Vitrification freezing reagent and vitrification unfreezing reagent as well as use method and application of vitrification freezing reagent and vitrification unfreezing reagent

Abstract

The invention discloses a vitrification reagent and a vitrification unfreezing reagent as well as a use method and application thereof, and belongs to the technical field of freezing and unfreezing, the vitrification reagent comprises a freezing liquid 1, a freezing liquid 2 and a freezing liquid 3, and the components comprise ethylene glycol, dimethyl sulfoxide, a human serum protein substitute, penicillin-streptomycin double antibiotics and cane sugar; the vitrification unfreezing reagent comprises an unfreezing solution 1, an unfreezing solution 2, an unfreezing solution 3 and an unfreezing solution 4, and comprises the following components: a human serum protein substitute, penicillin-streptomycin double antibiotics, an M199 culture medium and cane sugar; according to the application, the quantity of ice crystals generated in the ovarian tissue freezing process can be remarkably reduced, the ovarian tissue is protected from ferroptosis to a certain extent by reducing the oxidative stress level of the ovarian tissue, meanwhile, the tissue activity of the ovarian tissue after freezing and unfreezing is improved, and the survival rate of the transplanted ovarian tissue is improved.

Description

Technical Field

[0001] This application belongs to the technical field of freezing and thawing, and specifically relates to a vitrification cryopreservation reagent, a vitrification thawing reagent, and their usage methods and applications. Background Art

[0002] With the continuous update of medical technology, the diagnosis of tumors has been advanced, and the cure rate of tumors has been continuously improved. Especially for children and adolescents with tumors, the long-term survival rate is relatively high. However, the radiotherapy and chemotherapy indispensable for tumor treatment cause irreversible damage to the reproductive system. Therefore, the preservation of fertility before reproductive toxicity treatment is of crucial importance.

[0003] The methods for in vitro preservation of fertility include: embryo cryopreservation, oocyte cryopreservation, ovarian tissue cryopreservation. For infants, children, and adolescents, the cryopreservation of ovarian tissue is the only method for fertility preservation. Compared with embryo cryopreservation and oocyte cryopreservation, the cryopreservation of ovarian tissue can preserve a large number of primordial follicles. After transplantation of the cryopreserved ovarian tissue, not only can fertility be restored, but also the endocrine function of women can be restored.

[0004] However, the existing cryopreservation and thawing solutions for ovarian tissue are very few. The most widely used one is the cryopreservation and thawing solution for ovarian tissue of Kato Company in Japan, which has problems such as high price and long order delivery period. With the continuous improvement of the awareness of fertility preservation among doctors and patients, a large number of young tumor patients in China need to preserve their fertility. The effect of ovarian cryopreservation determines the restoration of fertility after transplantation of patients, and the core key lies in the cryopreservation and thawing solution for ovarian tissue.

[0005] Current research shows that about 60% of primordial follicles will be lost after cryopreservation and thawing of ovarian tissue, and visible ice crystals will be generated during the freezing process of the current commercially available cryopreservation solutions, resulting in problems such as low tissue activity after cryopreservation and thawing of ovarian tissue and unsatisfactory ovarian survival rate after transplantation.

[0006] Content of the Application The purpose of this application is to provide a vitrification cryopreservation reagent, a vitrification thawing reagent, and their usage methods and applications, which can significantly reduce the number of ice crystals generated during the cryopreservation of ovarian tissue, protect ovarian tissue from ferroptosis to a certain extent by reducing the oxidative stress level of ovarian tissue, and at the same time improve the tissue activity of ovarian tissue after cryopreservation and thawing and improve the survival rate of ovarian tissue after transplantation.

