Application of a natural eutectic solvent in vitrification cryopreservation of ovarian tissue

Abstract

This application provides the application of a natural eutectic solvent in the vitrification of ovarian tissue. The vitrification cryoprotectant comprises ethylene glycol, dimethyl sulfoxide (DMSO), and NADES. NADES is a mixture of betaine, proline, trehalose, and water. The concentrations of ethylene glycol, DMSO, and NADES are 5-10%. This vitrification cryoprotectant can inhibit ice crystal formation and promote vitrification in biological tissues, especially ovarian tissue, during vitrification, reducing intracellular water damage during freezing and protecting the structure and function of proteins and enzymes in the cytoplasm, thereby enhancing the cell's antifreeze ability. Simultaneously, this invention reduces the concentrations of the permeable cryoprotectants EG and DMSO, decreasing cytotoxicity and making cryopreservation safer.

Description

Technical Field

[0001] This application relates to the field of biotechnology and cryomedicine, specifically to the application of a natural eutectic solvent in the vitrification of ovarian tissue. Background Technology

[0002] Any discussion of prior art throughout the specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0003] In recent years, with the continuous development of early diagnosis and treatment technologies for tumors, the survival period of patients with malignant tumors has been continuously extended. Many female patients still have the desire to have children when diagnosed with cancer. However, while radiotherapy and chemotherapy kill cancer cells and save lives, they can also severely damage ovarian function, leading to premature ovarian failure. Ovarian tissue cryopreservation utilizes the principles of cryobiology to freeze and preserve ovarian tissue. After a woman's fertility decreases or is lost, the ovarian tissue is transplanted back into the body to restore fertility and endocrine function. Ovarian tissue cryopreservation is one of the main ways to preserve the fertility of female cancer patients and is also the only way for women before puberty to preserve their fertility. However, ovarian tissue cryopreservation can lead to the apoptosis and loss of some follicles, resulting in a low success rate after ovarian cryopreservation and transplantation. Increasing the volume of frozen ovarian tissue and reducing cryoprotection are key factors in improving the efficiency of ovarian tissue cryopreservation. Vitrification uses rapid cooling and high-concentration cryoprotectants to create a glassy state inside and outside the cells without ice crystal formation, preventing mechanical damage from ice crystal formation. It has been widely used clinically for embryo and oocyte cryopreservation. However, vitrification requires the use of high concentrations of cryoprotectants to dehydrate cells and prevent the formation of intracellular ice crystals. However, the cryoprotectants used, such as ethylene glycol (EG) and dimethyl sulfoxide (DMSO), are toxic to cells and can affect cell metabolism and subsequent embryonic development potential. Therefore, it is particularly important to reduce the use of these substances or avoid the damage they may cause.

[0004] The object of this invention is to overcome or improve at least one disadvantage of the prior art, or to provide a useful alternative. Unless the context clearly requires otherwise, the words “comprising” and “including” should be interpreted in an inclusive sense rather than an exclusive or exhaustive sense throughout the specification and claims; that is, in the sense of “including, but not limited to”. Summary of the Invention

[0005] Therefore, the purpose of this application is to provide an ovarian tissue vitrification freezing reagent, a kit containing the reagent, and its application. This invention uses a mixture of betaine, proline, trehalose, and water as the main component of the vitrification cryoprotectant for ovarian tissue, which together with EG and DMSO constitutes the vitrification cryoprotectant of this invention. Betaine acts as a hydrogen bond acceptor (HBA), and proline as a hydrogen bond donor (HBD). Both can bind to water molecules through electrostatic induction, interfering with the formation of dense hydrogen bonds and inhibiting ice crystal formation. Trehalose inhibits ice crystal growth during freezing and ice crystal recrystallization during thawing. The use of water not only adjusts the viscosity but also liquefies the mixture at room temperature or near room temperature. The combination of these six components inhibits ice crystal formation, growth, and recrystallization, while also retaining water, binding proteins, protecting cell membranes, and resisting oxidative stress. In particular, experimental verification shows that when applied to the vitrification and thawing of ovarian tissue, the thawed ovarian tissue exhibits good tissue morphology, low apoptosis of granulosa cells, good estrogen secretion capacity, and antioxidant capacity. Simultaneously, it reduces the concentration of the permeability protectants EG and DMSO, decreasing cytotoxicity and making cryopreservation safer.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a vitrification cryoprotectant comprising EG, DMSO and NADES, wherein NADES is a mixture of betaine, proline, trehalose and water, wherein the volume percentage concentration of EG is 5-10%, the volume percentage concentration of DMSO is 5-10%, and the volume percentage concentration of NADES is 5-10%.

[0008] In some embodiments of the present invention, the molar ratio of betaine, proline, trehalose and water in the NADES is (1-2):(1-2):(1-2):(3-5).

[0009] In a preferred embodiment of the present invention, the molar ratio of betaine, proline, trehalose and water in the NADES is 1:1:1:3.

[0010] In some embodiments of the present invention, the vitrification cryoprotectant uses a base solution as a solvent, wherein the base solution is MEM base tissue culture medium containing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES).

