders the cooling means A1 to stop its drive.

On the contrary, for example, in the temperature control unit B1, the assumed temperature at a specific timing in the preset cooling process procedure is compared with the actual temperature detected by the temperature detecting means D, and the actual temperature is the actual temperature. When it is determined that the temperature is lower than the assumed temperature at the timing, the temperature control unit B1 instructs the cooling means A1 to stop the driving, or drives the heating means C, or determines the degree thereof. Command.

For example, by describing the above steps in a program and storing them in the memory B3 or the like, automatic processing in the mammalian embryo freezing device A is possible. As the heating means C and the temperature detecting means D, known ones can be widely adopted.

In the present invention, examples of the mammalian embryo to be frozen include an in vitro fertilized egg collected after artificial fertilization, an in vitro fertilized egg (embryo) obtained by in vitro fertilization, a sex discrimination embryo, and a cloned embryo. The stage of the mammalian embryo is not particularly limited, but in general, when freezing for embryo transfer, those in the blastocyst stage to the expanded blastocyst stage are often frozen. The animal species is not particularly limited, and the present invention can be applied to freezing embryos of mammals including humans, but more preferably to freezing embryos of non-human mammals (non-human mammals). Therefore, it is more preferable to apply it to the freezing of the embryos of industrial animals (cattle, pigs, horses, sheep, goats, etc.), and most preferably to apply it to the freezing of the embryos of cattle.

FIG. 6 is a schematic partial external perspective view showing an example of a mammalian embryo freezing device according to the present invention. FIG. 6 is a schematic partial longitudinal sectional view showing an example of a mammalian embryo freezing device according to the present invention. The figure which shows the example of the system structure in the mammalian embryo freezing apparatus which concerns on this invention.

1 Refrigerant tank
11 The outer surface of the refrigerant tank
12 Same bottom
13 Same side
14 Side surface at a position facing side surface 13
2 Cylindrical member
21 Hollow tubular member
211, 212 Tip part (of hollow 21) of tubular member 2
22 Outer circumference of tubular member
3 Insulation member
4 cushioning member
5 Stirring means
A Mammalian Embryo Freezer
A1 Cooling means
A11 Endothermic part of cooling means
A2 Cooling processing unit
B control means
B1 temperature control unit
B2 CPU
B3 memory
B4 input means
C heating means
D Temperature detection means
M cooling medium

Claims (4)

A mammalian embryo freezing device having a cooling means and a cooling processing unit.
The cooling processing unit includes a refrigerant tank for accommodating a cooling medium and an elongated tubular member into which a straw tube containing a mammalian embryo can be inserted into a hollow.
Since the plurality of tubular members are formed so as to penetrate the wall surface of the refrigerant tank and project into the tank, the outer peripheral side of each tubular member is in contact with the cooling medium, and the hollow inside is the cooling medium. An isolated mammalian embryo freezer. The mammalian embryo freezing device according to claim 1, wherein the heat absorbing portion of the cooling means is brought into contact with the outer surface of the refrigerant tank to cool the cooling medium contained in the refrigerant tank. The mammal according to claim 1 or 2, wherein the cooling medium in the refrigerant tank cools the tubular member from the outer peripheral side, thereby cooling the straw pipe inserted in the hollow of the tubular member. Animal embryo freezing device. The mammalian embryo freezing device according to any one of claims 1 to 3, wherein the cooling means is a Stirling cooler.

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