Ovarian tissue low-temperature cryopreservation liquid as well as preparation method and application thereof

Abstract

The invention discloses an ovarian tissue low-temperature cryopreservation solution as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The invention relates to an ovarian tissue low-temperature cryopreservation solution as well as a preparation method and application thereof. Every 100 ml of the ovarian tissue low-temperature cryopreservation solution comprises 15-25 ml of dimethyl sulfoxide, 15-25 ml of ethylene glycol, 15-20 g of polysucrose, 38-46 g of sucrose, 40-45 ml of a phosphate buffer solution and 0.12-0.13 g of human serum albumin. According to the low-temperature cryopreservation solution for the ovarian tissues, the dimethyl sulfoxide and the ethylene glycol are used as permeable low-temperature protective agents to prevent the ovarian tissues from being damaged at low temperature, and the polysucrose and the sucrose are used for regulating the cell osmotic pressure to improve the cell dehydration effect, reduce the formation of ice crystals in cells and increase the viscosity of the solution; according to the low-temperature cryopreservation solution for the ovarian tissue, the solution forms an amorphous glassy state instead of ice crystals in the freezing process, the human serum albumin maintains osmotic pressure and nutrition, the tissue survival rate and the functional integrity are guaranteed, the ovarian tissue can be efficiently cryopreserved through the low-temperature cryopreservation solution for the ovarian tissue, and the follicle survival rate and the functional integrity after ovarian transplantation are effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an ovarian tissue low-temperature freezing preservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Vitrification is a rapid, efficient, and low-cost technology for cryopreserving ovarian tissue. It converts the ovaries and their protective solution from a liquid phase to a glassy solid during rapid cooling, allowing for long-term storage at ultra-low temperatures of -196°C. However, existing freezing and thawing procedures, including tissue freeze-thaw recipes and freeze-thaw protocols, can cause irreversible damage to the structure and function of ovarian tissue, including toxicity, osmotic pressure, cold shock, and intracellular ice crystal formation. This can lead to decreased physiological activity of ovarian tissue after recovery, resulting in failure to survive after transplantation, or even significant damage to follicles after survival.

[0003] In addition, the current ovarian tissue cryopreservation fluid can easily cause the ovarian tissue to become supercooled along with the cryopreservation fluid after freezing during the freezing process. The vitrified state formed in this state is extremely unstable during the thawing process, and there are strict requirements on the thawing rate and the operation of the operator during the thawing process. If the operation is improper, the sample will undergo devitrification during the thawing process. Once devitrification occurs, the sample will form ice crystals again, thereby damaging the ovarian tissue.

[0004] Therefore, a low-temperature freezing preservation solution for ovarian tissue, as well as a preparation method and application thereof, is necessary to solve the problems of supercooling of ovarian tissue and freezing solution during freezing and devitrification of samples during thawing. Summary of the Invention

[0005] In order to overcome the above problems, the present invention provides an ovarian tissue low-temperature freezing preservation solution and a preparation method and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions: The ovarian tissue cryopreservation solution includes a permeable cryoprotectant, a non-permeable cryoprotectant for internal tissue dehydration, a buffer solution, and human serum albumin. The four core components work synergistically to construct a functional system suitable for ovarian tissue cryopreservation. The permeable cryoprotectant can penetrate the cell membrane and enter the cell interior to protect against low-temperature damage at the cellular level. The non-permeable cryoprotectant acts on the outside of the cell to achieve tissue dehydration by regulating the extracellular environment. The buffer solution maintains a stable physical and chemical environment of the system. The human serum albumin ensures tissue activity from the two aspects of osmotic pressure balance and nutrient supply. The four components have clear division of labor and cooperate with each other to provide all-round protection for ovarian tissue cryopreservation.

[0007] The permeable cryoprotectant includes dimethyl sulfoxide and ethylene glycol, and the non-permeable cryoprotectant includes polyethylene glycol and sucrose; the dimethyl sulfoxide and the ethylene glycol as the permeable cryoprotectant have good cell membrane penetration, can quickly enter the ovarian tissue cells in a low-temperature environment, reduce the freezing point of the intracellular liquid, reduce the protein denaturation and the organelle damage of the cells caused by low temperature, and inhibit the initial formation of the ice crystals in the cells; the polyethylene glycol and the sucrose as the non-permeable cryoprotectant cannot penetrate the cell membrane, but can form a high osmotic pressure between the inside and outside of the cells by increasing the liquid concentration outside the cells, so that the water in the cells migrates outward, realizes efficient dehydration of the tissue inside, and the combination of the two can significantly improve the viscosity of the solution, and lay a foundation for the subsequent formation of a stable glass state.

[0008] In 100 ml of the ovarian tissue cryopreservation solution, the dimethyl sulfoxide is 15-25 ml, the ethylene glycol is 15-25 ml, the polyethylene glycol is 15-20 g, the sucrose is 38-46 g, the buffer is 40-45 ml, and the human blood albumin is 0.12-0.13 g; the use amount of 15-25 ml of the dimethyl sulfoxide and 15-25 ml of the ethylene glycol can ensure sufficient low-temperature protection effect and avoid cytotoxicity caused by too high concentration of the cryoprotectant, 15-20 g of the polyethylene glycol and 38-46 g of the sucrose form a high-osmotic environment, can promote the dehydration of the ovarian tissue cells, reduce the formation of ice crystals in the cells, and improve the viscosity of the solution, 40-45 ml of the buffer can stabilize the pH value and ion concentration of the system, provide a suitable environment for the tissue, and 0.12-0.13 g of the human blood albumin cooperates with the buffer to maintain the osmotic pressure, ensuring the preliminary stability of the form and function of the tissue in the freezing process.

[0009] The ratio of the buffer to the human blood albumin is 1000 ml:3 g. 1000 ml of the buffer is matched with 3 g of the human blood albumin, so that the osmotic pressure of the system can always be maintained at a level close to the physiological osmotic pressure of the ovarian tissue cells, avoiding the swelling or shrinkage of the cells caused by the sharp fluctuation of the osmotic pressure, and at the same time, the human blood albumin at the ratio can continuously provide necessary nutritional support for the ovarian tissue cells, slow down the metabolic consumption of the cells in the low-temperature environment, and ensure the active state of the tissue before freezing, laying a foundation for the survival rate improvement in the subsequent freezing and thawing processes.

[0010] The ovarian tissue cryopreservation solution as described above, wherein the buffer is phosphate buffer, and each 100 ml of the phosphate buffer comprises 0.005-0.015 g of magnesium chloride, 0.008-0.018 g of calcium chloride, 1-3 μl of phenol red, 0.05-0.15 g of glucose, 0.003-0.004 g of sodium pyruvate, 0.007-0.008 g of gentamicin and purified water. The components of the phosphate buffer can work together to ensure the stability of the system. Magnesium chloride and calcium chloride can supplement the divalent cations required by the cells, which can not only maintain the balance between the osmotic pressure of the ovarian tissue low-temperature freezing solution and the osmotic pressure in the ovarian tissue cells, but also ensure the stability of the intracellular enzyme activity and cell membrane. Phenol red can be used as a pH indicator. Phenol red can intuitively reflect the change in the pH value of the solution through color change. It is yellow when the pH is acidic and red when the pH is alkaline, which is convenient for operators to monitor and adjust the pH of the system in time to ensure that the environmental pH is appropriate. Glucose and sodium pyruvate serve as energy substrates to slowly provide the ovarian tissue cells with the energy required for metabolism, slow down the energy consumption of the cells in the low-temperature pretreatment stage, and maintain the basic physiological functions of the cells. Gentamycin, as a broad-spectrum antibiotic, can effectively inhibit the growth and reproduction of microorganisms such as bacteria, and avoid microbial contamination of ovarian tissue during the processing and preservation process. Pure water is used as a solvent to ensure that the various components are evenly dissolved, construct a stable buffer system, and provide a suitable physical and chemical environment for ovarian tissue.

