DMSO-free cryopreservation solution and its preparation method and application

Abstract

The present invention discloses a DMSO-free cryopreservation solution. The cryopreservation solution contains, per 100 mL of volume, 0.01-50.0 g of a bionic ice-controlling material, 5.0-30 mL of a polyol, 1-30 g of a water-soluble sugar, and 0-30 mL of serum, with the remainder being a buffer solution. The bionic ice-controlling material is selected from PVA and / or amino acid bionic ice-controlling materials. The cryopreservation solution and cryo-equilibration solution of the present invention are DMSO-free. When used for cryopreservation of mouse oocytes and embryos, they achieve cell and tissue viability and functional expression stability equivalent to or superior to commercially available cryopreservation solutions (containing 15% DMSO), demonstrating high preservation efficiency. The DMSO- and serum-free cryopreservation solution further overcomes the problems of commercially available cryopreservation solutions currently in clinical use, such as the low stability and potential introduction of parasitic biological contaminants due to the presence of serum. The cryopreservation solution of the present invention has a simple composition, is made from readily available raw materials, is low cost, and can be widely applied to the cryopreservation of various cells.

Description

Detailed Description of the Invention

[0001] This application claims priority from two prior applications, filed with the State Intellectual Property Office of China on April 9, 2019, bearing patent application number 201910281978.2 and entitled "DMSO-free cryopreservation solution and preparation method thereof," and patent application number 201910281986.7 and entitled "Peptide compound and cryopreservation solution containing the compound." Both applications are incorporated herein by reference in their entireties.

[0002] Technical Field The present invention belongs to the technical field of biomedical materials, and specifically relates to a DMSO-free cryopreservation solution and a method for preparing the same.

[0003] Background technology Since its introduction, cryopreservation technology has become an essential research method in the natural sciences and is widely adopted. With the rise in living standards and advances in medical technology, cryopreservation of human germ cells (sperm, oocytes), gonadal tissue, and other tissues has become an important means of preserving fertility. Furthermore, as the world's population ages rapidly, the demand for cryopreserved donated human cells, tissues, or organs that can be used in regenerative medicine and organ transplantation is rapidly increasing. Therefore, how to efficiently cryopreserve precious cell, tissue, and organ resources has become an important challenge in the field of life sciences.

[0004] Currently, vitrification is the most commonly used cryopreservation method. Vitrification allows intracellular and extracellular fluids to be directly converted to a vitrified state during the rapid freezing process, thereby avoiding damage caused by ice crystal formation during the freezing process. However, conventional cryopreservation reagents are unable to effectively control ice crystal growth during the temperature recovery process, resulting in cell damage. Dimethyl sulfoxide (DMSO) is a commonly used fluxing agent and osmotic cell cryopreservation protectant for in vitro cell culture. However, DMSO has been shown to cause adverse side effects in clinical trials and is highly cytotoxic. Different cell types have different sensitivities to DMSO concentrations, leading to cellular toxicities and limiting the application of cryopreservation reagents containing DMSO as the main protectant. The high concentrations (≥15%) of DMSO commonly used in vitrification currently have serious effects on the survival rate of cryopreserved specimens after recovery, and ultimately on the safety and functional performance of progeny. In short, currently used cryopreservation reagents lack the ability to effectively control ice crystal growth during the rewarming process and suffer from high toxicity.

[0005] Summary of the Invention To improve upon the above-mentioned deficiencies of the prior art, the present invention provides a DMSO-free cryopreservation solution and a method for preparing the same.

[0006] The present invention provides the following technical solutions:

[0007] DMSO-free cryopreservation solution containing, per 100 mL, 0.01-50.0 g of bionic ice-controlling material, 5.0-45 mL of polyol, and 0.1-1 mol L of water-soluble sugar. -1 The cryopreservation solution contains 0-30 mL of serum, and the remaining volume is buffer solution. The bionic ice-controlling material is selected from polyvinyl alcohol (PVA) and / or amino acid bionic ice-controlling materials, and the cryopreservation solution is a DMSO-free cryopreservation solution that does not contain dimethyl sulfoxide (DMSO).

[0008] According to the present invention, the amino acid bionic ice-control material is selected from one or more of polyamino acids (degree of polymerization ≧2, preferred degree of polymerization is 8-40, for example, degree of polymerization is 8, 15, 20, etc.), amino acids, and peptide compounds.

