CN116725003B - Stem cell cryopreservation solution and stem cell cryopreservation method - Google Patents

Abstract

The invention relates to the technical field of stem cell cryopreservation, and specifically provides a stem cell cryopreservation solution and a stem cell cryopreservation method; the stem cell cryopreservation solution uses an antifreeze composition of 43 to 58% by weight and a stem cell culture medium to make up the balance. ; The antifreeze composition is 30 to 35% by weight of antifreeze extract, 3% by weight of protocatechuic acid, 2% by weight of coumarin, and 6% by weight. % of quercetin and make up the balance of glycerin. The present invention uses the unique proportion of the antifreeze composition of the present invention in the cryopreservation solution, and cooperates with the unique stem cell cryopreservation method of the present invention, by improving the freezing rate of the inner and outer crystal nuclei of endometrial stem cells and the morphology of ice crystals, protecting enzymes including The living intracellular environment allows endometrial stem cells to have a higher survival rate and proliferation ability after recovery and even after repeated freezing and thawing.

Description

Stem cell cryopreservation solution and stem cell cryopreservation method

Technical field

The present invention relates to the technical field of stem cell cryopreservation, and in particular to a stem cell cryopreservation solution and a stem cell cryopreservation method.

Background technique

Stem cells are a type of pluripotent cells with the ability to replicate themselves. Under certain conditions, it can differentiate into a variety of functional cells. According to the developmental stage of stem cells, they are divided into embryonic stem cells and adult stem cells. Stem cells are divided into three categories according to their developmental potential: totipotent stem cells, pluripotent stem cells and unipotent stem cells. Stem cells are cells that are not yet fully differentiated and immature and have the potential to regenerate various tissues, organs and the human body.

Endometrial stem cells are a type of adult stem cells with the potential of self-renewal, multi-directional differentiation and high proliferation. At present, a large number of studies have confirmed that the existence of stem cell-like cells in the endometrium gives the endometrium a strong regenerative ability; as early as 1978, studies suggested that the proliferation and repair of the functional layer of the endometrium may be due to the effect of endometrial stem cells. A series of subsequent clinical and basic studies confirmed this hypothesis. The potential of the endometrium to regenerate and differentiate into other tissues has received increasing attention and may become an alternative treatment for endometrial repair in patients with thin endometrium and endometrial lesions.

In order to temporarily remove the endometrial stem cells from the growth state, preserve their cell characteristics and resuscitate the cells when needed, cryopreservation technology is often used to store the cells in -196°C liquid nitrogen, which can prevent the cells from being cultured. Cells are lost due to contamination or other accidents. However, when freezing, water leaks out of the cells and ice crystals form, causing cell damage. When cells are revived, water seeps into the cells to form intracellular recrystallization, which also causes cell damage. Existing technologies mostly use DMSO or glycerol as protective agents for cryopreservation, but the survival rate of cells after recovery is low, and even if they survive, their proliferation ability is often not high; in addition, the existing cryopreservation solutions do not support repeated freezing and thawing, which is harmful to Storing, transporting and using stem cells in batches brings inconvenience. Therefore, it is of great practical significance to provide a cell cryopreservation solution that has a higher survival rate and proliferation ability of endometrial stem cells after recovery or even repeated freezing and thawing.

Contents of the invention

In order to solve the above problems, the purpose of the present invention is to provide a stem cell cryopreservation solution and a stem cell cryopreservation method.

On the one hand, the present invention provides a stem cell cryopreservation solution, which is composed of the following components:

The antifreeze composition is 43~58% by weight and the stem cell culture medium makes up the balance;

The antifreeze composition is 30 to 35% by weight of antifreeze extract, 3% by weight of protocatechuic acid, 2% by weight of coumarin, and 6% by weight. of quercetin and glycerin to make up the balance;

The extraction method of the antifreeze extract includes:

Step 1: Raise adult rainbow trout at 2°C for 2 weeks, collect 400ml of fish blood and 300g of fish meat;

