WO2020180067A1

WO2020180067A1 - Rapid-freezing storage device for tissue and rapid-freezing storage method for tissue

Info

Publication number: WO2020180067A1
Application number: PCT/KR2020/002948
Authority: WO
Inventors: 이상훈; 이장우; 김보람
Original assignee: 고려대학교 산학협력단
Priority date: 2019-03-04
Filing date: 2020-03-02
Publication date: 2020-09-10
Also published as: KR102146379B1

Abstract

A rapid-freezing storage device for tissue according to an embodiment of the present invention comprises: a tissue seating part which has a cylindrical shape with an open upper portion and on which tissue is placed; and a tissue storage part comprising a storage case which has a cylindrical shape with an open upper portion and on which the tissue seating part is mounted, a storage lid for sealing the internal space of the storage case, and a manipulation member for manipulating the movement of the storage lid to enable the storage lid to be fastened to the storage case while the storage lid moves up and down with respect to the storage case, wherein tissue is placed on the tissue seating part and vitrified by liquid nitrogen inside a liquid nitrogen barrel, and after the tissue seating part is inserted into the storage case containing liquid nitrogen inside the liquid nitrogen barrel, the vitrified tissue is frozen and stored in the tissue storage part to maintain the vitrification thereof by liquid nitrogen contained in the storage case as the storage lid is fastened to the storage case by manipulation of the manipulation member.

Description

Rapid freezing storage device for tissues and storage method for rapid freezing tissues

The present invention relates to a rapid freezing storage device for tissues and a method for storing rapid freezing tissues, and in detail, the process of vitrifying tissue and sealing the vitrified tissue is continuously carried out inside the liquid nitrogen communication system to seal the vitrified tissue. The present invention relates to a rapid freezing storage device for tissues and a rapid freezing storage method for tissues capable of preventing damage to tissues due to a temperature difference between the temperature of the vitrified tissues and room temperature that may occur during the process.

After fertilization in the mother's womb, reproductive cells develop in the egg yolk of the fetus and move into the fetus. In the case of women, they differentiate into ovaries through the process of componentization. The eggs created during this period are held in the ovary in a state of being stopped at the beginning of the first meiosis phase.

After birth, some of the eggs in the body are matured through a series of processes that go through a primary follicle, a primary follicle, a secondary follicle, and a tertiary follicle. The egg is released from the body and plays a role as a reproductive cell.

At the 20th week of pregnancy, there are about 7 million eggs in the fetal ovary, but the number of eggs after birth decreases to 2 million, and the number continues to decrease to about 500,000 at the beginning of puberty. It is reduced to dogs, and the number of eggs released during a woman's lifetime is generally limited to 300 to 500.

Freezing methods for preserving fertility in female cancer patients include embryo freezing, egg automatic freezing, and ovarian tissue freezing. The selection of the most appropriate method among these depends on the timing and drug of chemotherapy, the type of cancer, the age of the patient and the presence of a spouse.

The currently clinically established method is cryopreservation of embryos and eggs, but this is an impossible method in women who cannot delay treatment to induce superovulation. On the other hand, cryopreservation of the ovaries is possible in all women, including children before puberty and women who cannot delay chemotherapy.

The main goal of ovarian tissue freezing is to implant cryopreserved ovarian tissue in the orthotopic site or at a heterotopic site, such as the upper arm or abdominal wall, when the patient is healed or the disease has been treated.

When cryopreservation of ovarian tissue, it is necessary to prevent the formation of ice crystals to adequately permeate the cryoprotective agent (CPA) to not only granulosa cells and stromal cells, but also eggs. Together, the toxicity of high concentrations of cryoprotectants (CPA) should be considered.

Until now, a standardized method of slow-freezing using propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol as a cryoprotectant (CPA) for cryopreservation of human ovaries. Was being used. However, a device that implements slow-freezing uses expensive equipment of tens of thousands of won, so it is difficult to use the equipment except for a large specialized hospital, and it takes a long time to freeze the tissue.

