Blastocyst vitrification refrigerating fluid set and application

Abstract

The invention relates to the technical field of cell cryopreservation, and discloses a blastocyst vitrification refrigerating fluid set and application, the refrigerating fluid set comprises a basic buffer solution, a composite permeable protective agent, a non-permeable protective agent, a thermosensitive macromolecule regulating agent, a molecular state locking agent, a zwitterionic osmotic pressure buffer agent, a cell apoptosis inhibitor and an efficient antioxidant, the invention further provides a preparation method of the blastocyst vitrification refrigerating fluid set. The preparation method comprises the following steps: weighing HEPES in a clean container; gradually adding ethylene glycol and dimethyl sulfoxide into the basic buffer solution according to a ratio of 1: 1; slowly adding trehalose into the mixed liquid; slowly adding poloxamer into the transparent solution; then adding chondroitin sulfate; proline is gradually added; the preparation method comprises the following steps: adding a generic caspase inhibitor Z-VAD-FMK; and then adding the L-glutathione. According to the method disclosed by the invention, the blastocyst is subjected to cryoprotection by adopting the impermeable protective agent trehalose, so that the cell survival rate is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell cryopreservation, and in particular to a blastocyst vitrification cryopreservation solution kit and application. BACKGROUND

[0002] In the field of cell cryopreservation, traditional methods generally rely on the use of permeable or non-permeable protective agents, but often fail to achieve ideal results in cell survival rate. For example, a lower concentration of trehalose cannot provide sufficient mechanical protection during ice crystal formation, resulting in cell damage. Simply increasing the permeable protective agent is not enough to solve this problem. As a result, cells are extremely susceptible to death during the freezing process, with a significant reduction in survival rate, which is obviously a significant disadvantage.

[0003] Existing technologies have relatively limited protection for cell membranes during the preservation process. Most experiments use simple cryopreservation fluid combinations, lacking comprehensive regulation of the freezing environment. As a result, the stability of the cell membrane is threatened and frequent damage occurs. The integrity of the cell membrane directly affects the survival and function of the cell. Therefore, the protection effect of the traditional scheme is not good, which damages the cell structure and reduces the possibility of recovering cell activity.

[0004] Current technologies generally do not consider the adaptability of cells to external environmental changes. Many researchers directly add proline in one go, causing the cells to face sudden osmotic pressure changes, resulting in mechanical damage to the cell membrane. This rough handling method often leads to an increase in cell apoptosis rate and a decrease in survival rate, making it impossible to achieve ideal freezing results. In sharp contrast, the present application uses a gradual addition method to improve the ability of cells to adapt to environmental changes, fundamentally improving the conditions for cell survival. SUMMARY

[0005] The purpose of the present application is to provide a blastocyst vitrification cryopreservation solution kit and application, which solves the problems of insufficient cryoprotection and low cell survival rate in the prior art.

[0006] To achieve the above purpose, the present application is implemented by the following technical scheme: a blastocyst vitrification cryopreservation solution kit, comprising: a basic buffer solution with a concentration of 5-25mM; a composite permeable protective agent with a concentration range of 7.5%-12.5%(v / v); a non-permeable protective agent with a concentration of 1.0-1.5M(v / v); a heat-sensitive macromolecular regulator with a concentration range of 0.5%-3.0%(w / v); a molecular state locking agent with a concentration range of 0.1%-0.5%(w / v); zwitterionic osmotic pressure buffer, concentration of 0.1%-0.5%(w / v); apoptosis inhibitor, concentration range of 10-50μM; high-efficiency antioxidant, concentration range of 1-10mM.

[0007] Preferably, the basic buffer is HEPES buffer, and the complex osmotic protective agent comprises ethylene glycol and dimethyl sulfoxide.

[0008] Further, HEPES buffer is selected to reliably maintain the pH value of the system in the physiological range under extreme temperature changes in freezing, which is a prerequisite for ensuring the stability of the basic metabolic environment of cells. Meanwhile, ethylene glycol and dimethyl sulfoxide are used as the complex osmotic protective agent, which takes advantage of the characteristics of small molecular weight and strong permeability, can quickly enter the cell interior to replace water, effectively reduce the freezing point, and can reduce the concentration of each other when achieving the same vitrification effect, thereby reducing the potential toxicity of high concentration of a single protective agent to cells.