Step 2: Put the fish blood and fish meat into the flash extractor at 2°C, rotate at 3600~4700r/min, extract for 17~28s and then filter through a 50-mesh filter to obtain a coarse-filtered extract;

Step 3: At 4°C, add an equal volume of glycerin to the coarse filter mixture, mix it in a SV-type static mixer for 60 to 70 seconds, and filter it with 150 mesh to obtain a fine filter extract. The void ratio of the mixer is 0.909%;

Step 4: Put the mixed solution into a centrifuge, centrifuge for 3 minutes per 5ml at a speed of 6200~6900r/min, and take the supernatant;

Step 5: Distill the supernatant under reduced pressure at 40°C with a vacuum of 600~800mbar until no liquid continues to flow out, and collect the distillate to obtain the antifreeze extract.

Further, the weight portion of the antifreeze composition is 54%.

Further, the weight portion of the antifreeze extract is 33%.

Further, the flash extraction process in step two is 4400r/min, and the extraction takes 23 seconds.

Further, the mixing time in step three is 66 seconds.

Further, the centrifugal speed in step 4 is 6700 r/min.

Further, the vacuum degree in step five is 700mbar.

On the other hand, the present invention provides a stem cell cryopreservation method, which method includes:

Step 1: Mix endometrial stem cells and the stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of the endometrial stem cells in the cooling system is 7×105/mL;

Step 2: Use the programmed cooling device to lower the temperature to -25~-30°C at a cooling rate of -2°C/min, and to -75~-80°C at a cooling rate of -4~-6°C/min. , reduce the temperature to -110~-115°C at a cooling rate of -10~-12°C/min;

Step 3: Move into -196°C liquid nitrogen to complete freezing.

Further, the stem cell cryopreservation method includes:

Step 1: Mix endometrial stem cells and the stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of the endometrial stem cells in the cooling system is 7×105/mL;

Step 2: Use the programmed cooling device to lower the temperature to -27°C at a cooling rate of -2°C/min, to -78°C at a cooling rate of -5°C/min, and to -11°C/min. rate reduces the temperature to -114°C;

Step 3: Move into -196°C liquid nitrogen to complete freezing.

The present invention can bring the following beneficial effects:

Organisms such as rainbow trout living in low-temperature environments need to prevent their body fluids from freezing. The long-term evolutionary process has resulted in the emergence of a wide range and diverse antifreeze substances in their bodies. The flash extraction method of the present invention fully crushes fish tissue while reducing damage to its antifreeze substances, especially long-term high-temperature damage, so that the antifreeze substances have sufficient activity to pass subsequent processing; the present invention uses a static mixer The mixing method utilizes the different regional ratios of turbulent flow and laminar flow to fully mix the coarse filtration mixture and glycerin, thereby avoiding the agglomeration of antifreeze substances and reducing the loss of antifreeze substances in subsequent filtration; the centrifugal speed of the present invention retains appropriate antifreeze. While freezing substances, larger impurities are removed, improving the purity of subsequent distillation of antifreeze substances, and also reducing rejection reactions after contact with stem cells and microbial contamination caused by complex environmental components; the vacuum distillation method of the present invention reduces High temperatures destroy antifreeze substances and distill a suitable antifreeze extract.

The present invention adopts a three-stage cooling method with a unique cooling rate and temperature, and cooperates with the cryopreservation solution of the present invention, so that the cryopreservation solution can fully integrate into the internal and external environment of the cells to protect the cells, and avoid excessive damage to the cells in a low temperature state for a long time, reducing the It eliminates the damage caused by low temperature and ice crystals formed at low temperatures to the internal and external environment of stem cells, and combined with the antibacterial effect of protocatechuic acid, the anticoagulant effect of coumarin, and the antioxidant effect of quercetin, it creates conditions for repeated freezing and thawing of stem cells.

Therefore, the present invention uses the unique proportion of the antifreeze composition of the present invention in the cryopreservation solution, and cooperates with the unique stem cell cryopreservation method of the present invention to improve the freezing rate of the inner and outer crystal nuclei of endometrial stem cells and the morphology and protection of ice crystals. The intracellular environment, including enzyme activity, allows endometrial stem cells to have a higher survival rate and proliferation ability after recovery and even after repeated freezing and thawing.

