JP2005040073A - Method and container for freeze-storing reproductive cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for freeze-storing reproductive cells in high viability, and to provide a container therefor. <P>SOLUTION: The method comprises the following practice: Reproductive cells 2 immersed in a vitrified liquid are dripped together with the vitrified liquid onto an absorptive material 3 to absorb the vitrified liquid adhered to the cells 2 into the absorptive material 3. The reproductive cells 2 are then immersed in a liquid nitrogen while holding the cells 2 on the absorptive material 3, vitrified and freeze-stored. In this method, the absorptive material 3 consists of filter paper, and the reproductive cells 2 consist of e.g. bovine blastocyst. The freeze-storage container 1 comprises a container body 4 having a large-diameter recess 6 mounted with the absorptive material 3, a small-diameter recess 7 at the bottom of the large-diameter recess 6, a wall member 8 and a groove 9, and a lid 5 having a top plate 11 for closing the container body 4, a skirt 12 and a recess 13. In the container body 4, the outer surface of its bottom is provided with an engagement groove 10 for fixing the container body 4 through engaging it on a projection 27 provided at a storage place 26. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for cryopreserving germ cells such as sperm, eggs, embryos, etc. taken out of the body for artificial insemination or embryo transfer of mammals, and a container for cryopreservation.

  In mammals, in order to inject and fertilize germ cells such as sperm, eggs, and embryos taken out of the body into the female body, the female sexual cycle must be in a state suitable for conception. Therefore, in order to perform artificial insemination or embryo transfer at an appropriate time, it is necessary to store the germ cells alive in a state where metabolism is stopped, for a long period of time. Has been done. As a method for freezing the germ cells, for example, a slow freezing method is known.

  In the slow freezing method, first, the germ cells are immersed in a preservation solution in which a freezing agent having a concentration of 1 to 2 mol / l is added to a physiological solution such as phosphate buffered saline (PBS) at room temperature. To do. As the antifreeze agent, compounds such as glycerol and ethylene glycol are used. The antifreeze has the effect of lowering the freezing point of the solution and making it difficult to freeze by increasing the molar concentration of the solution inside and outside the cell.

  When the germ cells are immersed in the preservation solution, the cell membrane is more permeable to moisture than the freezing agent, so that the water in the germ cells flows out into the preservation solution immediately after the immersion, The osmotic pressure is maintained at isobaric pressure. The germ cells initially contract due to the outflow of water, but then gradually expand as the cryoprotectant permeates into the germ cells, restoring volume. The germ cells are immersed in the preservation solution until the cryoprotectant concentration inside and outside the cells reaches equilibrium.

  Next, the germ cells are filled in a container such as a thin straw together with the preservation solution, cooled to a temperature slightly lower than the freezing point of the preservation solution, and then ice crystals are formed in the preservation solution outside the germ cells. (Ice planting). The ice planting can be performed, for example, by bringing tweezers immersed in liquid nitrogen into contact with the container.

  When ice crystals are formed in the preservation solution outside the germ cells by the ice planting, the ice crystals are composed only of water, so that the preservation solution is concentrated, resulting in a difference in osmotic pressure inside and outside the germ cells. . As a result, moisture in the germ cells flows into the preservation solution, and a function of maintaining the osmotic pressure at an equal pressure occurs, and the solution in the germ cells is also concentrated. In addition, the water | moisture content which flowed out in the said preservation | save liquid from the said germ cell forms an ice crystal with the water | moisture content in the said preservation | save liquid.

  Therefore, by cooling the container to −30 to −35 ° C. at a rate of 0.3 to 0.5 ° C./min, the solution inside and outside the germ cells is sufficiently cooled and the viscosity becomes high. In such a state, when the container is further cooled to the temperature of liquid nitrogen (-196 ° C.), vitrification in which both the germ cell and the surrounding micro solution are amorphous and remain solid. Happens. When the inside and outside of the cell are solidified by vitrification, the movement of the molecule is substantially lost. Therefore, it is considered that the vitrified germ cell can be stored semipermanently by storing it in liquid nitrogen.