[0009] Further, trehalose as a non-osmotic protective agent forms a hypertonic environment outside the cell, prompting the cell to dehydrate moderately, and its excellent vitrification characteristics can form a non-crystalline solid after cooling, providing physical support for the cell. Poloxamer as a heat-sensitive macromolecular regulator, its core function is to form a colloidal network at a specific temperature, physically hindering the free flow of water molecules, and fundamentally inhibiting the nucleation and growth of ice crystals. Chondroitin sulfate as a molecular state locking agent, by increasing the viscosity of the system, it further stabilizes the vitrification state and protects the cell membrane in cooperation with the former two.

[0010] Preferably, the zwitterionic osmotic pressure buffer is proline, the apoptosis inhibitor is pancaspase inhibitor Z-VAD-FMK, and the high-efficiency antioxidant is L-glutathione.

[0011] Further, proline as a zwitterion can effectively alleviate the osmotic pressure impact felt by the cell in a hypertonic environment, helping the cell to maintain normal volume and morphology. Pancaspase inhibitor Z-VAD-FMK is a broad-spectrum apoptosis inhibitor that directly inhibits programmed cell death induced by freezing and recovery stress by blocking the Caspase pathway. L-glutathione as an endogenous high-efficiency antioxidant is used to scavenge reactive oxygen free radicals generated during freezing and recovery due to metabolic disorders, and to reduce oxidative damage.

[0012] A preparation method of a blastocyst vitrification solution kit, comprising the following steps: In a clean container, weigh HEPES, add deionized water to make the concentration of the base buffer in the range of 5-25 mM, fully dissolved by stirring, and keep the pH value at 7.2-7.4; Gradually add 1:1 ratio of ethylene glycol and dimethyl sulfoxide to the base buffer, fully stir until a uniform mixed liquid is formed; Slowly add trehalose to the mixed liquid, continue to stir gently until the trehalose is completely dissolved to form a transparent solution; Slowly add poloxamer to the transparent solution and fully stir to form a colloidal state; Add chondroitin sulfate again and stir until it is completely dissolved; Add proline gradually and continue to stir gently at a temperature of 0-4℃; Add pan-caspase inhibitor Z-VAD-FMK to the mixed solution and stir gently to fully dissolve and uniform; Add L-glutathione again with a concentration of 1 mM-10 mM, fully stir for 5-10 minutes to obtain a uniform freezing solution, and then divide the prepared freezing solution into sterile freezing tubes and store in liquid nitrogen.

[0013] Preferably, after adding trehalose, continue to stir gently for 5-10 minutes to ensure complete dissolution, and the ambient temperature is 18℃-25℃.

[0014] Further, the operation is carried out at room temperature in the range of 18℃-25℃ to ensure sufficient dissolution rate of trehalose, and the gentle stirring and 5-10 minutes of time limit are to ensure complete dissolution and prevent the formation of small crystals or lumps that affect subsequent steps, while preventing excessive mechanical shear force from damaging the structure of other added components in the solution.

[0015] Preferably, the final concentration of the added poloxamer is kept at 0.1%-3.0%, and stirred for 5-20 minutes to form a transparent solution in a colloidal state.

[0016] Further, the concentration range of 0.1%-3.0% is a key parameter for forming an effective colloidal network structure, and a lower concentration will result in a sparse network and poor ice crystal inhibition, while a higher concentration will result in too high viscosity and is not conducive to operation. The stirring time of 5-20 minutes ensures that the poloxamer molecules can be fully dispersed and self-assembled into a uniform colloid.

[0017] Preferably, the final concentration of proline is 0.1%-0.5%, and the stirring time is 10-20 minutes, and the addition is kept at a cooling condition of 0-4℃.