[0009] According to the present invention, the peptide compound is a polypeptide (preferably a peptide consisting of 2 to 8 different amino acids, such as a dipeptide, tripeptide, or tetrapeptide), a glycopeptide derivative, or a compound represented by formula (I),

[0010] [ka]

[0011] wherein R is selected from substituted or unsubstituted alkyl groups, and the substituents may be selected from -OH, -NH2, -COOH, -CONH2, etc., e.g., R is substituted or unsubstituted C 1-6 Preferably, R is an alkyl group, -CH3, -CH2CH3, or -CH2CH2COOH, and n is an integer greater than or equal to 1 and less than or equal to 1000, such as an integer in the range of 1 to 100. In some embodiments of the invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0012] According to the present invention, the polyol may be a C2-5 polyol, preferably a C2-C3 diol or triol, such as any one of ethylene glycol, propylene glycol, and glycerol.

[0013] According to the present invention, the water-soluble saccharide may be at least one of non-reducing disaccharides, water-soluble polysaccharides, water-soluble cellulose, and glycosides, such as sucrose, trehalose, ficoll, and hydroxypropylmethylcellulose, which can protect the cell membrane and prevent cell sedimentation.

[0014] According to the present invention, the buffer may be at least one of DPBS, Hepes-buffered HTF buffer, and other cell buffers.

[0015] According to the present invention, the serum can be selected from human serum albumin or a substitute thereof, such as sodium dodecyl sulfonate (SDS), for cryopreserved objects of human origin, and fetal bovine serum or bovine serum albumin for cryopreserved objects of non-human origin. According to the cryopreservation solution of the present invention, the bionic ice-controlling material may be PVA, and the content of the PVA may be 0.1-6.0 g, for example, 0.5-5.0 g, specifically, 1.0 g, 2.0 g, 3.0 g, or 4.0 g.

[0016] According to the cryopreservation solution of the present invention, the bionic ice-controlling material may be a polyamino acid or an amino acid, and the content of the polyamino acid or amino acid may be 0.01-50 g, for example, 1.5-50 g, specifically, 8.0 g, 10 g, 15 g, 20 g, 30 g, or 40 g.

[0017] According to the cryopreservation solution of the present invention, the ice-controlling material may be a combination of PVA and polyamino acid, for example, 0.1-5.0 g of PVA and 1.0-9.0 g of polyamino acid.

[0018] According to the cryopreservation solution of the present invention, the ice-controlling material may be a combination of PVA and amino acid, for example, 0.1-5.0 g of PVA and 8.0-35 g of amino acid.

[0019] According to the cryopreservation solution of the present invention, the content of the polyol is 6.0-28 mL, for example, 7.0-20 mL, or 10-15 mL, calculated per 100 mL.

[0020] According to the cryopreservation solution of the present invention, the content of the serum is 0.1-30 mL, for example, 5.0-20 mL, 10-15 mL, calculated per 100 mL.

[0021] According to the cryopreservation solution of the present invention, the content of serum in the cryopreservation solution is preferably 0 when calculated per 100 mL.

[0022] According to the cryopreservation solution of the present invention, the content of the water-soluble saccharides in 100 mL of the cryopreservation solution is 0.1-1.0 mol L -1 , e.g., 0.1-0.8 mol L -1 , 0.2-0.6 mol L -1 , specifically, for example, 0.25 mol L -1 , 0.5 mol L -1 , 1.0 mol L -1 is.

[0023] The cryopreservation solution according to the present invention has a pH of 6.5-7.6, for example 6.9-7.2.

[0024] In one embodiment of the present invention, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0025] PVA 0.01-6.0 g Polyol 5.0-45 mL Serum 0.1-30 mL Water-soluble sugars 0.1-1.0 mol L -1 Buffer remaining.

[0026] Preferably, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0027] PVA 1.0-6.0 g Ethylene glycol 5-30 mL Serum 0.1-20 mL Sucrose 0.2-0.8 mol L-1 DPBS remaining.

[0028] In one embodiment of the present invention, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0029] PVA 1.0-5.0 g Polyol 10-45 mL Water-soluble sugars 0.1-1.0 mol L -1 Buffer remaining.

[0030] Preferably, it consists of the following components calculated by volume per 100 mL:

[0031] PVA 1.0-5.0 g Ethylene glycol 10-30 mL Sucrose 0.2-0.8 mol L -1 DPBS remaining.