Recently, studies showing good results using the vitrification method, a rapid freezing method that can prevent damage caused by ice crystals during freezing of ovarian tissue, have been published. Vitrification freezing is a process in which ovarian tissue is immersed in liquid nitrogen and rapidly cooled to -180°C.

Vitrification freezing requires tissues to be placed in liquid nitrogen within a very short period of time, so the most suitable tool for this procedure is required. In the case of freezing eggs or embryos, tools such as Kitazato's Cryotop ^TM and CooperSurgical's Cryoleaf ^TM are used.

Referring to FIG. 1, a process of freezing ovarian tissue using a Kitazato product, which is one of the products used for vitrification, will be described as follows.

First, as shown in Fig. 1 (a), a tissue for cryopreservation is prepared. Next, as shown in Fig. 1 (b), the tissue is contained in a vitrification media. This vitrification media treatment is for preventing moisture from escaping from the tissue.

As shown in Figure 1 (c), the media-treated tissue for vitrification is placed in a kit. Then, the tissue placed in the kit is inserted into the liquid nitrogen communication system, and the process of vitrification by the liquid nitrogen proceeds. When the tissue is vitrified, the kit comes out of the liquid nitrogen cylinder at -180°C and at room temperature, the kit is sealed in a case containing liquid nitrogen as shown in FIG. 1(d).

However, as the process shown in FIGS. 1(a) to 1(d) proceeds, the following problems may occur.

In the process of the kit coming out of the liquid nitrogen communication, there is a possibility that the tissue may be deformed due to the rapid temperature change between the liquid nitrogen (-180°C) and room temperature. The main goal of ovarian tissue freezing is to transplant cryopreserved ovarian tissue into the pelvis (orthotopic site) or at a heterotopic site such as the upper arm or abdominal wall when the patient is cured or the disease is over. Ovarian tissue has a problem that cannot be used for transplantation.

In addition, since the temperature of the case containing liquid nitrogen is very low at -180℃, if liquid nitrogen flows out of the case while the kit is sealed to the case, an accident such as a burn may occur on the hand of the operator holding the case. The possibility exists.

[Prior technical literature]

(Patent Document 1) Japanese Patent No. 5825571 discloses a cryopreservation tool for cryopreserving a biological sample.

In the present invention, the process of vitrifying the tissue and the process of sealing the vitrified tissue are continuously carried out inside the liquid nitrogen communication, and damage to the tissue due to the temperature difference between the temperature of the vitrified tissue and room temperature that can occur in the process of sealing the vitrified tissue The purpose of this study is to provide a quick-freezing storage device for tissues and a rapid-freezing storage method for tissues.

A tissue fast-freezing storage device according to an embodiment of the present invention has a cylindrical shape with an open top, and a tissue seating portion on which the tissue is placed; And a storage case with an open top and a tissue seat, a storage lid that seals the internal space of the storage case, and the storage lid is moved up and down with respect to the storage case so that the storage lid is coupled to the storage case. It includes a tissue storage unit including an operation member for manipulating the movement of the storage lid, wherein the tissue is placed on the tissue seating unit and is vitrified by liquid nitrogen in the liquid nitrogen communication unit, and the vitrified tissue is the tissue seating unit in the liquid nitrogen communication unit. After being inserted into the storage case containing liquid nitrogen, it is preferable to be frozen and stored in the tissue storage unit so that vitrification is continued by the liquid nitrogen contained in the storage case as the storage lid is coupled to the storage case by operation of the operating member.

In one embodiment of the present invention, the tissue storage unit is connected to the storage case and the storage lid sealing the inner space of the storage case and the storage case and the storage lid, the storage case having an open top and a tissue seat The storage lid is coupled to the storage case and includes an operation member operated to seal the internal space, and the tissue storage unit is mounted in the storage case so that the tissue is immersed in the liquid nitrogen in the liquid nitrogen communication unit, It is preferable that the storage lid is operated to be coupled to the storage case by the operation member, so that the tissue seating portion is sealed in the inner space of the storage case.