[0018] Further, 0-4℃ is to reduce the metabolic activity of cells, making them insensitive to changes in the external environment, while slowing down the rate of osmotic pressure change. The concentration of 0.1%-0.5% is an optimized range, aiming to provide effective osmotic pressure buffering without excessively changing the properties of the solution. Gradual addition of proline and 10-20 minutes of stirring ensures that proline is slowly and uniformly distributed in the system, giving cells enough time to adapt to the gradual change in osmotic pressure, thereby avoiding cell membrane damage or rupture caused by rapid changes.

[0019] Preferably, the concentration of proline after addition is 0.1%-0.5%, and stirring for 3-5 minutes.

[0020] A blastocyst vitrification cryopreservation solution kit is provided, which is used for cryopreservation of blastocysts to improve cell survival rate and developmental potential after cryopreservation.

[0021] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application realizes significant improvement in cell survival rate by using non-permeable protective agent trehalose for blastocyst cryopreservation. High concentration of trehalose can effectively inhibit ice crystal formation and reduce mechanical damage, thereby improving survival rate. Compared with the low concentration scheme used in the prior art, this method effectively overcomes the problem of cell damage during the freezing process, ensuring higher cell survival ability.

[0022] 2. The present application introduces a heat-sensitive macromolecular regulator, poloxamer, to enhance the cryopreservation effect of blastocysts. Experimental results show that the cryopreservation solution containing poloxamer significantly improves membrane integrity and reduces the proportion of damaged cells. Compared with some simple solvent technical solutions, this method solves the cell damage caused by unstable environment during cryopreservation, ensuring the integrity of cell structure.

[0023] 3. The present application optimizes the survival rate and developmental potential of blastocysts by controlling the addition of proline. The gradual addition of proline significantly improves cell adaptability and reduces oxidative stress. This completely avoids the cell impact caused by direct addition, and compared with traditional methods, reduces the risk of cell damage and apoptosis. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The present application provides a method flowchart. DETAILED DESCRIPTION

[0025] The following will be described in detail in conjunction with the accompanying Figure 1 The present application will be further described in detail.

[0026] The present application provides a blastocyst vitrification cryopreservation solution kit, embodiment 1 Component ratio and concentration: HEPES: 15mM Ethylene glycol: 10% (v / v) Dimethyl sulfoxide: 10% (v / v) Trehalose: 2% (w / v) Poloxamer: 1.5% (w / v) Chondroitin sulfate: 0.3% (w / v) Proline: 0.3% (w / v) Z-VAD-FMK: 5μM L-glutathione: 5mM Step: In a clean container, weigh HEPES, add deionized water to make the concentration of the basic buffer 15mM, and adjust the pH value to 7.2-7.4.

[0027] Add 10% volume of ethylene glycol and 10% volume of dimethyl sulfoxide in a ratio of 1:1 to the basic buffer, and stir well to form a uniform mixture.

[0028] Add 2% trehalose gradually, and continue to stir gently for 5-10 minutes until the trehalose is completely dissolved to form a transparent solution.

[0029] Slowly add 1.5% poloxamer to the transparent solution, stir well for 5-20 minutes to form a uniform colloidal state.

[0030] Add 0.3% chondroitin sulfate and stir until it is completely dissolved.

[0031] Add 0.3% proline to the mixture gradually at a temperature of 0-4°C, and continue to stir gently for 10-20 minutes to make it completely dissolved.

[0032] Add 5μM Z-VAD-FMK to the mixture, and stir gently to make it completely dissolved and uniform.

[0033] Finally, add 5mM L-glutathione to the mixture, stir well for 5-10 minutes to obtain a uniform freezing solution, and store it in a sterile freezing tube in liquid nitrogen.

[0034] Example 2 Component ratio and concentration: HEPES: 5mM Ethylene glycol: 5% (v / v) Dimethyl sulfoxide: 5% (v / v) Trehalose: 1% (w / v) Poloxamer: 0.1% (w / v) Chondroitin sulfate: 0.1% (w / v) Proline: 0.1% (w / v) Z-VAD-FMK: 2 μΜ L-glutathione: 1 mM Procedure: In a clean container, weigh HEPES and add deionized water to make the base buffer at a concentration of 5 mM, pH adjusted to 7.2-7.4.