  However, in the slow freezing method, the process of cooling the container to −30 to −35 ° C. at a rate of 0.3 to 0.5 ° C./min must be performed by computer control, and a large-scale apparatus is required. There is a problem. In the slow freezing method, since ice crystals are formed in the preservation solution outside the germ cells, the germ cells may be physically damaged by the ice crystals.

  Then, in order to solve the said problem, the vitrification freezing method is proposed (for example, refer nonpatent literature 1). In the vitrification freezing method, the germ cells are immersed in a vitrification solution obtained by adding a high-concentration antifreezing agent of about 8M to a physiological solution such as PBS, and kept at a predetermined temperature for a predetermined time. Is cooled in liquid nitrogen together with the vitrification solution.

  The vitrification liquid easily vitrifies because it contains a high concentration of antifreeze. Therefore, in the vitrification freezing method, the solution inside and outside the germ cells can be easily cooled by cooling to the temperature of liquid nitrogen while allowing the vitrification solution in an amount suitable for vitrification to pass through the germ cells. There is an advantage that it can be vitrified and does not require a large-scale device such as computer control. Further, since the vitrification solution contains a high concentration of antifreezing agent and no ice crystals are formed even when cooled to the temperature of liquid nitrogen, there is no possibility that the germ cells are physically damaged by the ice crystals.

However, the vitrification freezing method has a disadvantage that the germ cells stored in liquid nitrogen may be damaged.
Sonzaburo Kasai, “Frozen preservation of fertilized eggs-slowing and vitrification method”, livestock artificial insemination, Japan Livestock Artificial Insemination Association, November 1997, No. 183, p. 17-19

  An object of the present invention is to provide a method capable of eliminating such inconvenience and cryopreserving germ cells such as sperm, ovum and embryo taken out of the body with excellent viability.

  Another object of the present invention is to provide a container for cryopreserving the germ cells.

  In the vitrification freezing method, a cryoloop method, a gel loading tip method, a nylon mesh method, a cryotop method, a microdrop method, an open pull are used as a method for immersing the germ cells immersed in the vitrification solution in liquid nitrogen. Various methods such as the Do method and the minimum drop size technique have been proposed. In each of the above methods, the germ cells are immersed in liquid nitrogen together with the vitrification solution.

  In each of the above methods, the germ cells before being immersed in liquid nitrogen are present in the vitrification solution, but the vitrification solution is also solidified in the liquid nitrogen. In addition, when the germ cells stored in liquid nitrogen are melted, the solidified vitrification solution becomes a liquid. At this time, if the phase transition between the liquid phase and the solid phase occurs abruptly, a fracture surface is generated due to a difference in expansion rate or contraction rate between the vitrification solution and the germ cell, and the fracture surface causes the germ cell to pass through the germ cell. The germ cells are believed to be damaged when passed.

  Therefore, in order to achieve the above object, the germ cell cryopreservation method of the present invention drops germ cells immersed in a vitrification solution onto the absorbent together with the vitrification solution and adheres to the periphery of the germ cells. After the vitrification solution is absorbed by the absorbent material, the germ cells are vitrified by being immersed in liquid nitrogen while being retained on the absorbent material.

  According to the present invention, the germ cells immersed in the vitrification solution are dropped onto the absorbent together with the vitrification solution, so that the vitrification solution adhering to the periphery of the germ cells is the absorbent material. To be absorbed. As a result, the germ cells are vitrified by being immersed in liquid nitrogen in the absence of the vitrification solution outside the cell, and only the vitrification solution contained in the germ cells is cooled. So it can be cooled rapidly. Moreover, since the vitrification liquid does not exist outside the germ cells, the germ cells are not damaged by the phase transformation between the solid phase and the liquid phase of the vitrification solution outside the cells.

  Therefore, according to the present invention, germ cells such as sperm, ovum and embryo taken out of the body can be cryopreserved with excellent viability.

  As the absorbent, filter paper can be used, and filter paper for filter sterilization can be particularly preferably used. The method of the present invention can be applied to cryopreservation of germ cells such as bovine blastocysts.