[0011] The freezing method of ovarian tissue uses the ovarian tissue low-temperature freezing preservation solution described above, and the freezing method comprises the following steps: S1: Place the ovarian tissue in a cryovial containing equilibration solution and incubate at room temperature for 10 minutes; S2: Transfer the ovarian tissue to ovarian tissue cryopreservation solution and incubate at 4°C for 20 min; S3: Fill the cryovial with ovarian tissue cryopreservation solution; S4: The ovarian tissue was transferred into a cryovial, and the cryovial was immersed in liquid nitrogen at -196°C for cryopreservation.

[0012] The 10-minute incubation at room temperature in step S1 is a pretreatment stage for adapting the ovarian tissue to the cryoprotectant. The room temperature environment allows the ovarian tissue cells to gradually absorb the balancing solution, avoiding toxic stimulation to the cells due to a sudden increase in the concentration of the balancing solution. At the same time, it allows the tissue cells to slowly adapt to changes in osmotic pressure, reducing damage to the cells caused by drastic fluctuations in osmotic pressure, and preparing for subsequent entry into the high-concentration cryopreservation solution.

[0013] The low temperature environment of 4°C in step S2 can slow down the metabolic rate of ovarian tissue cells and reduce the consumption of cell activity. At the same time, the incubation time of 20 minutes can ensure that the cells fully absorb the permeable cryoprotectant in the cryopreservation solution, so that the permeable cryoprotectant reaches an effective concentration in the cells, laying a protective foundation for subsequent ultra-low temperature freezing. In addition, the 4°C environment can also reduce the toxic effects of non-permeable cryoprotectants on cells, balance the protective effect, and reduce the risk of cell damage.

[0014] The ovarian tissue low-temperature freezing preservation solution is loaded in advance in step S3 to ensure that the ovarian tissue can adapt to the environment more quickly after being transferred to the cryopreservation tube, avoid exposure of the tissue to air or other unsuitable environments during the transfer process, reduce the risks of temperature fluctuations, water loss or contamination, and ensure the activity and structural integrity of the tissue.

[0015] The -196°C liquid nitrogen in step S4 is an ultra-low temperature environment that can rapidly cool the ovarian tissue and the cryopreservation solution, quickly passing the dangerous temperature range for ice crystal formation. Combined with the glass-forming ability of the cryopreservation solution, the tissue and the preservation solution are rapidly transformed into a stable glass state, minimizing the formation of ice crystals in cells, thereby maintaining the structural and functional integrity of the ovarian tissue and achieving long-term preservation of the tissue. At the same time, the ultra-low temperature environment can almost completely stagnate cell metabolism, preventing tissue degeneration or damage during long-term preservation.

[0016] The application of the ovarian tissue low-temperature cryopreservation solution includes the ovarian tissue low-temperature cryopreservation solution described above, which is used for cryopreservation of ovarian tissue.

[0017] In the above-described method for freezing ovarian tissue, the balancing solution comprises 8-12 ml of dimethyl sulfoxide, 8-12 ml of ethylene glycol, 75-85 ml of buffer, and 0.2-0.3 g of human albumin per 100 ml. The low concentrations of dimethyl sulfoxide and ethylene glycol allow for gradual penetration of the protective agent, preventing cellular toxicity caused by rapid penetration. The buffer maintains the osmotic pressure and pH of the balancing solution, providing a suitable environment for the tissue. The human albumin helps maintain osmotic pressure and provides nutritional support for tissue cells.

[0018] In the above-described method for freezing ovarian tissue, the cryotube is a graphene-heated ovarian tissue cryotube, comprising a tube body, a tube cap, and a fixing member. The fixing member comprises a spoon-shaped surface for placing the ovarian tissue and a fixing pin disposed on the spoon-shaped surface for fixing the ovarian tissue. The fixing member is provided with a graphene heating layer within the fixing member, and the tube cap is provided with an electrical component electrically connected to an external power supply terminal, the graphene heating layer being electrically connected to the electrical component. The shape of the spoon-shaped surface allows the ovarian tissue to be laid flat, thereby preventing mechanical damage to the tissue due to shaking within the tube body. The fixing pin can stably fix the ovarian tissue, ensuring the tissue's position during freezing and thawing, thereby preventing uneven local protection or heating due to tissue displacement. The graphene heating layer has the characteristics of uniform heating and high thermal conductivity. In conjunction with the electrical component on the tube cap, precise temperature control and heating can be achieved during the thawing process, solving the problem of uneven heating in traditional thawing methods. At the same time, the stability of the graphene material ensures the service life of the cryotube in alternating environments between ultra-low temperature and room temperature, ensuring the reliability of the cryotube function.

[0019] The freezing method for ovarian tissue described above further comprises a thawing process, wherein the thawing process comprises the following steps: P1: Take the cryotube containing ovarian tissue out of liquid nitrogen and keep it in air for 15 seconds; P2: Place the cryovial in a sterilization tank filled with 37°C sterilized water and connect the cryovial to an electric source until the liquid in the cryovial is completely thawed; P3: Remove the ovarian tissue from the cryopreservation tube and place it in resuscitation solution 1, and keep it at room temperature for 3-4 minutes; P4: Remove the ovarian tissue from the resuscitation solution 1 and place it in the resuscitation solution 2, and keep it at room temperature for 3-4 minutes; P5: Remove the ovarian tissue from the resuscitation solution 2 and place it in the resuscitation solution 3, and keep it at room temperature for 3-4 minutes; P6: The ovarian tissue in the resuscitation solution 3 is removed and washed at room temperature.

[0020] The 15-second stay in the air in step P1 is the pre-warming stage of thawing, which can initially raise the temperature of the outer wall of the cryopreservation tube to avoid the cryopreservation tube from rupturing due to excessive temperature difference when directly placed in 37°C sterilized water. At the same time, it can also initially soften the low-temperature freezing preservation solution of the ovarian tissue in the cryopreservation tube, making transition preparations for subsequent thawing and reducing the impact of sudden temperature changes on the ovarian tissue.

[0021] In the P2 step, 37°C is the physiological temperature of the human body, which is consistent with the suitable temperature of ovarian tissue cells. It can thaw the low-temperature cryopreservation solution of ovarian tissue and avoid damaging cells due to excessive temperature. Sterile water and sterilization tanks can ensure a sterile environment during the thawing process and prevent microbial contamination. After the cryopreservation tube is connected to the power supply, the graphene heating layer starts heating and cooperates with the external 37°C water bath to achieve synchronous temperature increase inside and outside the cryopreservation tube, ensuring that the ovarian tissue is heated evenly, quickly crossing the dangerous temperature zone for ice crystal formation, avoiding the occurrence of devitrification during the thawing process, and ensuring tissue activity.

[0022] In the P3 step, the resuscitation solution 1 contains a relatively high concentration of sucrose. Maintaining it at room temperature for 3-4 minutes can utilize the hypertonic environment to further promote the ovarian tissue cells to expel a small amount of water that may remain during the freezing process. At the same time, it gradually dilutes the cryoprotectant in the cells, reducing the subsequent toxic effects of the protectant on the cells. The room temperature environment can prevent temperature fluctuations from causing additional damage to the fragile cells that have just been thawed, providing a gentle transition environment for the cells to restore their physiological functions.

