JP2006149231A - Narrow tube for freeze preservation of biological specimen, freeze preservation method for biological specimen and method for melting after freeze preservation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a freeze preservation technique of a biological specimen, keeping the simplicity of conventional technique, to perform the freeze preservation of a biological specimen of an animal in a narrow tube such as a straw, carry out the melting and dilution of the biological specimen in the narrow tube and keep high survival ratio of the biological specimen. <P>SOLUTION: The narrow tube for freeze preservation comprises the freeze preservation of a vitrifying preservation liquid containing a biological specimen of an animal and a dilution liquid, the melting of the preserved specimen and the transplantation of the biological specimen to an animal. The dilution liquid 5 is filled from one end of the narrow tube body 2 to the other end in frozen state, and a linear supporting tool 3 holding the vitrifying preservation liquid 4 containing the biological specimen on the tip end is placed in the narrow tube body 2 in a manner to place the tip end of the linear supporting tool 3 adjacent to the end face of the frozen layer of the diluting liquid 5. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an artificial insemination technique for mammals, and more specifically, a cryopreservation tubule for storing and transplanting biological specimens such as animal eggs, embryos and tissue cells, a cryopreservation method using the same, and The present invention relates to a thawing method after cryopreservation.

  At present, for example, embryos used in bovine embryo transfer technology are transferred in accordance with the estrous cycle of the recipient cow. In order to match the embryo transfer with this estrous cycle, the produced embryo is stored in a frozen state in a cooling substance such as liquid nitrogen, and then the embryo is thawed and transplanted. As a freezing method for preserving the embryo in a frozen state, a slow cooling method is known in which the embryo preservation solution is slowly frozen to approximately −30 ° C. and slowly cooled while dehydrating free water in the cells of the embryo. ing. When preserving embryos frozen by this method, embryos that have been preserved in tubules such as plastic straws and thawed in the tubules are used for transplantation. Thus, tubules obtained by cryopreserving embryos can be injected into a female uterus of an animal and transplanted with embryos, so that they are used in many places as an industry.

  However, it is known that the viability of the embryo after thawing is reduced if the bovine embryo, especially in vitro fertilized embryo is frozen and stored by the slow cooling method. For this reason, there is a problem that the conception rate is reduced as compared with a fresh embryo that is not frozen. As a method of freezing embryos to solve the problem of such slow cooling method, embryo preservation solution containing high-concentration antifreeze agent is rapidly cooled with a cooling substance such as liquid nitrogen together with the embryo to form a glass. Vitrification methods that solidify are known. Embryos frozen and stored by this vitrification method can be stored for a long period of time while maintaining high viability, and several inventions relating to storage methods and storage devices for embryos frozen by vitrification have been made. (For example, refer to Patent Documents 1 to 3.)

  In Patent Document 1, a diluted solution is put into a straw for embryo transfer and cooled and frozen, and a vitrification cryopreservation solution is injected directly on it, and an embryo is injected at the same time or before and after that. A straw for transplantation of a mammalian embryo that has been cooled and vitrified with nitrogen and a method for producing this straw are described.

  Patent Document 2 discloses an intra-stray dilution type mammal in which a vitrification preservation liquid layer having a mammalian embryo inside and a dilution liquid layer are provided adjacent to each other in the straw, and an air layer is disposed at both ends thereof. An embryo vitrification storage straw is described.

  In Patent Document 3, a minimum amount of vitrification sufficient to encapsulate these embryos or eggs on the inner surface of a cryopreservation container such as a frozen straw, a frozen vial or a freezing tube in which mammalian embryos or eggs are sterilized is disclosed. A method for preserving mammalian embryos or ova that has been affixed with a liquid, the cryopreservation container sealed, and the container brought into contact with liquid nitrogen and rapidly cooled is described. In addition, a melting method is described in which the container is taken out from liquid nitrogen, one end of the container is opened, and a diluent is injected into the container to dissolve the freezing of the embryo or egg.

JP-A-5-176946 JP-A-10-248860 JP 2000-189155 A

  When cryopreserved using a straw for mammalian embryo transfer described in Patent Document 1, a large amount of vitrification cryopreservation solution that is highly toxic to the embryo is used, and it is vitrified after being immersed in this preservation solution. For this reason, the time required for cooling becomes long, and a highly toxic vitrification solution is touched in a non-vitrified state. For this reason, there is a possibility that the survival rate of the embryo decreases and becomes unstable. Moreover, since there are many preservation | save solutions, the survival time of an embryo may fall and it may become unstable by extending dilution time also at the time of a melt | dissolution.

  The mammalian embryo vitrification preservation straw described in Patent Document 2 is said to be able to increase the survival rate of mouse embryos, but the operation to be performed until vitrification is complicated. In addition, since it takes time to vitrify, the viability of the embryo is not stable and the viability may be lowered, as in the straw of Patent Document 1.