  Next, the germ cell cryopreservation container of the present invention is a germ cell cryopreservation container that contains an absorbent material that retains vitrified germ cells and is immersed in liquid nitrogen. A large-diameter recess on which the material is placed, a small-diameter recess provided at the bottom of the large-diameter recess corresponding to the position of the germ cell held by the absorbent material, and a peripheral edge of the large-diameter recess A container main body comprising a wall member, a part of the wall member and a part of the bottom of the large-diameter recess, and a groove communicating the large-diameter recess and the outside, and closing the container main body A top plate, a skirt portion that hangs down from the periphery of the top plate and surrounds the outer peripheral side of the wall member, and a position corresponding to the position of the germ cells held in the absorbent material accommodated in the container body It consists of a cover body provided with the recessed part provided in the inner surface of the top plate.

  In the container of the present invention, the germ cells vitrified by the method of the present invention are placed in the large-diameter recess of the container body while being held by the absorbent, and the container body is closed by the lid. To be done. At this time, a small-diameter recess is provided at the bottom of the large-diameter recess corresponding to the position of the germ cell held by the absorbent material. Further, the top plate of the lid body is provided with a recess corresponding to the position of the germ cell held by the absorbent material accommodated in the container body.

  Therefore, according to the container of the present invention, the germ cells held in the absorbent material are between the small-diameter concave portion provided at the bottom of the large-diameter concave portion and the concave portion provided in the top plate of the lid. Thus, there is no direct contact with the container body or lid, and no physical damage is caused by the container body or lid.

  In addition, in the state where the lid is removed, the germ cells held by the absorbent material are placed in the large-diameter concave portion of the container body, and are surrounded by a wall member that rises from the peripheral edge of the large-diameter concave portion. Protected. At this time, since the container main body is provided with a groove portion that breaks a part of the wall member and a part of the bottom portion of the large-diameter recess, and communicates the large-diameter recess with the outside. The tip of the tweezers can be inserted into the end edge of the absorbent material.

  Therefore, according to the container of the present invention, the absorbent material accommodated in the container body can be easily taken out without damaging the germ cells.

  Moreover, the container of this invention WHEREIN: The said container main body is equipped with the groove part which engages and fixes to the protrusion provided in the storage place, It is characterized by the above-mentioned. According to the container of the present invention having the above-described configuration, the reproductive body is fixed in a state where the container itself is fixed by engaging the groove provided on the bottom outer surface of the container main body with the protrusion provided in the storage place. Cells can be preserved and germ cell damage can be prevented.

  Next, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. FIG. 1 is an explanatory cross-sectional view of a container for cryopreserving germ cells treated by the cryopreservation method of the present embodiment, and FIGS. 2 and 3 are explanatory views showing a method for using the storage container shown in FIG. .

  In the cryopreservation method of the present embodiment, first, bovine blastocysts as germ cells are immersed in a freezing basal medium at 30 to 39 ° C., for example, 39 ° C., and at room temperature, for example, 15 ° C. for 5 to 15 minutes. For example, hold for 10 minutes. Examples of the frozen basal medium include polyvinylpyrrolidone in an amount of 1 mg / ml, trehalose in an amount of 0.2 mol / l, and an amount of 14.8 mmol / l with respect to a known composition-modified PB1 medium. And the like added with L-proline. The trehalose is a saccharide having an effect of preventing frost damage.

  Next, the bovine blastocyst is immersed in a pre-freezing medium at room temperature, eg, 15 ° C., and held at room temperature, eg, 15 ° C., for 3-5 minutes, eg, 5 minutes. As the pre-freezing treatment medium, for example, a medium obtained by adding 7% by weight of ethylene glycol and 0.5% by weight of glycerol to the frozen basic medium can be used. The ethylene glycol and glycerol are antifreezing agents.