[0032] In one embodiment of the present invention, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0033] Amino acids 2.0-50 g PVA 0.1-6 g Polyol 10-30 ml Water-soluble sugars 0.1-1.0 mol L -1 Serum 10-20 ml Buffer remaining.

[0034] Preferably, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0035] L-Arg 5.0-18 g L-Thr 3.0-12 g PVA 1.0-6.0 g 10-20 ml of ethylene glycol Sucrose 0.2-0.8 mol L -1 Serum 10-20ml DPBS remaining.

[0036] In one embodiment of the present invention, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0037] Polyamino acids 0.1-9.0 g PVA 0.01-6.0 g Polyol 10-30ml Water-soluble sugars 0.1-1.0 mol L -1 Buffer remaining.

[0038] Preferably, the cryopreservation solution comprises the following components calculated by volume per 100 mL:

[0039] Polyproline or polyarginine 0.1-5.0 g PVA 1.0-6.0 g 10-20ml ethylene glycol Sucrose 0.2-0.8 mol L -1 DPBS remaining.

[0040] The present invention further provides a method for preparing the cryopreservation solution, which includes the steps of dissolving a bionic ice-control material in DPBS, adjusting the pH after cooling to room temperature, dissolving other components other than serum in the remaining DPBS, mixing after cooling, checking or adjusting the pH again, supplementing the remaining amount of buffer, and adding serum when in use.

[0041] According to the preparation method of the present invention, (1) dissolving PVA in a portion of a buffer solution, cooling to room temperature, and then adjusting the pH to obtain solution 1; (2) optionally dissolving the polyamino acid or amino acid in a portion of a buffer solution and adjusting the pH after cooling to room temperature to form solution 2; (3) dissolving the water-soluble saccharide in another portion of the buffer solution, and after all the water-soluble saccharide is dissolved, adding other components other than serum to obtain solution 3; (4) Mixing Solution 1, optional Solution 2, and Solution 3 after they have cooled to room temperature, adjusting the pH, and supplementing the remaining amount of buffer to a predetermined volume to obtain the cryopreservation solution.

[0042] According to the preparation method of the present invention, (1) dissolving a polyamino acid or an amino acid in a buffer solution, cooling to room temperature, and then adjusting the pH to form a solution 1; (2) optionally dissolving PVA in a portion of the buffer solution and adjusting the pH after cooling to room temperature to obtain solution 2; (3) dissolving the water-soluble saccharide in another portion of the buffer solution, and after all the water-soluble saccharide is dissolved, adding other components other than serum to obtain solution 3; (4) Mixing Solution 1, optional Solution 2, and Solution 3 after they have cooled to room temperature, adjusting the pH, and supplementing the remaining amount of buffer to a predetermined volume to obtain the cryopreservation solution.

[0043] According to the preparation method of the present invention, when the cryopreservation solution contains serum, the serum is added when the cryopreservation solution is used.

[0044] According to the preparation method of the present invention, in the step (1), PVA is dissolved by heating in a warm bath such as an oil bath or a water bath, for example, the temperature of the water bath is 60-95° C., preferably 80° C. In the step (1), the dissolving includes a stirring step.

[0045] According to the preparation method of the present invention, in step (2), the dissolution is ultrasonically assisted dissolution.

[0046] The volume per 100 mL is calculated as DMSO-free frozen equilibration solution containing 0-5.0 g of PVA, 5.0-45 mL of polyol, 0-30 mL of serum, and the remaining amount of buffer.

[0047] According to the freezing equilibrium solution of the present invention, the content of the PVA is 0.1-5.0 g, for example, 0.1 g, 0.5 g, 1.0 g, or 2.0 g.

[0048] According to the freezing equilibration solution of the present invention, the content of the polyol is 6.0-28 mL, for example, 7.0-20 mL, 10-15 mL.

[0049] According to the freezing equilibration solution of the present invention, the content of the serum is 0.1-30 mL, for example, 5.0-20 mL, 10-15 mL. In one embodiment of the present invention, the content of the serum is 0 mL.

[0050] In one embodiment of the present invention, the freezing equilibration solution contains, in terms of volume per 100 mL, 7.5-15 mL of polyol, 10-20 mL of serum, and the remaining amount of DPBS.

[0051] In one embodiment of the present invention, the freezing equilibration solution contains, calculated by volume per 100 mL, 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and the remaining amount of buffer solution.

