Low-toxicity cryopreservation solution based on membrane protection and ice control and application thereof in oocyte cryopreservation

Abstract

The application discloses a low-toxicity cryopreservation solution based on membrane protection and ice control as well as a preparation method and application thereof. The cryopreservation solution comprises a membrane protection molecule, an ice control material, a permeable amphiphilic ionic molecule, a water-soluble disaccharide, DMSO, ethylene glycol and serum. The cryopreservation solution can realize efficient preservation of mature oocytes, and maintain the membrane flow stability of the cell membrane before and after resuscitation, so that the structural function and genetic stability of the oocyte are maintained. The application realizes membrane state protection by adding metal zinc ions, the introduction of PVA increases the glassification ability and ice control effect of the cryopreservation system, and the addition of betaine small molecules can further improve the osmotic pressure. The concentration of DMSO in the traditional cryopreservation solution is reduced from 15% to 5%, and the concentration of EG in the traditional cryopreservation solution is reduced from 15% to 10%, so that the toxicity problem of the cryopreservation solution is greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a low-toxicity cryopreservation solution based on membrane protection and ice control, its preparation method and application, specifically to a low-toxicity cryopreservation solution based on membrane protection and ice control, its preparation method and its application in oocyte cryopreservation. Background Technology

[0002] Cryopreservation technology involves placing biological samples such as cells, tissues, or organs in an ultra-low temperature environment, significantly reducing or even stopping their metabolic rate to achieve long-term storage and restore their physiological functions after thawing. Currently, the fertility problems faced by young cancer patients, patients with low fertility, and infertile patients, especially women with premature ovarian failure and loss of ovarian function, urgently need solutions. The development of assisted reproductive technologies (ART) relying on the cryopreservation of oocytes, sperm, and embryos can not only meet the fertility needs of women of childbearing age but also further alleviate the pressure of declining birth rates and optimize population structure. It is worth noting that, unlike embryo freezing which involves some ethical, religious, and legal issues, oocyte cryopreservation has a wider range of applications, allowing women to make more suitable fertility choices. Oocytes are among the most difficult cell types to cryopreserve because their size is on the order of hundreds of micrometers, their specific surface area is low, and their water content is high, making them extremely sensitive to intracellular freezing and resulting in more severe cryopreservation damage.

[0003] Vitrification is currently the most efficient method for oocyte cryopreservation. Organic small-molecule cryoprotectants based on dimethyl sulfoxide (DMSO) and ethylene glycol (EG) have excellent cell permeability. During cooling, these small molecules can displace some intracellular water across the membrane. Simultaneously, based on hydrogen bonding with water molecules, they can vitrify the cytoplasm, preventing excessive solute accumulation and intracellular ice crystal formation, thus protecting the cell from fatal damage. However, DMSO, EG, and other permeable small molecules also have significant limitations when functioning as cryoprotectants (CPAs). When a large amount of bound water on the outer layer of biomacromolecules is bonded by DMSO through strong hydrogen bonds, its hydration shell is disrupted, leading to structural instability and even irreversible damage to subcellular structures (such as mitochondria, microtubules, and microfilaments). In severe cases, it can even disrupt intercellular junctions. Cryoprotectants containing more than 2% w / w DMSO can significantly damage the internal structure of cells, causing protein aggregation and, in severe cases, genomic damage, while also exhibiting epigenetic toxicity to oocytes. Meanwhile, clinical implementation faces a bottleneck challenge: low survival rate, low clinical pregnancy rate, low live birth rate, and high miscarriage rate. Existing cryopreservation technologies have long suffered from the cytotoxicity of high concentrations of chemical molecules (EG, DMSO) and the mechanical damage to cells caused by ice crystals during thawing, limiting the usability of oocytes. Furthermore, the cell membrane, as the interface between oocytes and sperm, is crucial for maintaining its structural and functional stability during cryopreservation, yet current research often neglects cell membrane protection. Therefore, proactive cell membrane protection combined with ice crystal growth inhibition, along with vitrification techniques that reduce the concentration of osmotic CPAs such as DMSO, will be an effective solution for optimizing oocyte vitrification cryopreservation in the future. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, the present invention provides a cryopreservation solution comprising: membrane protection molecules, ice-controlling materials, permeable amphiphilic molecules, water-soluble disaccharides, DMSO, ethylene glycol, and serum.

