Efficient sperm freeze-drying preserving fluid and preparation method thereof

Abstract

The invention relates to the technical field of sperm cryopreservation, in particular to an efficient sperm freeze-drying preservation solution and a preparation method thereof. The efficient sperm freeze-drying preserving fluid is prepared from the following components in percentage by weight: 0.06%-0.1% of amino trimethylolmethane, 0.06%-0.1% of amino trimethylolmethane, 0.02%-0.04% of citric acid, 1%-2% of lecithin, 2%-5% of vitamin C, 8%-15% of trehalose, 3%-5% of mannitol, 10%-20% of polyvinylpyrrolidone, 0.3%-0.4% of disodium EDTA (Ethylene Diamine Tetraacetic Acid), 3%-5% of fetal calf serum, 20%-25% of dimethyl sulfoxide and the balance of water. 20-25% of a phosphate buffer solution and 0.05-0.1% of stem cell factors; the sperm freeze-drying agent can protect sperms in the freeze-drying process, reduce sperm apoptosis in the freeze-storage process, enhance the adaptability of the sperms to the freeze-storage environment, help the sperms to recover activity and functions after unfreezing, effectively improve the survival rate and activity of the sperms after freeze-drying and resuscitation, reduce oxidative stress reaction caused by freezing and improve the survival rate of the sperms after freeze-drying and resuscitation. The cryopreserved sperm freeze-drying agent can improve the quality of cryopreserved sperms, improve the vitality and fertilization ability of the cryopreserved sperms, is suitable for a freeze-drying method, and enables the sperms to be transported and stored in a room temperature environment.

Description

Technical Field

[0001] The present invention relates to the technical field of sperm cryopreservation, and particularly relates to an efficient sperm freeze-drying preservation solution and a preparation method thereof. Background Art

[0002] With the development of infertility treatment, especially the popularization of artificial insemination and in vitro fertilization techniques, sperm cryopreservation technology has been increasingly widely used clinically. Particularly, sperm preservation technology has played an important role in male and female infertility, sperm preservation for cancer patients before treatment, and animal breed improvement. Early artificial insemination techniques mainly relied on fresh sperm, but in practice, there is a strong need to store sperm for future use in many cases. Traditional cryopreservation methods are difficult to ensure the biological activity of sperm. The formation of ice crystals and the expansion of intracellular water can cause sperm membrane rupture and structural damage. Therefore, an effective sperm protection solution is needed to reduce the damage to sperm during freezing, protect sperm from damage during low temperature or freezing, so that it can be successfully thawed and maintain its activity in the future. Sperm preservation solution is crucial in sperm preservation technology.

[0003] Currently, commonly used sperm cryoprotectants include glycerol, egg yolk, and sodium citrate. However, the effect of a single sperm cryoprotectant is not ideal, and often multiple components need to be combined to improve the survival rate and vitality of sperm during freezing and thawing. Chinese Patent with publication number CN117965433A provides a method, product, and application for improving the survival rate and activity of sperm in vitro. The method of mixing an artificial synthetic protein with the N-terminal amino acid sequence of protein QRICH2 with the existing semen preservation solution can significantly improve the activity of cryopreserved semen and can effectively preserve sperm for a long time. However, its preservation conditions are demanding, and it cannot be transported and stored at room temperature. Chinese Patent with publication number CN106818712A discloses a sperm drying solution and a sperm preservation method, which can preserve sperm at room temperature, but cannot preserve sperm for a long time, and the survival rate and activity of sperm preserved with this sperm drying solution are limited.

[0004] In view of the above problems, the present invention provides a solution. Summary of the Invention

[0005] The object of the present invention is to provide an efficient sperm freeze-drying preservation solution and a preparation method thereof, which are applicable to the freeze-drying method, can enable sperm to be transported and stored at room temperature, and the sperm preserved has good survival rate and activity.

[0006] To achieve the above object, the present invention adopts the following technical solution: An efficient sperm freeze-drying preservation solution is composed of the following percentage components: 0.06%-0.1% tris(hydroxymethyl)aminomethane, 0.02-0.04% citric acid, 1-2% lecithin, 2-5% vitamin C, 8-15% trehalose, 3-5% mannitol, 10-20% polyvinylpyrrolidone, 0.3-0.4% disodium EDTA, 3-5% fetal bovine serum, 20-25% dimethyl sulfoxide, 20-25% phosphate buffer solution, and 0.05-0.1% stem cell factor;

[0007] Among them, the preparation method of the stem cell factor includes the following steps:

[0008] A1: Disinfect the operating table with ultraviolet rays for 30 minutes, turn on the water bath and heat it to 37°C. Subsequently, prepare a culture medium of 44.5 ml DMEM + 5 ml fetal bovine serum + 0.5 ml double antibody. Quickly take out the mesenchymal stem cells from liquid nitrogen with forceps and immediately place them in the water bath to thaw and melt.