  Next, the bovine blastocyst is immersed in a vitrification solution at 3 to 10 ° C., for example, 4 ° C., and held for 20 to 60 seconds, for example, 30 seconds. Examples of the vitrification liquid include sucrose in an amount of 0.5 mol / l, ethylene glycol in an amount of 30% by weight, and an amount of 0.5% by weight with respect to the frozen basal medium. What added glycerol and the like can be used. The sucrose is a saccharide having an effect of preventing frost damage.

  When the time for immersing the bovine blastocyst in the vitrification solution is shorter than the above range, the permeation amount of the vitrification solution to the bovine blastocyst is small, and ice crystals are formed in the cells when cooled. There is a fear. Further, if the time for immersing the bovine blastocyst in the vitrification solution is longer than the above range, the viability of the bovine blastocyst may be lowered due to the toxicity of the antifreezing agent such as ethylene glycol and glycerol. .

  Next, the bovine blastocyst is sucked into a glass capillary or the like together with the vitrification solution and dropped onto a sterilized filter paper, and the vitrification solution adhering to the periphery of the bovine blastocyst is applied to the filter paper. Absorb. As the filter paper, for example, a dry filter sterilization membrane filter (manufactured by Nihon Millipore Corporation) can be used. The membrane filter for dry filtration sterilization has a diameter of 13 mm and can hold a large number of germ cells on one filter paper.

  Next, when it is confirmed that the vitrification solution adhering to the periphery of the bovine blastocyst has been absorbed by the filter paper, the filter paper holding the bovine blastocyst is immediately washed with liquid nitrogen. Immerse in. As a result, bovine blastocysts vitrified on the filter paper are obtained.

  Next, the bovine blastocyst is accommodated in a cryopreservation container 1 shown in FIG.

  A cryopreservation container 1 shown in FIG. 1 is made of a metal such as stainless steel, and includes a container body 4 that houses a filter paper 3 that holds a bovine blastocyst 2 and a lid body 5 that closes the container body 4. Become. The container body 4 includes a large-diameter recess 6 on which the filter paper 3 holding the bovine blastocyst 2 is placed, and a large-diameter recess 6 corresponding to the position of the bovine blastocyst 2 held on the filter paper 3. A small-diameter recess 7 provided at the bottom, a wall member 8 rising from the peripheral edge of the large-diameter recess 6, a part of the wall member 8 and a part of the bottom of the large-diameter recess 6, A groove portion 9 that communicates the small-diameter concave portion 7 with the outside, and an engagement groove portion 10 that fixes the container body 4 to a storage member described later are provided. On the other hand, the lid 5 includes a top plate 11 that closes the container main body 4, a skirt portion 12 that hangs down from the periphery of the top plate 11 and surrounds the outer peripheral side of the wall member 8, and filter paper accommodated in the container main body 4. 3 is provided with a recess 13 provided on the inner surface of the top plate 11 corresponding to the position of the bovine blastocyst 2 held by 3.

  According to the cryopreservation container 1, when the filter paper 3 holding the bovine blastocyst 2 is placed in the large-diameter recess 6 of the container body 4 and the container body 4 is closed with the lid 5, Above and below the bovine blastocyst 2 are a small-diameter recess 7 in the container body 4 and a recess 13 in the lid 5. Therefore, the bovine blastocyst 2 is not in direct contact with the container body 4 or the lid 5, and physical damage due to contact with the container body 4 or the lid 5 can be prevented.

  When accommodating the filter paper 3 holding the bovine blastocyst 2 in the cryopreservation container 1, as shown in FIG. 2, the edge of the filter paper 3 is gripped by tweezers 14 or the like, and the lid 5 is removed. The filter paper 3 is placed in the large-diameter recess 6 of the container body 4 in the state. At this time, since the groove portion 9 is provided in the container body 4, the operation of placing the filter paper 3 in the large-diameter concave portion 6 can be easily performed by inserting the tip of the tweezers 14 into the groove portion 9. . Further, when the filter paper 3 holding the bovine blastocyst 2 is taken out from the cryopreservation container 1, it can be carried out by a procedure completely opposite to the operation for accommodation.