  In the storage method described in Patent Document 3, when the vitrification solution and the diluent are incorporated in the straw, the straw is poured into the liquid nitrogen, so that the diluent in the straw is rapidly cooled and frozen. It will be. At this time, there is a possibility of freezing with bubbles in the diluted solution, many bubbles are generated in the straw at the time of melting, the vitrification solution is insufficiently diluted and the viability of the embryo etc. decreases There is a risk that the straw may break during melting. In addition, when the straw is thin, pasting the embryo or egg in the vitrification solution to the inner surface of the straw is a cumbersome operation and it is uncertain whether the embryo in the vitrification solution has adhered to the straw. It is necessary to attach a lot of vitrification solution together with the embryo etc. in order to ensure the attachment. Furthermore, since vitrification with liquid nitrogen is performed through a straw, there is a possibility that the transfer of cooling heat is dull and the viability is lowered.

  As described above, conventional devices such as straws for freezing and storing bovine embryos and storage methods cannot maintain the viability of embryos after storage and thawing, and the conception rate after transplantation is reduced. There is. Such a problem is similarly caused not only in bovine embryos but also in biological specimens such as mammalian embryos.

  The problem to be solved by the present invention is that a biological specimen of an animal is cryopreserved in a thin tube such as a straw while maintaining the convenience in that a conventional straw is used, and the biological sample is stored in the thin tube. The objective is to establish a cryopreservation technique for biological specimens that can thaw and dilute biological specimens and maintain high viability of biological specimens.

  The tubule for cryopreservation of a biological specimen of the present invention stores a vitrification preservation solution and a diluent containing a biological specimen of an animal in a frozen state, and then thaws to transplant the biological specimen to an animal. For cryopreservation, in a frozen state, the diluent is filled from one end side to the other end side in the capillary body, and a vitrification storage solution containing a biological specimen is attached to the tip portion The linear support is characterized in that it is arranged in the thin tube main body so that the front end side of the linear support is adjacent to the end face of the diluted solution frozen layer.

  The amount of vitrification solution to be used is required to cover the specimen by attaching a vitrification solution containing biological specimens of animals (hereinafter referred to as “specimen”) to the tip of the linear support. Therefore, it is possible to shorten the time until the specimen is vitrified after being included in the vitrification preservation solution. Furthermore, at the time of thawing after cryopreservation, the vitrification stock solution can be quickly thawed and reliably diluted with a diluent. Therefore, since the time required for freezing and thawing is short and the dilution after thawing is reliable, the viability of the specimen can be kept high.

  Here, if an air layer that divides the diluent is provided in the capillary body, the diluent including the specimen can be reliably isolated in the capillary body from the time after dilution until transplantation. Further, it is possible to reliably prevent impurities from entering from the lower part of the thin tube.

  The specimen cryopreservation method of the present invention is a method of cryopreserving a vitrification preservation solution containing a specimen of an animal contained in a thin tube under a frozen state and a diluting solution, wherein the diluting solution is placed in the main body of the thin tube. The diluting solution freezing step that sucks and freezes, the vitrification freezing step in which the vitrification preserving solution containing the specimen is attached to the tip of the linear support tool and rapidly freezes, and the linear support tool after the vitrification freezing process are the same. A support arrangement process in which the tip side of the linear support is arranged in the thin tube main body so as to be adjacent to a terminal surface of the dilution liquid freezing layer in the thin tube main body, a vitrification storage solution containing the frozen specimen, and a dilution solution; A sealing step of sealing the narrow tube containing the gas, and a storage step of storing the thin tube after the sealing step in a cooling substance and storing it at a low temperature.

  The cryopreservation method according to the present invention is a method for cryopreserving a specimen using the aforementioned cryopreservation capillary. In cryopreservation, first, the diluted solution is sucked into the thin tube body and frozen. Then, the linear support tool which made the vitrification preservation solution adhere and frozen is arrange | positioned in a thin tube main body. Accordingly, the specimen can be reliably introduced into the thin tube main body, and the vitrification preserving solution and the diluent containing the sample can be reliably separated and arranged in the frozen state within the thin tube main body. In addition, it goes without saying that the above-mentioned effects can be obtained by using the cryopreservation capillary.

  Here, if the movement of the linear support tool in the thin tube main body in the support tool arranging step is performed by guiding a linear support tool made of a ferromagnetic material partially or entirely with a magnet, The linear support tool can be easily and reliably moved.