[0035] Add 5% volume of ethylene glycol and 5% volume of dimethyl sulfoxide in a 1:1 ratio to the base buffer, mix well until a homogeneous mixture is formed.

[0036] Add 1% trehalose gradually and continue gentle stirring for 5-10 minutes until the trehalose is completely dissolved to form a clear solution.

[0037] Slowly add 0.1% poloxamer to the clear solution, mix well for 5-20 minutes to form a homogeneous colloidal state.

[0038] Add 0.1% chondroitin sulfate and stir until it is completely dissolved.

[0039] At a temperature of 0-4°C, add 0.1% proline to the mixture gradually, continue to stir gently for 10-20 minutes until it is completely dissolved.

[0040] Add 2 μΜ Z-VAD-FMK to the mixture and gently stir until it is completely dissolved and uniform.

[0041] Finally, add 1 mM L-glutathione to the mixture, mix well for 5-10 minutes to obtain a homogeneous freezing solution, and aliquot into sterile freezing tubes and store in liquid nitrogen.

[0042] Example 3 Component ratio and concentration: HEPES: 25 mM Ethylene glycol: 15% (v / v) Dimethyl sulfoxide: 15% (v / v) Trehalose: 3% (w / v) Poloxamer: 3.0% (w / v) Chondroitin sulfate: 0.5% (w / v) Proline: 0.5% (w / v) Z-VAD-FMK: 10 μΜ L-glutathione: 10 mM Procedure: In a clean container, weigh HEPES, add deionized water to make the concentration of the basic buffer 25 mM, and adjust the pH value to 7.2-7.4.

[0043] Add 15% volume of ethylene glycol and 15% volume of dimethyl sulfoxide in a ratio of 1:1 to the basic buffer step by step, and fully stir until a uniform mixture is formed.

[0044] Add 3% trehalose step by step, and continue to stir gently for 5-10 minutes until the trehalose is completely dissolved to form a transparent solution.

[0045] Slowly add 3.0% poloxamer to the transparent solution, fully stir for 5-20 minutes to form a uniform colloidal state.

[0046] Add 0.5% chondroitin sulfate, and stir until it is completely dissolved.

[0047] Add 0.5% proline to the mixture step by step at a temperature of 0-4°C, continue to stir gently for 10-20 minutes, and make it completely dissolved.

[0048] Add 10 μM Z-VAD-FMK to the mixture, and fully dissolve it uniformly by gentle stirring.

[0049] Finally, add 10 mM L-glutathione to the mixture, fully stir for 5-10 minutes to obtain a uniform freezing solution, and store it in a sterile freezing tube in liquid nitrogen.

[0050] Comparative Example 1; compared with Example 1, the difference is that the concentration of the non-permeable protective agent trehalose is reduced to 1% (w / v), and the rest is the same.

[0051] Comparative Example 2; compared with Example 1, the difference is that the addition of the heat-sensitive macromolecular regulator poloxamer is omitted, and the rest is the same.

[0052] Comparative Example 3; compared with Example 1, the difference is that the step of adding proline step by step at a temperature of 0-4°C is omitted, and it is directly added, and the rest is the same.

[0053] Comparative Example 4; compared with Example 1, the difference is that the high-efficiency antioxidant L-glutathione is removed, and the addition step of this component is omitted, and the rest is the same.

[0054] Experiment 1: Effect of Trehalose Concentration on Cell Survival Rate Experiment Description Purpose of the experiment: To test the effect of changes in the concentration of the non-permeable protective agent trehalose on the survival rate of blastula.

[0055] Experimental materials: Prepared Example 1 freezing solution (trehalose concentration of 2% (w / v)), prepared Comparative Example 1 freezing solution (trehalose concentration of 1% (w / v)), blastocysts (derived from laboratory-cultured mouse embryos), sterile cryovials, culture medium (e.g., DMEM + 10% fetal bovine serum), trypan blue stain, constant-temperature incubator.