Detailed ways

In order to explain the overall concept of the present invention more clearly, the overall solution of the present invention is described in detail below in the form of examples; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. ; However, it will be obvious to those skilled in the art that the present invention may be practiced without one or more of these details; in other instances, in order to avoid confusion with the present invention, some technical features that are well known in the art are Not described.

In the present invention: glycerin, protocatechuic acid, coumarin and quercetin are all purchased from Merck Chemical Technology (Shanghai) Co., Ltd., and the product numbers are G5516, PHL89766, Y0000438 and PHR1488 respectively; stem cell culture medium and endometrial stem cells Both were purchased from Wuhan Pronosai Life Technology Co., Ltd., with the product numbers of CM-H230 and CP-H230 respectively; Type III HPLC-12 antifreeze protein was purchased from Albertson Biotechnology Co., Ltd., expressed by its strain Arctic, and conforms to the protein database UniProt The standard of registration number P19614; the programmable cooling device was purchased from Jiangsu Aisike Refrigeration Technology Co., Ltd., model Cryobox-17s; the flash extractor was purchased from Beijing Jinyang Wanda Technology Co., Ltd., model JHBE-50T; the SV-type static mixer was purchased from Shanghai Chongye Industrial Co., Ltd., model SV-3.5/65; the centrifuge was purchased from Changzhou Jintan Sanhe Instrument Co., Ltd., with a power of 600W; the vacuum distiller was purchased from Zhengzhou Yarong Instrument Co., Ltd., model RE-2000E.

Unless otherwise specified, each raw material component in the following examples can be purchased through commercial channels, the experimental instruments used are all conventional laboratory experimental instruments, and the performance test methods are test methods known in the art.

The preferred implementation is as follows:

Example 1:

The antifreeze extract is prepared using the following method:

The first step: raise adult rainbow trout at 2°C for 2 weeks, collect 400ml of fish blood and 300g of fish meat;

Step 2: Put the fish blood and fish meat into the flash extractor at 2°C, rotate at 4400r/min, extract for 23 seconds and then filter through a 50-mesh filter to obtain a coarse-filtered extract;

Step 3: At 4°C, add an equal volume of glycerin to the coarse filter mixture, mix it cyclically for 66 seconds through an SV-type static mixer, and filter it with 150 mesh to obtain the fine filter extract. The void ratio of the mixer is 0.909%;

Step 4: Put the mixed solution into a centrifuge, centrifuge for 3 minutes per 5ml at a speed of 6700r/min, and take the supernatant;

Step 5: Distill the supernatant under reduced pressure at 40°C and a vacuum of 700 mbar until no liquid continues to flow out, and collect the distillate to obtain the antifreeze extract.

Use the following method to prepare stem cell cryopreservation solution:

Step 1: Prepare an antifreeze composition of 54% by weight and make up the remaining amount of stem cell culture medium; the antifreeze composition is an antifreeze extract of 33% by weight and a raw material of 3% by weight. Catechin, 2% by weight of coumarin, 6% by weight of quercetin and glycerol to make up the balance;

Step 2: Mix and stir the above components at 25°C for 20 seconds to obtain a dry cell cryopreservation solution with a volume of 200 ml.

Stem cells are cryopreserved using the following methods:

The first step: stir and mix the endometrial stem cells and stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of endometrial stem cells in the cooling system is 7×10 ⁵ /mL;

Step 2: Use the programmable cooling device, move the cooling system into each 5ml cryogenic tube provided with the programmable cooling device, and then put it into the programmable cooling device. Start the programmable cooling device to reduce the temperature to -2°C/min at a cooling rate. 27℃, reduce the temperature to -78℃ at a cooling rate of -5℃/min, and reduce the temperature to -114℃ at a cooling rate of -11℃/min;

Step 3: Move the cryopreservation tube into -196°C liquid nitrogen to complete cryopreservation.