[0005] According to an embodiment of the present invention, the membrane protection molecule is selected from zinc salts, such as zinc gluconate, which can ionize into divalent zinc ions in aqueous solution, thereby protecting cell membrane fluidity at low temperatures.

[0006] According to an embodiment of the present invention, the zinc ion content in the zinc salt is 0.001g-0.0325g per 100mL of cryopreservation solution, preferably 0.0065g-0.0325g, for example 0.001g, 0.002g, 0.003g, 0.005g, 0.0065g, 0.008g, 0.01g, 0.0162g, 0.02g, 0.025g or 0.0325g; most preferably 0.0162g.

[0007] According to an embodiment of the present invention, the ice-controlling material is selected from polyvinyl alcohol (PVA).

[0008] In some embodiments, the PVA has a molecular weight of 5-50 kDa and a degree of hydrolysis of 85-99%, which helps to inhibit the growth of ice crystals during heating and cooling processes.

[0009] According to an embodiment of the present invention, the content of PVA is 0.3-1g per 100mL of cryopreservation solution, for example, 0.3g, 0.5g, 0.7g or 1.0g, preferably 0.5g.

[0010] According to an embodiment of the present invention, the permeable amphiphilic molecule is selected from betaine. Betaine is a quaternary ammonium salt, widely found in plants, animals, and microorganisms, and plays a role in regulating osmotic pressure and maintaining glass transition level through permeation across membranes.

[0011] According to an embodiment of the present invention, the content of betaine is 3-10g per 100mL of cryopreservation solution, for example, 3g, 5g, 7g or 10g, preferably 5g.

[0012] According to an embodiment of the present invention, the water-soluble disaccharide may be at least one of sucrose, trehalose and maltose; preferably sucrose.

[0013] According to an embodiment of the present invention, the content of the water-soluble disaccharide is 10-20g per 100mL of cryopreservation solution, for example 10g, 12g, 15g, 17.12g or 20g.

[0014] According to an embodiment of the present invention, the DMSO content is 0 to 10 mL per 100 mL of cryopreservation solution, for example, 1 mL, 2 mL, 5 mL, 8 mL or 10 mL.

[0015] According to an embodiment of the present invention, the ethylene glycol content is 5 to 15 mL per 100 mL of cryopreservation solution, for example, 5 mL, 8 mL, 10 mL, 12 mL or 15 mL.

[0016] According to an embodiment of the present invention, the serum may be human serum albumin for human cryopreservation subjects, and fetal bovine serum (FBS) or bovine serum albumin for non-human cryopreservation subjects.

[0017] According to an embodiment of the present invention, the serum content is 15 to 30 mL per 100 mL of cryopreservation solution, for example, 15 mL, 20 mL or 30 mL.

[0018] According to an embodiment of the present invention, the cryopreservation solution further includes a base solution. In this invention, the amount of the base solution is not specifically limited and can be adjusted to the required amount as needed. Preferably, the base solution is an aqueous solution and may be selected from at least one of PBS (Phosphate Buffer Saline) and HEPES solution.

[0019] According to an embodiment of the present invention, the pH value of the cryopreservation solution is in the range of 6.5-7.5, preferably 7.0-7.4, for example 7.0, 7.2 or 7.4.

[0020] According to an embodiment of the present invention, the cryoprotectant solution contains, per 100 mL volume: zinc salt, wherein the zinc ion content is 0.0065 g-0.0325 g; PVA, 0.3-1 g; betaine, 3-10 g; water-soluble disaccharide, 17.12 g; DMSO, 5 mL; ethylene glycol, 10 mL; serum, 20 mL; the balance being the base solution.