[0011] In a preferred embodiment of the present invention, the vitrification cryoprotectant is composed of a base solution, ethylene glycol, dimethyl sulfoxide, and NADES; wherein the base solution, as a solvent, is a MEM basic tissue culture medium containing HEPES; the volume percentage concentration of EG is 5-10%, the volume percentage concentration of DMSO is 5-10%, and the volume percentage concentration of NADES is 5-10%; NADES is a mixture of betaine, proline, trehalose, and water, and the molar ratio of betaine, proline, trehalose, and water is 1:1:1:3.

[0012] In some embodiments of the present invention, the vitrification cryoprotectant is composed of a base solution, ethylene glycol, dimethyl sulfoxide, and NADES; wherein the base solution, as a solvent, is a MEM basic tissue culture medium containing HEPES; the concentration of ethylene glycol is 5% (v / v), the concentration of dimethyl sulfoxide is 5% (v / v), and the concentration of NADES is 5% (v / v); NADES is a mixture of betaine, proline, trehalose, and water, and the molar ratio of betaine, proline, trehalose, and water is 1:1:1:3.

[0013] In some embodiments of the present invention, the vitrification cryoprotectant is composed of a base solution, ethylene glycol, dimethyl sulfoxide, and NADES; wherein the base solution, as a solvent, is a MEM basic tissue culture medium containing HEPES; the concentration of ethylene glycol is 10% (v / v), the concentration of dimethyl sulfoxide is 10% (v / v), and the concentration of NADES is 10% (v / v); the NADES is a mixture of betaine, proline, trehalose, and water, and the molar ratio of betaine, proline, trehalose, and water is 1:1:1:3.

[0014] In a second aspect, the present invention provides a vitrification thawing agent comprising NADES, wherein the NADES is a mixture of betaine, proline, trehalose and water, wherein the volume percentage concentration of NADES is 5-15%.

[0015] In some embodiments of the present invention, the molar ratio of betaine, proline, trehalose and water in the NADES is (1-2):(1-2):(1-2):(3-5).

[0016] In a preferred embodiment of the present invention, the molar ratio of betaine, proline, trehalose and water in the NADES is 1:1:1:3.

[0017] In some embodiments of the present invention, the vitrification cryo-thawing agent uses a base solution as a solvent, and the base solution is MEM base tissue culture medium containing HEPES.

[0018] In a preferred embodiment of the present invention, the vitrification cryo-thawing agent is composed of a base solution and NADES; wherein, the base solution, as a solvent, is a MEM basic tissue culture medium containing HEPES; the volume percentage concentration of NADES is 5-15%; the NADES is a mixture of betaine, proline, trehalose and water, and the molar ratio of betaine, proline, trehalose and water is 1:1:1:3.

[0019] In a third aspect of the present invention, the present invention provides a method for preparing the vitrification cryoprotectant described in the first aspect above, comprising: adding ethylene glycol, dimethyl sulfoxide and NADES to a base solution, adjusting the volume with the base solution, and filtering to remove bacteria, thereby obtaining the product.

[0020] Furthermore, the present invention provides a method for preparing the vitrification cryo-thawing agent described in the second aspect above, which includes adding NADES to a base solution, making up the volume with the base solution, and filtering to remove bacteria, thereby obtaining the agent.

[0021] Meanwhile, the present invention provides a method for preparing NADES, which includes: gently mixing betaine, proline, trehalose and water, heating and stirring continuously until a transparent viscous liquid is obtained, wherein the heating temperature is 40-60°C.

[0022] Aseptic techniques must be used during the preparation of the vitrified cryoprotectant and vitrified cryothrottle. The prepared vitrified cryoprotectant and vitrified cryothrottle should be refrigerated when not in use, with a refrigeration temperature preferably between 2-8°C.

[0023] In a fourth aspect, the present invention provides a kit comprising the vitrification cryoprotectant described in the first aspect and / or the vitrification thawing agent described in the second aspect. For example, the vitrification cryoprotectant can be used as an equilibration solution and / or freezing solution in the kit, and the vitrification thawing agent can be used as a thawing solution and / or diluent and / or cleaning solution in the kit.

[0024] In a fifth aspect of the invention, the invention provides an ovarian tissue vitrification and thawing kit, comprising a freezing kit and a thawing kit, wherein the freezing kit contains the vitrification cryoprotectant described in the first aspect above; and / or, the thawing kit contains the vitrification thawing agent described in the second aspect above.

[0025] In some embodiments of the present invention, the freezing kit includes a balancing fluid and a freezing fluid;

[0026] The equilibration solution and / or cryosol uses a base solution as a solvent, and the solute is composed of ethylene glycol, dimethyl sulfoxide and NADES; the base solution is MEM basic tissue culture medium containing HEPES; and NADES is a mixture of betaine, proline, trehalose and water.

[0027] In some embodiments of the present invention, the resuscitation kit includes thawing fluid, diluent, and cleaning fluid, wherein the cleaning fluid may contain multiple components, such as cleaning fluid 1 and cleaning fluid 2.