[0009] A2: Aspirate and transfer the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml DMEM(+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left and right evenly, and then place the culture dish in a cell culture incubator with 5% CO2 for culture.

[0010] A3: When the cell density reaches 85% under the microscope, discard the original culture medium, then wash the cells 2 times with phosphate buffer solution. After completely removing the culture medium with a pipette, use 5 ml of 9% trypsin to digest the mesenchymal stem cells, add 5 ml of culture medium to the culture dish to terminate the digestion after the mesenchymal stem cells are detached from the bottom of the culture dish, and use a pipette to aspirate and blow the mixture of culture medium and trypsin to completely mix the mesenchymal stem cells to make a mesenchymal stem cell suspension.

[0011] A4: Aspirate and transfer the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml DMEM(+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left and right evenly, and then place the culture dish in a cell culture incubator with 5% CO2 for culture.

[0012] A5: When the cell density reaches 85% under the microscope, discard the original culture medium, wash the cells 2 times with phosphate buffer solution. After washing, add serum-free culture medium, and then place the culture dish in a cell culture incubator with 5% CO2 for 24 h.

[0013] A6: Collect the serum-free medium after the culture is completed. Add 5% mannitol to the medium, mix well, and place it on a freeze dryer for 12 hours of freeze-drying. After the freeze-drying is completed, stem cell factor is obtained.

[0014] Further, the double antibody described in step A1 is a mixed antibiotic of ampicillin and streptomycin, where the mass ratio of ampicillin to streptomycin is 1:1; the mass ratio of the serum-free medium to 5% mannitol described in step B6 is 20:1.

[0015] Further, a preparation method of an efficient sperm freeze-drying preservation solution includes the following steps:

[0016] B1: Disinfect the operating table with ultraviolet rays for 30 minutes. Turn on the water bath and set the temperature to 25 - 30 °C. Then add tris(hydroxymethyl)aminomethane, polyvinylpyrrolidone, disodium EDTA, and dimethyl sulfoxide into a non-enzymatic centrifuge tube and heat it in a water bath. During the water bath heating, gently shake the centrifuge tube until it is completely mixed to obtain the pre-liquid A of the freeze-drying preservation solution.

[0017] B2: Mix and dissolve citric acid, lecithin, vitamin C, and mannitol in phosphate buffer solution. Then transfer the mixture to a non-enzymatic centrifuge tube, set the temperature of the water bath to 30 - 35 °C, and heat it in a water bath. During the water bath heating, gently shake the centrifuge tube until it is completely mixed to obtain the pre-liquid B of the freeze-drying preservation solution.

[0018] B3: Mix the pre-liquid A of the freeze-drying preservation solution and the pre-liquid B of the freeze-drying preservation solution. Then add fetal bovine serum and stem cell factor, and use a pipette to blow and suck to mix evenly to obtain the sperm freeze-drying preservation solution.

[0019] Further, the polyvinylpyrrolidone is water-soluble polyvinylpyrrolidone with a molecular weight of 40; the citric acid is white citric acid powder with a specification of 200 mesh; the lecithin is yellow egg yolk lecithin powder with a specification of 200 mesh; the vitamin C is white water-soluble L-ascorbic acid powder with a specification of 200 mesh.

[0020] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: The present invention can protect sperm from damage during the freeze-drying process, reduce sperm apoptosis during the cryopreservation process, enhance the adaptability of sperm to the cryopreservation environment, help sperm recover activity and function after thawing, can effectively improve the survival rate and vitality of sperm after freeze-drying and resuscitation, and at the same time reduce the oxidative stress response caused by freezing, can improve the quality of cryopreserved sperm, improve the vitality and fertilization ability of cryopreserved sperm, and is applicable to the freeze-drying method, which can enable sperm to be transported and stored at room temperature. Specific embodiments

[0021] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0022] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods, carried out according to the technologies or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0023] Example 1

[0024] 1: Disinfect the ultraviolet operation table for 30 minutes, turn on the water bath to heat to 37°C, then prepare the culture medium: 44.5 ml of DMEM + 5 ml of fetal bovine serum + 0.5 ml of double antibody. Quickly take out the mesenchymal stem cells from liquid nitrogen with forceps and immediately place them in the water bath to thaw and melt.