[0023] In the P4 step, the sucrose concentration in the resuscitation solution 2 is lower than that in the resuscitation solution 1, forming a gradient osmotic pressure environment. The 3-4 minute incubation at room temperature allows the cells to gradually adapt to the decrease in osmotic pressure and slowly absorb water, thereby avoiding cell swelling and rupture due to a sudden drop in osmotic pressure. At the same time, the protective agent remaining in the cells is further diluted, reducing the toxicity of the protective agent, and creating conditions for the cells to subsequently recover their metabolic function.

[0024] The resuscitation solution 3 in step P5 does not contain sucrose and is composed mainly of phosphate buffer and human serum albumin. Its osmotic pressure is close to the physiological osmotic pressure of the cells. Incubation for 3-4 minutes at room temperature allows the cells to fully adapt to the physiological osmotic pressure environment and completely removes residual cryoprotectants in the cells. At the same time, human serum albumin provides nutritional support for the cells, helping them to restore their normal morphology and physiological functions, thereby preparing for subsequent ovarian tissue transplantation or further processing.

[0025] Washing at room temperature in the P6 step can remove residual components of the resuscitation fluid attached to the surface of the ovarian tissue, preventing the residual components from having adverse effects on the subsequent transplantation process. At the same time, it further ensures the cleanliness of the tissue, reduces potential contamination risks, and provides guarantees for the survival and functional recovery of the ovarian tissue after transplantation back into the body.

[0026] In the freezing method for ovarian tissue described above, during the thawing process, the electrical component of the cryotube is electrically connected to the external power supply, and the graphene heating layer heats the ovarian tissue fixed to the spoon core surface by the fixing needle. This heating method achieves uniform heating of the ovarian tissue. When the fixing needle fixes the ovarian tissue, the tissue can be in close contact with the fixing member. The heat generated by the graphene heating layer can be evenly transferred to various parts of the ovarian tissue through the fixing member, avoiding local damage caused by the excessive temperature difference between the tissue surface and the interior during traditional water bath thawing. At the same time, the heating temperature can be regulated by controlling the external power supply, ensuring that the thawing process is always within the appropriate temperature range, thereby maximizing the protection of the activity and structural integrity of the ovarian tissue.

[0027] The freezing method of ovarian tissue as described above, Each 100 ml of the resuscitation solution 1 includes 32-37 g of sucrose, 70-80 ml of phosphate buffer and 0.2-0.4 g of human albumin; Each 100 ml of the resuscitation solution 2 includes 15-20 g of sucrose, 85-95 ml of phosphate buffer and 0.2-0.4 g of human albumin; Every 100 ml of the resuscitation fluid 3 includes 95-99.9 ml of phosphate buffer and 0.2-0.4 g of human albumin.

[0028] The high concentration of sucrose (32-37 g) in the resuscitation solution 1 can form a hypertonic environment outside the cells, drive the discharge of residual water in the cells, and reduce the risk of cell swelling. At the same time, the phosphate buffer maintains the stability of the system pH and ion concentration, and human serum albumin helps maintain osmotic pressure and provides nutrition.

[0029] The concentration of 15-20 g of sucrose in the resuscitation solution 2 is between that of the resuscitation solution 1 and the resuscitation solution 3, forming an osmotic pressure gradient. The phosphate buffer and human serum albumin work together to maintain the stability of the system.

[0030] The 95-99.9 ml phosphate buffer in the resuscitation solution 3 simulates the physiological environment of cells, and human serum albumin provides nutrition and osmotic pressure support.

[0031] The application of the freezing method for ovarian tissue includes the freezing method for ovarian tissue as described above, which is applied to cryopreservation of ovarian tissue.

[0032] Compared with the existing technology, the beneficial effects of this technical solution are: The low-temperature cryopreservation solution for the ovarian tissue provided by the application uses dimethyl sulfoxide and ethylene glycol as the permeable cryoprotectant, and adds high-concentration poly-sucrose and sucrose as the non-permeable cryoprotectant, thereby providing better internal dehydration effect of the tissue through the combination of poly-sucrose and sucrose, further increasing the viscosity of the solution, promoting the formation of amorphous glass state instead of ice crystals in the solution during the freezing and thawing process, better protecting the ovarian tissue, maintaining the osmotic pressure and nutrition of human blood albumin, ensuring the survival rate and functional integrity of the tissue, and enabling the low-temperature cryopreservation solution for the ovarian tissue to efficiently cryopreserve the ovarian tissue, so that the ovarian tissue can reach an ideal balanced glass state during freezing, avoid overcooling state, reduce the formation of intracellular ice crystals during freezing and thawing, better avoid the damage of ice crystals and the like to the ovarian tissue during the cryopreservation process, and effectively improve the follicle survival rate and functional integrity and other physiological functions after ovarian transplantation.

[0033] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and / or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without any creative effort.

[0035] Figure 1 is a freezing step flow chart in the freezing method for the ovarian tissue of the present application; Figure 2 is a thawing step flow chart in the freezing method for the ovarian tissue of the present application; Figure 3 is a structure perspective view of the cryopreservation tube used in the present application; Figure 4 is a sectional view of the cryopreservation tube used in the present application; Figure 5 is Figure 4 is a partial enlarged view of A in FIG. 8; Figure 6 is an exploded schematic view of the cryopreservation tube used in the present application. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Example: Figures 1-6 As shown, this embodiment discloses an ovarian tissue cryopreservation solution, its preparation method, and application, relating to the field of biomedical materials technology. The ovarian tissue cryopreservation solution includes a permeable cryoprotectant, a non-permeable cryoprotectant for internal tissue dehydration, a buffer, and human serum albumin. The four core components work synergistically to construct a functional system adapted for ovarian tissue cryopreservation. The permeable cryoprotectant can penetrate the cell membrane and enter the cell interior, protecting it from low-temperature damage at the cellular level. The non-permeable cryoprotectant acts on the outside of the cell, achieving tissue dehydration by regulating the extracellular environment. The buffer maintains a stable physical and chemical environment of the system. The human serum albumin ensures tissue activity from the two aspects of osmotic pressure balance and nutrient supply. The four components have clear division of labor and cooperate with each other to provide all-round protection for ovarian tissue cryopreservation.

[0038] The permeable cryoprotectant includes dimethyl sulfoxide and ethylene glycol, and the non-permeable cryoprotectant includes polysucrose and sucrose. As permeable protectants, dimethyl sulfoxide and ethylene glycol have good cell membrane permeability and can quickly enter ovarian tissue cells under low temperature conditions, lowering the freezing point of the intracellular fluid, reducing protein denaturation and organelle damage caused by low temperature, and inhibiting the initial formation of ice crystals in cells. As non-permeable protectants, although polysucrose and sucrose cannot penetrate the cell membrane, they can form a higher osmotic pressure inside and outside the cell by increasing the liquid concentration outside the cell, causing the water in the cell to migrate outward, thereby achieving efficient dehydration inside the tissue. The combined use of the two can significantly increase the viscosity of the solution, laying the foundation for the subsequent formation of a stable glassy state.

[0039] Every 100 ml of ovarian tissue cryopreservation solution contains 15-25 ml of dimethyl sulfoxide, 15-25 ml of ethylene glycol, 15-20 g of polysucrose, 38-46 g of sucrose, 40-45 ml of buffer, and 0.12-0.13 g of human serum albumin. The 15-25 ml of dimethyl sulfoxide and 15-25 ml of ethylene glycol ensure sufficient cryoprotection while avoiding cytotoxicity caused by excessive concentration of the protective agent. The 15-20 g of polysucrose and 38-46 g of sucrose form a hypertonic environment, which promotes dehydration of ovarian tissue cells, reduces intracellular ice crystal formation, and increases solution viscosity. The 40-45 ml of buffer stabilizes the pH value and ion concentration of the system, providing a suitable environment for the tissue. The 0.12-0.13 g of human serum albumin cooperates with the buffer to maintain osmotic pressure, ensuring initial morphological and functional stability of the tissue during cryopreservation.