[0052] In the freezing equilibration solution of the present invention, the PVA, polyol and serum can be selected from the corresponding components of the cryopreservation solution.

[0053] The present invention further provides a method for preparing the above-mentioned frozen equilibration solution, which comprises dissolving each component in a buffer solution, storing the serum separately, and adding the serum at the time of use.

[0054] The cryopreservation reagent is a DMSO-free reagent containing the above-mentioned freezing equilibrium solution and the above-mentioned freezing preservation solution, wherein the freezing equilibrium solution and the freezing preservation solution exist independently.

[0055] According to the cryopreservation reagent of the present invention, the serum content of the cryopreservation solution is 0, and the freezing equilibrium solution contains, calculated by volume per 100 mL, 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and the remaining amount of buffer solution.

[0056] According to the cryopreservation reagent of the present invention, the freezing equilibrium solution contains the following components when calculated by volume per 100 mL:

[0057] PVA 0-5.0 g Polyamino acids 0-15 g Polyol 5.0-45 mL Serum 0-30 mL Buffer remaining.

[0058] The cryopreservation solution contains the following components, calculated based on a total volume of 100 mL:

[0059] PVA 0.01-6.0 g Amino acids or polyamino acids 0-50 g Polyol 5.0-45 mL Serum 0-30 mL Water-soluble sugars 0.1-1.0 mol L -1 Buffer remaining.

[0060] According to the present invention, the PVA is isotactic PVA, syndiotactic PVA and Atactic The PVA may be selected from one or a combination of two or more PVAs, for example, the syndiotacticity of the PVA is 15%-60%, preferably 45%-60%, for example 50%-55%.

[0061] According to the present invention, the PVA may be selected from PVAs with a molecular weight of 10-500 kDa or higher, such as 10-30 kDa, 30-50 kDa, 80-90 kDa, 200-500 kDa. According to the present invention, the PVA may be selected from PVAs with a degree of hydrolysis higher than 80%, such as PVAs with a degree of hydrolysis of 80%-99%, 82-87%, 87%-89%, 89%-99%, 98%-99%.

[0062] According to the present invention, the polyamino acid may be selected from homopolymers (degree of polymerization ≧2) of at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, and the like.

[0063] According to the present invention, the peptide compound is a polypeptide consisting of two or more amino acids, which may be selected from one or more of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), and L-Ala-L-Ala-L-Thr (AAT). These polypeptides can be synthesized by polypeptide synthesis methods known in the art, such as solid-phase synthesis.

[0064] According to the present invention, the glycopeptide derivatives are synthesized from sugars and amino acids, such as molecules formed by chemical bonds between glucolactone (GDL) and glycophilic amino acids, such as GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Thr, etc. The glycopeptide compounds can be prepared by methods known in the art for reacting sugars and amino acids, such as solid-phase synthesis or by reacting sugars and amino acids in an organic solvent.

[0065] According to the present invention, the peptide compound has any one of the structures shown in formula (1)-formula (8).

[0066] [ka]

[0067] JPEG0007748063000003.jpg218169

[0068] According to the present invention, the compound represented by formula (I) has any one of the structures shown below.

[0069] [ka]

[0070] According to the present invention, the compound represented by the formula (9) is prepared by the following synthetic route.

[0071] [ka]

[0072] In the cryopreservation solution and the cryo-equilibration solution according to the present invention, the amount of each component used is based on the total volume of the solution of 100 mL, with the remainder being the buffer solution.

[0073] The present invention further provides the application of the above cryopreservation solution in the cryopreservation of various cells, organs and tissues, including the cryopreservation of oocytes, embryos, various stem cells, organs and tissues, including but not limited to ovarian organs and tissues.

[0074] The present invention further provides a method for freezing and recovering cells or embryos, comprising the steps of:

[0075] (1) Cells or embryos are placed in the cryopreservation solution of the present invention to prepare a cell suspension, which is then frozen.

[0076] (2) The frozen cells or embryos are placed in a thawing solution and thawed to recover them.

[0077] In the freezing and recovery method of the present invention, the cells or embryos are first equilibrated in an equilibration solution before being placed in a cryopreservation solution.

[0078] The present invention further provides a method for cryopreserving stem cells, which employs the microdroplet method, for example, the method for cryopreserving stem cells includes the following steps: adding a cryopreservation solution to stem cells, dispersing them by spraying, preparing a stem cell suspension, placing the stem cell suspension on a freezing carrier sheet, and cryopreserving it in liquid nitrogen (-196°C).