[0028] Wherein, at least one of the thawing solution, diluent, and washing solution is composed of a base solution and NADES; wherein, the base solution is a solute, which is a MEM basic tissue culture medium containing HEPES; and the NADES is a mixture of betaine, proline, trehalose, and water.

[0029] In some of the above embodiments, the molar ratio of betaine, proline, trehalose and water in the NADES is (1-2):(1-2):(1-2):(3-5), preferably 1:1:1:3; the volume percentage concentration of NADES is 5-15%.

[0030] In some of the above embodiments, the concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the equilibration fluid and / or refrigerant are 5-10% (v / v).

[0031] In some of the above embodiments, the concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the equilibration solution are all 5% (v / v); and the concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the cryogenic solution are all 10% (v / v).

[0032] In some of the above embodiments, the concentration of NADES in at least one of the thawing solution, diluent, and cleaning solution is 5-15% (v / v).

[0033] In some of the above embodiments, the concentration of NADES in the thawing solution is 15.0% (v / v), the concentration of NADES in the diluent is 10.0% (v / v), and the concentration of NADES in the cleaning solution 1 is 5.0% (v / v).

[0034] In some of the above embodiments, when the washing solution contains two components, washing solution 1 consists of a base solution and NADES; wherein, the base solution is a solute, which is MEM basic tissue culture medium containing HEPES; NADES is a mixture of betaine, proline, trehalose and water; washing solution 2 is prepared with the base solution and contains 0.5 IU / mL r-FSH (recombinant follicle-stimulating hormone), 1% ITS (insulin iron selenide transfer protein), and 0.5% penicillin and streptomycin.

[0035] For example, in one of the above embodiments, the freezing kit includes a balancing fluid and a freezing fluid, and the revival kit includes a thawing fluid, a diluent, a cleaning fluid 1, and a cleaning fluid 2;

[0036] The equilibration solution and / or cryosol uses a base solution as a solvent, and the solute is composed of ethylene glycol, dimethyl sulfoxide, and NADES; the base solution is MEM basic tissue culture medium containing HEPES; the volume percentage concentration of ethylene glycol is 5-10%, the volume percentage concentration of dimethyl sulfoxide is 5-10%, and the volume percentage concentration of NADES is 5-10%; NADES is a mixture of betaine, proline, trehalose, and water, and the molar ratio of betaine, proline, trehalose, and water is (1-2):(1-2):(1-2):(3-5), preferably 1:1:1:3;

[0037] The thawing solution, diluent, and washing solution 1 are all composed of a base solution and NADES. The base solution is a solute, which is a MEM basic tissue culture medium containing HEPES. The NADES is a mixture of betaine, proline, trehalose, and water. The molar ratio of betaine, proline, trehalose, and water is (1-2):(1-2):(1-2):(3-5), preferably 1:1:1:3. The volume percentage concentration of NADES is 5-15%. The washing solution 2 is prepared with the base solution and contains 0.5 IU / mL r-FSH (recombinant follicle-stimulating hormone), 1% ITS (insulin iron selenide transfer protein), and 0.5% penicillin and streptomycin.

[0038] In a sixth aspect of the invention, the invention provides the application of the vitrification cryoprotectant described in the first aspect, or the vitrification cryo-thawing agent described in the second aspect, or the kit described in the fourth aspect, or the ovarian tissue vitrification freezing and thawing kit described in the fifth aspect, in the vitrification freezing and / or thawing of biological tissues; preferably, the biological tissue is ovarian tissue.

[0039] In a seventh aspect, the present invention provides a method for vitrifying and thawing ovarian tissue, comprising treating ovarian tissue using the vitrifying cryoprotectant described in the first aspect, or the vitrifying cryothrottle agent described in the second aspect, or the kit described in the fourth aspect, or the ovarian tissue vitrification and thawing kit described in the fifth aspect.

[0040] When processing ovarian tissue, the vitrification cryoprotectant can be used as a balancing solution and / or freezing solution to treat the ovarian tissue, which is then immersed in liquid nitrogen for preservation. The vitrification thawing agent can be used as a thawing solution and / or diluent and / or washing solution to thaw and restore the ovarian tissue preserved in liquid nitrogen.

[0041] In some embodiments of the present invention, taking the vitrification and thawing kit for ovarian tissue as an example, the method includes:

[0042] The ovarian tissue was cryopreserved using a cryotherapy kit, which included immersing the ovarian tissue in a balanced solution and a cryotherapy solution in sequence, then placing it into an ovarian cryopreservation carrier, sealing it, and immersing it in liquid nitrogen for cryopreservation.

[0043] The revival kit for reviving frozen ovarian tissue includes: taking the cryogenic carrier containing the ovarian tissue out of liquid nitrogen and quickly immersing it in thawing solution; detaching the ovarian tissue from the carrier; removing the cryogenic carrier from the thawing solution; pressing the tissue appropriately to ensure it is completely submerged in the thawing solution; and then sequentially passing it through diluent, washing solution 1, and washing solution 2.