[0056] Experimental procedure Freezing solution preparation: In a clean environment, divide the freezing solutions of Example 1 and Comparative Example 1 into sterile cryovials, about 1 mL per vial.

[0057] Blastocyst treatment: On a sterile workbench, use a pipette to take the same number of blastocysts (e.g., 30) and add them to each cryovial, respectively.

[0058] Cryopreservation: Place the cryovials in a liquid nitrogen freezer and follow the conventional freezing procedure, ensuring an appropriate freezing rate to protect the cell structure.

[0059] Thawing treatment: According to the requirements, take the frozen tube out of the liquid nitrogen and place it in a 37°C water bath for rapid thawing (about 2-3 minutes), ensuring that the thawing process is fast to reduce damage to the cells.

[0060] Cell culture: Transfer the thawed blastocysts to the culture medium containing DMEM and 10% fetal bovine serum, mix gently, and then divide into culture dishes and place them in a constant-temperature incubator at 37°C for 48 hours.

[0061] Cell viability detection: After the culture ends, add a certain concentration of trypan blue stain (e.g., 0.4%), and after about 5 minutes of staining reaction, use a microscope to observe.

[0062] Count the number of surviving blastocysts and the total number of blastocysts, and calculate the survival rate.

[0063] The experimental data are shown in Table 1; Table 1 Blastocyst survival rate experimental results Sample Total blastocyst number Number of surviving blastocysts Survival rate (%) Example 1 30 27 90 Comparative Example 1 30 24 80 Example 1 30 26 86.67 Comparative Example 1 30 21 70 Example 1 30 28 93.33 Comparative Example 1 30 23 76.67 Summary; The application of different concentrations of the impermeable cryoprotectant trehalose in the cryopreservation of blastocysts significantly affected cell survival. In Example 1, the higher concentration of trehalose (2%) significantly improved cell survival compared to the lower concentration (1%) of the comparative Example 1. Trehalose provides protection to cells at low temperatures, and its impermeable nature prevents the formation of ice crystals, thereby reducing mechanical damage to cell membranes and organelles during the freezing process. The improved survival rate means that cells retain better structural integrity and functionality during thawing and recovery, which is closely related to the biological mechanisms of cell survival.

[0064] In addition, the experiment also showed that the absence of the heat-sensitive macromolecular regulator, poloxamer, may have affected the stability of the cells, as the physical and chemical properties of the environment in which the cells were placed in the freezing solution changed greatly without the formation of a stable colloidal state. This change can cause cells to experience greater stress during freezing and thawing, leading to additional damage to cell membranes, ultimately reflected in a decrease in survival rate. Therefore, the importance of the synergistic effect of poloxamer in cell protection cannot be ignored, as it maintains the homeostasis of the extracellular environment, optimizing the effect of cryopreservation.

[0065] The experimental results reinforce the possibility of achieving superior protection and survival of blastocysts by precisely adjusting the concentrations of different components. In the process of cell cryopreservation, the use of appropriate combinations of permeable and impermeable cryoprotectants not only improves cell survival rate, but also enhances subsequent culture and developmental potential.

[0066] Experiment 2: Effect of poloxamer absence on cell status Experiment description Purpose of the experiment: To test the effect of omitting the heat-sensitive macromolecular regulator, poloxamer, on blastocyst survival and membrane integrity.

[0067] Materials: Prepared Example 1 freezing solution (with poloxamer), prepared comparative Example 2 freezing solution (without poloxamer), blastocysts (derived from laboratory-cultured mouse embryos), sterile cryovials, culture medium (e.g., DMEM + 10% fetal bovine serum), cell membrane integrity detection reagents (such as fluorescent dyes for flow cytometry), constant temperature incubator (37°C, 5% CO2).

[0068] Experimental steps Freezing solution preparation: In a clean environment, divide the freezing solutions of Example 1 and comparative Example 2 into sterile cryovials, each containing about 1 mL.

[0069] Blastocyst treatment: On a sterile workbench, take the same number of blastocysts (e.g., 30) and add them to each cryovial.

[0070] Cryopreservation: Place the cryovials into a liquid nitrogen freezer following the regular freezing procedure to maximize the protection of cell structure.