[0040] The ratio of buffer to human albumin is 1000ml:3g. This combination of 1000ml of buffer and 3g of human albumin consistently maintains the system's osmotic pressure close to the physiological osmotic pressure of ovarian tissue cells, preventing cell swelling or shrinkage caused by drastic fluctuations in osmotic pressure. Furthermore, this ratio of human albumin continuously provides essential nutritional support for ovarian tissue cells, slowing their metabolic consumption at low temperatures and ensuring tissue activity prior to cryopreservation, laying the foundation for improved survival during subsequent freezing and thawing.

[0041] The ovarian tissue low-temperature freezing preservation solution provided by the present invention uses dimethyl sulfoxide and ethylene glycol as permeable cryoprotectants, and adds high-concentration polysucrose and sucrose as non-permeable cryoprotectants. The combined use of polysucrose and sucrose provides a better dehydration effect inside the tissue, further increases the viscosity of the solution, promotes the solution to form an amorphous glassy state rather than ice crystals during the freezing and thawing process, and better protects the ovarian tissue. Human serum albumin maintains osmotic pressure and nutrition, ensures tissue survival rate and functional integrity. The ovarian tissue low-temperature freezing preservation solution can efficiently freeze ovarian tissue, so that the ovarian tissue reaches an ideal balanced vitrified state when frozen, avoids the supercooling state, thereby reducing the formation of ice crystals in cells of the tissue during freezing and thawing, better avoiding damage to the ovarian tissue by ice crystals and the like during the freezing process, and effectively improving various physiological functions such as the survival rate and functional integrity of follicles after ovarian transplantation.

[0042] In one embodiment, per 100 ml of ovarian tissue cryopreservation solution, the solution comprises 20 ml of dimethyl sulfoxide, 20 ml of ethylene glycol, 18 g of polysucrose, 10.3 g of sucrose, 42.1 ml of phosphate buffer, and 0.126 g of human serum albumin. The polysucrose in the ovarian tissue cryopreservation solution is Ficoll 70, and the optimal effect is achieved when the content of the solution is 18 g of polysucrose per 100 ml of the solution.

[0043] Furthermore, the buffer is a phosphate buffer, and each 100 ml of the phosphate buffer includes 0.005-0.015 g magnesium chloride, 0.008-0.018 g calcium chloride, 1-3 μl phenol red, 0.05-0.15 g glucose, 0.003-0.004 g sodium pyruvate, 0.007-0.008 g gentamicin and purified water. The components of the phosphate buffer can work together to ensure the stability of the system. Magnesium chloride and calcium chloride can supplement the divalent cations required by the cells, which can not only maintain the balance between the osmotic pressure of the ovarian tissue low-temperature freezing solution and the osmotic pressure in the ovarian tissue cells, but also ensure the stability of the intracellular enzyme activity and cell membrane. Phenol red can be used as a pH indicator. Phenol red can intuitively reflect the change in the pH value of the solution through color change. It is yellow when the pH is acidic and red when the pH is alkaline, which is convenient for operators to monitor and adjust the pH of the system in time to ensure that the environmental pH is appropriate. Glucose and sodium pyruvate serve as energy substrates to slowly provide the ovarian tissue cells with the energy required for metabolism, slow down the energy consumption of the cells in the low-temperature pretreatment stage, and maintain the basic physiological functions of the cells. Gentamycin, as a broad-spectrum antibiotic, can effectively inhibit the growth and reproduction of microorganisms such as bacteria, and avoid microbial contamination of ovarian tissue during the processing and preservation process. Pure water is used as a solvent to ensure that the various components are evenly dissolved, construct a stable buffer system, and provide a suitable physical and chemical environment for ovarian tissue.

[0044] In one embodiment, phosphate buffer solution is composed of 0.01 g magnesium chloride, 0.013 g calcium chloride, trace amount of phenol red, 0.1 g glucose, 0.0036 g sodium pyruvate, 0.0075 g gentamicin and purified water per 100 ml.

[0045] This embodiment also provides a method for freezing ovarian tissue, comprising the ovarian tissue cryopreservation solution according to any one of claims 1 to 2, comprising the following steps: S1: Place the ovarian tissue in a cryovial containing equilibration solution and incubate at room temperature for 10 minutes; S2: Transfer the ovarian tissue to ovarian tissue cryopreservation solution and incubate at 4°C for 20 min; S3: Fill the cryovial with ovarian tissue cryopreservation solution; S4: The ovarian tissue was transferred into a cryovial, and the cryovial was immersed in liquid nitrogen at -196°C for cryopreservation.

[0046] The 10-minute incubation at room temperature in step S1 is a pretreatment stage for adapting the ovarian tissue to the cryoprotectant. The room temperature environment allows the ovarian tissue cells to gradually absorb the balancing solution, avoiding toxic stimulation to the cells due to a sudden increase in the concentration of the balancing solution. At the same time, it allows the tissue cells to slowly adapt to changes in osmotic pressure, reducing damage to the cells caused by drastic fluctuations in osmotic pressure, and preparing for subsequent entry into the high-concentration cryopreservation solution.

[0047] The low temperature environment of 4°C in step S2 can slow down the metabolic rate of ovarian tissue cells and reduce the consumption of cell activity. At the same time, the incubation time of 20 minutes can ensure that the cells fully absorb the permeable cryoprotectant in the cryopreservation solution, so that the permeable cryoprotectant reaches an effective concentration in the cells, laying a protective foundation for subsequent ultra-low temperature freezing. In addition, the 4°C environment can also reduce the toxic effects of non-permeable cryoprotectants on cells, balance the protective effect, and reduce the risk of cell damage.

[0048] The ovarian tissue low-temperature freezing preservation solution is loaded in advance in step S3 to ensure that the ovarian tissue can adapt to the environment more quickly after being transferred to the cryopreservation tube, avoid exposure of the tissue to air or other unsuitable environments during the transfer process, reduce the risks of temperature fluctuations, water loss or contamination, and ensure the activity and structural integrity of the tissue.

[0049] The -196°C liquid nitrogen in step S4 is an ultra-low temperature environment that can rapidly cool the ovarian tissue and the cryopreservation solution, quickly passing the dangerous temperature range for ice crystal formation. Combined with the glass-forming ability of the cryopreservation solution, the tissue and the preservation solution are rapidly transformed into a stable glass state, minimizing the formation of ice crystals in cells, thereby maintaining the structural and functional integrity of the ovarian tissue and achieving long-term preservation of the tissue. At the same time, the ultra-low temperature environment can almost completely stagnate cell metabolism, preventing tissue degeneration or damage during long-term preservation.

[0050] This embodiment also provides the use of a low-temperature cryopreservation solution for ovarian tissue, including the low-temperature cryopreservation solution for ovarian tissue according to any one of claims 1 to 2, wherein the low-temperature cryopreservation solution is used for cryopreservation of ovarian tissue.

[0051] Furthermore, per 100 ml of the balancing solution, the solution comprises 8-12 ml of dimethyl sulfoxide, 8-12 ml of ethylene glycol, 75-85 ml of a buffer solution, and 0.2-0.3 g of human albumin. The low concentrations of dimethyl sulfoxide and ethylene glycol allow for gradual penetration of the protective agent, preventing cellular toxicity caused by rapid penetration. The buffer solution maintains stable osmotic pressure and pH value of the balancing solution, providing a suitable environment for the tissue. The human albumin assists in maintaining osmotic pressure and provides nutritional support for tissue cells.