[0044] Compared with existing technologies, the advantages of this invention are:

[0045] This invention provides cryoprotectants and thawing agents for vitrification and thawing of ovarian tissue. Both the cryoprotectant and thawing agent contain NADES, a mixture of betaine, proline, trehalose, and water. The cryoprotectant also contains ethylene glycol and dimethyl sulfoxide. The cryoprotectant of this invention can inhibit ice crystal formation, promote vitrification, reduce intracellular water damage during freezing and thawing, protect the structure and function of proteins and enzymes in the cytoplasm, thereby enhancing the cell's cryoprotective capacity. Furthermore, it reduces the concentration of permeable cryoprotectants, lowers cytotoxicity, and makes cryopreservation safer. This invention solves the damage problems caused by low temperature, intracellular and extracellular ice crystal formation, and the high permeability and toxicity of cryoprotectants during ovarian tissue vitrification. This invention also provides a kit or apparatus containing the aforementioned cryoprotectant and / or thawing agent for ovarian tissue. In particular, the present invention provides a kit containing specific components for vitrification and thawing of ovarian tissue and its method of use. The ovarian tissue vitrified and thawed by the kit has good tissue morphology (manifested as a large number of primordial follicles with intact basement membranes, clear oocyte nuclear membranes, uniform and normal morphology of granulosa cells, with a normal morphology rate of primordial follicles higher than 70% and a normal morphology rate of primary follicles higher than 61%), very low degree of apoptosis of granulosa cells, good estrogen secretion capacity and antioxidant capacity, which is a significant improvement over the prior art.

[0046] Meanwhile, this invention reduces the concentrations of the permeability protectants EG and DMSO, thereby decreasing cytotoxicity and making cryopreservation safer. Furthermore, the method of this invention is simple and easy to operate, time-efficient, and provides good cryopreservation results, reducing ice crystal formation, decreasing the degree of granulosa cell apoptosis after vitrification of ovarian tissue, and improving the efficiency of ovarian tissue vitrification. Attached Figure Description

[0047] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. The embodiments of this application will be described in detail below with reference to the accompanying drawings, wherein:

[0048] Figure 1 Comparative images of morphological observation of ovarian tissue after vitrification and thawing.

[0049] Figure 2 Comparison of ovarian tissue apoptosis detection results.

[0050] Figure 3 Comparison of the contents of multiple antioxidant indicators CAT, MDA and GSH-PX measured after homogenization of ovarian tissue, where each mgprot represents one milligram of protein.

[0051] Figure 4 The results show the hormone levels in the culture medium of ovarian tissues in each group on days 2, 4, 6, 8, and 10 after in vitro culture. Detailed Implementation

[0052] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this application. The preferred methods and materials described herein are for illustrative purposes only. In the experiments below, where each group involves multiple data results, unless otherwise specified, results are expressed as Mean or Mean ± SD. The form of presentation or processing based on it includes calculations and / or drawing.

[0054] Example 1 Preparation of ovarian tissue vitrification kit

[0055] This embodiment provides an ovarian tissue vitrification kit, which includes a freezing kit and a thawing kit. The freezing kit includes equilibration fluid (ES) and cryosol (VS), and the thawing kit includes thawing fluid (TS), diluent (DS), washing solution 1 (WS1), and washing solution 2 (WS2). The specific components are as follows:

[0056] Equilibrium solution (ES): Prepared with base solution, containing 5.0% EG, 5.0% DMSO, and 5.0% NADES.

[0057] Preparation method of ES: Perform aseptic operation in a clean bench, add 5.0 mL EG, 5.0 mL DMSO and 5.0 mL NADES to the base solution, make up to 100 mL with base solution, filter through a 0.22 μm filter for sterilization and store at 4°C.

[0058] Coolant (VS): Prepared with base fluid, containing 10.0% EG, 10.0% DMSO, and 10.0% NADES.

[0059] Preparation method of VS: Perform aseptic operation in a clean bench, add 10.0 mL EG, 10.0 mL DMSO and 10.0 mL NADES to the base solution, and make up to 100 mL with base solution. After filtration through a 0.22 μm filter for sterilization, store at 4°C.

[0060] Thawing fluid (TS): Prepared with base fluid and contains 15.0% NADES.

[0061] Preparation method of TS: Perform aseptic operation in a clean bench, add 15.0 mL of NADES to the base solution, make up to 100 mL with base solution, filter through a 0.22 μm filter for sterilization, and store at 4°C.

[0062] Diluent (DS): Prepared with base solution and contains 10.0% NADES.

[0063] Preparation method of DS: Perform aseptic operation in a clean bench, add 10.0 mL of NADES to the base solution, make up to 100 mL with base solution, filter through a 0.22 μm filter for sterilization, and store at 4°C.

[0064] Cleaning solution 1 (WS1): Prepared with base solution, containing 5.0% NADES.

[0065] Preparation method of WS1: Perform aseptic operation in a clean bench, add 5.0 mL of NADES to the base solution, make up to 100 mL with base solution, filter through a 0.22 μm filter for sterilization, and store at 4°C.