[0079] According to an embodiment of the present invention, thawing cryopreserved stem cells involves placing the frozen carrier sheet on which the stem cells are placed in α-MEM medium and thawing at 37°C.

[0080] According to an embodiment of the present invention, the stem cells are various stem cells having differentiation functions known in the art, such as totipotent stem cells, pluripotent stem cells, or unipotent stem cells, including, but not limited to, embryonic stem cells, various mesenchymal stem cells (e.g., umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.), hematopoietic stem cells, etc.

[0081] The present invention further provides a method for cryopreserving organs and / or tissues, comprising the steps of: equilibrating the organs and / or tissues in a freezing equilibration solution, placing the organs and / or tissues in the cryopreservation solution, and then placing the organs and / or tissues on a freezing carrier sheet and cryopreserving them in liquid nitrogen.

[0082] In one embodiment, the organ and / or tissue is ovarian tissue or an ovarian organ, which may be an ovarian tissue section or a complete ovarian tissue.

[0083] In the present invention, "cryopreservation" and "cryopreservation" have the same meaning and can be used interchangeably, and refer to the preservation of a substance, cell, tissue, or organ at a low temperature so that it retains its original physicochemical and / or biological activity and physiological and biochemical functions.

[0084] In the present invention, the type of "stem cells" is not particularly limited, but the cryopreservation solution of the present invention can be used for cryopreserving various stem cells in this field, including, but not limited to, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, hematopoietic stem cells, etc.

[0085] In the present invention, biological tissue can be derived from animals, including warm-blooded mammals such as humans and primates; birds; domestic or farm animals such as cats, dogs, sheep, goats, cows, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; reptiles; zoo animals; wild animals, etc.

[0086] Beneficial effects The cryopreservation solution and cryo-equilibration solution provided by the present invention are DMSO-free. When used to cryopreserve mouse oocytes and embryos, they achieve cell and tissue viability and functional expression stability equivalent to or superior to commercially available cryopreservation solutions (containing 15% DMSO by volume), demonstrating high preservation efficiency. The DMSO- and serum-free cryopreservation solution further overcomes the problems of commercially available cryopreservation solutions currently commonly used in clinical practice, such as the low stability and susceptibility to the introduction of parasitic biological contaminants due to the presence of serum. The cryopreservation solution of the present invention has a simple composition, is made from readily available raw materials, and is inexpensive, making it widely applicable to the cryopreservation of oocytes, cell-like cells (e.g., embryos), stem cells, tissues, and organs.

[0087] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Images of stained sections of fresh (not frozen) ovarian organs from 3-day-old mice.

[0088] [Figure 2] Images of stained sections of the cryopreserved intact ovarian organs of Comparative Example 7 after thawing.

[0089] [Figure 3] Images of stained sections of the cryopreserved complete ovarian organs of Application Example 14 after thawing.

[0090] [Figure 4] A stained section image of the cryopreserved complete ovarian organ of Application Example 15 after thawing.

[0091] [Figure 5] Images of stained sections of cryopreserved ovarian organs from Application Example 16 after thawing.

[0092] [Figure 6] Images of stained sections of fresh (not frozen) ovarian tissue from sexually mature mice.

[0093] [Figure 7] Images of stained sections of cryopreserved ovarian tissue sections from Comparative Example 8 after thawing.

[0094] [Figure 8] A stained image of a section of ovarian tissue that has been cryopreserved in Application Example 17 after thawing.

[0095] [Figure 9] A stained image of a section of ovarian tissue that has been cryopreserved in Application Example 18 after thawing.

[0096] [Figure 10] A stained image of a section of ovarian tissue that has been cryopreserved in Application Example 19 after thawing.

[0097] MODE FOR CARRYING OUT THE INVENTION The preparation method of the present invention will be described in more detail below with reference to specific examples.It should be understood that the following examples are merely intended to illustrate and interpret the present invention, and should not be interpreted as limiting the scope of the claims of the present invention.Any technology realized based on the above content of the present invention is included in the scope of the claims of the present invention.

[0098] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents, materials, etc. used in the following examples are commercially available.

[0099] The PVA used in the examples of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23 kDa, and a degree of hydrolysis of 98%.