[0052] In one embodiment, each 100 ml of the equilibration solution contains 10 ml of dimethyl sulfoxide, 10 ml of ethylene glycol, 80 ml of phosphate buffer and 0.24 g of human albumin.

[0053] Furthermore, if Figure 3-6 As shown, the cryopreservation tube is a graphene-heated ovarian tissue cryopreservation tube, which includes a tube body 1, a tube cap 2 and a fixing part 3. The fixing part 3 includes a spoon-shaped surface 31 for placing ovarian tissue and a fixing needle 32 provided on the spoon-shaped surface 31 for fixing the ovarian tissue. A graphene heating layer is provided in the fixing part 3, and an electrical component 21 electrically connected to an external power supply end is provided on the tube cap 2. The graphene heating layer is electrically connected to the electrical component 21. The shape of the spoon center surface 31 allows the ovarian tissue to be laid flat, which can prevent the tissue from being mechanically damaged due to shaking in the tube body. The fixing needle 32 can stably fix the ovarian tissue, ensuring that the tissue is in a stable position during the freezing and thawing process, avoiding local uneven protection or uneven heating due to tissue displacement. The graphene heating layer has the characteristics of uniform heating and high thermal conductivity. Combined with the electrical component 21 on the tube cap 2, it can achieve precise temperature control and heating during the thawing process, solving the problem of uneven heating in traditional thawing methods. At the same time, the stability of the graphene material can ensure the service life in an alternating environment of ultra-low temperature and normal temperature, and ensure the reliability of the cryopreservation tube function.

[0054] Specifically, the cryopreservation tube uses high-impact polypropylene (HIPP). By adding a rubber phase, such as ethylene-propylene copolymer, to homopolymer PP, the HIPP improves the toughness of PP and can withstand ultra-low temperatures of -196°C without cracking. At the same time, the HIPP has excellent chemical stability and will not react with the components in the reagents, nor will it precipitate small molecules to contaminate the ovarian tissue cryopreservation solution or damage the ovarian tissue. In addition, the HIPP has high impact strength and is not easily damaged during the process of clamping and transferring with tweezers. The HIPP tube body is also highly transparent, making it easier for operators to observe the status of the tissue inside the tube.

[0055] In addition, if Figures 4 and 5 As shown, the graphene heating layer includes three layers, which are composed of two layers of metal copper sandwiching a layer of graphene film. The graphene film is also provided with a temperature sensor on the side close to the center of the spoon to monitor the temperature during the thawing process, and cooperates with the control module provided at the external power supply end to control the temperature to prevent the temperature from being too high or too low and affecting the ovarian tissue.

[0056] Furthermore, the cryopreservation method further includes a thawing process, and the thawing process includes the following steps: P1: Take the cryotube containing ovarian tissue out of liquid nitrogen and keep it in air for 15 seconds; P2: Place the cryovial in a sterilization tank filled with 37°C sterilized water and connect the cryovial to an electric source until the liquid in the cryovial is completely thawed; P3: Remove the ovarian tissue from the cryopreservation tube and place it in resuscitation solution 1, and keep it at room temperature for 3-4 minutes; P4: Remove the ovarian tissue from the resuscitation solution 1 and place it in the resuscitation solution 2, and keep it at room temperature for 3-4 minutes; P5: Remove the ovarian tissue from the resuscitation solution 2 and place it in the resuscitation solution 3, and keep it at room temperature for 3-4 minutes; P6: The ovarian tissue in the resuscitation solution 3 is removed and washed at room temperature.

[0057] The 15-second stay in the air in step P1 is the pre-warming stage of thawing, which can initially raise the temperature of the outer wall of the cryopreservation tube to avoid the cryopreservation tube from rupturing due to excessive temperature difference when directly placed in 37°C sterilized water. At the same time, it can also initially soften the low-temperature freezing preservation solution of the ovarian tissue in the cryopreservation tube, making transition preparations for subsequent thawing and reducing the impact of sudden temperature changes on the ovarian tissue.

[0058] In the P2 step, 37°C is the physiological temperature of the human body, which is consistent with the suitable temperature of ovarian tissue cells. It can thaw the low-temperature cryopreservation solution of ovarian tissue and avoid damaging cells due to excessive temperature. Sterile water and sterilization tanks can ensure a sterile environment during the thawing process and prevent microbial contamination. After the cryopreservation tube is connected to the power supply, the graphene heating layer starts heating and cooperates with the external 37°C water bath to achieve synchronous temperature rise inside and outside the cryopreservation tube, ensuring that the ovarian tissue is heated evenly, quickly crossing the dangerous temperature zone for ice crystal formation, avoiding the occurrence of devitrification during the thawing process, and ensuring tissue activity.

[0059] In the P3 step, the resuscitation solution 1 contains a relatively high concentration of sucrose. Maintaining it at room temperature for 3-4 minutes can utilize the hypertonic environment to further promote the ovarian tissue cells to expel a small amount of water that may remain during the freezing process. At the same time, it gradually dilutes the cryoprotectant in the cells, reducing the subsequent toxic effects of the protectant on the cells. The room temperature environment can prevent temperature fluctuations from causing additional damage to the fragile cells that have just been thawed, providing a gentle transition environment for the cells to restore their physiological functions.

[0060] In the P4 step, the sucrose concentration in the resuscitation solution 2 is lower than that in the resuscitation solution 1, forming a gradient osmotic pressure environment. The 3-4 minute incubation at room temperature allows the cells to gradually adapt to the decrease in osmotic pressure and slowly absorb water, thereby avoiding cell swelling and rupture due to a sudden drop in osmotic pressure. At the same time, the protective agent remaining in the cells is further diluted, reducing the toxicity of the protective agent, and creating conditions for the cells to subsequently recover their metabolic function.

[0061] The resuscitation solution 3 in step P5 does not contain sucrose and is composed mainly of phosphate buffer and human serum albumin. Its osmotic pressure is close to the physiological osmotic pressure of the cells. Incubation for 3-4 minutes at room temperature allows the cells to fully adapt to the physiological osmotic pressure environment and completely removes residual cryoprotectants in the cells. At the same time, human serum albumin provides nutritional support for the cells, helping them to restore their normal morphology and physiological functions, thereby preparing for subsequent ovarian tissue transplantation or further processing.

[0062] Washing at room temperature in the P6 step can remove residual components of the resuscitation fluid attached to the surface of the ovarian tissue, preventing the residual components from having adverse effects on the subsequent transplantation process. At the same time, it further ensures the cleanliness of the tissue, reduces potential contamination risks, and provides guarantees for the survival and functional recovery of the ovarian tissue after transplantation back into the body.

[0063] Furthermore, during the thawing process, the electrical component 21 of the cryotube is electrically connected to an external power supply, and the graphene heating layer heats the ovarian tissue fixed to the spoon core 31 via the fixing needles 32. This heating method achieves uniform heating of the ovarian tissue. When the fixing needles 32 fix the ovarian tissue, the tissue can be in close contact with the fixing member 3. The heat generated by the graphene heating layer can be evenly transferred to all parts of the ovarian tissue through the fixing member 3, avoiding local damage caused by the large temperature difference between the tissue surface and the interior during traditional water bath thawing. At the same time, the heating temperature can be regulated by controlling the external power supply, ensuring that the thawing process is always within the appropriate temperature range, maximizing the protection of the activity and structural integrity of the ovarian tissue.