  The specimen thawing method of the present invention uses the cryopreservation tubule, and when thawing an animal specimen cryopreserved by the cryopreservation method, the sealed portion of the tubule previously immersed in a cooling substance is drawn out into the air. In this state, it is possible to discharge the gas generated during melting from the sealed part by heating and loosening the sealed part. After that, the tubule is taken out of the cooled substance and heated to contain the frozen specimen. The vitrification storage solution and the dilution solution are melted, and the vitrification storage solution is diluted with the dilution solution.

  The thawing method according to the present invention is a method in which a specimen is cryopreserved and then thawed using the cryopreservation capillary described above. Before the tubules after cryopreservation are heated and thawed, the capillaries of the capillaries are loosened to facilitate the discharge of the gas generated during thawing, and the clogging that occurs when the gas in the capillaries expands during thawing. It is possible to prevent protrusions of objects and breakage of thin tubes. In addition, it goes without saying that the above-mentioned effects can be obtained by using the cryopreservation capillary.

  Here, when the thin tube is heated to melt the vitrification stock solution and the diluted solution containing the frozen biological specimen, and the vitrification stock solution is diluted with the diluting solution, a line is drawn in the thawed dilution solution. By moving the shaped support, the vitrification preservation solution adhering to the linear support can be diluted with a surely melted diluent.

  In addition, if the linear support device is moved into the melted diluted liquid by guiding the linear support device, which is partially or entirely made of a ferromagnetic material, with a magnet, the linear support device is reliably melted. It can be moved into the diluting solution, further ensuring the melting and dilution of the vitrification stock solution. Further, when the linear support after the vitrification preservation solution is completely removed is taken out from the thin tube, it can be taken out by being guided out of the thin tube by a magnet.

  The greatest feature of the present invention is that the vitrification preserving solution containing the specimen is attached to the tip of the linear support, and is rapidly frozen and vitrified. It is to be introduced together with a linear support tool. In this way, the vitrification preservation solution containing the specimen is attached to the tip of the linear support, frozen, and introduced into the narrow tube, thereby reducing the amount of vitrification preservation solution used. The amount of time required for freezing can be reduced during freezing, the time required for thawing can be reduced during thawing, and dilution with a diluted solution after thawing can be performed in a short time and reliably. Can be achieved. Since the time required for freezing and thawing is short and the dilution after thawing is reliable, the viability of the specimen can be kept high.

  FIG. 1 is a diagram showing a cryopreservation capillary 1 according to a first embodiment of the present invention. As shown in FIG. 1, a cryopreservation capillary (hereinafter referred to as “capillary”) 1 in the present embodiment has a dilute solution 5 directed from one end to the other end in the capillary body 2 in a frozen state. The linear support 3 filled with the vitrification preservation solution 4 containing the sample attached to the tip 3a is a capillary tube so that the tip of the linear support 3 is adjacent to the end surface of the frozen layer of the diluent 5. Arranged in the main body 2.

  One end of the thin tube 1 on the side of the thin tube main body 2 on which the diluent 5 is filled is closed with a cotton plug 8, and the other end on the side on which the linear support 3 is disposed is covered with a sealing member 7. The entire narrow tube body 2 is sealed and frozen. Two air layers 6 are provided on the filling side of the diluting solution 5 near the cotton plug 8, and the diluting solution 5 is divided by the air layers 6a and 6b to provide a small amount of diluting solutions 5a and 5b. It has been.

  Here, the linear support tool 3 is demonstrated using FIG. 2A and 2B are diagrams showing the linear support 3, wherein FIG. 2A is a side view, FIG. 2B is a plan view, and FIG. 2C shows a state in which a specimen E encapsulated with the vitrification preservation solution 4 is held. A side view and (d) are top views which show the state which hold | maintained the sample E covered with the vitrification preservation | save liquid 4. FIG. The linear support tool 3 is a tool for freezing the vitrification preservation solution 4 containing the specimen E and introducing it into the thin tube main body 2, and as shown in FIG. 2, as shown in FIG. It is comprised by the metal part 3c.

  The main body portion 3b is a linear yarn-like fiber material made of polyamide fiber (for example, smooth surface nylon, etc.) having low temperature resistance, liquid nitrogen resistance, stretch resistance, and alteration resistance. The main body 3b has a total length of 5 to 40 mm and a diameter of 0.2 to 0.4 mm. The distal end portion 3a is an extension of the main body portion 3b, and is formed in a flat shape by providing a gradient of 20 to 40 ° at one end of the main body portion 3b. The width | variety of the front-end | tip part 3a is 0.3-0.6 mm, and length is 0.5-2.0 mm. In addition, the inner side of the front-end | tip part 3a (The side of the surface where the angle | corner with the main-body part 3b is an obtuse angle) is made into planar shape. Moreover, the front-end | tip part 3a is the same material as the main-body part 3b, and the thickness of the front-end | tip part 3a is about 0.1 mm.