[0066] Washing solution 2 (WS2): Prepared with base solution, containing 0.5 IU / mL r-FSH, 1% ITS, and 0.5% penicillin antibody.

[0067] Preparation method of WS2: Perform aseptic operation in a clean bench, add 1.0 mL ITS, 0.5 mL double antibody, and 50 IU r-FSH to the base solution, and make up to 100 mL with base solution. After filtration through a 0.22 μm filter for sterilization, store at 4°C.

[0068] NADES is a mixture of betaine, proline, trehalose, and water in a molar ratio of betaine:proline:trehalose:water = 1:1:1:3. The preparation method for NADES includes: gently mixing 58.57g betaine, 57.50g proline, 171.15g trehalose, and 27mL water, heating to 40-60℃, and stirring constantly until a clear, viscous liquid is obtained.

[0069] The basal medium is MEM basal tissue culture medium containing 25 mmol / L HEPES buffer and 10% human serum albumin (HSA). The preparation method for the basal medium includes: 0.59 g HEPES, 10 mL HSA, diluted to 100 mL with MEM, adjusting the pH to 7.2-7.4 with 1 N HCl and 1 N NaOH, filtering through a 0.22 μm filter for sterilization, and storing at 4°C.

[0070] Example 2

[0071] The difference from Example 1 is that the molar ratio of betaine:proline:trehalose:water in this example is 2:2:2:3.

[0072] Example 3

[0073] The difference from Example 1 is that the molar ratio of betaine:proline:trehalose:water in this example is 1:1:1:5.

[0074] Comparative Example 1

[0075] The difference from Example 1 is that the molar ratio of betaine:proline:trehalose:water in this comparative example is 5:2:2:21.

[0076] Comparative Example 2

[0077] The difference from Example 1 is that betaine was not added to the composition of NADES in this comparative example.

[0078] Comparative Example 3

[0079] The difference from Example 1 is that trehalose was not added to the composition of NADES in this comparative example.

[0080] Comparative Example 4

[0081] The difference from Example 1 is that proline was not added to the composition of NADES in this comparative example.

[0082] Comparative Example 5

[0083] The difference from Example 1 is that glucose is used instead of trehalose in the composition of NADES in this comparative example.

[0084] Comparative Example 6

[0085] The difference from Example 1 is that the thawing fluid, diluent, and cleaning fluid 1 in this comparative resuscitation kit have different compositions. The thawing fluid is prepared with a base solution and contains 1M sucrose; the diluent is prepared with a base solution and contains 0.5M sucrose; and the cleaning fluid 1 is prepared with a base solution and contains 0.25M sucrose.

[0086] Comparative Example 7

[0087] The difference from Example 1 is that EG and DMSO were not added to the equilibrium liquid and the cryogenic liquid in this comparative example.

[0088] Comparative Example 8

[0089] This comparative example provides a vitrification freezing kit, which includes a balancing fluid, a freezing fluid, and a thawing fluid. The compositions of the balancing fluid, freezing fluid, and thawing fluid (including thawing fluid 1, thawing fluid 2, and thawing fluid 3) are as follows:

[0090] Equilibration solution: DPBS buffer containing 0.25M trehalose, 0.2M betaine and 1.5mM HSA, pH=7.2.

[0091] Cryosol: DPBS buffer containing 1M trehalose, 0.5M proline, 0.3M betaine and 1.5mM HSA, pH=7.2.

[0092] Thawing solution 1: DPBS buffer of 0.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0093] Thawing solution 2: DPBS buffer containing 0.2M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0094] Thawing solution 3: DPBS buffer containing 0.2M betaine and 1.5mM HSA, pH=7.2.

[0095] Experimental Example

[0096] Ovarian tissue acquisition and processing: Ovaries were obtained from non-pregnant ewes aged 8-10 months and weighing approximately 30 kg from the slaughterhouse. The acquired ovarian tissue was transported to the laboratory in pre-cooled PBS. The medulla was removed, preserving the ovarian cortex, resulting in a ovarian tissue thickness of approximately 1 mm after processing. The tissue was processed on an ice plate, cut into 10 mm × 5 mm × 1 mm pieces, washed, and set aside for use.

[0097] I. Experimental grouping and freezing / thawing procedures:

[0098] The tissue blocks were randomly divided into 13 groups, with 6 tissue blocks set as parallels in each group. 3 of these were used for tissue section observation and tissue homogenization, and 3 were used for tissue culture.

[0099] 1. Fresh control group: No freezing or thawing treatment was performed;

[0100] 2. KITA Group: The freezing and thawing of ovarian tissue was performed according to the product instructions using a commercially available ovarian tissue vitrification freezing and thawing kit (OvaCryo Device Type M, KITAZATO).

[0101] 3. Experimental Group (NADES Group): This group was divided into three subgroups: NADES1, NADES2, and NADES3. Each subgroup underwent freezing and thawing using the vitrification kits for ovarian tissue prepared in Examples 1-3 of this application. The freezing and thawing methods were as follows:

[0102] (1) Ovarian tissue freezing: The freezing solution (VS) was removed from the freezer and brought to room temperature. Large pieces of sheep ovarian tissue were successively immersed in balanced solution (ES) for 15 min and freezing solution (VS) for 10 min, and then placed in an ovarian freezing carrier, sealed, and directly frozen in liquid nitrogen at -196℃ for preservation.