[0064] Furthermore, each 100 ml of the resuscitation solution 1 includes 32-37 g of sucrose, 70-80 ml of phosphate buffer and 0.2-0.4 g of human albumin; Each 100 ml of the resuscitation solution 2 includes 15-20 g of sucrose, 85-95 ml of phosphate buffer and 0.2-0.4 g of human albumin; Every 100 ml of the resuscitation fluid 3 includes 95-99.9 ml of phosphate buffer and 0.2-0.4 g of human albumin.

[0065] The high concentration of sucrose (32-37 g) in the resuscitation solution 1 can form a hypertonic environment outside the cells, drive the discharge of residual water in the cells, and reduce the risk of cell swelling. At the same time, the phosphate buffer maintains the stability of the system pH and ion concentration, and human serum albumin helps maintain osmotic pressure and provides nutrition.

[0066] The concentration of 15-20 g of sucrose in the resuscitation solution 2 is between that of the resuscitation solution 1 and the resuscitation solution 3, forming an osmotic pressure gradient. The phosphate buffer and human serum albumin work together to maintain the stability of the system.

[0067] The 95-99.9 ml phosphate buffer in the resuscitation solution 3 simulates the physiological environment of cells, and human serum albumin provides nutrition and osmotic pressure support.

[0068] In one embodiment, 100 ml of resuscitation fluid 1 contains 34.23 g of sucrose, 78.4 ml of phosphate buffer and 0.235 g of human albumin; 100 ml of resuscitation fluid 2 contains 17.12 g of sucrose, 89.2 ml of phosphate buffer and 0.268 g of human albumin; and 100 ml of resuscitation fluid 3 contains 99.7 ml of phosphate buffer and 0.3 g of human albumin.

[0069] This embodiment also provides an application of a freezing method for ovarian tissue, including the freezing method for ovarian tissue according to any one of claims 5 to 9, which is applied to cryopreservation of ovarian tissue.

[0070] In one embodiment, the ovarian tissue cryopreservation solution described above is used and applied to the ovarian tissue freezing method described above, wherein the freezing process: Human ovarian tissue obtained surgically (Beijing University of Chinese Medicine, Shenzhen Hospital) was placed in a cryovial containing equilibration solution and incubated at room temperature for 10 minutes. Subsequently, the ovarian tissue was transferred to cryopreservative solution and incubated at 4°C for 20 minutes. Finally, the ovarian tissue was transferred to a cryovial containing cryopreservative solution and cryopreserved by immersion in liquid nitrogen at -196°C.

[0071] Thawing process: The cryovial containing ovarian tissue was removed from liquid nitrogen, exposed to air for 15 seconds, and then placed in a sterilizer filled with 37°C sterile water until the liquid in the cryovial was completely thawed. The ovarian tissue was removed from the cryovial and placed in the prepared resuscitation solution 1 at room temperature for 3 minutes, resuscitation solution 2 at room temperature for 3 minutes, and resuscitation solution 3 at room temperature for 4 minutes. The tissue was then washed thoroughly and allowed to equilibrate at room temperature for 10 minutes.

[0072] The treated ovarian tissue was transplanted back into the patient's body. One month later, testing revealed normal hormone secretion, follicular development, and other normal ovarian physiological functions, compared to normal ovarian function. Furthermore, follow-up results indicate that three patients (at Beijing University of Chinese Medicine Shenzhen Hospital) have successfully conceived through the ovarian cryotransplantation treatment described in this example, including one live birth.

[0073] The present invention further provides Example 1 and Example 2, which are described as follows: Example 1 includes a pre-treatment process and a thawing process. In the pre-treatment process, a 10×10 mm piece of ovarian tissue is treated with ovarian tissue balancing solution, cryopreservation solution, etc. 2After the ovarian tissue is taken out, use tweezers to flatten it onto the spoon center surface 311 of the placement part 31, so that the first fixing part 32 pierces the ovarian tissue to achieve the fixation effect. Then put the ovarian tissue into the tube body 1 containing 1 ml of cryopreservative solution and tighten it, and store it in liquid nitrogen. Thawing process: Take out the cryotube from the liquid nitrogen, quickly insert the power transmission port 211 on the cryotube electrical connection component 21 into the socket of the external power supply end of the cryotube, and the graphene film 351 in the placement part 31 begins to heat and keep it constant at 37°C. Hold the cryotube cap 2 and immerse the cryotube in 37°C constant temperature sterilized water to thaw. After thawing is completed, proceed to the subsequent steps.

[0074] Example 2 includes a pre-treatment process and a thawing process. In the pre-treatment process, a 10×10 mm piece of ovarian tissue is treated with ovarian tissue balance solution, cryopreservation solution, etc. 2 After the ovarian tissue is removed, tweezers are used to flatten it onto the spoon center surface 311 of the placement part 31, so that the first fixing part 32 pierces the ovarian tissue to achieve the fixation effect. Then, the tube cap 2 is held to place the ovarian tissue in liquid nitrogen for freezing. After freezing, it is placed in the empty tube body 1 and tightened, and the cryotube is placed in liquid nitrogen for storage. During the thawing process, the cryotube is taken out of the liquid nitrogen, and the power transmission port 211 on the cryotube electrical connection assembly 21 is quickly inserted into the socket of the external power supply end of the cryotube. At this time, the graphene film 351 in the placement part 31 quickly provides heat to the ovarian tissue. Then, the cryotube is unscrewed to take out the placement part 31 and the ovarian tissue. The cryotube cap 2 is held to completely immerse the ovarian tissue on the fixing spoon 31 in the thawing solution to thaw. After thawing, the subsequent steps are carried out.

[0075] Furthermore, the method for using the graphene-heated ovarian tissue cryopreservation tube includes a pre-treatment step and a thawing step: The freezing steps are as follows: Q1. Processing of ovarian tissue sections; Q2. Spread the processed ovarian tissue slices flat on the placement portion 31 for fixation; Q3. Freeze the ovarian tissue first; Q4. After the ovarian tissue is frozen, the placement portion 31 and the ovarian tissue are moved into the tube body 1 and the tube cap 2 is tightened; Q5. Preserving the cryovial in liquid nitrogen; The thawing steps are as follows: Q1: Take out the cryovial from liquid nitrogen; Q2: Connect the electrical connection component 21 on the pipe cap 2 to the external power supply terminal and power on, and the heating component starts working; Q3: Open the tube cap 2 and remove the ovarian tissue; Q4: The placement part 31 and the ovarian tissue are immersed in a constant-temperature thawing solution for thawing.

[0076] In the freezing step, step Q1 processing of the ovarian tissue section can remove impurities, adjust the tissue size and ensure that the tissue meets the cryopreservation requirements; Step Q2 fixes the tissue on the placement part, which can ensure the stability of the ovarian tissue position and the uniformity of stress and heat during subsequent operations; Step Q3 first freezes the ovarian tissue, which can make the tissue preliminarily enter a low-temperature state before being moved into the tube body, reducing the damage to the tissue caused by temperature fluctuations during the subsequent moving into the tube body process; Step Q4 moves the tube body and tightens the tube cap after the ovarian tissue is frozen, which can avoid the influence of air in the tube body on the freezing effect, while ensuring the sealing of the tube body to prevent subsequent liquid nitrogen infiltration or storage liquid leakage; Step Q5 places the cryopreservation tube in liquid nitrogen to maintain the tissue in a stable glass state at ultra-low temperature for long-term storage.