Example 2-25:

The only difference between Example 2 and Example 1 is the proportion of the components of the dry cell cryopreservation solution. The weight portion of the antifreeze composition of Example 2 is 43%;

The only difference between Example 3 and Example 1 is the proportion of the components of the dry cell cryopreservation solution. The weight portion of the antifreeze composition of Example 3 is 58%;

The only difference between Example 4 and Example 1 is that the proportion of the components of the dry cell cryopreservation solution is different. The weight portion of the antifreeze extract in Example 4 is 30%;

The only difference between Example 5 and Example 1 is the proportion of the components of the dry cell cryopreservation solution. The weight portion of the antifreeze extract in Example 5 is 35%;

The only difference between Embodiment 6 and Embodiment 1 is that the rotation speed of the flash extractor in Embodiment 6 is 3600 r/min;

The only difference between Embodiment 7 and Embodiment 1 is that the rotation speed of the flash extractor in Embodiment 7 is 4700r/min;

The only difference between Embodiment 8 and Embodiment 1 is that the extraction time of the flash extractor in Embodiment 8 is 17 seconds;

The only difference between Embodiment 9 and Embodiment 1 is that the extraction time of the flash extractor in Embodiment 9 is 28 seconds;

The only difference between Example 10 and Example 1 is that the SV-type static mixer of Example 10 circulates for 60 seconds;

The only difference between Example 11 and Example 1 is that the SV-type static mixer of Example 11 circulates mixing for 70 seconds;

The only difference between Example 12 and Example 1 is that the centrifuge speed of Example 12 is 6200 r/min;

The only difference between Example 13 and Example 1 is that the centrifuge speed of Example 13 is 6900 r/min;

The only difference between Example 14 and Example 1 is that the vacuum degree of vacuum distillation in Example 14 is 600 mbar;

The only difference between Example 15 and Example 1 is that the vacuum degree of vacuum distillation in Example 15 is 800 mbar;

The only difference between Example 16 and Example 1 is that the stem cell cryopreservation method is different. In Example 16, the temperature is lowered to -25°C at a cooling rate of -2°C/min;

The only difference between Example 17 and Example 1 is that the stem cell cryopreservation method is different. In Example 17, the temperature is lowered to -30°C at a cooling rate of -2°C/min;

The only difference between Example 18 and Example 1 is that the stem cell cryopreservation method is different. In Example 18, the temperature is lowered to -78°C at a cooling rate of -4°C/min;

The only difference between Example 19 and Example 1 is that the stem cell cryopreservation method is different. In Example 19, the temperature is lowered to -78°C at a cooling rate of -6°C/min;

The only difference between Example 20 and Example 1 is that the stem cell cryopreservation method is different. In Example 20, the temperature is lowered to -75°C at a cooling rate of -5°C/min;

The only difference between Example 21 and Example 1 is that the stem cell cryopreservation method is different. In Example 21, the temperature is lowered to -80°C at a cooling rate of -5°C/min;

The only difference between Example 22 and Example 1 is that the stem cell cryopreservation method is different. In Example 22, the temperature is lowered to -114°C at a cooling rate of -10°C/min;

The only difference between Example 23 and Example 1 is that the stem cell cryopreservation method is different. In Example 23, the temperature is lowered to -114°C at a cooling rate of -12°C/min;

The only difference between Example 24 and Example 1 is that the stem cell cryopreservation method is different. In Example 24, the temperature is lowered to -110°C at a cooling rate of -11°C/min;

The only difference between Example 25 and Example 1 is the cryopreservation method of stem cells. In Example 25, the temperature was lowered to -115°C at a cooling rate of -11°C/min.

Comparative Example 1-3:

Comparative Example 1 used the universal serotype procedure cryopreservation solution purchased from Wuhan Punosai Life Technology Co., Ltd., product number PB180436;

Comparative Example 2 used dental pulp stem cells purchased from Wuhan Pronosai Life Technology Co., Ltd., product number CP-H231;

Comparative Example 3 used adipose mesenchymal stem cells purchased from Wuhan Pronosai Life Technology Co., Ltd., product number CP-H202.