[0025] 2: Pipette the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes in total. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish evenly up, down, left and right, and then place the culture dish in a 5% CO2 cell culture incubator for culture.

[0026] 3: When the cell density reaches 85% observed under the microscope, discard the original culture medium, wash the cells 2 times with phosphate buffer solution. After completely removing the culture medium with a pipette, use 5 ml of 9% trypsin to digest the mesenchymal stem cells, add 5 ml of culture medium to the culture dish to terminate the digestion after the mesenchymal stem cells are detached from the bottom of the culture dish, and use a pipette to blow and suck the mixture of culture medium and trypsin to completely mix the mesenchymal stem cells to make a mesenchymal stem cell suspension.

[0027] 4: Pipette the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes in total. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish evenly up, down, left and right, and then place the culture dish in a 5% CO2 cell culture incubator for culture.

[0028] 5: When the cell density reaches 85% observed under the microscope, discard the original culture medium, wash the cells 2 times with phosphate buffer solution. After washing, add 20 g of serum-free culture medium, and then place the culture dish in a 5% CO2 cell culture incubator for 24 h.

[0029] 6: Collect the serum-free medium after the culture is completed. Add 1 g of 5% mannitol to the medium, mix well, place it on a freeze dryer and freeze-dry for 12 h. After freeze-drying, 0.8 g of the stem cell factor prepared in Example 1 is obtained.

[0030] Table 1, Reagent Parameter Table for Example 1

[0031]

[0032]

[0033] Example 2

[0034] 1: Disinfect the operating table with ultraviolet light for 30 minutes. Turn on the water bath and heat it to 37 °C. Then prepare 44.5 ml of DMEM + 5 ml of fetal bovine serum + 0.5 ml of double antibody. Quickly take out the mesenchymal stem cells from liquid nitrogen with forceps and immediately place them in the water bath to thaw and melt.

[0035] 2: Aspirate and transfer the mesenchymal stem cell suspension into a 15 ml centrifuge tube. Add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left and right evenly, and then place the culture dish in a cell culture incubator with 5% CO2 for culture.

[0036] 3: When the cell density reaches 85% observed under the microscope, discard the original medium, wash the cells 2 times with phosphate buffer solution. After completely removing the medium with a pipette, use 5 ml of 9% trypsin to digest the mesenchymal stem cells, add 5 ml of medium to the culture dish to terminate the digestion when the mesenchymal stem cells detach from the bottom of the culture dish, and use a pipette to aspirate and blow the medium and trypsin mixture to completely mix the mesenchymal stem cells to make a mesenchymal stem cell suspension.

[0037] 4: Aspirate and transfer the mesenchymal stem cell suspension into a 15 ml centrifuge tube. Add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left and right evenly, and then place the culture dish in a cell culture incubator with 5% CO2 for culture.

[0038] 5: When the cell density reaches 85% observed under the microscope, discard the original medium, wash the cells 2 times with phosphate buffer solution. After washing, add 40 g of serum-free medium, and then place the culture dish in a cell culture incubator with 5% CO2 for culture for 24 h.

[0039] 6: Collect the serum-free medium after the culture is completed. Add 2 g of 5% mannitol to the medium, mix well, place it on a freeze dryer and freeze-dry for 12 h. After freeze-drying, 1.56 g of the stem cell factor prepared in Example 2 is obtained.

[0040] Table 2, Reagent Parameter Table for Example 2

[0041] Drug Name Source Model Parameter DMEM ThermoFisher 11320033 500ml Fetal Bovine Serum BBI E510002 500ml Ampicillin BBI A430258 200mg Streptomycin BBI A430977 500mg Trypsin Diamond A100458 50g Phosphate Buffered Solution Aladdin P492453 500ml Serum-Free Medium Cytiva SH30941.02 1L Mannitol Aladdin L130871 100mg

[0042] Example 3

[0043] 1: Disinfect the operating table with ultraviolet light for 30 minutes. Turn on the water bath and set the temperature to 25°C. Then add 0.6 g of aminotrimethylmethane, 100 g of polyvinylpyrrolidone, 3 g of disodium EDTA, and 200 g of dimethyl sulfoxide to a non-enzymatic centrifuge tube and heat it in a water bath. Gently shake the centrifuge tube during the water bath heating until it is completely mixed to obtain the pre-liquid A of the freeze-drying preservation solution.