[0007] To achieve the above technical effects, this application is implemented as follows: In the first aspect, this application provides a vitrification cryopreservation reagent, which includes: cryopreservation solution 1, cryopreservation solution 2, and cryopreservation solution 3, calculated by volume percentage. The cryoprotectant 1 includes: 5% - 7% ethylene glycol, 5% - 7% dimethyl sulfoxide, 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin - streptomycin double antibiotic, and 62.5% - 71.5% M199 medium; The cryoprotectant 2 includes: 11% - 13% ethylene glycol, 11% - 13% dimethyl sulfoxide, 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin - streptomycin double antibiotic, and 50.5% - 59.5% M199 medium; The cryoprotectant 3 includes: 23% - 25% ethylene glycol, 23% - 25% dimethyl sulfoxide, 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin - streptomycin double antibiotic, 26.5% - 35.5% M199 medium and sucrose; the concentration of sucrose in the cryoprotectant 3 is 0.9M - 1.1M.

[0008] The vitrification cryoprotectant provided by this application has a pH closer to the human body pH value, has a relatively higher osmotic pressure, and is more conducive to the replacement of water during the freezing process of ovarian tissue; and compared with the existing commercial cryopreservation and thawing reagent sets, after the ovarian tissue is treated with the reagent of this application, the number and area of the formed ice crystals are significantly reduced.

[0009] Further, the cryoprotectant 1 includes: 6% ethylene glycol, 6% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin - streptomycin double antibiotic, and 67% M199 medium; The cryoprotectant 2 includes: 12% ethylene glycol, 12% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin - streptomycin double antibiotic, and 55% M199 medium; The cryoprotectant 3 includes: 24% ethylene glycol, 24% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin - streptomycin double antibiotic, 31% M199 medium and sucrose; the concentration of sucrose in the cryoprotectant 3 is 1M.

[0010] Further, the human serum albumin substitute is a salt solution including human serum albumin and human serum globulin; the mass ratio of human serum albumin to human serum globulin in the salt solution is 4.5 - 5.5:0.5 - 1.5.

[0011] In the second aspect, this application provides a method for using a vitrification cryoprotectant, and the method for using includes the following steps: Obtain the processed ovarian tissue, place the ovarian tissue in the cryoprotectant 1 to equilibrate for 4 minutes - 6 minutes at room temperature, then transfer it to the cryoprotectant 2 to equilibrate for 4 minutes - 6 minutes, and finally transfer it to the cryoprotectant 3 to equilibrate for 10 minutes - 15 minutes until the ovarian tissue becomes translucent and then immediately plunge it into liquid nitrogen for cryopreservation.

[0012] In a third aspect, the present application provides an application of a vitrification cryopreservation reagent for cryopreserving human or animal ovarian tissue.

[0013] In a fourth aspect, the present application further provides a vitrification thawing reagent for thawing ovarian tissue. The ovarian tissue is cryopreserved using the above-mentioned vitrification cryopreservation reagent. The vitrification thawing reagent includes: thawing solution 1, thawing solution 2, thawing solution 3, and thawing solution 4. Calculated by volume percentage, thawing solution 1 includes: 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin-streptomycin double antibiotic, 76.5% - 81.5% M199 medium, and sucrose; the concentration of sucrose in thawing solution 1 is 0.9M - 1.1M; Thawing solution 2 includes: 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin-streptomycin double antibiotic, 76.5% - 81.5% M199 medium, and sucrose; the concentration of sucrose in thawing solution 2 is 0.4M - 0.6M; Thawing solution 3 includes: 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin-streptomycin double antibiotic, 76.5% - 81.5% M199 medium, and sucrose; the concentration of sucrose in thawing solution 3 is 0.2M - 0.3M; Thawing solution 4 includes: 18% - 22% human serum albumin substitute, 0.5% - 1.5% penicillin-streptomycin double antibiotic, 76.5% - 81.5% M199 medium.

[0014] Further, thawing solution 1 includes: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 1 is 1M; Thawing solution 2 includes: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 2 is 0.5M; Thawing solution 3 includes: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 3 is 0.25M; Thawing solution 4 includes: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium.

[0015] Further, the human serum albumin substitute is a salt solution including human serum albumin and human serum globulin; the mass ratio of human serum albumin to human serum globulin in the salt solution is 4.5 - 5.5:0.5 - 1.5.