[0100] In the present embodiment, the poly-L-proline used in the freezing solution has a degree of polymerization of 8 or 15 and a molecular weight of 795 or 1475, and the poly-L-arginine has a degree of polymerization of 8 and a molecular weight of 1267. The poly-L-proline in the melting solution has a degree of polymerization of 8 and a molecular weight of 795. In the examples of the present invention, the survival rate is the average survival rate of 3-12 repeated experiments.

[0101] Example 1. Cryopreservation of mouse oocytes and embryos 1. Preparation of cryopreservation solution: Prepare cryopreservation solution according to the following formula: Cryopreservation solution A: Contains the following ingredients per 100 ml.

[0102] [Table 1]

[0103] 2.0 g of PVA was heated in a water bath at 80 °C and dissolved in 25 mL of DPBS by magnetic stirring and adjusted to pH 7.0 to give solution 1. 1.5 g of poly-L-proline was dissolved in another 20 mL of DPBS by sonication and adjusted to pH 7.0 to give solution 2. 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5 mol L−1) was added. -1 ) was dissolved in 25 mL of DPBS by ultrasonication. After all the sucrose was dissolved, 10 mL of ethylene glycol was added in order to obtain solution 3. After solutions 1, 2, and 3 were returned to room temperature, the three solutions were mixed uniformly, the pH was adjusted to 7.0, and the volume was adjusted to 100 mL with DPBS to make up the remaining amount, and the solution was then ready for use.

[0104] Cryopreservation solution B: Contains the following ingredients per 100 ml.

[0105] [Table 2]

[0106] Preparation of cryopreservation solutions: 2.0 g of PVA was heated in a water bath at 80 °C and dissolved in 20 mL of DPBS by magnetic stirring. The pH was adjusted to 7.1 to form solution 1. 8.0 g of L-Arg and 4.0 g of L-Thr were dissolved in 20 mL of DPBS and the pH was adjusted to 7.1 to form solution 2. 17 g (0.05 mol) of sucrose was added (the final concentration of sucrose in the cryopreservation solution was 0.5 mol L). -1 ) was dissolved in 20 mL of DPBS by ultrasonication. After all the sucrose was dissolved, 10 mL of ethylene glycol was added to make solution 3. After solutions 1, 2, and 3 were returned to room temperature, the three solutions were mixed uniformly, the pH was adjusted to 7.1, and the volume was adjusted to 80% of the total volume with DPBS to make up the remaining volume. 20 mL of serum was added at the time of use.

[0107] Cryopreservation solution C: Contains the following ingredients per 100 ml.

[0108] [Table 3]

[0109] 2.0 g of PVA was heated in a water bath at 80 °C and dissolved in 25 mL of DPBS by magnetic stirring. The pH was adjusted to 6.9 to give solution 1, and 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution was 0.5 mol L−1). -1 ) was dissolved in 25 mL of DPBS by ultrasonication. After all the sucrose was dissolved, 10 mL of ethylene glycol was added to make solution 2. After solution 1 and solution 2 were returned to room temperature, the two solutions were mixed uniformly, the pH was adjusted, and the remaining volume was adjusted to 80% of the total volume. 20 mL of serum was stored separately and added to the storage solution when it was used.

[0110] Cryopreservation solution D: Contains the following ingredients per 100 ml.

[0111] [Table 4]

[0112] 2.0 g of PVA was heated in a water bath at 80 °C and dissolved in 30 mL of DPBS by magnetic stirring. The pH was adjusted to 7.0 to give solution 1, and 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution was 0.5 mol L−1). -1 ) was dissolved in 25 mL of DPBS by ultrasonication. After all the sucrose was dissolved, 10 mL of ethylene glycol was added to make solution 2. After solution 1 and solution 2 were returned to room temperature, the two solutions were mixed uniformly, the pH was adjusted, and the total volume was adjusted to 100 mL, and the remaining amount was supplemented. It was then ready for use.

[0113] 2. Preparation of freezing equilibration solution: Prepare freezing equilibration solution according to the following formula: Freezing equilibration solution a: Heat 2.0 g of PVA in an 80°C water bath and dissolve in 50 mL of DPBS using magnetic stirring. Once the PVA is completely dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix evenly, and make up to 100 mL with DPBS. Ready for use.

[0114] Freezing equilibration solution b: For a total volume of 100 mL, dissolve 7.5 mL of ethylene glycol in 72.5 mL of DPBS, mix evenly, and add 20 mL of serum at the time of use.