[0044] 2: Mix and dissolve 0.2 g of citric acid, 1 g of lecithin, 20 g of vitamin C, 80 g of trehalose, and 30 g of mannitol in 250 g of phosphate buffer solution. Then transfer the mixture to a non-enzymatic centrifuge tube, set the water bath temperature to 30°C, and heat it in a water bath. Gently shake the centrifuge tube during the water bath heating until it is completely mixed to obtain the pre-liquid B of the freeze-drying preservation solution.

[0045] 3: Mix the pre-liquid A of the freeze-drying preservation solution and the pre-liquid B of the freeze-drying preservation solution. Then add 30 g of fetal bovine serum and 0.5 g of the stem cell factor prepared in Example 1, and use a pipette to blow and suck to mix evenly to obtain 620 g of the sperm freeze-drying preservation solution prepared in Example 3.

[0046] Table 3, Reagent Parameter Table for Example 3

[0047] Drug Name Source Model Parameter Tris(hydroxymethyl)aminomethane Shandong Huayao Biotechnology Co., Ltd. 03532 1kg Polyvinylpyrrolidone Shandong Zhongwei New Materials Co., Ltd. 9003 1kg EDTA Disodium Shaanxi Changji Auxiliary Biotechnology Co., Ltd. JFE1 500g Dimethyl Sulfoxide Shandong Rongsheng New Materials Co., Ltd. 67 500ml Citric Acid Shandong Ruigang Chemical Industry Co., Ltd. 024 1kg Lecithin Xi'an Yuankui Biotechnology Co., Ltd. YK-01 500g Vitamin C Shandong Juxinlong Biotechnology Co., Ltd. SY 500g Mannitol Aladdin L130871 100mg Phosphate Buffered Solution Aladdin P492453 500ml Fetal Bovine Serum BBI E510002 500ml Trehalose Henan Shengteng Biotechnology Co., Ltd. 0201 500g

[0048] Example 4

[0049] 1: Disinfect the operating table with ultraviolet light for 30 minutes. Turn on the water bath and set the temperature to 30°C. Then add 1 g of aminotrimethylmethane, 200 g of polyvinylpyrrolidone, 4 g of disodium EDTA, and 250 g of dimethyl sulfoxide to a non-enzymatic centrifuge tube and heat it in a water bath. Gently shake the centrifuge tube during the water bath heating until it is completely mixed to obtain the pre-liquid A of the freeze-drying preservation solution.

[0050] 2: Mix and dissolve 0.4 g of citric acid, 20 g of lecithin, 50 g of vitamin C, and 50 g of mannitol in 250 g of phosphate buffer solution. Then transfer the mixture to a non-enzymatic centrifuge tube, set the water bath temperature to 35°C, and heat it in a water bath. Gently shake the centrifuge tube during the water bath heating until it is completely mixed to obtain the pre-liquid B of the freeze-drying preservation solution.

[0051] 3: Mix the pre-liquid A of the freeze-dried preservation solution and the pre-liquid B of the freeze-dried preservation solution, then add 50 g of fetal bovine serum and 1 g of the stem cell factor prepared in Example 2, and use a pipette to blow and suck to mix evenly to obtain the sperm freeze-dried preservation solution.

[0052] Table 4, Reagent Parameter Table for Example 4

[0053] Drug Name Source Model Parameter Tris(hydroxymethyl)aminomethane Shandong Huayao Biotechnology Co., Ltd. 03532 1kg Polyvinylpyrrolidone Shandong Zhongwei New Materials Co., Ltd. 9003 1kg EDTA Disodium Shaanxi Changji Auxiliary Biotechnology Co., Ltd. JFE1 500g Dimethyl Sulfoxide Shandong Rongsheng New Materials Co., Ltd. 67 500ml Citric Acid Shandong Ruigang Chemical Industry Co., Ltd. 024 1kg Lecithin Xi'an Yuankui Biotechnology Co., Ltd. YK-01 500g Vitamin C Shandong Juxinlong Biotechnology Co., Ltd. SY 500g Mannitol Aladdin L130871 100mg Phosphate Buffered Solution Aladdin P492453 500ml Fetal Bovine Serum BBI E510002 500ml Trehalose Henan Shengteng Biotechnology Co., Ltd. 0201 500g

[0054] Comparative Example 1

[0055] Select Example 1 of the Chinese patent with the publication number CN106818712A as Comparative Example 1.