[0077] In the thawing step, step Q1 removes the cryopreservation tube from the liquid nitrogen, and the tube body and the ovarian tissue are at ultra-low temperature; Step Q2 connects the external power supply end to the electrical connection assembly and supplies power, and the heating assembly heats the tissue from the inside of the placement part to gradually restore the temperature of the tissue; Step Q3 opens the cryopreservation tube to take out the ovarian tissue, which can avoid the continuous influence of the residual low-temperature environment in the tube body on the tissue; Step Q4 immerses the placement part and the ovarian tissue fixed thereon in a constant-temperature thawing solution, which cooperates with the external thawing solution and the internal heating assembly to thaw the tissue quickly and uniformly. The placement part fixes the position of the ovarian tissue during thawing to avoid uneven heating caused by the floating of the tissue in the thawing solution, thereby protecting the activity of the tissue and preparing for subsequent transplantation or other processing.

[0078] In one embodiment, step Q3 first freezes the ovarian tissue, which means that the tissue fixed on the placement part 31 is exposed to air for 15 seconds for pre-warming, then quickly immersed in liquid nitrogen for rapid freezing to preliminarily vitrify, then immediately perform step Q4 to move it into the tube body 1 which has been filled with a small amount of cryopreservation solution and tighten the tube cap 2, and finally put the entire cryopreservation tube into liquid nitrogen for long-term storage. In the thawing step, the constant-temperature thawing solution in step Q4 is 37℃ constant-temperature sterile water, the entire placement part 31 is immersed in the constant-temperature sterile water together with the tissue, and the internal and external heating cooperate to thaw the tissue. After thawing, the tissue is transferred to the recovery solution 1, the recovery solution 2 and the recovery solution 3 in sequence for gradient osmotic pressure balance.

[0079] Further, the use method of the graphene-heated ovarian tissue cryopreservation tube includes a pre-processing step and a thawing step. The freezing steps are as follows: L1. Process ovarian tissue sections; L2, laying the processed ovarian tissue slices flat on the placement portion 31 for fixation; L3, adding ovarian tissue cryopreservation solution into the tube body 1; L4, move the placement portion 31 and the ovarian tissue together into the tube body 1 and cover it with the tube cap 2; L5. Place the cryovial in liquid nitrogen for storage; The thawing steps are as follows: L1: Take out the cryotube from liquid nitrogen; L2: Connect the electrical connection component 21 on the pipe cap 2 to the external power supply terminal and power on, and the heating component starts working; L3: Immerse the cryotube in a constant temperature thawing liquid for thawing.

[0080] In the freezing step, step L1 processes the ovarian tissue slices to remove impurities, adjust the tissue size, and ensure that the tissue meets the freezing requirements; Step L2: Flattening and fixing the tissue on the placement part to ensure that the ovarian tissue is in a stable position and evenly stressed and heated during subsequent operations; In step L3, a cryopreservative solution is added to the cryopreservation tube so that the solution fully wraps the tissue and penetrates into the ovarian tissue to provide protection for the ovarian tissue in a low-temperature environment; Step L4: The placement portion and the ovarian tissue thereon are moved into the tube body and capped to achieve a seal, prevent leakage of the preservation solution, and prevent external contamination of the ovarian tissue and the cryopreservation solution; In step L5, the cryovial is placed in liquid nitrogen, and the ultra-low temperature of -196°C is used to rapidly form a glassy state between the tissue and the cryopreservative solution, thereby arresting cell metabolism and achieving long-term preservation of the ovarian tissue.

[0081] In the thawing step, after the cryotube is taken out from liquid nitrogen in step L1, the tube body is in an ultra-low temperature state; Step L2: connecting the external power supply terminal to the electrical connection component and applying power, thereby activating the heating component and starting to heat the ovarian tissue from the inside; In step L3, the cryovial is immersed in a constant-temperature thawing liquid to heat the cryovial from the outside. The coordinated heating inside and outside can quickly and evenly heat the ovarian tissue, avoiding the formation of ice crystals due to excessive local temperature differences. At the same time, the precise temperature control of the heating component combined with the constant-temperature thawing liquid can effectively prevent the ovarian tissue from undergoing devitrification in the temperature danger zone of -140°C to -60°C, thereby ensuring tissue activity.

[0082] In one embodiment, the processing of step L1 is specifically as follows: fresh ovarian tissue is cut into slices and washed with physiological saline. In step L2, the slices are spread flat on the spoon core surface 311 and punctured and fixed with four needle-shaped first fixing parts 32. In step L3, 5 ml of ovarian tissue cryopreservation solution prepared by the formula described in claim 8 is added to the tube body 1. In step L4, the fixing part 3 that fixes the tissue is screwed together with the tube cap 2 on the tube body 1 filled with freezing solution. In step L5, the assembled cryopreservation tube is directly immersed in liquid nitrogen for storage. In the thawing step, the constant temperature thawing liquid of step L3 is sterile water at 37°C. After the cryopreservation tube is immersed, the external water bath and the internal graphene heating work together to usually complete the thawing within 60-90 seconds.

[0083] This embodiment improves the solubility of solution components by using the solubilizing effect of high-concentration sucrose, polysucrose, and phosphate buffer. Polysucrose can form hydrogen bonds with water molecules, inhibiting the aggregation of sucrose molecules. Experimental verification shows that after refrigeration at -20°C for 72 hours and freezing at -196°C for 3 months, no visible crystals are found in the preservation solution. Even if trace amounts of microcrystals are occasionally present under extreme conditions, the Mohs hardness of the microcrystals is far lower than the mechanical strength of the cell membrane. Moreover, when heated to 4°C, the microcrystals completely dissolve within 5 minutes. Cell metabolism recovery takes 10-15 minutes, which precedes the recovery of cell activity, without causing physical damage to the cells or affecting the puncture operation.

[0084] In addition, all ingredients in the formula have been verified for compatibility. Dimethyl sulfoxide and ethylene glycol have no chemical reaction with polysucrose and sucrose, and will not form precipitation with magnesium chloride and calcium chloride in phosphate buffer, thus avoiding the reaction of calcium salts with high concentrations of alcohols. When human serum albumin coexists with ingredients such as gentamicin and sodium pyruvate, the protein structure is stable, and no denatured bands are shown in SDS-PAGE electrophoresis. No harmful substances such as protein degradation products and antibiotic complexes will be produced due to the repulsion of ingredients, thus ensuring the stability of the solution components throughout the entire freezing period.

[0085] The present invention adopts a sequence of dissolving the liquid components first, adding the buffer, and finally adding the solid components and human albumin. Mixing the liquid components first creates a uniform solvent environment, preventing localized concentrations of the solid components. Adding the human albumin last prevents denaturation caused by prolonged contact with the high-concentration protective agent. Experiments have shown that the preservation solution prepared in this order exhibits minimal osmotic pressure fluctuations after storage at 4°C for 24 hours, while solutions prepared in the reverse order exhibit greater fluctuations.

[0086] Although the best effect of the preservation solution is to prepare it for immediate use, it can be extended to 24 hours by sterilizing it and storing it in a sealed and light-proof place at 4°C. The gentamicin in the phosphate buffer inhibits the growth of microorganisms, and the light-proof environment slows down the photolysis of dimethyl sulfoxide to produce toxic substances. If long-term standby is required, polysucrose and sucrose can be stored separately from dimethyl sulfoxide, ethylene glycol and buffer. They should be mixed and human serum albumin should be added 10 minutes before use to avoid the loss of stability caused by the long-term coexistence of the components, thus resolving the contradiction between the cumbersome operation of immediate preparation and the attenuated effect of early preparation.