[0071] Thawing process: Remove the cryovials from liquid nitrogen and place them in a 37°C water bath for rapid thawing (about 2-3 minutes).

[0072] Cell culture: Transfer the thawed blastocysts into the culture medium containing DMEM and 10% fetal bovine serum, gently mix, then dispense into culture dishes and place in a 37°C incubator for 48 hours.

[0073] Cell membrane integrity detection: After the culture, use specific fluorescent dyes to stain the cells to detect the integrity of the cell membrane.

[0074] Analyze the stained cells by flow cytometry and count the number of normal membrane cells and damaged membrane cells respectively The experimental data is shown in Table 2; Table 2 Cell membrane integrity experiment results Summary: Removing the thermosensitive macromolecular regulator poloxamer will adversely affect the survival rate and cell membrane integrity of the blastocyst. The freezing solution containing poloxamer in Example 1 shows a higher proportion of intact membranes, indicating that poloxamer plays a key stabilizing role in the cryopreservation process. The colloidal properties of poloxamer can effectively maintain the uniformity of the environment around the cells, alleviate the mechanical pressure caused by temperature gradients and ice crystal formation during the freezing process, and thus protect the integrity of the cell membrane. In contrast, the blastocysts in Comparative Example 2, which removed poloxamer, showed a significant decrease in survival rate and membrane integrity, demonstrating the importance of poloxamer.

[0075] The integrity of the cell membrane is an important indicator for evaluating the survival and function of cells. The experiment shows that the higher proportion of intact membranes and fewer damaged membrane cells in Example 1 confirm that by combining scientifically designed freezing solution components, the survival ability of blastocysts under low temperature conditions can be significantly enhanced. Through the synergistic effect of specific components, the stress of cells during freezing and thawing is buffered, ensuring the stability of their physiological state. This shows that in the cell cryopreservation system, the synergy of components directly affects the survival and functional recovery of cells, reflecting the effectiveness of the invention in optimizing cryopreservation technology.

[0076] This experiment further verifies the importance of optimizing the combination of each component in the freezing solution. Intelligent selection and configuration can significantly improve the safety of cells during the freezing process and the activity after recovery, providing substantial technical support for the cryopreservation of blastocysts.

[0077] Experiment 3: Effect of proline addition method on cell activity Experiment Description Experiment Purpose: To test the effect of direct addition and gradual addition of proline on blastocyst survival rate and developmental potential.

[0078] Experimental Materials: Prepared Example 1 freezing solution (proline added gradually), prepared Comparative Example 3 freezing solution (proline added directly), blastocysts (derived from laboratory-cultured mouse embryos), sterile cryovials, culture medium (e.g., DMEM + 10% fetal bovine serum), microscope, counting plate, culture dish.

[0079] Experimental Steps Freezing solution preparation: In a sterile environment, divide the freezing solutions of Example 1 and Comparative Example 3 into sterile cryovials, each containing about 1 mL.

[0080] Blastocyst collection: On a sterile operating table, take the same number of blastocysts (e.g., 30) and add them to each cryovial.

[0081] Cryopreservation: Place the cryovials in a liquid nitrogen freezer and use the standard freezing program to freeze them to the maximum extent to protect the cell structure.

[0082] Thawing process: Remove the cryovials from liquid nitrogen and quickly place them in a 37°C water bath for thawing (control the time to 2-3 minutes).

[0083] Cell culture: Transfer the thawed blastocysts to a culture medium containing DMEM and 10% fetal bovine serum, mix gently, and then divide them into culture dishes and place them in a 37°C incubator for 48 hours.

[0084] Morphological observation: After 48 hours of culture, use a microscope to observe the development state of the blastocysts and record the morphological characteristics, including cell number, structural integrity, and developmental stage.