In an embodiment of the present invention, the tissue storage unit further includes a case holder on which the storage case is mounted, and a lid holder on which the case holder is placed facing the case holder and on which the storage lid is mounted, and an operation member It is preferable to connect the lid holder and the case holder to manipulate the gap between the case holder and the lid holder.

In one embodiment of the present invention, the tissue seating portion is made of a mesh net, and before the tissue is vitrified, when chemically treated by the vitrification media, the tissue mounting portion is in a state in which the tissue is seated, and the vitrification media is It is preferable that it is inserted into the contained medicine container and contained in the vitrification media for chemical treatment.

In one embodiment of the present invention, it is preferable that the bottom surface of the tissue seating portion is made of a material having high thermal conductivity.

On the other hand, the method for storing the tissue for rapid freezing according to an embodiment of the present invention comprises the steps of: (A) the tissue is placed on the tissue seating unit, and is treated with a drug by a vitrification media; (B) a step in which the tissue seating portion is inserted into the liquid nitrogen communication and the tissue is rapidly frozen and vitrified by the liquid nitrogen in the liquid nitrogen communication; (C) the tissue storage unit including the storage case, the storage lid and the operation member is inserted into the interior of the liquid nitrogen communication, and the liquid nitrogen is contained in the storage case; (D) the tissue seating portion is inserted into the storage case and mounted, so that the tissue is immersed in liquid nitrogen in the inner space of the storage case; (E) the storage lid is operated to be coupled to the storage case by the operation member, and sealing the tissue in a state immersed in liquid nitrogen in the internal space; And (F) the tissue storage unit in a state in which cold air is maintained by the liquid nitrogen in the storage case, comes out of the liquid nitrogen communication and is inserted into the freezing storage tank, and the tissue storage in the freezing storage tank is preferably included.

In one embodiment of the present invention, it is preferable that steps B to E are sequentially performed in liquid nitrogen communication.

In an embodiment of the present invention, in step F, it is preferable that the tissue storage unit is seated in a dipper mounted on a freezing storage tank and positioned so as to be immersed in liquid nitrogen in the freezing storage tank.

In the present invention, the vitrification process of the tissue and the process of sealing the vitrified tissue are continuously performed inside the liquid nitrogen communication system, and the vitrified tissue, which can occur during the process of sealing the existing vitrified tissue in a container containing liquid nitrogen at room temperature. It is possible to prevent damage to the tissue caused by the temperature difference between the temperature and the temperature at which the tissue is sealed.

The present invention prevents tissue damage during the process of freezing and sealing the tissue, and after chemotherapy, it is possible to increase the success rate of tissue transplantation using the thawed tissue.

In addition, the present invention eliminates the process of transferring the drug-treated tissue by the vitrification media to the kit used for vitrification of the tissue compared to the prior art by being chemically treated by the vitrification media while the tissue is placed on the tissue seat. Thus, it is possible to improve the work efficiency of the pretreatment process before freezing the tissue.

In addition, the present invention allows the storage lid to be coupled to the storage case with only a simple operation of the operating member, so that the tissue is sealed in the storage case containing liquid nitrogen, so that the operator can remove the vitrified tissue without contacting the storage case, which is a part containing liquid nitrogen. It can be sealed in the tissue storage area.

Due to this, in the process of sealing the kit with the vitrified tissue, which was discussed as a problem in the prior art, in a case containing liquid nitrogen, that is, in a state in which the operator holds the case containing liquid nitrogen, the lid connected to the kit is coupled to the case. In the process, as liquid nitrogen flows out of the case, the liquid nitrogen comes into contact with the hand of a worker holding the case, thereby preventing an accident in which the worker is burned by the liquid nitrogen.

1 is a view for explaining a tissue freezing method according to the prior art.

Figure 2 is a schematic diagram showing the configuration of a tissue storage device for rapid freezing according to an embodiment of the present invention.

3 and 4 are views for explaining a process in which the tissue seating unit is sealed to the tissue storage unit.

5 is a view for explaining a tissue storage unit according to another example.