  A metal part 3c made of a ferromagnetic material is provided at the other end opposite to the tip part 3a with respect to the main body part 3b. The metal part 3c is a steel pipe having an outer diameter of 0.5 to 0.8 mm and an inner diameter of 0.3 to 0.5 mm and having a length of 1 to 2 mm wrapped around the end part of the main body part 3b. In addition, since this metal part 3c is attracted | sucked to a magnet etc., the linear support tool 3 can be easily guide | induced into the thin tube main body 2 using a magnet etc.

  Therefore, in this linear support tool 3, since the flat tip portion 3a is formed with a gradient at one end of the main body portion 3b, the main body is set so that the plane of the flat tip portion 3a is substantially horizontal. While maintaining the state in which the portion 3b is held obliquely downward, the specimen E encapsulated with the vitrification preservation solution 4 is dropped and held on the upper surface of the flat tip portion 3a, and into the liquid nitrogen which is a cooling substance. By soaking, the vitrification preservation solution 4 containing the specimen E can be vitrified.

  Further, the vitrification preservation solution 4 containing the specimen E is attached to the tip 3a of the linear support 3 so that the amount of the vitrification preservation liquid used is the minimum amount necessary for the covering of the specimen E. Therefore, the time until the specimen E is vitrified after being included in the vitrification preservation solution 4 can be shortened. Furthermore, since the vitrification preservation solution 4 containing the specimen E and the diluent 5 are stored in a frozen state and then melted, the linear support 3 can be easily guided into the diluent 5, The vitrification stock solution 4 can be quickly melted and reliably diluted with the diluent 5. Therefore, since the time required for freezing and thawing is short and the dilution after thawing is reliable, the viability of the specimen E can be maintained high, and the conception rate can be improved while maintaining simplicity. It becomes. In this embodiment, the linear support tool 3 having the shape and material shown in FIG. 2 is used. However, as long as the vitrification preservation solution 4 including the specimen E can be adhered and guided to the inside and outside of the thin tube main body 2. However, the present invention is not limited to this. Moreover, although liquid nitrogen is used as a cooling substance, since there is a possibility that other substances can be used, it is not limited to this.

  Furthermore, by providing the air layer 6b, the diluted solution 5 including the specimen E can be surely divided in the thin tube main body 2 from the time after dilution until the transplantation. Since the nearest dilution liquid 5b retains contaminants such as cotton fibers in the cotton plug 8, contamination of the dilution liquid 5 can be reliably prevented, and the dilution liquid 5b flows into the animal body at the time of transplantation. Can be prevented. In addition, when the specimen E is sedimented by gravity and is present near the lower end of the diluent 5, the air layer 6 a and the diluent 5 a can be provided to prevent an extreme descent in the narrow tube 1. Can be positioned upward. For this reason, when extruding the diluent 5 at the time of transplantation, it becomes easy to extrude the specimen E from the tubule 1 and a decrease in conception rate can be suppressed. Further, if the diluent 5a is further pushed out after the diluent 5 containing the specimen E is pushed out at the time of transplantation, the diluent 5 attached to the inner wall of the thin tube main body 2 can be pushed out reliably. Here, the amount of the diluent 5a is only required to prevent the specimen E from being extremely lowered in the narrow tube 1, and is approximately 20% of the diluent 5 in this embodiment.

(Method for cryopreserving specimens)
A method for cryopreserving a specimen will be described with reference to FIG. FIG. 3 is a diagram showing a step of cryopreserving the vitrification preservation solution 4 and the dilution solution 5 containing the specimen E in a state where they are built in the thin tube 1. In this cryopreservation method, a diluting solution freezing step in which the diluting solution 5 is sucked into the thin tube body 2 and frozen, and a vitrification freezing solution in which the vitrification preserving solution 4 including the specimen E attached to the linear support 3 is vitrified is used. This is a cryopreservation method including a process, a support arrangement process for introducing and arranging the linear support 3 in the thin tube main body 2, a sealing step for sealing the thin tube main body 2, and a storage step for preserving the thin tube 1 at a low temperature. . Details will be described below.

1) Diluent freezing step (dilution liquid aspiration and freezing)
First, the diluted solution 5 is absorbed by the cotton plug 8 on one end side of the 0.25 ml-capillary main body 2, and the diluted solution 5 is sucked so that the air layer 6 having a length of 15 mm to 20 mm can be taken on the opposite side of the cotton plug 8. To do. Next, as shown in FIG. 3 (a), the dilute solution 5 is placed on the approximately 10 mm-thick polystyrene foam plate 13 floated on the liquid nitrogen 12 in the container 11 with the thin tube main body 2 lying sideways. After slowly freezing in the liquid nitrogen gas 12 a, it is immersed in the liquid nitrogen 12. By slowly freezing the diluent 5, the diluent 5 can be frozen without containing bubbles, and the generation of bubbles at the time of melting can be suppressed.
Here, the diluent 5 is referred to as Dulbecco's phosphate buffer (hereinafter referred to as “D-PBS”) to which 0.5 M sucrose and 20% calf serum (hereinafter referred to as “CS”) are added. ). The concentrations of the diluents 5a and 5b are the same as the concentration of the diluent 5.