[0056] Comparative Example 2

[0057] Purchase the sperm cryopreservation solution produced by Danish CooperSurgical Company as Comparative Example 2.

[0058] In vitro cryopreservation experiment of rat sperm

[0059] 1: Dilute the freshly collected rat semen with a diluent until the sperm density in the semen is 1×10 6 cells / ml. Then use a centrifuge to remove seminal plasma. The centrifuge centrifugal force is set to 800 xg and centrifuged for 3 min. After centrifugation, mix the centrifuged semen with the sperm preservation solutions prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2 to obtain a sperm suspension. The sperm density in the sperm suspension is 1×10 6 cells / ml. Then freeze-dry the sperm suspension prepared by mixing with Example 3 and Example 4 for 3 h to make sperm freeze-dried powder. Pipette 20 μl of the sperm suspension prepared by mixing with Comparative Example 1 and drop it on a glass slide and air-dry it naturally for 30 min. Freeze the sperm suspension prepared by mixing with Comparative Example 2 in a liquid nitrogen tank;

[0060] 2: After 1 month, use the in situ end labeling method for DNA fragmentation to test the sperm preserved with the sperm preservation solutions prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2. Use a flow cytometer to detect the number of apoptotic sperm. The number of test sperm is 10,000, and calculate the percentage of apoptotic sperm to obtain the sperm apoptosis rate;

[0061] 3: After 1 month, use intracytoplasmic sperm injection technology to combine the sperm preserved with the sperm preservation solutions prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2 with oocytes, and culture the obtained embryos in vitro. Observe the cleavage rate at 48 h of culture. The cleavage rate = the number of cleaved embryos / the number of test oocytes × 100%. Observe the blastocyst rate at 168 h of culture. The blastocyst rate = the number of blastocysts / the number of test oocytes × 100%;

[0062] Table 5, Results of in vitro cryopreservation experiment of rat sperm

[0063] Grouping Sperm Apoptosis Rate Number of Tested Oocytes Cleavage Rate Blastocyst Rate Example 3 47.65％ 200 70.82％ 28.14％ Example 4 46.57％ 200 81.51％ 30.78％ Control Example 1 69.88％ 200 74.21％ 22.32％ Control Example 2 48.29％ 200 69.22％ 20.11％

[0064] In vitro cryopreservation experiment of human sperm

[0065] 1: Dilute the freshly collected rat semen with a diluent until the sperm density in the semen is 1×10 6 per ml. Subsequently, use a centrifuge to remove seminal plasma. Set the centrifugal force of the centrifuge to 800xg and centrifuge for 3 min. After centrifugation, mix the centrifuged semen with the sperm preservation solutions prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2 to obtain a sperm suspension. The sperm density in the sperm suspension is 1×10 6 per ml. Subsequently, lyophilize the sperm suspension prepared by mixing with Example 3 and Example 4 for 3 h to make sperm lyophilized powder. Pipette 20 μl of the sperm suspension prepared by mixing with Comparative Example 1 onto a glass slide and air-dry it naturally for 30 min. Store the sperm suspension prepared by mixing with Comparative Example 2 in a liquid nitrogen tank;

[0066] 2: After 1 month, 3 months, 6 months, and 12 months respectively, use the in situ end-labeling method for DNA fragmentation to test the sperm preserved with the sperm preservation solutions prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2. Use a flow cytometer to detect the number of apoptotic sperm. The number of test sperm is 10,000, and calculate the percentage of apoptotic sperm;

[0067] Table 6, Results of in vitro cryopreservation experiment of human sperm

[0068]

[0069]

[0070] By analyzing Table 5 and Table 6, it can be seen that the sperm preservation solutions prepared in Example 3 and Example 4 can effectively protect sperm from damage during the freeze-drying process in vitro, still have good survival rates after long-term cryopreservation, and at the same time, the motility of the surviving sperm is not significantly reduced. After fertilization of the eggs, there are good cleavage rates and blastocyst rates, which can enable sperm to be transported and stored at room temperature.