Figure 6 is a view for explaining the process of the inventors of the rapid freezing storage device of the tissue is inserted into a freezing storage tank, frozen storage.

Hereinafter, with reference to the accompanying drawings, a description will be given of a quick-freezing storage apparatus for tissues and a rapid-freezing storage method for tissues according to a preferred embodiment of the present invention.

A tissue storage device for rapid freezing of a tissue according to an embodiment of the present invention is for storing tissue that is rapidly cooled and vitrified. The tissue was obtained from the human body before chemotherapy, and is intended to be transplanted back into the human body after chemotherapy. In order for the tissue transplanted into the human body to achieve the desired effect, the tissue must not be deformed during the tissue storage process.

In the present invention, the process of vitrifying the tissue and sealing the tissue to the tissue storage unit proceeds continuously within the liquid nitrogen communication unit, thereby sealing the vitrified tissue to the tissue storage unit together with liquid nitrogen. Thereafter, the tissue storage unit in which the tissue is sealed comes out of the liquid nitrogen communication and is then stored in a freezing storage tank. The freezing storage tank is a tank containing liquid nitrogen.

As shown in Figure 2, the tissue storage device for rapid freezing 100 includes a tissue storage unit 110 and a tissue seating unit 130.

The tissue seating portion 130 has a cylindrical shape with an open top. The tissue seating unit 130 is placed on the tissue 150 on the bottom surface. The tissue seating unit 130 may have a bottom surface made of a mesh network, so that the tissue 150 is placed on the tissue seating unit 130 to be treated with chemicals by the vitrification media.

The tissue 150 is vitrified while being rapidly cooled by liquid nitrogen in a state seated on the tissue mounting portion 130. The bottom surface of the tissue seating unit 130 is made of a material having a high thermal conductivity, so that the rapid cooling efficiency of the tissue by liquid nitrogen may be increased. As the material having high thermal conductivity, any one of silver, copper, aluminum, stainless steel, aluminum nitride, silicon nitride, alumina, metal powder, aluminum nitride, and the like may be selected.

A handle 131 is provided on the upper end of the tissue seating part 130. This is, in the process of vitrifying the tissue 150, in the process of vitrifying the tissue 150, while the operator stably holds the tissue seat 130, gently shake the tissue seat 130 in a state immersed in liquid nitrogen. While it is possible to promote the vitrification of the tissue 150 more smoothly. In addition, by preventing the operator from contacting the liquid nitrogen, it is possible to prevent the operator from being injured by the liquid nitrogen during the vitrification process of the tissue 150. The handle 131 can be removed from the tissue mounting portion 130 in the process of sealing the tissue mounting portion 130 to the tissue storage portion.

After vitrification of the tissue 150, the tissue seating unit 130 is inserted into the storage case 114 of the tissue storage unit 110. The tissue seating portion 130 is mounted on the storage case 114 so that the upper end of the tissue seating portion 130 is caught on the upper end of the storage case 114 and the bottom surface of the tissue seating portion 130 is spaced apart from the bottom surface of the storage case 114. At this time, liquid nitrogen is contained in the storage case 114. The tissue seating unit 130 is sealed in the inner space of the tissue storage unit 110 while the storage case 114 and the storage lid 115 are assembled with each other by the manipulation of the operation member 113.

The tissue storage unit 110 is composed of a case holder 111, a lid holder 112, a storage case 114, a storage lid 115, and an operation member 113. The tissue storage unit 110 is a container for freezing the vitrified tissue 150. The vitrified tissue 150 is sealed and stored in the tissue storage unit 110 in a state placed on the tissue seating unit 130. At this time, the vitrified tissue 150 is stored in the tissue storage unit 110 in a state immersed in liquid nitrogen stored in the storage case 114.

The storage case 114 has a cylindrical shape with an open top. The storage case 114 is a container in which the tissue seating unit 130 is mounted. The storage case 114 is manufactured in a size in which the tissue seating portion 130 is inserted into the inner space and can be mounted. When the storage case 114 is combined with the storage lid 115, the inner space is sealed.