  In this embodiment, the groove 9 is formed so as to communicate the large-diameter recess 6 and the small-diameter recess 7 with the outside. However, the groove 9 is inserted into the tip of the tweezers 14 so that the edge of the filter paper 3 is What is necessary is just to be able to hold | grip, and the small diameter recessed part 7 does not need to be connected with the exterior. Further, in the cryopreservation container 1, there is no clearance between the container body 4 and the lid 5, and the liquid nitrogen remains in the cryopreservation container 1 even when the cryopreservation container 1 is immersed in liquid nitrogen as described later. Can not invade. Since liquid nitrogen is not aseptic, by preventing liquid nitrogen from entering the cryopreservation container 1, it is possible to prevent the bovine blastocyst 2 from being attacked by bacteria in the liquid nitrogen.

  The cryopreservation container 1 is stored in, for example, a storage cylinder 21 shown in FIG. 3 and stored in liquid nitrogen. The storage cylinder 21 is made of metal such as stainless steel, and includes a semi-cylindrical storage cylinder main body 22 and a semi-cylindrical lid body 23. The storage cylinder main body 22 is a concave groove along the length direction at the edge. 24, and the lid body 23 is slidable in the length direction by engaging a ridge 25 provided on the end edge portion along the length direction with the groove 24. The storage cylinder body 22 includes a plurality of shelf plates 26, and the shelf plates 26 are provided with protrusions 27 that engage with the engagement groove portions 10 provided in the container body 4 of the cryopreservation container 1.

  Therefore, the cryopreservation container 1 containing the filter paper 3 holding the bovine blastocyst 2 is fixed to the shelf plate 26 by engaging the engagement groove 10 with the protrusion 27 of the storage cylinder body 22. . And the storage cylinder 21 which stored the cryopreservation container 1 is closed by sliding the cover body 23 in the length direction of the storage cylinder main body 22.

  The storage cylinder 21 is immersed and stored in liquid nitrogen filled in a liquid nitrogen container. As the liquid nitrogen container, a container conventionally used for cryopreservation of germ cells can be used as it is.

  In the thawing of the bovine blastocyst 2 cryopreserved by the cryopreservation method of this embodiment, the filter paper 3 taken out from the cryopreservation container 1 in liquid nitrogen is immersed in a thawed medium at 38 to 40 ° C., for example, 39 ° C. It can be performed by holding for 5 to 10 minutes, for example, 5 minutes. As the melting medium, for example, a medium obtained by adding polyvinylpyrrolidone in an amount of 1 mg / ml and sucrose in an amount of 0.3 mol / l to a known composition-modified PB1 medium can be used. .

  The bovine blastocyst 2 held on the filter paper 3 can be simultaneously melted and recovered from the filter paper 3 by being immersed in the melting medium under the above conditions, and the vitrification solution is removed. . The bovine blastocyst 2 from which the vitrification solution has been removed is transferred to a culture medium and cultured.

  Next, an example is shown.

  First, embryos (bovine blastocysts) as germ cells were immersed in about 0.1 ml of a frozen basal medium at 39 ° C. and held for 10 minutes. As the frozen basal medium, polyvinylpyrrolidone in an amount of 1 mg / ml, trehalose in an amount of 0.2 mol / l, and L in an amount of 14.8 mmol / l with respect to a known composition-modified PB1 medium. -What added proline was used.

  Next, the bovine blastocyst was immersed in about 0.1 ml of a pre-freezing medium at 15 ° C. and held for 5 minutes. As the pre-freezing medium, a medium obtained by adding 7% by weight of ethylene glycol and 0.5% by weight of glycerol to the frozen basal medium was used.

  Next, the bovine blastocyst is immersed in about 2 ml of a vitrification solution at 4 ° C., and suction and discharge are repeated 2 to 3 times using a glass capillary for 20 to 30 seconds. Equilibrated with liquid. As the vitrification solution, sucrose in an amount of 0.5 mol / l, ethylene glycol in an amount of 30% by weight, and glycerol in an amount of 0.5% by weight with respect to the frozen basic medium. The thing which added was used.