[0071] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A highly efficient sperm freeze-dried storage solution, characterized in that: The composition is composed of the following percentage ingredients: 0.06%-0.1% aminotrimethylolmethane, 0.02-0.04% citric acid, 1-2% lecithin, 2-5% vitamin C, 8-15% trehalose, 3-5% mannitol, 10-20% polyvinylpyrrolidone, 0.3-0.4% disodium EDTA, 3-5% fetal bovine serum, 20-25% dimethyl sulfoxide, 20-25% phosphate buffer solution and 0.05-0.1% stem cell factor; The preparation method of stem cell factor comprises the following steps: A1: Disinfect the operating table with ultraviolet light for 30 minutes, turn on the water bath and heat it to 37°C, then prepare the culture medium 44.5ml DMEM + 5ml fetal bovine serum + 0.5ml double antibody, quickly take out the mesenchymal stem cells from the liquid nitrogen with tweezers and immediately put them in a water bath to thaw; A2: Pipette the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml DMEM (+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml culture medium to resuspend the cells, and evenly spread the resuspended cells in a 60 mm culture dish. Shake the dish evenly up and down and left and right, and then place the culture dish in a cell culture incubator with 5% CO2 for culture; A3: When the cell density reaches 85% under the microscope, discard the original culture medium, wash the cells twice with phosphate buffer solution, use a pipette to completely remove the culture medium, use 5 ml of 9% trypsin to digest the mesenchymal stem cells, and make the mesenchymal stem cells detach from the bottom of the culture dish. Add 5 ml of culture medium to the culture dish to terminate the digestion, and use a pipette to blow and aspirate the culture medium and trypsin mixture to completely mix the mesenchymal stem cells to prepare a mesenchymal stem cell suspension; A4: Pipette the mesenchymal stem cell suspension into a 15 ml centrifuge tube, add 6 ml DMEM (+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml culture medium to resuspend the cells, and evenly spread the resuspended cells in a 60 mm culture dish. Shake the dish evenly up and down and left and right, and then place the culture dish in a cell culture incubator with 5% CO2 for culture; A5: When the cell density reaches 85% under the microscope, discard the original culture medium, wash the cells twice with phosphate buffer solution, add serum-free culture medium after washing, and then place the culture dish in a cell culture incubator with 5% CO2 for 24 hours; A6: Collect the serum-free culture medium after the culture is completed, add 5% mannitol to the culture medium, mix well and place in a freeze dryer for lyophilization for 12 hours. After lyophilization, stem cell factor is obtained.

2. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The dual antibody described in step A1 is a mixed antibiotic of ampicillin and streptomycin, wherein the mass ratio of ampicillin to streptomycin is 1:

1.

3. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The mass ratio of the serum-free medium to 5% mannitol described in step B6 is 20:

1.

4. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The polyvinyl pyrrolidone is water-soluble polyvinyl pyrrolidone with a molecular weight of 40.

5. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The citric acid is white citric acid powder with a specification of 200 meshes.

6. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The lecithin is yellow egg yolk lecithin powder with a specification of 200 meshes.

7. The high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The vitamin C is white water-soluble L-ascorbic acid powder with a specification of 200 meshes.

8. The method for preparing a high-efficiency sperm freeze-dried storage solution according to claim 1, characterized in that: The following steps are involved: B1: UV disinfection of the operating table for 30 minutes, turn on the water bath and set the temperature to 25-30°C, then add aminotrimethylolmethane, polyvinylpyrrolidone, disodium EDTA and dimethyl sulfoxide into an enzyme-free centrifuge tube and heat it in a water bath. During the water bath heating, gently shake the centrifuge tube until it is completely mixed to obtain the freeze-dried storage solution pre-liquid A; B2: Mix and dissolve citric acid, lecithin, vitamin C and mannitol in a phosphate buffer solution, then transfer the mixture to an enzyme-free centrifuge tube, set the water bath temperature to 30-35°C, heat in a water bath, and gently shake the centrifuge tube during heating in the water bath until it is completely mixed to obtain the freeze-dried storage solution pre-solution B; B3: Mix the freeze-dried storage solution pre-liquid A and the freeze-dried storage solution pre-liquid B, then add fetal bovine serum and stem cell factor, and use a pipette to mix evenly to obtain the sperm freeze-dried storage solution.

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