The storage case 114 contains liquid nitrogen. The storage case 114 is provided with a plurality of openings 114a. The plurality of openings 114a are passages through which liquid nitrogen vaporized in the internal space of the storage case 114 exits to the outside of the storage case 114. As the vaporized liquid nitrogen is moved out of the storage case 114 through the plurality of openings 114a, the storage case 114 is sealed by the storage lid 115, and then the liquid nitrogen is vaporized and the storage case 114 ), the pressure in the inner space of the inner space is increased, so that the storage lid 115 can be prevented from being separated from the storage case 114.

2 to 4, the storage case 114 is detachably installed on the case holder 111. And, the storage lid 115 is detachably installed on the lid holder 112. The case holder 111 and the lid holder 112 are connected to each other by an operation member 113. On the other hand, as shown in Figure 5, the present invention (100a) is a tissue storage unit (110a) without a case holder 111 and/or a lid holder 112, the storage case 114 and the storage lid ( 115) may be directly installed on the operation member 113.

The operating member 113 is composed of a first operating member (113a) and a second operating member (113b). The first operation member 113a is installed to protrude upward from the side of the case holder 111. A first handle 113a1 is provided on an upper end of the first manipulation member 113a. In addition, a dipper hanger 113a2 is provided on the upper end of the first manipulation member 113a. The dipper hook portion 113a2 is a ring provided so that the tissue storage portion 110 is caught on the dipper 170.

The operator holds the first handle (113a1) and / or the second handle (113b1) provided on the top of the operation member 113, as if a ladle scoops the soup in a soup pot, and only the portion of the storage case 114 is liquefied nitrogen communication 160 ), and put the liquid nitrogen in the storage case 114.

The storage lid 115 is connected to the second operation member 113b. At this time, the storage lid 115 is located above the storage case 114. A second handle 113b1 is provided on the upper end of the second manipulation member 113b.

The second manipulation member 113b is coupled to the first manipulation member 113a so as to be movable up and down with respect to the first manipulation member 113a. The first manipulating member 113a is provided with a guide groove 113a3, and the second manipulating member 113b is provided with a locking protrusion 113b2 that can move up and down along the guide groove 113a3.

The operator presses the second handle 113b1 toward the storage case 114 so that the second operation member 113b moves downward relative to the first operation member 113a, so that the storage lid 115 is stored. It can be manipulated to be coupled to the case 114. In addition, the operator pulls the second handle (113b1) upward, so that the second operation member (113b) is moved upward relative to the first operation member (113a), so that the storage lid (115) is the storage case (114) Can be operated separately.

In a simple manner of pressing down or pulling the second handle 113b1 down, without holding the storage case 114 containing liquid nitrogen according to the present embodiment, the tissue storage case 114 Can be combined or separated. Accordingly, the present invention can prevent an image caused by contact with liquid nitrogen, which is generated in the process of sealing the tissue seating unit 130 to the storage case 114 in which liquid nitrogen is stored.

Hereinafter, a description will be given of a method of storing tissues for rapid freezing as follows.

First, in a state in which the tissue 150 is placed on the tissue seating unit 130, chemical treatment is performed by the vitrification media. The tissue seating unit 130 may have a bottom surface made of a mesh network, so that the tissue 150 is placed on the tissue seating unit 130 to be treated with chemicals by the vitrification media.

Compared with the prior art, the present invention can improve the work efficiency of the pretreatment process prior to freezing of the tissue 150 by omitting the process of transferring the tissue 150 treated by the vitrification media to the kit.

The reason why the tissue 150 is treated with chemicals is that the permeable cryoprotectant lowers the glass transition temperature of the solution, thus preventing the solution from being actually frozen, and inducing a fatal stress due to ice formation in the cells. In antifreeze solutions, buffers, carbohydrates (e.g., glucose, lactose, raffinose, sucrose and trehalose), salts (sodium citrate) (sodium citrate), citric acid), egg yolk and antibiotics permeable cryoprotectant glycerol. As a cryoprotectant, the osmotic pressure is balanced to protect the plasma membrane and the mitochondrial membrane from damage, and to prevent DNA damage from the formation of lipid peroxidation and reactive oxygen species. do.