[0016] In a fifth aspect, the present application provides a method for using a vitrification thawing reagent. The method for use includes the following steps: Take out the cryopreserved ovarian tissue from liquid nitrogen, put it into thawing solution 1 heated and rewarmed to 36°C - 40°C and immerse it completely for 1 - 2 minutes, then transfer it to thawing solution 2 and equilibrate at room temperature for 4 - 6 minutes, then transfer it to thawing solution 3 and equilibrate at room temperature for 4 - 6 minutes, and finally transfer it to thawing solution 4 and equilibrate at room temperature for 4 - 6 minutes to obtain the ovarian tissue after the equilibration is completed.

[0017] In the sixth aspect, the present application provides an application of a vitrification thawing reagent for thawing cryopreserved human or animal ovarian tissue.

[0018] The present application discloses a vitrification cryopreservation reagent, a vitrification thawing reagent, and their usage methods and applications, which have at least one of the following beneficial effects: (1) It can reduce the formation of ice crystals during the freezing process of ovarian tissue: The pH values of the vitrification cryopreservation reagent and the vitrification thawing reagent of the present application are closer to the human body pH value, have a relatively higher osmotic pressure, and are more conducive to the replacement of water during the freezing process of ovarian tissue; compared with the existing commercial cryopreservation and thawing reagent sets, after the ovarian tissue is treated with the reagents provided by the present application, the number and area of the formed ice crystals are significantly reduced.

[0019] (2) It has good antioxidant protection and anti-ferroptosis effects.

[0020] (3) After using the vitrification cryopreservation reagent and the vitrification thawing reagent of the present application, it can improve the tissue activity of ovarian tissue after freezing and thawing and improve the survival rate of transplanted ovarian tissue. Description of the Drawings

[0021] Figure 1 It is a physical picture of the ice crystal formation situation of ovarian tissue after being frozen by the existing commercial cryopreservation and thawing reagent set.

[0022] Figure 2 It is a physical picture of the ice crystal formation situation of ovarian tissue after being frozen by the vitrification cryopreservation reagent and the vitrification thawing reagent provided by Example 4 of the present application.

[0023] Figure 3 It is a graph showing the expression of oxidative stress-related proteins in ovarian tissue of different groups provided by Example 4 of the present application. Figure 4 It is a graph showing the ferritin expression in ovarian tissue of different groups provided by Example 4 of the present application.

[0024] Figure 5 It is a graph showing the expression of ferroptosis-related proteins in ovarian tissue of different groups provided by Example 4 of the present application.

[0025] Figure 6 It is a schematic diagram of the position of ovarian cortex slices in nude mice after transplantation provided by Example 5 of the present application.

[0026] Figure 7 This is a graph showing the follicle growth in ovarian tissues of different groups provided in Example 5 of this application. Detailed implementation manners

[0027] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the protection scope of this application.

[0028] Example 1 The formulation of a vitrification cryoprotectant reagent disclosed in this application is as follows: Cryoprotectant reagent: (calculated by volume ratio) Cryoprotectant solution 1: 6% ethylene glycol, 6% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 67% M199 medium; Cryoprotectant solution 2: 12% ethylene glycol, 12% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 55% M199 medium; Cryoprotectant solution 3: 24% ethylene glycol, 24% dimethyl sulfoxide, 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 31% M199 medium and sucrose; the concentration of sucrose in cryoprotectant solution 3 is 1M.

[0029] It should be noted that ethylene glycol and dimethyl sulfoxide are selected as permeating cryoprotectants in this application. However, high concentrations of ethylene glycol and dimethyl sulfoxide are toxic to cells. By gradually increasing the concentration, cells can gradually adapt to the presence of these cryoprotectants, reach an appropriate concentration, thereby reducing the occurrence of toxic damage and improving the survival rate of ovarian cells.

[0030] It should be noted that penicillin-streptomycin double antibiotic is selected in this application. Among them, penicillin can target Gram-positive bacteria, and streptomycin targets Gram-negative bacteria, covering a wide range of bacterial species and providing more comprehensive bactericidal protection.