[0115] Comparative Example 1: Freezing equilibration solution 1#: Contains 7.5% (v / v) DMSO, 7.5% (v / v) ethylene glycol, and 20% (v / v) fetal bovine serum per mL, with the remainder being DPBS.

[0116] Cryopreservation solution 1#: Contains 15% (v / v) DMSO, 15% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and 0.5 M sucrose per mL, with the remainder being DPBS.

[0117] Freezing equilibration solution b: Contains 7.5% (v / v) ethylene glycol and 20% (v / v) fetal bovine serum per mL, with the remainder being DPBS.

[0118] Cryopreservation solution 2#: Contains 10% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and 0.5 M sucrose per mL, with the remainder being DPBS.

[0119] The melt formulations used in Example 1 of the present invention and Comparative Example 1 include the following three types.

[0120] Melt 1#: Melt I (1.0 mol L -1 of sucrose, 20% serum, and the remainder is DPBS), melting solution II (0.5 mol L -1 of sucrose, 20% serum, and the balance is DPBS), melting solution III (0.25 mol L -1 sucrose, 20% serum, the remainder is DPBS), and thaw solution IV (20% serum, the remainder is DPBS).

[0121] Melt 2#: Melt I (1.0 mol L -1 of sucrose, 20 mg mL -1 of PVA, the balance being DPBS), melting solution II (0.5 mol L -1 of sucrose, 20 mg mL -1 of PVA, the balance being DPBS), melting solution III (0.25 mol L -1 of sucrose, 20 mg mL -1 PVA, the remaining volume is DPBS), melting solution IV (20 mg mL -1 PVA, the remainder is DPBS).

[0122] Melt 3#: Melt I (1.0 mol L -1 of sucrose, 20 mg mL -1 PVA, 10 mg mL -1 of polyproline, the balance being DPBS), melting solution II (0.5 mol L-1 of sucrose, 20 mg mL -1 of PVA, 5.0 mg mL -1 of polyproline, the balance being DPBS), melting solution III (0.25 mol L -1 of sucrose, 20 mg mL -1 of PVA, 2.5 mg mL -1 of polyproline, the remaining volume is DPBS), melting solution IV (20 mg mL -1 PVA and the remainder DPBS).

[0123] Application example 1: Using the freezing equilibration solution and cryopreservation solution of the above examples and comparative examples, oocytes and embryos are cryopreserved according to the methods in Tables 1 and 2, respectively.

[0124] 1. Oocyte Cryopreservation First, mouse oocytes were placed in a cryopreservation solution and allowed to equilibrate for 5 minutes. They were then placed in the prepared cryopreservation solution for 1 minute. After equilibration in the cryopreservation solution, the oocytes were placed in a cryopreservation carrier lever and then quickly placed in liquid nitrogen (-196°C). The carrier lever was sealed and subsequently stored. Upon thawing, the cryopreserved oocytes were placed in thawing solution I at 37°C and allowed to equilibrate for 5 minutes. They were then sequentially equilibrated in thawing solutions II-IV for 3 minutes each. After thawing, the oocytes were cultured for 2 hours, and the number of surviving cells was counted and the survival rate was calculated (see Table 1).

[0125] 2. Embryo cryopreservation First, mouse embryos were placed in a freezing equilibration solution for 5 minutes, then placed in the prepared cryopreservation solution for 50 seconds. After equilibration in the freezing solution, the embryos were placed in a freezing carrier lever and then quickly placed in liquid nitrogen (-196°C). The carrier lever was sealed and subsequently stored. Upon thawing, the frozen embryos were placed in thawing solution I at 37°C for 3 minutes, and then equilibrated in thawing solutions II-IV for 3 minutes each. After thawing, the embryos were cultured for 2 hours, after which the number of surviving embryos was counted and the survival rate was calculated (see Table 2).

[0126] [Table 5]

[0127] [Table 6]

[0128] As can be seen from the data in Example 1 and Comparative Example 1, the DMSO-free cryopreservation solution and freezing equilibration solution of the present invention exhibit excellent cryopreservation effects for oocytes and embryos even without the addition of DMSO due to the synergistic effects of each component, thereby overcoming the drawback of conventional cryopreservation solutions, which contain high concentrations of DMSO and are toxic to cells or embryos. Furthermore, as can be seen from Application Examples 2, 6-8, even without the addition of serum and DMSO to the equilibration solution, freezing solution, and thawing solution, the synergistic effects of the bionic ice-control material and the penetrating protectant ethylene glycol of the present invention enable a superior survival rate for cryopreservation of oocytes and embryos compared to conventional commercially available cryopreservation solutions, further resolving the problems of commercially available cryopreservation solutions currently commonly used in clinical practice, such as the short storage period and susceptibility to the introduction of parasitic biological contaminants due to the presence of serum.