[0021] This invention also provides a method for preparing the above-mentioned cryopreservation solution, wherein the method includes adding a membrane protection molecule, an ice-controlling material, a permeable amphiphilic molecule, a water-soluble disaccharide, DMSO, ethylene glycol, and serum to a base solution to obtain the cryopreservation solution. The membrane protection molecule, ice-controlling material, permeable amphiphilic molecule, water-soluble disaccharide, DMSO, ethylene glycol, and serum have the meanings and dosages described above.

[0022] According to an embodiment of the present invention, the method for preparing the cryopreservation solution is as follows:

[0023] Step 1: Dilute the serum with a basic solution;

[0024] Step 2: Dissolve PVA in the base solution by heating to prepare PVA stock solution;

[0025] Step 3: Dissolve the membrane protection molecules, the osmotic amphiphilic molecules, and the water-soluble disaccharide in the solution obtained in Step 1;

[0026] Step 4: Add the PVA mother liquor from Step 2 to the solution obtained in Step 3, and add DMSO and EG. Make up the final amount with the base solution.

[0027] According to an embodiment of the present invention, the preparation method further includes a fifth step, filtering the cryopreservation solution obtained in step four. Preferably, the filtering can be performed using methods known in the art, such as using a 0.22 μm filter.

[0028] For example, the preparation method is as follows, per 100 mL of cryopreservation solution:

[0029] Step 1: Dilute fetal bovine serum (FBS) to 20% v / v using basal PBS solution, with at least 100 mL available.

[0030] Step 2: Add PVA to the basic solution PBS and heat to dissolve, preparing a 20X PVA stock solution;

[0031] Step 3: Dissolve betaine, zinc gluconate, and sucrose in the solution obtained in Step 1;

[0032] Step 4: Take the 20X PVA obtained in Step 2 and add it to the solution in Step 3 to obtain 1X PVA; add DMSO and EG; make up the final amount to 100mL with the basic solution from Step 1.

[0033] Step 5: Filter the cryopreservation solution obtained in Step 4 using a 0.22μm filter.

[0034] The present invention also provides the application of the above-described cryopreservation solution in cryopreserved mammalian oocytes.

[0035] The present invention also provides a cryopreservation method, the cryopreservation method comprising cryopreserving mammalian oocytes using the above-described cryopreservation solution.

[0036] The present invention also provides a method for protecting cell membranes. The method uses the above-mentioned cryopreservation solution to maintain the fluidity of cell membranes at low temperatures to avoid damage to cell membranes during cryopreservation, thereby achieving safe and efficient cryopreservation of oocytes.

[0037] According to an embodiment of the present invention, the cell membrane is primarily derived from biological samples sensitive to membrane damage, such as cells, tissues, or organs.

[0038] According to an embodiment of the present invention, the cells sensitive to membrane damage are selected from at least one of highly differentiated cells, cells with differentiation capacity, and tumor cell lines.

[0039] Preferably, the highly differentiated cells are selected from mature oocytes, corneal endothelial cells, etc. Preferably, the cells with differentiation capacity include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, etc. Preferably, the tumor cell line is selected from, for example, mouse lymphoma cells. Exemplarily, the oocytes are selected from oocytes extracted from various mammals in metaphase II of meiosis, having already expelled the first polar body.

[0040] According to embodiments of the present invention, the biological sample is derived from insects, animals, or plants, preferably mammals. More preferably, the mammal is selected from at least one of rats, mice, cattle, pigs, rabbits, monkeys, and humans.

[0041] The beneficial effects of this invention are:

[0042] This invention provides a cryopreservation solution for oocyte vitrification using cryoprotective membranes and ice-controlling materials. It enables efficient preservation of mature MII oocytes and maintains membrane flow stability before and after thawing. Compared to commercial reagents, using a fresh control group, the cryopreservation solution of this invention maintains the structural function and genetic stability of oocytes. This invention achieves membrane state protection by adding zinc ions, the introduction of PVA increases the vitrification capacity and ice-controlling effect of the cryopreservation system, and the addition of betaine molecules further improves osmotic pressure. The cryopreservation solution of this invention reduces the DMSO concentration in traditional cryopreservation solutions from 15% to 5% and the EG concentration from 15% to 10%, thereby significantly reducing the toxicity of cryopreservation solutions. Attached Figure Description

[0043] Figure 1 The morphology of oocytes before (Fresh) and after cryopreservation.