2) Vitrification freezing process (attachment and vitrification of the vitrification preservation solution including the specimen to the linear support)
The specimen E is immersed in a pretreatment A solution and a pretreatment B solution described later for 5 minutes each. Further, the specimen E is immersed in the vitrification preservation solution 4 and sucked into a Pasteur pipette 9, and while being confirmed with a stereomicroscope, as shown in FIG. The vitrification preservation solution 4 containing the specimen E is dropped on the tip portion 3a of 3. Thereafter, as shown in FIG. 3 (c), 30 seconds after the specimen E is immersed in the vitrification preservative 4, the linear support 3 is quickly put into the liquid nitrogen 12 and the vitrification preservative 4 Vitrification is performed. Here, it shows below about the composition of each liquid.
Pretreatment A solution is D-PBS supplemented with 10% glycerol, 0.1 M sucrose, 0.1 M xylose and 1% polyethylene glycol, and Pretreatment B solution is 10% glycerol, 10% ethylene glycol, 0% D-PBS supplemented with 2M sucrose, 0.2M xylose and 2% polyethylene glycol. Vitrification stock solution 4 is D-PBS supplemented with 20% glycerol, 20% ethylene glycol, 0.3M sucrose, 0.3M xylose and 3% polyethylene glycol.

3) Support tool placement process (introduction into the narrow tube body)
The thin tube main body 2 is held in a state of standing still horizontally in the liquid nitrogen 12, and the linear support tool 3 vitrified with the vitrification preservation solution 4 including the specimen E is passed from the distal end portion 3a side to the opening of the thin tube main body 2. Then, the linear support 3 is indirectly guided from the outside by the magnet 15 to the end face of the frozen layer of the diluent 5 in the thin tube body 2. The magnet 15 can be operated using tweezers 14.

4) Sealing step As shown in FIG. 3 (d), the liquid nitrogen 12 is previously filled in another dewar bottle 16 so as to have a depth corresponding to a length of about 15 mm shorter than the length of the thin tube main body 2. The thin tube body 2 is quickly transferred to the dewar 16 using the tweezers 14 with the cotton plug 8 side of the thin tube body 2 into which the linear support 3 is inserted. The liquid nitrogen present in the thin tube main body 2 is vaporized and sealed with the powder paste and the sealing member 7 to obtain the cryopreservation thin tube 1. Here, the sealing member 7 should just be a thing which can be pushed into a 0.25 ml plastic straw with frost resistance, and can be fixed with powder glue, and a thing with low temperature resistance, such as a polypropylene, is preferable.

5) Storage step The capillary tube 1 is placed with the cotton plug 8 side down, transferred to a liquid nitrogen cylinder (not shown), and stored in the liquid nitrogen cylinder.

  By the method as described above, the specimen E can be reliably introduced into the thin tube main body 2 and the vitrification preservation solution 4 and the diluent 5 containing the specimen E are reliably separated in the thin tube main body 2 in a frozen state. Can be placed and stored frozen. In addition, about component composition, such as the dilution liquid 5 used in this embodiment, the vitrification preservation | save liquid 4, etc., an example is shown and a component and a density | concentration can be adjusted according to a condition.

(Method of melting specimen)
A method for melting specimen E will be described with reference to FIG. FIG. 4 is a diagram illustrating a process of melting the specimen E in the thin tube 1. In this melting method, the sealed portion of the thin tube 1 is brought upright in a state where it is drawn out into the air, and the sealed portion is heated and loosened to allow the gas generated during melting to be discharged from the sealed portion. In this method, the vitrification preservation solution 4 and the dilution solution 5 are melted by heating, and the vitrification preservation solution 4 is diluted with the dilution solution 5. Details will be described below.

1) Excess gas discharge step As shown in FIG. 4 (a), only the sealed portion of the thin tube 1 is picked up from the liquid nitrogen of a cryopreserved cylinder (not shown), and is brought upright in a state of being drawn out into the air. While holding the sealing part with the finger 21, the glue of the sealing member 7 is warmed and loosened, and the gas generated during melting can be discharged from the sealing part.

2) Melting step As shown in FIG. 4B, the diluting solution 5 is melted by immersing it in the warm water 23 at 20 to 25 ° C. while keeping the narrow tube 1 upright. At this time, the vitrification preservation solution 4 also melts.