At room temperature, the tissue 150 is chemically treated in an Equilibration Solution and a Vitrification Solution, which are media for vitrification.

Equilibration Solution (ES) is formed by mixing HEPES solution, supplemented solution, ethylene glycol (Ethylene Glycol), dimethyl sulphoxide (DMSO), and SSS (or SPS) in a specific ratio.

Vitrification Solution (VS) is formed by mixing HEPES solution, sucrose, ethylene glycol (Ethylene Glycol), dimethyl sulphoxide (DMSO), and SSS (or SPS) in a specific ratio.

In the present invention, the tissue 150 is placed on the tissue seating unit 130 and treated with chemicals for 25 minutes in an Equilibration Solution (ES), which is one of the media for vitrification, and then the tissue 150 using sterile gauze. ) From the Equilibration Solution (ES). Thereafter, the tissue 150 is placed on the tissue seating unit 130 and is treated with chemicals for 15 minutes in a vitrification solution (VS). Thereafter, the vitrification solution is removed from the tissue 150 using sterile gauze.

Next, the tissue seating unit 130 is inserted into the liquid nitrogen communication unit 160 and the tissue 150 is rapidly frozen and vitrified by the liquid nitrogen in the liquid nitrogen communication unit 160.

The chemically treated tissue 150 is inserted into the liquid nitrogen communication unit 160 as it is placed on the tissue seating unit 130, and is vitrified by the liquid nitrogen. The vitrification process of the tissue 150 is a process in which the chemically treated tissue 150 is wound in liquid nitrogen of the liquid nitrogen communication unit 160 and rapidly cooled, so that the tissue 150 is changed into a vitreous tissue 150.

This is, while the operator grasps the handle 131 of the tissue seating unit 130, and gently shakes the tissue seating unit 130 while only the tissue 150 is immersed in liquid nitrogen, the liquid nitrogen and liquid nitrogen and Proceed to contact. Liquid nitrogen is introduced into the tissue seating unit 130 through the mesh network 132. The tissue 150 is vitrified while being rapidly cooled by liquid nitrogen.

Subsequently, the tissue storage unit 110 is inserted into the liquid nitrogen communication unit 160. Then, liquid nitrogen is contained in the storage case 114. Inside the liquid nitrogen communication 160, the tissue mounting portion 130 is inserted into the storage case 114 and mounted. At this time, the tissue 150 is immersed in liquid nitrogen in the inner space of the storage case 114.

Then, inside the liquid nitrogen communication 160, the operator couples the storage lid 115 to the tissue storage case 114 in a simple manner to press down the second handle 113b1. Accordingly, the present invention seals the tissue storage unit 110 without holding the storage case 114 containing liquid nitrogen by hand, so that the tissue seating unit 130, which has been pointed out as a problem in the prior art, is a storage case 114 in which liquid nitrogen is stored. ), which frequently occurs in the process of sealing, can prevent burns caused by contact with liquid nitrogen.

Thereafter, the inventors' tissue rapid freezing storage device 100 comes out of the liquid nitrogen communication unit 160, is mounted on a dipper, and stored frozen in a freezing storage tank 180. As described above, the freezing storage tank 180 is a tank in which liquid nitrogen is stored, and the inner space is opened and closed by the tank lid.

In the process of moving out of the liquid nitrogen communication 160 to the freezing storage tank 180 at room temperature, the tissue 150 is stored in the liquid nitrogen stored in the tissue storage unit 110. As a result, vitrification of the tissue 150 is maintained, and thermal deformation of the tissue 150 due to a temperature difference between liquid nitrogen and room temperature, which has been pointed out as a problem in the prior art, can be prevented.

As a result, the present invention prevents damage to the tissue 150 during the freezing process of the tissue 150, so that when a tissue 150 transplantation surgery is performed by defrosting the frozen tissue 150 later, the surgical performance can be improved. I can.