[0044] Figure 2 For the quality assessment of oocytes.

[0045] Figure 3 To detect cell membrane fluidity in oocytes.

[0046] Figure 4 The blastocyst development rate after in vitro fertilization of oocytes. Detailed Implementation

[0047] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0048] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0049] The cryopreservation solution for mature oocytes described below includes the following components:

[0050] Betaine: Purchased from Bailingwei Company, product number: 214119.

[0051] Zinc gluconate: purchased from Bailingwei Company, product number: 965581.

[0052] PVA: Purchased from Aladdin Company, item number: P434374.

[0053] Sugar: Purchased from Sigma-Aldrich, product number: V900116.

[0054] Serum albumin: purchased from MCE, catalog number: HY-D0842.

[0055] Fetal bovine serum: purchased from Gibco.

[0056] PBS basal solution: The basal solution used for oocyte cryopreservation can be a simple isotonic solution, purchased from Gibco.

[0057] Cells: Using 3-4 week old C57 female mice, the mice were injected with gonadotropins PMSG and HCG successively to synchronize the reproductive cycle and ovulation time of the mice and increase the number of ovulations. Mature oocytes were taken from the upper part of the oviduct (ampullary) and placed in M2 medium for later use.

[0058] Example: Cryopreservation of Mature Mouse Oocytes

[0059] 1. Materials

[0060] Low-toxicity cryoprotectant based on membrane protection and ice control

[0061] A cryopreservation solution was prepared using the following components and base solution. The preparation method is as follows, based on 100 mL of cryopreservation solution:

[0062] Step 1: Dilute fetal bovine serum (FBS) to 20% v / v using basal solution PBS (i.e., phosphate-buffered saline solution: containing 0.01M sodium dihydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 0.137M sodium chloride, 2.7mM potassium chloride, pH 7.4), with at least 100mL available.

[0063] Step 2: Weigh 1.0g PVA (molecular weight 13-23kDa, degree of hydrolysis 98%), use PBS as the basic solution and heat in a water bath to prepare 10mL of PVA stock solution with a concentration of 100mg / mL;

[0064] Step 3: Weigh 5.0g betaine, 0.1139g zinc gluconate and 17.12g sucrose;

[0065] Step 4: Take 60 mL of the solution obtained in Step 1 and dissolve the solid powder in Step 3;

[0066] Step 5: Add 5 mL of 100 mg / mL PVA stock solution to the solution from Step 4, and add 5 mL of DMSO and 10 mL of EG respectively; the final volume is made up to 100 mL with the basal solution PBS from Step 1, and the final PVA concentration is 5 mg / mL, i.e. 0.5% w / v;

[0067] Step 6: After filtering the cryopreservation solution obtained in Step 5 through a 0.22μm filter, it is used for oocyte cryopreservation experiments.

[0068] 2. Methods

[0069] The main method for cryopreservation of oocytes is the standard vitrification cryopreservation method based on Cryotop rods, which is a rapid cooling method involving direct immersion in liquid nitrogen. The specific cryopreservation steps are as follows:

[0070] (1) First, place the oocytes in a solution containing equilibration solution for at least 3 minutes to perform preliminary water molecule replacement. The equilibration solution consists of 7.5% DMSO-7.5% EG-20% FBS (dissolve 7.5 mL DMSO, 7.5 mL EG, and 20 mg FBS in 100 mL basal solution PBS).

[0071] (2) Afterward, the oocytes were transferred to a high-concentration cryopreservation solution and kept there for 1 to 3 minutes. The treatment time depends on the experimental design.

[0072] (3) Finally, the oocytes were rapidly transferred to a cryotope with a droplet volume of 1 μL and immediately immersed in liquid nitrogen for ultra-rapid freezing to achieve vitrification.

[0073] (4) Store the carrier rod in a liquid nitrogen tank.

[0074] The above operations are performed using an oral pipette and under an optical microscope.