The stem cell cryopreservation method of Comparative Examples 4-11 is the same as that of Example 1:

The only difference between Comparative Example 4 and Example 1 is that the stem cell cryopreservation solution in Comparative Example 4 is an antifreeze composition of 70% by weight and a stem cell culture medium that makes up the balance;

The only difference between Comparative Example 5 and Example 1 is that the weight portion of the antifreeze extract in the antifreeze composition of Comparative Example 5 is 45%;

The only difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, the antifreeze extract was replaced by type III HPLC-12 antifreeze protein, and protocatechuic acid, coumarin and quercetin were replaced by resveratrol;

The only difference between Comparative Example 7 and Example 1 is that the rotation speed of the flash extractor in Comparative Example 7 is 6000r/min;

The only difference between Comparative Example 8 and Example 1 is that the flash extractor extraction time of Comparative Example 8 is 60 minutes;

The only difference between Comparative Example 9 and Example 1 is that the SV-type static mixer of Comparative Example 9 circulates mixing for 100 seconds;

The only difference between Comparative Example 10 and Example 1 is that the centrifuge speed of Comparative Example 10 is 8000r/min;

The only difference between Comparative Example 11 and Example 1 is that the vacuum degree of vacuum distillation in Comparative Example 11 is 1000 mbar.

Comparative Examples 12-14 are the same as the stem cell cryopreservation solution of Example 1:

The only difference between Comparative Example 12 and Example 1 is that Comparative Example 12 lowered the temperature to -27°C at a cooling rate of -2°C/min and to -78°C at a cooling rate of -5°C/min; move into - 196℃ liquid nitrogen, complete cryopreservation;

The only difference between Comparative Example 13 and Example 1 is that Comparative Example 13 lowered the temperature to -114°C at a cooling rate of -2°C/min; moved it into -196°C liquid nitrogen to complete freezing;

The only difference between Comparative Example 14 and Example 1 is that Comparative Example 14 lowered the temperature to -25°C at a cooling rate of -2°C/min and to -100°C at a cooling rate of -5°C/min; move into - Liquid nitrogen at 196°C to complete cryopreservation.

Comparative Example 15 is as follows:

The antifreeze extract is prepared using the following method:

The first step: raise adult rainbow trout at 2°C for 2 weeks, collect 400ml of fish blood and 300g of fish meat;

Step 2: Put the fish blood and fish meat into the flash extractor at 2°C, rotate at 4400r/min, extract for 23 seconds and then filter through a 50-mesh filter to obtain a coarse-filtered extract;

Step 3: At 4°C, add an equal volume of glycerin to the coarse filter mixture, circulate it through an SV-type static mixer for 66 seconds, and filter it with 150 mesh to obtain a fine filter extract. The void ratio of the mixer is 0.88%;

Step 4: Put the mixed solution into a centrifuge, centrifuge for 3 minutes per 5ml at a speed of 6700r/min, and take the supernatant;

Step 5: Distill the supernatant under reduced pressure at 50°C and a vacuum of 700 mbar until no liquid continues to flow out, and collect the distillate to obtain the antifreeze extract.

Use the following method to prepare stem cell cryopreservation solution:

Step 1: Prepare an antifreeze composition of 54% by weight and make up the remaining amount of stem cell culture medium; the antifreeze composition is an antifreeze extract of 33% by weight and 5% of the original Catechin, 4% by weight of coumarin, 9% by weight of quercetin and glycerin to make up the balance;

Step 2: Mix and stir the above components at 25°C for 20 seconds to obtain a dry cell cryopreservation solution with a volume of 200 ml.