3) Dilution step As shown in FIG. 4 (c), the linear support 3 is guided by the magnet 15 from the outside of the thin tube body 2 and immersed in the diluted solution 5 in which the tip 3a of the linear support 3 is melted. The specimen E is melted and the vitrification stock solution 4 is diluted. At this time, since the case where the vitrification preservation solution 4 including the specimen E adheres to the inner wall surface of the thin tube main body 2 immediately after melting and is not diluted is considered, as shown in FIG. Then, the diluted solution 5 is pushed up by pushing the cotton plug 8 about 5 mm with the metal fitting 24, and the vitrification storage solution 4 is completely immersed in the diluted solution 5, so that it can be surely diluted. Further, if the vitrification preservation solution 4 including the specimen E has a specific gravity higher than that of the dilution solution 5, the vitrification preservation solution 5 is lowered by itself and the vitrification preservation solution 5 is diluted in the dilution solution 5.

4) Lifting step of the linear support tool After confirming that the vitrification preservation solution 4 including the specimen E completely separated from the linear support tool 3 and moved into the dilution liquid 5, it is shown in FIG. 4 (e). As described above, the sealing portion of the thin tube 1 is cut with a cutter, and the linear support 3 is guided using the magnet 15 to be taken out of the thin tube 1.

  As described above, by loosening the sealing portion of the thin tube 1 before heating the thin tube 1 after cryopreservation, the gas generated during melting can be easily discharged, and the gas in the thin tube 1 expands during melting. This makes it possible to prevent the thin tube 1 from being damaged. Moreover, the vitrification preservative 4 can be rapidly thawed and diluted by melting and dilution using the linear support 3 that has been frozen by attaching a small amount of the vitrification preservative 4 containing the specimen E. Thereby, it is possible to perform melting, dilution and transplantation work even at the production site while maintaining the viability of the specimen E high.

  Next, the thin tube 31 which is the 2nd Embodiment of this invention is demonstrated using FIG. As shown in FIG. 5, since the thin tube 31 has the same reference numerals in the frozen state except for the diluents 25, 25a, and 25b, description thereof is omitted. The dilution liquid 25 is the same composition liquid as the dilution liquid 5 (first embodiment), and the dilution liquids 25a and 25b are the dilution liquid 25 that does not contain 0.5M sucrose and have a low concentration. .

  In the present embodiment, the dilution liquid 25 and the dilution liquid 25a are set to substantially the same volume (liquid length A = liquid length A). At the time of melting, the vitrification preservation solution 4 is diluted with the diluent 25 as a first step, and the specimen E remains in the diluent 25 until transplantation. Next, as a second stage, the diluent 25 and the diluent 25a are mixed at the time of being pushed out into the animal body (in the uterus). Here, the vitrification preservation solution 4 containing the specimen E is diluted from the thick dilution liquid 25 to the mixed liquid of the dilution liquid 25 and the dilution liquid 25a having a low concentration step by step, thereby osmotic stress of the specimen E. Can be eliminated and the survival can be improved. Further, the amount of each diluted solution is not limited to this, and the composition and the amount of each diluted solution can be changed, and dilution suitable for each specimen becomes possible.

  By virtue of vitrification using the tubule 1 in the first embodiment of the present invention, the vitrification preservation solution containing the in vitro fertilized embryo as a specimen is vitrified, cryopreserved, thawed, and survival of the in vitro fertilized embryo The sex was investigated. As a control, the slow-cooling method was used to investigate the viability of the in vitro fertilized embryos that had been frozen and stored after thawing the slow-chilled preservation solution containing the in vitro fertilized embryos.

(1) In vitro fertilized embryo production Within 6 hours of slaughter from cow ovaries, cumulus cell ovum complex was aspirated together with D-PBS supplemented with bovine serum albumin. Morphologically good cumulus cell ovary complex was washed several times in 199 medium supplemented with CS (hereinafter referred to as “ovum maturation liquid”) and then matured in vitro with this oocyte maturation liquid for 20 to 22 hours. .
For in vitro fertilization, thawed frozen semen was diluted with bracket orphanant solution supplemented with theophylline and caffeine, centrifuged twice, washed and concentrated, and sperm concentration with bracket orphanant solution supplemented with bovine serum albumin and heparin Was prepared, and mature eggs were introduced for 4 hours.
Oocytes fertilized in vitro were developed and cultured until 8 days of fertilization. The culture gas phase conditions for embryo production were 5% CO ₂ , 95% air and 38.5 ° C.

(2) Test Embryo For in vitro fertilized embryos, expanded embryos with morphologically uniform monolayers of trophoblast cells and a solid inner cell mass on the 7th or 8th day after in vitro fertilization A blastocyst stage embryo was used as a specimen, washed with 20% CS-added D-PBS (hereinafter referred to as “preservation solution”), and then subjected to cryopreservation by a slow cooling method and a vitrification method.
As for in-vivo fertilized embryos, high-quality in-vivo fertilized embryos collected by artificial insemination after superovulation were washed with a preservation solution, and then subjected to freezing preservation by a slow cooling method and a vitrification method.