[0075] 3. Oocyte cryopreservation and thawing protocol - thawing rate - cell morphology detection

[0076] Cells can theoretically be preserved in liquid nitrogen indefinitely. However, in order to shorten the experimental period, cells are revived and subjected to subsequent functional testing experiments after being preserved in liquid nitrogen for more than 30 minutes. The specific cell revival steps are as follows:

[0077] (1) Remove the vitrified oocyte carrier from liquid nitrogen and place it in thawing solution 1 at 37°C for 1 min; thawing solution 2 for 3 min; thawing solution 3 for 3 min; and thawing solution 4 twice for 3 min each time to quickly restore cell activity. Gradually remove the cryopreservation solution to prevent cell damage caused by rapid osmotic pressure impact.

[0078] (2) Evaluate oocyte morphology under a microscope (surviving oocytes should exhibit characteristics such as intact zona pellucida, homogeneous cytoplasm, and intact polar bodies), and calculate the oocyte viability after thawing. Analyze the data of each group using statistical methods to compare the oocyte viability and morphological changes under different cryopreservation conditions.

[0079] 4. Quality testing of oocytes after cryopreservation and thawing

[0080] Using fresh oocytes as a control, multiple functional assays were performed on frozen-thawed oocytes using immunofluorescence (IF) and live cell staining methods, including mitochondrial membrane potential, spindle morphology, ATP content, and ROS level.

[0081] (1) Detection of mitochondrial membrane potential

[0082] After washing oocytes with PBS, they were placed in M2 medium (for in vitro culture of pre-implantation mouse embryos) containing 2.0 μM JC-1 probe and incubated at 37°C for 30 min. The oocytes were washed three times with PBS to remove free dye, and the red / green fluorescence intensity ratio was observed using laser confocal microscopy to assess changes in mitochondrial membrane potential. JC-1 forms a red fluorescent polymer (590 nm) at high membrane potential and a green fluorescent monomer (530 nm) at low membrane potential. Fluorescence intensity was measured using ImageJ software, and the red / green fluorescence intensity ratio was calculated to determine the mitochondrial membrane potential level. A lower red / green fluorescence intensity ratio in the oocyte indicates a lower mitochondrial membrane potential and more severe mitochondrial damage.

[0083] (2) Spindle morphology detection

[0084] Immunofluorescence staining was used to detect the spindle apparatus and chromosome conformation of oocytes after vitrification. Oocytes were fixed in 3.7% paraformaldehyde (PFA) at 25°C for 30 min; subsequently, they were permeabilized with 5% Triton X-100 for 20 min; blocked with 3.0% bovine serum albumin (BSA) for 30 min; then incubated with anti-α-tubulin antibody (1:1000 dilution) for at least 1 hour, washed, and co-incubated with secondary antibody (Alexa Fluor 488-labeled rabbit anti-mouse IgG (H+L), 1:1000 dilution) for 30 min, while simultaneously staining DNA with Hoechst 33342 (5.0 μg / mL). After three washes, at least 1.5 μL of anti-fluorescence quencher was added to each oocyte, which was then mounted on a glass slide for observation and statistical analysis. Fluorescence images were captured using a confocal laser scanning microscope. In mature oocytes, a normal spindle is defined as a compact bipolar organelle with a typical barrel-shaped structure and neatly arranged metaphase chromosome plates. Abnormal spindles exhibit structural changes such as multipolar spindles, disordered spindles, or chromosome translocations. The normal spindle rate was calculated, with at least 50 oocytes counted in each group.

[0085] (3) ATP content detection

[0086] Each group of oocytes was transferred to a 96-well black detection plate (5 oocytes / well) (Corning, 3922), and 100 μL of LG1 PLUS culture medium was added. 100 μL of CellTiter-Glo reagent was added to the culture medium containing oocytes in each well, and the plates were incubated at 25°C for 10 min. The chemiluminescence signal was detected using a multi-mode microplate reader, and the ATP content of each group of oocytes was calculated based on the ATP luminescence standard curve.