Stem cells are cryopreserved using the following methods:

Step one: Mix endometrial stem cells and stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of endometrial stem cells in the cooling system is 7×10 ⁶ /mL;

Step 2: Use the programmable cooling device, move the cooling system into each 5ml cryogenic tube provided with the programmable cooling device, and then put it into the programmable cooling device. Start the programmable cooling device to reduce the temperature to -2°C/min at a cooling rate. 20℃, reduce the temperature to -85℃ at a cooling rate of -8℃/min, and reduce the temperature to -100℃ at a cooling rate of -15℃/min;

Step 3: Move into -196°C liquid nitrogen to complete freezing.

Prepare 12 groups of endometrial stem cells from each of the above examples for cryopreservation for 40 days. Place cryopreservation tubes in warm water at 37°C and gently shake them for thawing and recovery within 90 seconds. Take 1 to 6 groups to test the survival rate and proliferation ability, and 7 to 12 groups. Groups were frozen again for 40 days according to the cryopreservation method of the present invention. After repeated freezing and thawing until the third recovery, the survival rate and proliferation ability of groups 7 to 12 were tested after repeated freezing and thawing.

Survival test:

Take out the cryovials from groups 1 to 3 and 7 to 9 in each example, open the lid, suck out the cell suspension with a pipette, add it to the centrifuge tube and add 10 times more stem cell culture medium, mix well; centrifuge at 1000 rpm for 5 minutes; discard the supernatant To the supernatant, add 10% calf serum culture medium purchased from Merck Chemical (Shanghai) Co., Ltd. (Cat. No. RAB1347) to resuspend the cells. Add trypan blue purchased from Wuhan Pronosei Life Technology Co., Ltd. to every 1 ml of cell suspension. Dye 2 drops of dye solution at room temperature for 4 minutes. For the stained cell material, take a drop of cell suspension and place it on a glass slide. Add a cover slip and observe under a high-power microscope; dead cells are light blue and enlarged and dull; viable cells are light blue and enlarged and dull. The cells are not stained and maintain normal shape and are shiny; count the number of live and dead cells, and calculate the cell viability according to the formula: cell viability (%) = number of unstained cells/total number of cells observed × 100%.

The survival rate test results of each example are shown in Table 1. In Table 1, the survival rate test results take the average of the results of groups 1 to 3 and the average of the results of groups 7 to 9 of each example and keep them to integers.

It can be seen from the data in Table 1 that compared with other examples, the cell survival rate of Examples 1 to 25 of the present invention is higher. The endometrial stem cells of Examples 1 to 25 of the present invention have better survival rates after recovery, especially after repeated freezing and thawing. Outstanding survival rate compared to other technical solutions.

Proliferation ability test:

Embodiment 1 of the present invention has the highest cell survival rate. In order to further screen the implementation scope and verify the superiority of the embodiment of the present invention over other examples, 4 to 6 groups and 10 to 12 groups of endometrial stem cells cryopreserved in each example were Resuscitate, add to the centrifuge tube and add 10 times more stem cell culture medium, mix well; centrifuge at 1000rpm for 5 minutes, rinse with PBS buffer with product number P3744-12PAK purchased from Merck Chemical (Shanghai) Co., Ltd. to remove the freeze. Reserve liquid. After resuspension, seed the cells in a 96-well plate at a density of 1×10 ³ cells/well, and add an appropriate amount of stem cell culture medium to allow them to grow adherently. The time for the cell proliferation rate to reach 1.3 was calculated to 100 h.

The proliferative ability test results of each example are shown in Table 2. In Table 2, the proliferation ability test results are taken as the average of the results of groups 4 to 6 and the average of the results of groups 10 to 12 of each example and are kept to the integer.

It can be seen from the data in Table 2 that compared with other examples, the time for the cell proliferation rate of Examples 1 to 25 of the present invention to reach 1.3 is shorter. After the endometrial stem cells of Examples 1 to 25 of the present invention are revived and repeatedly frozen After fusion, it has outstanding proliferative capabilities compared with other technical solutions. Furthermore, the time required for the cell proliferation rate to reach 1.3 in Example 1 of the present invention is shorter than that of other embodiments of the present invention. The endometrial stem cells of Example 1 of the present invention have outstanding proliferation ability after recovery compared with other embodiments. In summary, the best implementation mode of the present invention is Embodiment 1.