[0103] (2) Ovarian tissue resuscitation: Remove the ovarian tissue resuscitation solution from the freezer, warm the thawing solution (TS) to 37°C, and restore the remaining resuscitation solutions to room temperature. Take the cryocart containing the ovarian tissue out of liquid nitrogen and quickly immerse it in the thawing solution (TS) at 37°C. Shake the cryocart to detach the ovarian tissue from the carrier. Then remove the cryocart from the thawing solution (TS), and use ophthalmic forceps to press the tissue appropriately to ensure that the tissue is completely immersed in the thawing solution (TS) for 1 minute. Then, rinse with diluent (DS) for 3 minutes, wash solution 1 (WS1) for 5 minutes, and wash solution 2 (WS2) for 5 minutes.

[0104] 4. Control Group: This group includes 8 groups, named control groups 1, 2, 3, 4, 5, 6, 7, and 8 respectively. Each of the eight groups was subjected to freezing and thawing using the vitrification kits of ovarian tissue prepared in Comparative Examples 1-8 of this application. Among them, the freezing and thawing methods used in Comparative Examples 1-7 were the same as those used in the NADES group. The freezing and thawing method for control group 8 was as follows: a large piece of sheep ovarian tissue was soaked in a balanced solution for 5 minutes, then soaked in a freezing solution for 25 minutes, placed in an ovarian cryopreservation carrier, sealed, and directly placed in liquid nitrogen at -196°C for cryopreservation. The cryopreservation carrier containing the ovarian tissue was removed from the liquid nitrogen and quickly placed in thawing solution 1 at 37°C. The cryopreservation carrier was shaken to detach the ovarian tissue from the carrier. Then, the cryopreservation carrier was removed from thawing solution 1, and the tissue was gently pressed with ophthalmic forceps to ensure that the tissue was completely immersed in the thawing solution (placed in thawing solutions 1-3 for 5 minutes each).

[0105] II. Ovarian tissue sections and staining:

[0106] Methods: Ovarian tissues from each group were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and then prepared into 4μm thick slides.

[0107] 1. HE staining: 25 sections were selected intermittently from each group for hematoxylin-eosin (HE) staining. Normal and abnormal follicles were classified under an optical microscope.

[0108] Results: Table 1 shows the comparison results of the normal morphology rate of follicles in each group. HE staining results showed that the total number of follicles in the ovarian tissue of the fresh control group, NADES1-3 group and KITA group was relatively large, and many primordial follicles had intact basement membranes, clear oocyte nuclear membranes, uniform granulosa cells and normal morphology. In contrast, the control group 1-8 showed many abnormal follicles with oocyte shrinkage, nuclear pyknosis, vacuoles in the cytoplasm, and disordered arrangement of granulosa cells.

[0109] As an example, Figure 1 The morphological observations of some groups (fresh control group, KITA group, NADES1 group and control group 8) are shown.

[0110] Table 1 Comparison of the rate of normal follicle morphology in different groups after ovarian tissue resuscitation

[0111]

[0112] Note: n represents the number of morphologically normal follicles, m represents the number of morphologically abnormal follicles, and * indicates that P < 0.05 compared with the fresh control group.

[0113] Primordial follicles are follicles with a single layer of flattened granulosa cells or flattened granulosa cells containing some cuboidal granulosa cells surrounding the primary oocyte; primary follicles are follicles with a single layer of cuboidal granulosa cells surrounding the primary oocyte.

[0114] Among them, follicles with normal morphology are regular in shape, round or oval, with intact oocytes, visible round nuclei, and evenly distributed granulosa cells without condensation.

[0115] Abnormal follicle morphology: The follicle morphology and structure are incomplete, vacuoles and nuclear pyknosis appear in the oocyte, and the granulosa cells are arranged in a disordered manner, with obvious absence or nuclear pyknosis.

[0116] 2. Ovarian tissue apoptosis detection:

[0117] Ovarian tissue apoptosis was detected using the TUNEL assay (terminal deoxynucleotidyltransferase-mediated dUTP nick-end-labeling, Roche, Switzerland), with DAPI staining used to label cell nuclei (blue). The procedure was strictly performed according to the kit instructions. Apoptotic cells (including follicles and stromal cells) were defined as TUNEL-positive and appeared green in the TUNEL stain. Five slides were prepared for each group, and two high-power fields were randomly selected from each slide. Apoptotic cells were counted using Image-pro Plus 6.0 software.

[0118] Results: The degree of apoptosis in granulosa cells was determined by the number of TUNEL-positive cells. A higher number of TUNEL-positive cells may indicate a higher degree of apoptosis in the ovarian tissue. Results are shown in Table 2. Figure 2 .

[0119] Table 2. TUNEL-positive cell count in ovarian tissue of each group (cell count / ×400 fields)

[0120]

[0121] Note: * indicates P < 0.05 compared to the fresh control group.