[0087] 5. Cell membrane fluidity detection

[0088] Oocyte membranes were labeled with the fluorescent probe DilC12, and membrane fluidity was detected using FRAP (fluorescent bleaching recovery) technology. The zona pellucida was removed within 3–5 seconds using acidic M2 medium, followed by rapid washing with neutral medium. Oocytes were stained for 30 min with 10 μM of lipophilic DiIC12(3) perchlorate (1,1-docodecyl-3,3,3,3-tetramethylindocyanine perchlorate). After three washes with PBS, the cells were transferred to a confocal glass dish. Oocyte membrane fluidity was characterized using FRAP technology, as follows: A region of interest (ROI) and a background region were set using the ZEN system software of a laser scanning confocal microscope. The ROI was photobleached with 100% laser power at a wavelength of 561 nm, and the fluorescence signal was monitored. The fluorescence recovery time was at least 5 min. The fluorescence intensity was corrected for and normalized according to the background fluorescence, and the fluorescence recovery curve was fitted using a single exponential model with Origin2024 software. Fi represents the fluorescence intensity before bleaching. F0 represents the fluorescence intensity after bleaching, and F1 represents the fluorescence recovery intensity after bleaching. After data normalization, the fluorescence recovery rate is calculated using the formula R = (F1 - F0) / (Fi - F0). The higher the R value, the higher the membrane fluidity.

[0089] 6. In vitro fertilization and blastocyst development

[0090] Oocytes from each group were incubated in HTF medium at 37°C and 5% CO2. Epididymal sperm from 12-week-old adult male C57BL / 6 mice were cultured for 1 hour to achieve capacitation. The sperm and oocytes were then co-incubated for 6 hours to confirm the formation of two pronuclei. Fertilized eggs were transferred to KSOM medium, and embryonic development from 2-cell stage to blastocyst stage was monitored to assess cellular reproductive function.

[0091] Experiment 1. Effects of Addition of Various Components on Oocyte Cryopreservation and Recovery Rate

[0092] Cryopreservation solutions were prepared using PBS as a base solution, with betaine, zinc gluconate, sucrose, fetal bovine serum (FBS), DMSO, EG, and PVA added sequentially. These solutions were compared with conventional commercially available reagents to obtain cryopreservation solutions with different specific compositions. The mass of each component is shown in Table 1. Table 1 also presents the experimental results using conventional commercially available cryopreservation solutions. The recovery rate obtained using the above oocyte vitrification cryopreservation method was used as a control and denoted as the VM group.

[0093] Table 1. Composition of cryopreservation solution and oocyte recovery rate after cryopreservation in each group (based on 100 mL cryopreservation solution).

[0094]

[0095]

[0096] Note: The remaining amount should be made up with PBS basal solution.

[0097] The equilibration solution and thawing solution used in this invention, according to the standard vitrification cryopreservation method, are shown in Table 2.

[0098] Table 2. Composition of the equilibration solution and gradient thawing solution used in the 100mL volumetric meter.

[0099]

[0100] Results: Mature oocytes from 4-week-old C57BL / 6 mice were cryopreserved using standard Cryotop. Table 1 shows that the VM1 group, which introduced osmotic betaine to reduce the concentrations of DMSO and EG, had a recovery rate comparable to the commercial VM group (approximately 85%). Further introducing zinc ions, a membrane-protecting molecule, into the VM1 group (VM2 group) increased the recovery rate to 92%. Adding PVA ice-controlling material to the cryopreservation solution resulted in VM3, with a recovery rate of approximately 96%, significantly higher than the recovery rates of cells cryopreserved in VM2-1 to VM2-4 groups (all below 85%). This demonstrates that the present invention, through the synergistic effect of osmotic molecule substitution, membrane protection, and ice-controlling material, can reduce the concentrations of DMSO and EG in traditional cryopreservation solutions while maintaining a high cell recovery rate. Therefore, the cryopreservation solution of the present invention has practical application value in maintaining oocyte quality.

[0101] Experiment 2. Morphological examination of oocytes after thawing

[0102] The morphological changes of oocytes after cryopreservation in each group were observed in Table 1. The specific experimental steps are as follows: Cell state and morphology were photographed using a fluorescence inverted microscope (NIKON Ti2-U), namely Fresh (not cryopreserved), VM group, VM1 group, VM2 group, VM2-1 group, VM2-2 group, VM2-3 group, VM2-4 group and VM3 group.