[0085] The experimental data is shown in Table 3; Table 3 Experimental results of the effect of proline addition method on cell activity Sample Total blastocyst number Number of surviving blastocysts Developmental condition Developmental ratio (%) Example 1 30 28 Normal 93.33 Comparative Example 3 30 24 Abnormal 80 Example 1 30 27 Normal 90 Comparative Example 3 30 20 Abnormal 66.67 Example 1 30 26 Normal 86.67 Comparative Example 3 30 21 Abnormal 70 Example 1 30 29 Normal 96.67 Comparative Example 3 30 22 Abnormal 73.33 Summary; The addition of proline significantly affected the survival rate and developmental status of the blastocysts. In Example 1, the stepwise addition of proline exhibited a higher survival rate and normal developmental status, reflecting that this addition method effectively reduced the stress experienced by the cells during freezing. This stepwise addition strategy helps cells gradually adapt to changes in the external environment, thereby reducing the impact on cell membranes and organelles and protecting the physiological functions of the cells.

[0086] In contrast, the direct addition of proline in Comparative Example 3 resulted in a significant decrease in survival rate and normal development rate, which may be due to the sudden increase in proline concentration causing a sharp change in extracellular osmotic pressure, leading to instant damage to the cell membrane. Cells often struggle to quickly adapt to sudden changes in chemical environment, affecting their survival and development. Therefore, this experiment highlights the importance of precise control over the addition steps and methods to ensure proper protection of cells during cryopreservation.

[0087] The results of this experiment further verify the importance of optimizing the cryopreservation system through scientific configuration and addition methods.

[0088] Experiment 4: Effect of missing antioxidants on oxidative stress Experiment Description Purpose of the experiment: Compare the effect of removing the high-efficiency antioxidant L-glutathione on cell survival rate and oxidative stress.

[0089] Experimental materials: Prepared Example 1 cryopreservation solution (containing L-glutathione), prepared Comparative Example 4 cryopreservation solution (without L-glutathione), blastocysts (derived from laboratory-cultured mouse embryos), sterile cryovials, culture medium (e.g., DMEM + 10% fetal bovine serum), oxidative stress detection reagents (such as dihydrofluorescein or other active oxygen probes), constant temperature incubator (37°C, 5% CO2).

[0090] Experimental steps Cryopreservation solution preparation: In a sterile environment, divide the cryopreservation solutions of Example 1 and Comparative Example 4 into sterile cryovials, each containing about 1 mL.

[0091] Blastocyst collection: On a sterile operating table, take the same number of blastocysts (e.g., 30) and add them to each cryovial, respectively.

[0092] Cryopreservation: Place the cryovials in a liquid nitrogen freezer and follow the standard freezing procedure to ensure maximum protection of the structure and function of the cells.

[0093] Thawing treatment: The cryo tube was removed from liquid nitrogen and immediately placed in a 37°C water bath for thawing (holding time is about 2-3 minutes).

[0094] Cell culture: The thawed blastocyst was transferred to the culture medium containing DMEM and 10% fetal bovine serum, gently mixed, and aliquoted in the culture dish, and placed in a 37°C incubator for 48 hours.

[0095] Oxidative stress detection: After the end of the culture, the cells were stained with an oxidative stress detection reagent to assess the level of reactive oxygen species (ROS) in the cells.

[0096] The cells were analyzed using a flow cytometer or a fluorescence microscope to count the proportion and number of reactive oxygen species positive cells.

[0097] The experimental data is shown in Table 4; Table 4 Oxidative stress experiment results Summary: The absence of the antioxidant L-glutathione had a significant impact on the survival rate and oxidative stress level of the blastocyst. In Example 1, the cryopreservation solution containing L-glutathione played an important role in protecting the cells, significantly reducing the production of reactive oxygen species (ROS). This phenomenon indicates that L-glutathione can effectively capture free radicals and reduce the damage of oxidative stress to cells. With the reduction of ROS level, the integrity and functionality of the cell membrane are maintained, thereby improving the survival rate of cells and the developmental potential after recovery.

[0098] In contrast, Comparative Example 4 failed to effectively inhibit the generation of reactive oxygen species, resulting in a significant increase in the proportion of ROS positive cells. This condition often triggers adverse reactions such as apoptosis and necrosis, affecting cell survival and development. The removal of L-glutathione makes the cells more susceptible to oxidative damage during freezing and thawing, and thus can be considered as a decrease in the self-repairing ability of cells under stress conditions. This observation highlights the importance of antioxidants in the process of cell cryopreservation, especially in the face of oxidative stress.