[0122] 3. Antioxidant capacity test:

[0123] After ovarian tissue was resuscitated and homogenized, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were detected using appropriate kits according to the instructions. MDA levels can indirectly indicate the severity of free radical attack on cells, and combined analysis of CAT, GSH-Px, and MDA levels can evaluate changes in oxidative stress levels.

[0124] Results: The NADES group showed higher levels of CAT and GSH-PX, slightly lower than the fresh control group but comparable to the KITA group, although the MDA level was relatively lower. This indicates that the experimental group was able to increase the levels of related antioxidant enzymes CAT and GSH-Px in cryopreserved ovarian tissue, reduce the level of the oxidative metabolite MDA, inhibit excessive oxidative stress in ovarian tissue, and improve antioxidant capacity. Figure 3 The comparison of antioxidant index content between the KITA group, NADES1 group and control group 8 is shown.

[0125] III. In vitro culture of ovarian tissue and detection of hormone levels

[0126] Methods: Fresh ovarian tissue (3 pieces per group) from the control group and other groups after resuscitation was collected and processed into 2mm×2mm×1mm pieces. The small tissues from each group were randomly divided into 6 subgroups with 7 pieces per subgroup and placed in 12-well plates. 2 mL of culture medium was added to each well and the plates were incubated at 37℃ in a 5% incubator. Hormone levels in the incubator were measured every other day. 0.5 mL of culture medium was collected for each measurement and an equal amount of culture medium was added. The estradiol (E2) content in the culture medium was detected using a Roche automated biochemical analyzer on days 2, 4, 6, 8, and 10 after incubation.

[0127] The ovarian tissue culture medium consisted of α-MEM + 5 mg / mL HSA + 0.5 IU / mL rFSH + 1% ITS + 0.5% antibiotic antifungal solution. The preparation method (based on 50 mL) was as follows: 2.5 mL HSA, 0.5 mL ITS, 0.25 mL double antibiotic, 0.5 IU / mL rFSH, and α-MEM were added to bring the volume to 50 mL. The mixture was then mixed, filtered for sterilization, aliquoted, and stored at 4°C.

[0128] Results: Ovarian tissues in all groups secreted estradiol. The estradiol growth trend in the ovarian tissues of the NADES1-3 and KITA groups was consistent with that in the fresh control group. Hormone levels increased with increasing culture time. The estradiol content in the NADES1-3 groups was comparable (especially the highest content in the NADES1 group, reaching 1059.90 pg / mL on day 10, while the other two groups were also above 850 pg / mL), second only to the fresh culture group (reaching 1308.83 pg / mL on day 10), superior to the KITA group (reaching 906.35 pg / mL on day 10), and significantly superior to the control groups 1-8 (where control group 5 reached 915.78 pg / mL on day 10, control group 6 reached 803.95 pg / mL, and the remaining groups were all far below 700 pg / mL, especially control group 8, which had the lowest level at only 312.67 pg / mL). Figure 4Hormone level-time curves for the fresh control group, NADES1-3 group, KITA group, and control group 1-8, plotted based on the results.

[0129] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A reagent kit, characterized in that, The kit contains a vitrification thawing agent and a vitrification cryoprotectant; The vitrification cryoprotectant is composed of a base solution, ethylene glycol, dimethyl sulfoxide, and NADES. The base solution is a solvent and is a MEM basic tissue culture medium containing HEPES. The NADES is a mixture of betaine, proline, trehalose, and water. The concentrations of ethylene glycol, dimethyl sulfoxide, and NADES are 5-10% v / v. In NADES, the molar ratio of betaine, proline, trehalose and water is (1-2):(1-2):(1-2):(3-5); Vitrification cryoprotectants are used for vitrification and thawing of ovarian tissue; The kit also contains a vitrification cryo-thawing agent, which consists of a base solution and NADES; The vitrification cryo-thawing agent contains MEM basic tissue culture medium containing HEPES as the base solution; the volume percentage concentration of NADES is 5-15%; NADES is a mixture of betaine, proline, trehalose and water, wherein the molar ratio of betaine, proline, trehalose and water is (1-2):(1-2):(1-2):(3-5).

2. The reagent kit according to claim 1, characterized in that, In NADES, a vitrification thawing agent, the molar ratio of betaine, proline, trehalose, and water is 1:1:1:

3.

3. The reagent kit according to claim 1, characterized in that, The vitrification cryoprotectant contains ethylene glycol at a concentration of 5% v / v, dimethyl sulfoxide at a concentration of 5% v / v, and NADES at a concentration of 5% v / v; NADES is a mixture of betaine, proline, trehalose, and water, with a molar ratio of 1:1:1:

3. Alternatively, the concentration of ethylene glycol, dimethyl sulfoxide, and NADES in the vitrification cryoprotectant is 10% v / v, 10% v / v, and 10% v / v, respectively; NADES is a mixture of betaine, proline, trehalose, and water, with a molar ratio of 1:1:1:

3.