(3A) Embryo cryopreservation method by slow cooling method Each embryo (in vitro fertilized embryo, in vivo fertilized embryo) obtained as described above is introduced into a slow cooling preservation solution, equilibrated for 15 minutes, and then a 0.25 ml tubule body (plastic) (This is a straw made and referred to as “straw” hereinafter.) Using a program freezer, a straw was set in a refrigerant at −7 ° C., and forced ice planting was performed using tweezers cooled with liquid nitrogen after 2 minutes. After 10 minutes of forced ice planting, the mixture was slowly cooled at 0.3 ° C./min. After reaching −30 ° C., a straw was introduced into liquid nitrogen and stored.
Here, the slow cooling storage solution is a storage solution (20% CS-added D-PBS) added with 10% ethylene glycol and 0.1M sucrose, but the concentration and the like can be changed depending on the situation.

(3B) Embryo cryopreservation method by vitrification Each embryo (in vitro fertilized embryo, in vivo fertilized embryo) obtained as described above was subjected to the above-described specimen cryopreservation method.

(4A) Thawing method of embryos cryopreserved by the slow cooling method (slow cooling frozen embryos) In order to investigate the viability of the embryos after thawing, the straws cryopreserved by the slow cooling method were taken out from liquid nitrogen and kept at room temperature for 6 seconds. Then, the embryo was thawed with a slow cooling preservation solution by maintaining it in 30 ° C. hot water for 20 seconds. Next, hold the thawed straw at room temperature for 5 minutes, cut the sealed side with a straw cutter, apply pressure from the cotton plug side of the straw, push the embryo into the petri dish with a slow cooling stock solution, and store the embryo at 37 ° C. Was introduced to dilute the slow cooling stock solution.

(4B) Thawing Method of Embryo Cryopreserved by Vitrification Method (Vitrified Frozen Embryo) Thawing was performed by the above-described method for thawing a specimen. In order to investigate viability, pressure was applied to the cotton plug 5 minutes after the start of melting (when the straw was taken out from the liquid nitrogen cylinder), the embryo was pushed into the petri dish with the diluent, and the embryo was stored at 37 ° C. Introduced and saved.

(5) Thawed Embryo Culture Thawed and diluted slow-chilled frozen embryos and vitrified frozen embryos were washed with a culture solution and cultured for 72 hours in a culture solution drop of 20 μl per embryo. The culture gas phase conditions were 5% CO ₂ , 95% air and 38.5 ° C.
Here, the culture solution is a 199 medium supplemented with 100 μM β-mercaptoethanol and 20% fetal calf serum.

(6) Survivability of thawed embryos Survival, development and status of thawed in vitro fertilized embryos were observed 24 hours, 48 hours and 72 hours after the start of culture. Of the embryos with zona pellucida escape completed 72 hours after thawing, those with good growth of trophoblast cells and inner cell mass were designated as high quality zona pellucida escape embryos.

(7) Embryo transfer After thawing slow-cooled frozen embryos (in vitro fertilized embryos, in vivo fertilized embryos) and vitrified frozen embryos (in vitro fertilized embryos, in vivo fertilized embryos), attach a straw to the transplanter and thaw About 5 minutes after the start, the ovarian uterine horn was transplanted into the uterine horn of the luteal side 7 days after estrus. In addition, two embryos in the case of in vitro fertilized embryos and one embryo in the case of in vivo fertilized embryos were encapsulated at the time of cryopreservation and transferred to one straw.

(8) Survival after thawing of preserved embryos Table 1 shows the survival rate of embryos after warming or thawing of preserved embryos. The survival rate over time of in vitro fertilized embryos vitrified and frozen by vitrification is 96% (slow cooling: 69%) in culture for 24 hours and 96% in 48 hours compared to slow-cooled frozen embryos by slow cooling (Slow cooling: 69%) and 94% (slow cooling: 66%) at 72 hours.

(9) Zona pellucida escape after thawing of preserved embryos Table 2 shows the zona pellucida escape rate after thawing of in vitro fertilized embryos. The zona pellucida escape rate of vitrified embryos thawed and diluted in a straw is 67% (slow cooling: 29%) at 48 hours of culture and 89% (slow cooling: 51%) at 72 hours compared to the slow cooling method. It was significantly higher and the growth was good. Also, the morphology of zona pellucida escaped embryos had a significantly high quality zona pellucida escape rate (quality zona pellucida escape rate; 84%: 32%).