[0099] The results of this experiment further demonstrate the importance of properly selecting and applying antioxidants in the cell cryopreservation system. By introducing L-glutathione and other low-toxicity and high-efficiency antioxidant ingredients, the survival ability of cells can be effectively enhanced, thereby providing more reliable support for the preservation technology of blastocysts.

[0100] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A blastocyst vitrification solution kit, characterized by, include: Basic buffer solution, concentration 5-25mM; Composite permeability protectant, concentration range 7.5%-12.5% ​​(v / v); Non-permeable protective agent, concentration 1.0-1.5M (v / v); Thermosensitive macromolecular regulator, concentration range 0.5%-3.0% (w / v); Molecular state-locking agent, concentration range 0.1%-0.5% (w / v); Amphoteric osmotic buffer with a concentration of 0.1%-0.5% (w / v); Inhibitors of apoptosis, with a concentration range of 10-50 μM; Highly effective antioxidant, with a concentration range of 1-10 mM.

2. The blastocyst vitrification solution kit of claim 1, wherein, The base buffer is HEPES buffer, and the composite permeability protectant includes ethylene glycol and dimethyl sulfoxide.

3. The blastocyst vitrification solution kit of claim 1, wherein, The non-permeable protective agent is trehalose, the thermosensitive macromolecular regulator is poloxamer, and the molecular state locking agent is chondroitin sulfate.

4. The blastocyst vitrification solution kit of claim 1, wherein, The zwitterionic osmotic pressure buffer is proline, the apoptosis inhibitor is the pan-cysteine ​​inhibitor Z-VAD-FMK, and the highly effective antioxidant is L-glutathione.

5. A method for preparing a blastocyst vitrification cryoprotectant kit, used to prepare a blastocyst vitrification cryoprotectant kit as described in any one of claims 1-4, characterized in that, Includes the following steps; Weigh HEPES in a clean container, add deionized water to make the concentration of the basic buffer solution range from 5 to 25 mM, stir to dissolve it completely, and keep the pH value at 7.2-7.

4. Gradually add ethylene glycol and dimethyl sulfoxide in a 1:1 ratio to the basal buffer solution and stir thoroughly until a homogeneous mixture is formed. Slowly add trehalose to the mixed liquid and continue to stir gently until the trehalose is completely dissolved and a clear solution is formed; Slowly add poloxamer to the clear solution and stir thoroughly to form a colloidal state; Add chondroitin sulfate and stir until it is completely dissolved. Gradually add proline while continuously stirring gently at 0-4℃; Add the pan-cysteine ​​inhibitor Z-VAD-FMK to the mixture and stir gently until it is fully dissolved and homogeneous. Add L-glutathione at a concentration of 1mM-10mM, stir thoroughly for 5-10 minutes to obtain a homogeneous cryosol, dispense the prepared cryosol into sterile cryotubes, and store in liquid nitrogen.

6. The method of claim 5, wherein the method further comprises, After adding the trehalose, continue to stir gently for 5-10 minutes to ensure it dissolves completely, with the ambient temperature at 18℃-25℃.

7. The method for preparing a blastocyst vitrification cryosol kit according to claim 5, characterized in that, The final concentration of the added poloxamer is maintained at 0.1%-3.0%, and the mixture is stirred for 5-20 minutes to form a transparent colloidal solution.

8. The method of claim 5, wherein the method further comprises, The final concentration of proline is 0.1%-0.5%, and the stirring time is 10-20 minutes, while maintaining a cooling condition of 0-4℃.

9. The method of claim 5, wherein the method further comprises, After adding proline to a concentration of 0.1%-0.5%, stir for 3-5 minutes.

10. A blastocyst vitrification solution kit of any one of claims 1 to 3, wherein, The cryosol is used to cryopreserve blastocysts to improve cell survival rate and developmental potential after cryopreservation.

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