[0031] It should be noted that sucrose in the cryoprotectant reagent can enable the ovarian tissue to be fully dehydrated, reduce the formation of ice crystals during freezing, reduce the freezing damage of the ovarian tissue, and improve the survival effect of the tissue after freeze-thawing.

[0032] The formulation of a vitrification thawing reagent disclosed in this application is as follows: Thawing reagent: (calculated by volume ratio) Thawing solution 1: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 1 is 1 M; Thawing solution 2: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 2 is 0.5 M; Thawing solution 3: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium, and sucrose; the concentration of sucrose in thawing solution 3 is 0.25 M; Thawing solution 4: 20% human serum albumin substitute, 1% penicillin-streptomycin double antibiotic, 79% M199 medium.

[0033] It should be noted that in the vitrification thawing reagent, sucrose is used as a non-permeating protective agent and its concentration gradually decreases, resulting in the decrease of ethylene glycol and dimethyl sulfoxide in the cell with the decrease of the sucrose concentration in the extracellular fluid, thereby driving the diffusion of ethylene glycol and dimethyl sulfoxide out of the cell and improving the survival effect of ovarian tissue.

[0034] Example 2 The usage method of the vitrification freezing reagent disclosed in this application is as follows: Place the processed ovarian cortex slices with a thickness of 1 mm into thawing solution 1 and equilibrate at room temperature for 5 minutes. After equilibration for 5 minutes, transfer them to thawing solution 2 and equilibrate for 5 minutes. After 5 minutes, transfer them to thawing solution 3 and equilibrate for 10 - 15 minutes. Specifically, observe the transparency of the ovarian tissue. Once the ovarian tissue is transparent, quickly place it on the carrier rod and immediately immerse it in liquid nitrogen for cryopreservation. During the equilibration process, place it on a shaker at 50 r / min throughout, which helps the permeated water leave the tissue surface and improves the dehydration effect.

[0035] It should be noted that the dehydration time of thawing solution 3 can be controlled within 10 - 15 minutes according to the thickness and dehydration degree of the ovarian cortex slices. When the tissue is transparent, it is considered successful dehydration, and there is basically no ice crystal formation or only trace ice crystal formation after freezing.

[0036] The usage method of the vitrification thawing reagent disclosed in this application is as follows: Place thawing solution 1 in a constant temperature water bath and keep the water temperature at 38°C. Immediately place the ovarian cortex slices cryopreserved in liquid nitrogen together with the carrier rod into thawing solution 1 and immerse them completely for 1 minute. After 1 minute, immediately transfer the ovarian tissue to thawing solution 2 and equilibrate at room temperature for 5 minutes, then transfer it to thawing solution 3 and equilibrate at room temperature for 5 minutes. The equilibrated ovary is transferred to thawing solution 4 and equilibrated at room temperature for 5 minutes. Subsequently, transfer the ovary to the transplantation solution at 37°C for preparation of transplantation. During the equilibration process, place it on a shaker at 50 r / min throughout.

[0037] It should be noted that in order to ensure the effects of the vitrification cryopreservation reagent and the vitrification thawing reagent, they need to be equilibrated at room temperature for 30 minutes before use, and the equilibration process should be carried out on a shaker at 50 r / min.

[0038] Comparative Example 1 The existing commercial cryopreservation and thawing reagent set (manufacturer: Kato Corporation, Japan) includes: Cryoprotectant 1, Cryoprotectant 2, Cryoprotectant 3, Thawing Solution 1, Thawing Solution 2, and Thawing Solution 3; The usage method is as follows: Put the processed ovarian cortex slices with a thickness of 1 mm into Cryoprotectant 1 and equilibrate at room temperature for 5 minutes, then put the tissue into Cryoprotectant 2 and equilibrate for 5 minutes, and finally equilibrate in Cryoprotectant 3 for 15 minutes and immediately place it on a carrier rod and freeze it in liquid nitrogen for preservation.

[0039] When thawing, put the ovarian tissue preserved by liquid nitrogen freezing together with the carrier rod into Thawing Solution 1 at 37 degrees for equilibration for 1 minute, then put the ovarian tissue into Thawing Solution 2 for equilibration for 3 minutes, and finally put the ovarian tissue into Thawing Solution 3 for equilibration for 5 minutes to complete the thawing. The equilibration process should be carried out on a shaker at 50 r / min.