[0103] Depend on Figure 1 Fluorescence inverted microscopy imaging results showed that, compared with the fresh group (Fresh non-frozen group), the oocytes in the VM3 group maintained normal structure and morphology after thawing and resuscitation, and the zona pellucida remained stable. This indicates that the cryopreservation solution of the present invention can effectively protect cell membrane structure.

[0104] Experiment 3. Oocyte quality assessment

[0105] Compared with the fresh group (Fresh non-frozen group), the quality of oocytes after cryopreservation and thawing using the traditional cryopreservation solution (VM group) and the cryopreservation solution with the controlled ice and membrane protection strategy of this invention (VM3 group) was assessed. This included measuring mitochondrial membrane potential, spindle morphology, and ATP content. The specific experimental steps are as follows:

[0106] (1) Detection of mitochondrial membrane potential

[0107] After washing oocytes with PBS, they were placed in culture medium containing 2.0 μM JC-1 probe and incubated at 37°C for 30 min. The cells were washed three times with PBS to remove free dye, and the red / green fluorescence intensity ratio was observed using a laser confocal microscope to assess changes in mitochondrial membrane potential. Fluorescence intensity was measured using ImageJ software to determine the mitochondrial membrane potential level.

[0108] (2) Spindle morphology detection

[0109] Immunofluorescence staining was used to detect the spindle apparatus and chromosome conformation of oocytes after vitrification. Oocytes were fixed in 3.7% paraformaldehyde (PFA) at 25°C for 30 min; subsequently, they were permeabilized with 5% Triton X-100 for 20 min; blocked with 3.0% bovine serum albumin (BSA) for 30 min; then incubated with anti-α-tubulin antibody (1:1000 dilution) for at least 1 hour, washed, and co-incubated with secondary antibody (Alexa Fluor 488-labeled rabbit anti-mouse IgG (H+L), 1:1000 dilution) for 30 min, while simultaneously staining DNA with Hoechst 33342 (5.0 μg / mL). After three washes, 1.5 μL of anti-fluorescence quencher was added to each oocyte, and the cells were mounted for observation. Abnormal spindles are characterized by structural changes such as multipolar spindles, disordered spindles, or chromosome translocations. The normal rate of spindles is statistically analyzed.

[0110] (3) ATP content detection

[0111] Each group of oocytes was transferred to a 96-well black detection plate (5 oocytes / well) and 100 μL of G1 PLUS culture medium was added. 100 μL of CellTiter-Glo reagent was added to the culture medium containing oocytes in each well, and the plates were incubated at 25°C for 10 min. The chemiluminescence signal was detected using a multi-mode microplate reader, and the ATP content of each group of oocytes was calculated based on the ATP luminescence standard curve.

[0112] The results are as follows Figure 2 As shown in the figure, the mitochondrial membrane potential, spindle morphology, and ATP level of oocytes in the VM3 group after thawing are comparable to those in the fresh group. This indicates that the VM3-co-protected cryopreservation system can effectively maintain its function at the same level as the fresh group.

[0113] Experiment 4. Cell membrane fluidity detection

[0114] The zona pellucida was removed within 3–5 seconds using acidic M2 medium, followed by rapid washing with neutral medium. Oocytes were stained with 10 μM lipophilic DiIC12(3) perchlorate (1,1-docodecyl-3,3,3,3-tetramethylindocyanine perchlorate) for 30 min. After three washes with PBS, the cells were transferred to a confocal glass dish. Oocyte membrane fluidity was characterized using FRAP technology, as follows: A region of interest (ROI) and a background region were set using the ZEN system software of a laser scanning confocal microscope. The ROI was photobleached with 100% laser power at a wavelength of 561 nm, and the fluorescence signal was monitored. The fluorescence recovery time was at least 5 min. The fluorescence intensity was corrected and normalized according to the background fluorescence, and the fluorescence recovery curve was fitted using a single exponential model with Origin2024 software.