It should be noted that the present invention is a preferred experiment for human endometrial stem cells. Compared with Comparative Examples 2 and 3, the effect of the embodiments of the present invention on human endometrial stem cells is significantly better than other types of stem cells. Compared with Comparative Example 1, the present invention also solves the problem of poor cryopreservation effect of universal cryopreservation solution on human endometrial stem cells.

The above are only examples of the present invention and are not intended to limit the present invention; for those skilled in the art, the present invention may have various modifications and changes; any modifications made within the spirit and principles of the present invention Modifications, equivalent substitutions, improvements, etc. shall be included in the scope of the claims of the present invention.

Claims (9)

1. A stem cell cryopreservation solution, characterized in that the stem cell cryopreservation solution consists of the following components: The antifreeze composition is 43~58% by weight and the stem cell culture medium makes up the balance; The antifreeze composition is 30 to 35% by weight of antifreeze extract, 3% by weight of protocatechuic acid, 2% by weight of coumarin, and 6% by weight. of quercetin and glycerin to make up the balance; The extraction method of the antifreeze extract includes: Step 1: Raise adult rainbow trout at 2°C for 2 weeks, collect 400ml of fish blood and 300g of fish meat; Step 2: Put the fish blood and fish meat into the flash extractor at 2°C, rotate at 3600~4700r/min, extract for 17~28s and then filter through a 50-mesh filter to obtain a coarse-filtered extract; Step 3: At 4°C, add an equal volume of glycerin to the coarse filter mixture, mix it in a SV-type static mixer for 60 to 70 seconds, and filter it with 150 mesh to obtain a fine filter extract. The void ratio of the mixer is 0.909%; Step 4: Put the mixed solution into a centrifuge, centrifuge for 3 minutes per 5ml at a speed of 6200~6900r/min, and take the supernatant; Step 5: Distill the supernatant under reduced pressure at 40°C with a vacuum of 600~800mbar until no liquid continues to flow out, and collect the distillate to obtain the antifreeze extract. 2. The stem cell cryopreservation solution according to claim 1, wherein the weight portion of the antifreeze composition is 54%. 3. The stem cell cryopreservation solution according to claim 1, wherein the weight portion of the antifreeze extract is 33%. 4. The stem cell cryopreservation solution according to claim 1, characterized in that the flash extraction process in step 2 is 4400 r/min and the extraction takes 23 seconds. 5. The stem cell cryopreservation solution according to claim 1, characterized in that the mixing time in step three is 66 seconds. 6. The stem cell cryopreservation solution according to claim 1, wherein the centrifugal speed in step 4 is 6700 r/min. 7. The stem cell cryopreservation solution according to claim 1, wherein the vacuum degree in step five is 700 mbar. 8. A stem cell cryopreservation method using the stem cell cryopreservation solution according to any one of claims 1 to 7, characterized in that the stem cell cryopreservation method includes: Step 1: Mix endometrial stem cells and the stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of the endometrial stem cells in the cooling system is 7×10 ⁵ /mL; Step 2: Use the programmed cooling device to lower the temperature to -25~-30°C at a cooling rate of -2°C/min, and to -75~-80°C at a cooling rate of -4~-6°C/min. , reduce the temperature to -110~-115°C at a cooling rate of -10~-12°C/min; Step 3: Move into -196°C liquid nitrogen to complete freezing. 9. The stem cell cryopreservation method according to claim 8, characterized in that the stem cell cryopreservation method includes: Step 1: Mix endometrial stem cells and the stem cell cryopreservation solution at 25°C to obtain a cooling system. The final number density of the endometrial stem cells in the cooling system is 7×10 ⁵ /mL; Step 2: Use the programmed cooling device to lower the temperature to -27°C at a cooling rate of -2°C/min, to -78°C at a cooling rate of -5°C/min, and to -11°C/min. rate reduces the temperature to -114°C; Step 3: Move into -196°C liquid nitrogen to complete freezing.