[0040] Example 3 Use a blood gas analyzer to test the pH value of the liquid and a freezing point osmometer to test the osmotic pressure level of the liquid. Compare the physical and chemical parameters of the existing commercial cryopreservation and thawing reagent set with the vitrification cryopreservation reagent and the vitrification thawing reagent of this application, which are pH, PCO 2 / PO 2 and osmotic pressure, as shown in Table 1: Table 1 Physical and Chemical Parameters (pH, PCO 2 / PO 2 and Osmotic Pressure) It should be noted that the pH of the cryopreservation and thawing solution of this application is closer to the physiological pH value of the human body (7.3 - 7.4). In addition, the osmotic pressure of the cryoprotectant of the vitrification cryopreservation reagent of this application is relatively higher, which is beneficial to the replacement of water during the freezing process of the tissue. At the same time, the osmotic pressure of the cryoprotectant of the vitrification cryopreservation reagent of this application has more gradients, and the degree of decrease in osmotic pressure is more gentle, which is beneficial to the re-entry of water during the thawing process of the tissue and reduces the risk of tissue cell damage.

[0041] Example 4 In this experiment, fresh bovine ovarian tissue was taken and treated in PBS solution at 4°C. The medulla of the bovine ovarian tissue was carefully removed, and after removal, the cortex with a thickness of 1 mm was retained. The cortex was cut into cortical slices with a uniform size of about 1 cm × 1 cm. The cortical slices were divided into three groups: 1. Fresh untreated group (Group F); 2. Treated with an existing commercial cryopreservation and thawing reagent kit according to the method in Comparative Example 1 (Group C); 3. Treated with the vitrification cryopreservation reagent and vitrification thawing reagent in Example 1 of this application and according to the method in Example 2 (Group V). Among them, the ice crystal formation of the ovarian tissue in Group C and Group V is as Figure 1 , Figure 2 shown.

[0042] The experimental results of the expression of oxidative stress-related proteins, ferritin expression, and ferroptosis-related protein expression in ovarian tissue of different groups are as Figure 3 , Figure 4 , Figure 5 shown.

[0043] Example 5 Take fresh bovine ovarian tissue, treat the ovarian tissue in PBS solution at 4°C, carefully remove the medulla of the bovine ovarian tissue, and after removal, retain the cortex with a thickness of 1 mm. Cut the cortex into cortical slices with a uniform size of about 1 cm × 1 cm. Subsequently, the ovarian tissue was transplanted onto the back of nude mice after being treated with an existing commercial cryopreservation and thawing reagent kit and the vitrification cryopreservation reagent and vitrification thawing reagent of this application, and the transplantation position is as Figure 6 shown.

[0044] Three months after transplantation, the ovarian tissue was taken out, and indicators such as the volume change of ovarian tissue and the number of follicles at all levels in different groups were measured. The specific grouping is as follows: 1. Fresh ovarian tissue was directly transplanted onto the back of nude mice (Group F); 2. Ovarian tissue was transplanted onto the back of nude mice after being treated with an existing commercial cryopreservation and thawing reagent according to the method in Comparative Example 1 (Group C); 3. Ovarian tissue was transplanted onto the back of nude mice after being treated with the vitrification cryopreservation reagent and vitrification thawing reagent in Example 1 of this application and according to the method in Example 2 (Group V). The experimental results are shown in Table 2, Table 3, Figure 7 shown.

[0045] Table 2 Growth of ovarian tissue in different groups 3 months after transplantation Table 3 Growth of ovarian tissue in different groups 3 months after transplantation * indicates compared with Group F (fresh group), *=p < 0.05; **=p < 0.01; ***=p < 0.001 By analyzing the experimental results of the above embodiments, it can be concluded that the vitrification cryopreservation reagent and the vitrification thawing reagent of the present application have at least one of the following beneficial effects: 1. The reagent of the present application can reduce ice crystal formation during ovarian tissue cryopreservation: ① As shown in Table 1, the pH of the cryopreservation and thawing solution of the present application is closer to the human pH value, and the relatively higher osmotic pressure is more conducive to the replacement of water during ovarian tissue cryopreservation.