Although some embodiments of the present invention have been shown and described, those skilled in the art of ordinary skill in the art to which the present invention pertains will appreciate that the present embodiments can be modified without departing from the principles or spirit of the present invention. . The scope of the invention will be determined by the appended claims and their equivalents.

[Explanation of code]

100: tissue fast freezing storage device

110: organizational storage unit 111: case holding unit

112: lid holder 113: operation member

114: storage case 115: storage lid

130: tissue seating unit 160: liquid nitrogen communication

170: dipper 180: freezing storage tank

The present invention is applicable to the field of frozen storage of tissues such as ovaries.

Claims

The upper portion has an open cylindrical shape, the tissue seating portion on which the tissue is placed; And

A storage case having an open top and a storage case in which the tissue mounting part is mounted, a storage lid sealing the inner space of the storage case, and the storage lid as the storage lid is moved up and down with respect to the storage case. Including a tissue storage unit including an operation member for operating the movement of the storage lid to be coupled to the storage case,

The tissue is placed on the tissue seat and is vitrified by liquid nitrogen in the liquid nitrogen communication,

The vitrified tissue is contained in the storage case as the tissue seating portion is inserted into the storage case containing liquid nitrogen in the liquid nitrogen communication container, and then the storage lid is coupled to the storage case by operation of the operation member. Rapid freezing storage apparatus for tissues, characterized in that frozen storage in the tissue storage unit to continue vitrification by the liquid nitrogen.
The method of claim 1,

The operating member includes a first operating member installed on the storage case, and a second operating member installed on the storage lid,

The second operation member is coupled to the first operation member so that the storage case and the storage lid are arranged vertically, and is operated to couple the storage lid to the storage case while moving downward along the first operation member. Rapid freezing storage device for tissues characterized by.
The method of claim 1, wherein the tissue storage unit,

A case holder on which the storage case is mounted,

Further comprising a lid holder positioned at the top of the case holder to face the case holder and on which the storage lid is mounted,

The operation member connects the lid holder and the case holder to manipulate a gap between the case holder and the lid holder.
The method of claim 1,

The tissue seating portion is made of a mesh net at the bottom,

Before the tissue is vitrified, when chemically treated by the vitrification media,

The tissue seating unit is inserted into a medicine container containing the vitrification media in a state in which the tissue is seated, and is contained in the vitrification media to be treated with chemicals.
The method of claim 4,

The tissue seating unit, characterized in that the bottom surface is made of a material having high thermal conductivity.
(A) in a state in which the tissue is placed on the tissue seating portion, the step of chemical treatment by the media for vitrification;

(B) a step in which the tissue seating portion is inserted into the liquid nitrogen communication and the tissue is rapidly frozen and vitrified by the liquid nitrogen in the liquid nitrogen communication;

(C) inserting a tissue storage unit including a storage case, a storage lid and an operating member into the liquid nitrogen communication unit, and containing the liquid nitrogen in the storage case;

(D) the tissue seating portion is inserted into the storage case and mounted so that the tissue is immersed in the liquid nitrogen in the inner space of the storage case;

(E) a step of sealing the tissue in a state immersed in the liquid nitrogen in the inner space by operating the storage lid to be coupled to the storage case by the operation member; And

(F) the tissue storage unit in a state in which cold air is maintained by the liquid nitrogen in the storage case, and is inserted out of the liquid nitrogen communication and inserted into a freezing storage tank, and storing tissue in the freezing storage tank. Fast freezing storage method of tissue
The method of claim 6,

The step B to the step E is a rapid freezing storage method of the tissue, characterized in that it proceeds sequentially in the liquid nitrogen communication.
The method of claim 7,

In the step F, the tissue storage unit is mounted on a dipper mounted on the freezing storage tank, and is positioned to be immersed in liquid nitrogen in the freezing storage tank.
The method of claim 6,

The tissue seating portion is made of a mesh net at the bottom,

When the tissue is chemically treated by the vitrification media before vitrification,

The tissue seating portion is inserted into a medicine container containing the vitrification media in a state in which the tissue is seated, and is contained in the vitrification media to be treated with chemicals.