[0087] In addition, dimethyl sulfoxide may degrade under high temperature and light to produce cytotoxic formaldehyde. The present invention is incubated at a low temperature of 4°C, stored in the dark, and used within a short period of time. Experimental tests show that the formaldehyde content of the preservation solution is less than 0.1μg / ml after 24 hours of storage at 4°C. This content is far below the cytotoxic threshold of 1μg / ml. Ethylene glycol may oxidize to produce oxalic acid if it comes into contact with metal ions for a long time. The pH buffering capacity of the phosphate buffer in the formula can inhibit the oxidation reaction, and the use of PP cryopreservation tubes without metal ion dissolution further avoids the production of harmful substances. Ovarian tissue may release a small amount of lactic acid during the low-temperature pretreatment stage. Excessive accumulation will lower the local pH value. The pH buffering capacity of the phosphate buffer in the formula can neutralize lactic acid and maintain pH stability. At the same time, glucose and sodium pyruvate provide cells with alternative energy substrates, reducing the amount of lactic acid produced by anaerobic respiration and inhibiting the accumulation of toxic substances from the source. This design can further improve the survival of tissue cryopreservation.

[0088] The specific working principle of the present invention is: In the ovarian tissue cryopreservation solution provided by the present invention, dimethyl sulfoxide and ethylene glycol serve as permeability protectants, which can penetrate the cell membrane to lower the freezing point of the intracellular fluid, inhibit ice crystal formation, and avoid cell damage. Polysucrose and sucrose serve as non-permeability protectants, creating a hypertonic environment outside the cells to promote dehydration, while increasing the viscosity of the solution and assisting in the formation of a stable glassy state during freezing. The phosphate buffer maintains pH and ion balance. In addition, glucose and sodium pyruvate provide energy, gentamicin inhibits bacteria, and human serum albumin and the buffer work together in a ratio of 1000ml:3g to maintain osmotic pressure stability, provide nutrition, and reduce freeze-thaw damage.

[0089] When freezing, the ovarian tissue is first incubated in a balanced solution at room temperature for 10 minutes, and then gradually penetrated by a permeable protective agent to allow the ovarian tissue to adapt to the change in osmotic pressure. It is then transferred to a cryopreservation solution and incubated at 4°C for 20 minutes. The low temperature slows down metabolism, allowing the protective agent to be fully loaded to an effective concentration. Finally, it is immersed in -196°C liquid nitrogen, quickly crossing the ice crystal danger zone of -10°C to -60°C, forming a glassy state and stagnating cell metabolism, thereby achieving long-term preservation.

[0090] During thawing, the cryovials are first pre-heated in air for 15 seconds to prevent them from breaking due to temperature difference after being directly placed in 37°C sterilized water. The contents of the cryovials are then thawed in 37°C water. The graphene-heated cryovials can be connected to electricity synchronously, and the internal and external temperatures are raised synchronously to prevent the ovarian tissue from devitrifying. The ovarian tissue is then immersed in hypertonic resuscitation solution 1, mesosmotic resuscitation solution 2, and physiological osmotic resuscitation solution 3 in sequence. The low-temperature freezing preservation solution of the ovarian tissue is removed through gradient concentration to allow the ovarian tissue to adapt to the osmotic pressure. Finally, the residue is washed away at room temperature to ensure its availability for transplantation.

[0091] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A low-temperature freezing preservation solution for ovarian tissue, characterized in that: Including permeable cryoprotectants, non-permeable cryoprotectants for internal tissue dehydration, buffers and human albumin; The permeable cryoprotectant includes dimethyl sulfoxide and ethylene glycol, and the non-permeable cryoprotectant includes polysucrose and sucrose; Each 100 ml of ovarian tissue cryopreservation solution contains 15-25 ml of dimethyl sulfoxide, 15-25 ml of ethylene glycol, 15-20 g of polysucrose, 38-46 g of sucrose, 40-45 ml of buffer and 0.12-0.13 g of human serum albumin; The ratio of the buffer solution to the human serum albumin is 1000 ml:3 g.

2. The ovarian tissue cryopreservation solution according to claim 1, characterized in that: The buffer is a phosphate buffer, and each 100 ml of the phosphate buffer includes 0.005-0.015 g of magnesium chloride, 0.008-0.018 g of calcium chloride, 1-3 μl of phenol red, 0.05-0.15 g of glucose, 0.003-0.004 g of sodium pyruvate, 0.007-0.008 g of gentamicin, and purified water.

3. A method for freezing ovarian tissue, comprising the ovarian tissue cryopreservation solution according to any one of claims 1 to 2, characterized in that: The steps include: S1: Place the ovarian tissue in a cryovial containing equilibration solution and incubate at room temperature for 10 minutes; S2: Transfer the ovarian tissue to ovarian tissue cryopreservation solution and incubate at 4°C for 20 min; S3: Fill the cryovial with ovarian tissue cryopreservation solution; S4: The ovarian tissue was transferred into a cryovial, and the cryovial was immersed in liquid nitrogen at -196°C for cryopreservation.

4. The use of a cryopreservation solution for ovarian tissue, characterized in that: The invention comprises the ovarian tissue low-temperature freezing preservation solution according to any one of claims 1 to 2, and the low-temperature freezing preservation solution is used for cryopreservation of ovarian tissue.

5. The method for freezing ovarian tissue according to claim 3, characterized in that: Every 100 ml of the equilibrated solution includes 8-12 ml of dimethyl sulfoxide, 8-12 ml of ethylene glycol, 75-85 ml of buffer and 0.2-0.3 g of human serum albumin.

6. The method for freezing ovarian tissue according to claim 3, characterized in that: The cryotube is a graphene-heated ovarian tissue cryotube, comprising a tube body (1), a tube cap (2) and a fixing member (3), wherein the fixing member (3) comprises a spoon-shaped surface (31) for placing the ovarian tissue and a fixing needle (32) provided on the spoon-shaped surface (31) for fixing the ovarian tissue, a graphene heating layer (33) is provided in the fixing member (3), and an electrical component (21) electrically connected to an external power supply end is provided on the tube cap (2), and the graphene heating layer is electrically connected to the electrical component (21).

7. The method for freezing ovarian tissue according to claim 6, characterized in that: The cryopreservation method further includes a thawing process, which includes the following steps: P1: Take the cryotube containing ovarian tissue out of liquid nitrogen and keep it in air for 15 seconds; P2: Place the cryovial in a sterilization tank filled with 37°C sterilized water and connect the cryovial to an electric source until the liquid in the cryovial is completely thawed; P3: Remove the ovarian tissue from the cryopreservation tube and place it in resuscitation solution 1, and keep it at room temperature for 3-4 minutes; P4: Remove the ovarian tissue from the resuscitation solution 1 and place it in the resuscitation solution 2, and keep it at room temperature for 3-4 minutes; P5: Remove the ovarian tissue from the resuscitation solution 2 and place it in the resuscitation solution 3, and keep it at room temperature for 3-4 minutes; P6: The ovarian tissue in the resuscitation solution 3 is removed and washed at room temperature.

8. The method for freezing ovarian tissue according to claim 7, characterized in that: During the thawing process, the electrical component (21) of the cryotube is electrically connected to an external power supply terminal, and the graphene heating layer (33) heats the ovarian tissue fixed on the spoon core surface (31) through the fixing needle (32).

9. The method for freezing ovarian tissue according to claim 7, wherein: Each 100 ml of the resuscitation solution 1 includes 32-37 g of sucrose, 70-80 ml of phosphate buffer and 0.2-0.4 g of human albumin; Each 100 ml of the resuscitation solution 2 includes 15-20 g of sucrose, 85-95 ml of phosphate buffer and 0.2-0.4 g of human albumin; Every 100 ml of the resuscitation fluid 3 includes 95-99.9 ml of phosphate buffer and 0.2-0.4 g of human albumin.

10. Application of a method for freezing ovarian tissue, characterized in that: The invention relates to a freezing method for ovarian tissue according to any one of claims 5 to 9, wherein the freezing method is applied to cryopreservation of ovarian tissue.

Images