[0046] ② As Figure 1 、 Figure 2 shown, compared with the existing commercial cryopreservation and thawing reagent set, after the ovarian tissue is treated with the reagent of the present application, the number and area of ice crystals formed are significantly reduced.

[0047] 2. The reagent of the present application has good antioxidant stress and anti-ferroptosis effects: ① As Figure 3 shown, compared with the existing commercial cryopreservation and thawing reagent set, after the ovarian tissue is cryopreserved and thawed with the reagent of the present application, the expression of the oxidative stress marker malondialdehyde (MDA) is significantly reduced, and there is no statistical difference from the fresh group. In addition, the expression of the marker (GPX4) that inhibits lipid peroxidation increases, and the expression of the DNA break marker (γ-H2AX) also decreases significantly. It can be seen that compared with the existing commercial cryopreservation and thawing reagent set, the reagent of the present application has better antioxidant protection.

[0048] It should be noted that among them, glycylproline dipeptidyl aminopeptidase (GAPDH) is one of the key enzymes of cell metabolism, stably expressed in most cells, and can be used to standardize the expression levels of other proteins and eliminate the influence of experimental conditions; anti-β-actin (β-actin) is one of the main components of the cytoskeleton, which is crucial for maintaining cell morphology and stability. Similar to GAPDH, β-actin is stably expressed in most cells and can also be used to standardize the expression levels of other proteins; the important factor (Nrf2) of the oxidative defense system reflects the defense ability of ovarian tissue against oxidative stress.

[0049] ② As Figure 4 shown, ferritin mainly includes ferritin heavy chain protein (FTH), ferritin light chain protein (FTL), and mitochondrial ferritin (FTMT). FTL has a more stable structure and can store more iron. FTH has significant antioxidant activity, and FTMT can protect cells from iron-dependent oxidative damage.

[0050] ③ As Figure 5As shown, the expressions of ferroptosis suppressor protein 1 (FSP1) and ferroptosis inhibitor (VKORL) are significantly increased in the group of this application. It can be seen that compared with the existing commercial freeze-thaw reagent set, the reagent of this application shows a better anti-ferroptosis effect and more effectively preserves ovarian tissue.

[0051] 3. The freeze-thaw function of ovarian tissue recovers well after transplantation: ① As shown in Table 2, compared with the existing commercial freeze-thaw reagent set, the degree of volume atrophy of ovarian tissue after freeze-thaw treatment with the reagent of this application is lighter and there is no significant difference from the fresh group.

[0052] ② As shown in Table 3, Figure 7 compared with the existing commercial freeze-thaw reagent set, after freeze-thaw treatment of ovarian tissue with the reagent of this application, the numbers of follicles at all levels are significantly increased, and the conditions of follicle degeneration and atresia are lower than those of the commercial group.

[0053] The embodiments of this application have been described above in conjunction with the accompanying drawings. However, this application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of this application, those of ordinary skill in the art can also make many forms without departing from the purpose of this application and the scope protected by the claims, and all belong to the protection scope of this application.

Claims

1. A vitrification reagent, characterized in that: The vitrification freezing reagent includes: freezing liquid 1, freezing liquid 2 and freezing liquid 3, calculated by volume percentage, The freezing solution 1 comprises: 5% to 7% ethylene glycol, 5% to 7% dimethyl sulfoxide, 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin antibiotics, and 62.5% to 71.5% M199 culture medium; The freezing solution 2 comprises: 11% to 13% ethylene glycol, 11% to 13% dimethyl sulfoxide, 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin antibiotics, and 50.5% to 59.5% M199 culture medium; The freezing solution 3 comprises: 23%-25% ethylene glycol, 23%-25% dimethyl sulfoxide, 18%-22% human serum protein substitute, 0.5%-1.5% penicillin-streptomycin antibiotics, 26.5%-35.5% M199 culture medium and sucrose; the concentration of the sucrose in the freezing solution 3 is 0.9M-1.1M.