(10) Conception Result by Embryo Transfer Table 3 shows the conception result by in-situ melting transplantation of in vitro fertilized embryos. The results of fertilization of thawed embryos after 2 cold-cooled or vitrified embryos were stored in a straw and then thawed at the transplantation site were 35% (15/43) for slow cooling and 61% (17/28) for vitrification. And vitrification got significantly higher conception rate.
In addition, Table 4 shows the results of conception by in-situ melting transplantation of in-vivo fertilized embryos. As in the case of in vitro fertilized embryos, the results of thawing and transplanting one in-bovine fertilized embryo that had been slowly cooled or vitrified were 54% (7/13) with slow cooling and vitrification. Is 50% (4/8), and no significant difference is seen, but by this method, a high fertility rate was obtained by thawing and transplanting vitrified bovine embryos in a straw.

  The cryopreservation tubule of the present invention can be widely used for transplantation into mammals while maintaining high viability of biological specimens, and is particularly effective when used for transplantation of bovine embryos.

It is a figure which shows the thin tube which is the 1st Embodiment of this invention. It is a figure which shows a linear support, Comprising: (a) is a side view, (b) is a top view, (c) is a side view which shows the state holding the specimen covered with the vitrification preservation solution, (d) FIG. 3 is a plan view showing a state in which a specimen encapsulated with a vitrification preservation solution is held. It is a figure which shows the process of cryopreserving in the state which contained the vitrification preservation | save liquid containing a sample, and a dilution liquid in the thin tube. It is a figure which shows the process of melt | dissolving a sample within a thin tube. It is a figure which shows the thin tube which is the 2nd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,31 Narrow tube for cryopreservation 2 Narrow tube main body 3 Linear support 3a Tip part 3b Main body part 3c Metal part 4 Vitrification preservation solution 5, 5a, 5b, 25, 25a, 25b Dilution liquid 6a, 6b Air layer 7 Sealing member 8 Cotton plug 9 Pasteur pipette 11 Container 12 Liquid nitrogen 12a Liquid nitrogen gas 13 Styrofoam plate 14 Tweezers 15 Magnet 16 Dewar bottle 21 Finger 22 Gas 23 Mild hot water 24 Press fitting

Claims (7)

A cryopreservation tubule for storing a vitrification preservation solution and a diluent containing an animal biological specimen in a frozen state, and then thawing and transplanting the biological specimen to the animal,
In the frozen state, the linear support is filled with the dilute solution from one end to the other end in the main body of the capillary tube, and the vitrification solution containing the biological specimen is attached to the tip. A thin tube for cryopreservation of a biological specimen, characterized in that the distal end side of the device is disposed in the thin tube main body so as to be adjacent to the end surface of the frozen layer of the diluent.   The thin tube for cryopreservation of a biological specimen according to claim 1, wherein an air layer for dividing the diluent is provided in the thin tube main body. A method for cryopreserving a vitrification preservation solution and a diluting solution containing a biological specimen of an animal contained in a capillary under a frozen state,
Dilution freezing process that sucks and freezes the diluting liquid in the thin tube body, vitrification freezing process that rapidly freezes by attaching a vitrification preservation solution containing biological specimens to the tip of the linear support, and vitrification A supporting tool arranging step of placing the linear support after the freezing process in the thin tube main body so that the tip side of the linear support tool is adjacent to the end surface of the diluent freezing layer in the thin tube main body, and the living organism in the frozen state A sealing step of sealing a capillary tube containing a vitrification preservation solution containing a biological specimen and a diluent, and a storage step of storing the capillary tube after the sealing step in a cooling substance and storing it at a low temperature. To cryopreserve biological specimens.   4. The biological specimen according to claim 3, wherein the movement of the linear support in the thin tube main body in the support placement step is performed by guiding a linear support made of a ferromagnetic material partially or entirely by a magnet. Cryopreservation method.   In thawing the biological specimen of an animal cryopreserved by the cryopreservation method according to claim 3 or 4 using the cryopreservation capillary according to claim 1, a sealed portion of the capillary previously immersed in a cold substance is used. By letting it stand upright in the air and warming and loosening the sealing part, the gas generated during melting can be discharged from the sealing part, and then the capillary tube is taken out of the cooling substance and heated to freeze. A method for thawing a biological specimen after cryopreservation, characterized by melting a vitrification preservation solution and a diluent containing the biological specimen prepared, and diluting the vitrification preservation solution with the diluent.   When a thin tube is heated to melt the vitrification stock solution and the diluted solution containing the frozen biological specimen and dilute the vitrification stock solution with the diluting solution, the linear support is in the thawed dilution solution. The method for thawing a biological specimen after cryopreservation according to claim 5, wherein   The biological support after cryopreservation according to claim 6, wherein the linear support is moved into the melted diluent by inducing a linear support made of a ferromagnetic material partially or entirely by a magnet. How to melt the specimen.

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