[0115] The results are as follows Figure 3 As shown in the figure, the membrane fluidity of oocytes decreased after thawing from cryopreservation using the traditional vitrification cryopreservation system (VM group). However, cryopreservation using the VM3 cryopreservation solution (controlled ice + membrane protection synergy) of this invention effectively maintained the membrane fluidity of oocytes after thawing, restoring it to the level of fresh oocytes. Figure 3 Membrane phase response maintains membrane stability, which may provide important protection for improving fertilization rate and embryonic development.

[0116] Experiment 5. In vitro fertilization and blastocyst development rate

[0117] Oocytes from each group were incubated in HTF medium at 37°C and 5% CO2. Epididymal sperm from 12-week-old adult male C57BL / 6 mice were cultured for 1 hour to achieve capacitation. The sperm and oocytes were then co-incubated for 6 hours to confirm the formation of two pronuclei. Fertilized eggs were transferred to KSOM medium, and embryonic development from 2-cell stage to blastocyst stage was monitored to assess cellular reproductive function.

[0118] The results are as follows Figure 4 As shown in the figure, the results of in vitro fertilization and embryo culture show that the blastocyst development rate of the VM3 group is higher than that of the commercial VM group, which indicates that the cryopreservation solution of the present invention has practical application value in maintaining oocyte quality.

[0119] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cryopreservation solution, characterized in that, include: Membrane protection molecules, ice-controlling materials, permeable amphiphilic molecules, water-soluble disaccharides, DMSO, ethylene glycol, and serum.

2. The cryopreservation solution as described in claim 1, characterized in that, The membrane protective molecule is selected from zinc salts, such as zinc gluconate; And / or, the zinc ion content in the zinc salt is 0.0065g-0.0325g per 100mL of cryopreservation solution.

3. The cryopreservation solution as described in claim 1 or 2, characterized in that, The ice-controlling material is selected from polyvinyl alcohol (PVA); And / or, the PVA content is 0.3-1g per 100mL of cryopreservation solution.

4. The cryopreservation solution according to any one of claims 1-3, characterized in that, The permeable amphiphilic molecule is selected from betaine; And / or, the content of betaine is 3-10g per 100mL of cryopreservation solution; And / or, the water-soluble disaccharide may be at least one of sucrose, trehalose and maltose; And / or, the content of the water-soluble disaccharide is 10-20g per 100mL of cryopreservation solution.

5. The cryopreservation solution according to any one of claims 1-4, characterized in that, The DMSO content is 0–10 mL per 100 mL of cryopreservation solution; And / or, the ethylene glycol content is 5 to 15 mL per 100 mL of cryopreservation solution; And / or, the serum may be human serum albumin for human cryopreservation subjects, or fetal bovine serum (FBS) or bovine serum albumin for non-human cryopreservation subjects; And / or, the serum content is 15-30 mL per 100 mL of cryopreservation solution.

6. The cryopreservation solution according to any one of claims 1-5, characterized in that, The cryopreservation solution also includes a base solution; And / or, the base solution is selected from at least one of PBS solution and HEPES solution; And / or, the pH value of the cryopreservation solution is in the range of 6.5-7.5, preferably 7.0-7.

4.

7. The method for preparing the cryopreservation solution according to any one of claims 1-6, characterized in that, The preparation method includes adding membrane protection molecules, ice-controlling materials, permeable amphiphilic molecules, water-soluble disaccharides, DMSO, ethylene glycol and serum to a base solution to obtain the cryopreservation solution.

8. The use of the cryopreservation solution according to any one of claims 1-6 in cryopreserving mammalian oocytes.

9. A cryopreservation method, characterized in that, The cryopreservation method includes cryopreserving mammalian oocytes using the cryopreservation solution according to any one of claims 1-6.

10. A method for protecting cell membranes, characterized in that, The method uses the cryopreservation solution described in any one of claims 1-6 to maintain cell membrane fluidity at low temperature, thereby avoiding cell membrane damage during cryopreservation and achieving safe and efficient oocyte cryopreservation.

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