4. The reagent kit according to claim 1, characterized in that, The molar ratio of betaine, proline, trehalose, and water in the vitrification thawing agent NADES is 1:1:1:

3.

5. A method for preparing the kit according to any one of claims 1 to 4, comprising a method for preparing a vitrification cryoprotectant, comprising: Add ethylene glycol, dimethyl sulfoxide and NADES to the base solution, bring the volume up to the desired level, filter to remove bacteria, and the solution is ready. A method for preparing a vitrification cryo-thawing agent includes adding NADES to a base solution, making up the volume with the base solution, and filtering to remove bacteria. The preparation method of NADES includes: mixing betaine, proline, trehalose and water, heating and stirring until a clear viscous liquid is obtained.

6. An ovarian tissue vitrification cryopreservation and thawing kit, comprising a cryopreservation kit and a thawing kit, wherein, The cryoprotectant contained in the kit according to any one of claims 1 to 4; The refrigeration kit includes a balancing fluid and a freezing fluid; The equilibration solution and / or cryosol uses the base solution as a solvent, and the solute is composed of ethylene glycol, dimethyl sulfoxide and NADES; the base solution is MEM basic tissue culture medium containing HEPES; NADES is a mixture of betaine, proline, trehalose and water; The resuscitation kit includes thawing fluid, diluent, and cleaning fluid, wherein the cleaning fluid contains at least cleaning fluid 1. At least one of the thawing solution, diluent, and washing solution 1 is composed of a base solution and NADES; wherein, the base solution is a solute, and the base solution is MEM basic tissue culture medium containing HEPES; NADES is a mixture of betaine, proline, trehalose and water; The molar ratio of betaine, proline, trehalose, and water in NADES is (1-2):(1-2):(1-2):(3-5); the volume percentage concentration of NADES is 5-15%. The concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the equilibration fluid and / or refrigerant are 5-10% v / v; Cleansing solution 2 is prepared with a base solution and contains recombinant follicle-stimulating hormone, insulin iron-selenium transfer protein, and antibiotics.

7. The ovarian tissue vitrification and thawing kit according to claim 6, characterized in that, The molar ratio of betaine, proline, trehalose and water in NADES is 1:1:1:3; The concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the equilibration solution are all 5% v / v; the concentrations of ethylene glycol, dimethyl sulfoxide, and NADES in the cryogenic solution are all 10% v / v. The concentration of NADES in the thawing solution is 15.0% v / v, the concentration of NADES in the diluent is 10.0% v / v, and the concentration of NADES in the cleaning solution 1 is 5.0% v / v. The cleaning solution comprises cleaning solutions 1 and 2.

8. The ovarian tissue vitrification cryopreservation and thawing kit according to claim 6, characterized in that, The cryo-pack includes a balancing fluid and a cryo-fluid, and the revival kit includes a thawing fluid, a diluent, a cleaning fluid 1, and a cleaning fluid 2. The equilibration solution and / or cryosol uses a base solution as a solvent, and the solute is composed of ethylene glycol, dimethyl sulfoxide, and NADES. The base solution is MEM basic tissue culture medium containing HEPES. The volume percentage concentrations of ethylene glycol, dimethyl sulfoxide, and NADES are 5-10% and 5-10% respectively. NADES is a mixture of betaine, proline, trehalose, and water, and the molar ratio of betaine, proline, trehalose, and water is (1-2):(1-2):(1-2):(3-5). The thawing solution, diluent, and washing solution 1 are all composed of a base solution and NADES. The base solution is the solute, which is MEM basic tissue culture medium containing HEPES. The NADES is a mixture of betaine, proline, trehalose, and water, with a molar ratio of (1-2):(1-2):(1-2):(3-5). The volume percentage concentration of NADES is 5-15%. The washing solution 2 is prepared with the base solution and contains 0.5 IU / mL recombinant follicle-stimulating hormone, 1% insulin iron-selenium transfer protein, and 0.5% penicillin and streptomycin.

9. The kit according to any one of claims 1 to 4 or the ovarian tissue vitrification and thawing kit according to any one of claims 6 to 8, for use in vitrification and / or thawing of biological tissues; wherein the biological tissue is ovarian tissue.

10. A method for vitrifying and thawing ovarian tissue, comprising treating the ovarian tissue using a kit as described in any one of claims 1 to 4 or an ovarian tissue vitrification and thawing kit as described in any one of claims 6 to 8.

11. The method according to claim 10, characterized in that, The ovarian tissue was cryopreserved using a cryotherapy kit, which included immersing the ovarian tissue in a balanced solution and a cryotherapy solution in sequence, then placing it into an ovarian cryopreservation carrier, sealing it, and immersing it in liquid nitrogen for cryopreservation. The revival kit for reviving frozen ovarian tissue includes: removing the cryogenic carrier containing the ovarian tissue from liquid nitrogen and quickly immersing it in thawing solution; detaching the ovarian tissue from the carrier; removing the cryogenic carrier from the thawing solution; pressing the tissue appropriately to ensure it is completely submerged in the thawing solution; and then passing it through diluent and washing solution in sequence.

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