  Next, the bovine blastocyst is sucked into a glass capillary or the like together with about 5 μl of the vitrification solution, and dropped onto a sterilized membrane filter for dry filtration sterilization (manufactured by Nihon Millipore Corporation). The vitrification solution adhering to the periphery was absorbed by the filter. Absorption of the vitrification solution was confirmed visually.

  Next, after confirming the absorption of the vitrification solution, the filter holding the bovine blastocyst was immediately immersed in liquid nitrogen to vitrify the bovine blastocyst. And the said filter with which the said bovine blastocyst was hold | maintained was accommodated in the cryopreservation container 1 shown in FIG. 1, the cryopreservation container 1 was immersed in liquid nitrogen, and was preserve | saved for 1 to 14 days.

  Next, the bovine blastocysts stored as described above were thawed. The thawing was performed by immersing the filter holding the bovine blastocysts in a 39 ° C. melting medium and holding for 5 minutes. As the melting medium, a medium obtained by adding polyvinylpyrrolidone in an amount of 1 mg / ml and sucrose in an amount of 0.3 mol / l to a known composition-modified PB1 medium was used. As a result of the thawing, in this example, the number of viable embryos was 5 with respect to the number of test embryos 5 and all were alive.

  Next, when the total number of embryos (bovine blastocysts) that survived the thawing was cultured, the total number of 5 viable embryos hatched.

  Therefore, according to the cryopreservation method of the present embodiment, excellent viability can be obtained for germ cells treated by the cryopreservation method, and the quality of the surviving germ cells is maintained extremely well. Obviously it can be.

  In the present embodiment, a bovine blastocyst is described as an example of a germ cell. However, the cryopreservation method of the present embodiment adjusts the composition of each medium, the composition of the vitrification solution, the temperature obtained when immersed in the vitrification solution, the time, etc., so that bovine sperm, bovine ovum, and other mammals It can also be applied to other germ cells such as animal sperm, ovum and embryo.

Explanatory sectional drawing which shows the example of 1 structure of the cryopreservation container of this invention. Explanatory drawing which shows the usage method of the cryopreservation container shown in FIG. Explanatory drawing which shows the usage method of the cryopreservation container shown in FIG.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Cryopreservation container, 2 ... Germ cell, 3 ... Filter paper, 4 ... Container body, 5 ... Lid body, 6 ... Large diameter recessed part, 7 ... Small diameter recessed part, 8 ... Wall member, 9 ... Groove part, 10 ... Engaging groove part 11 ... Top plate, 12 ... Skirt part, 13 ... Recess, 26 ... Storage place, 27 ... Projection.

Claims (5)

  A germ cell immersed in a vitrification solution is dropped onto the absorbent material together with the vitrification solution, and the vitrification solution adhering to the periphery of the germ cell is absorbed by the absorbent material. A method for cryopreserving germ cells, characterized by vitrification by immersing in liquid nitrogen while being retained on the absorbent material and cryopreserving.   2. The germ cell cryopreservation method according to claim 1, wherein the absorbent material is filter paper.   The method for cryopreserving germ cells according to claim 1 or 2, wherein the germ cells are bovine blastocysts. A germ cell cryopreservation container that contains an absorbent material that retains vitrified germ cells and is immersed in liquid nitrogen,
A large-diameter recess where the stored absorbent material is placed, a small-diameter recess provided at the bottom of the large-diameter recess corresponding to the position of the germ cell held in the absorbent material, and the large-diameter recess A container body comprising: a wall member rising from the peripheral edge of the wall; and a groove portion that breaks a part of the wall member and a part of the bottom of the large-diameter recess to communicate the large-diameter recess with the outside;
A top plate that closes the container body; a skirt portion that hangs down from the periphery of the top plate and surrounds the outer peripheral side of the wall member; and the germ cells held in the absorbent housed in the container body A germ cell cryopreservation container comprising a lid provided with a recess provided on the inner surface of the top plate corresponding to the position of the top plate.   5. The germ cell cryopreservation container according to claim 4, wherein the container main body includes an engaging groove portion that engages and fixes a protrusion provided in a storage place on an outer surface of the bottom portion.

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