2. The vitrification reagent according to claim 1, characterized in that The freezing solution 1 comprises: 6% ethylene glycol, 6% dimethyl sulfoxide, 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, and 67% M199 culture medium; The freezing solution 2 comprises: 12% ethylene glycol, 12% dimethyl sulfoxide, 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, and 55% M199 culture medium; The freezing solution 2 comprises: 24% ethylene glycol, 24% dimethyl sulfoxide, 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, 31% M199 culture medium and sucrose; the concentration of the sucrose in the freezing solution 3 is 1M.

3. The vitrification reagent according to claim 1, characterized in that: The human serum protein substitute is a saline solution comprising human serum albumin and human serum globulin; the mass ratio of human serum albumin to human serum globulin in the saline solution is 4.5-5.5:0.5-1.

5.

4. A method for using the vitrification reagent according to any one of claims 1 to 3, characterized in that: The method of use comprises the following steps: The processed ovarian tissue was obtained, and the ovarian tissue was placed in freezing solution 1 for equilibration at room temperature for 4 to 6 minutes, then transferred to freezing solution 2 for equilibration for 4 to 6 minutes, and finally transferred to freezing solution 3 for equilibration for 10 to 15 minutes until the ovarian tissue was transparent, and then immediately put into liquid nitrogen for cryopreservation.

5. Use of the vitrification freezing agent according to any one of claims 1 to 3 for cryopreservation of human or animal ovarian tissue.

6. A vitrification thawing reagent for frozen ovarian tissue, wherein the ovarian tissue is frozen using the vitrification freezing reagent according to any one of claims 1 to 3, characterized in that: The vitrified thawing reagent includes: thawing solution 1, thawing solution 2, thawing solution 3 and thawing solution 4, calculated by volume percentage, The thawing solution 1 comprises: 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin antibiotics, 76.5% to 81.5% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 1 is 0.9M-1.1M; The thawing solution 2 comprises: 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin antibiotics, 76.5% to 81.5% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 2 is 0.4M to 0.6M; The thawing solution 3 comprises: 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin antibiotics, 76.5% to 81.5% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 3 is 0.2M-0.3M; The thawing solution 4 comprises: 18% to 22% human serum protein substitute, 0.5% to 1.5% penicillin-streptomycin double antibiotics, and 76.5% to 81.5% M199 culture medium.

7. The vitrified thawing reagent according to claim 6, characterized in that: The thawing solution 1 comprises: 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, 79% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 1 is 1M; The thawing solution 2 comprises: 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, 79% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 2 is 0.5M; The thawing solution 3 comprises: 20% human serum protein substitute, 1% penicillin-streptomycin antibiotics, 79% M199 culture medium and sucrose; the concentration of the sucrose in the thawing solution 3 is 0.25M; The thawing solution 4 comprises: 20% human serum protein substitute, 1% penicillin-streptomycin double antibiotics, and 79% M199 culture medium.

8. The vitrified thawing reagent according to claim 6, characterized in that: The human serum protein substitute is a saline solution comprising human serum albumin and human serum globulin; the mass ratio of human serum albumin to human serum globulin in the saline solution is 4.5-5.5:0.5-1.

5.

9. A method for using the vitrified thawing reagent according to any one of claims 6 to 8, characterized in that: The method of use comprises the following steps: The frozen ovarian tissue was taken out from liquid nitrogen, and completely immersed in thawing solution 1 heated to 36°C~40°C for 1 minute to 2 minutes, then transferred to thawing solution 2 to equilibrate at room temperature for 4 minutes to 6 minutes, then transferred to thawing solution 3 to equilibrate at room temperature for 4 minutes to 6 minutes, and finally transferred to thawing solution 4 to equilibrate at room temperature for 4 minutes to 6 minutes to obtain the ovarian tissue after equilibrium.

10. Use of the vitrified thawing reagent according to any one of claims 6 to 8 for thawing cryopreserved human or animal ovarian tissue.

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