[0129] Example 2: Cryopreservation of human umbilical cord mesenchymal stem cells 1. Preparation of cryopreservation solution: Prepare a cryopreservation solution with the following ingredients: Cryopreservation solution E: In a total volume of 100 mL, 10 mL of ethylene glycol, 20 mL of serum, and 17 g of sucrose (0.5 mol L−1) were added. -1 ), 4.0 g of poly-L-arginine (degree of polymerization: 8), 1.0 g of PVA, and the remaining amount of DPBS.

[0130] Cryopreservation solution F: In a total volume of 100 mL, 20 mL of ethylene glycol, 20 mL of serum, and 17 g of sucrose (0.5 mol L−1) were added. -1 ), L-Arg 16 g, L-Thr 8.0 g and the balance DPBS.

[0131] Cryopreservation solution G: In a total volume of 100 mL, 10 mL of ethylene glycol, 20 mL of serum, and 17 g of sucrose (0.5 mol L−1) were added. -1 ), 2.0 g of PVA and the remaining amount of DPBS.

[0132] Cryopreservation solution H: In a total volume of 100 mL, 10 mL of ethylene glycol, 20 mL of serum, and 17 g of sucrose (0.5 mol L−1) were added. -1 ), 28 g of TR and the balance of DPBS.

[0133] Cryopreservation solution I: In a total volume of 100 mL, 10 mL of ethylene glycol, 17 g of sucrose (0.5 mol L−1) -1 ), 2.0 g of PVA and the remaining amount of DPBS.

[0134] The method for preparing the cryopreservation solution was the same as in Example 1.

[0135] Here, the preparation method of TR is as follows.

[0136] (1) Place the 2-chlorotrityl chloride resin in a reaction tube and add DCM (20 mL g -1 Add 100 ml of ...

[0137] (2) Remove the solvent DMF and add 20% piperidine / DMF solution (10 mL g -1 ) was added, and after 5 min the solvent was removed, followed by the addition of 20% piperidine / DMF solution (10 mL g -1 After 15 minutes, remove the piperidine solution. Take a small amount of resin, wash it three times with ethanol, add ninhydrin reagent, and heat at 105-110°C for 5 minutes. A dark blue color indicates a positive reaction.

[0138] (3) The product obtained in the above reaction was dissolved in DMF (15 mL g-1 , twice), methanol (15 mL g -1 , twice) and DMF (15 mL g -1 After washing with 100 mL of HCl (100 mL, 100 mL, 100 mL, 100 mL), Fmoc-Arg(Pbf)-OH dissolved in as little DMF as possible was added to the reaction tube. A two-fold excess of Fmoc-Arg(Pbf)-OH and a two-fold excess of HBTU were then added. An eight-fold excess of DIEA was then added immediately, and the reaction was allowed to proceed for 30 minutes.

[0139] (4) After extracting the solution, take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, and heat it at 105-110°C for 5 minutes. When it becomes colorless, it is a negative reaction, that is, the reaction is complete.

[0140] (5) The product obtained in the above reaction was dissolved in DMF (15 mL g -1 , twice), methanol (15 mL g -1 , twice) and DMF (15 mL g -1 After washing with 20% piperidine / DMF solution (10 mL g -1 ) was added, and after 5 min the solvent was removed, followed by the addition of 20% piperidine / DMF solution (10 mL g -1 After 15 minutes, remove the piperidine solution, take a small amount of resin, wash with ethanol, add ninhydrin reagent, and heat at 105-110°C for 5 minutes. A positive reaction occurs when the resin turns dark blue.

[0141] (6) The product obtained in the above reaction was dissolved in DMF (15 mL g -1 , twice), methanol (15 mL g -1 , twice) and DCM (15 mL g -1 After washing with 100 mL of HCl (1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 1x, 2 ...

[0142] (7) Cutting solution (15 mL g -1 The product is cleaved with TFA:water:EDT:TiS=95:1:2:2, V / V) for 90 minutes. The cleavage solution is dried with nitrogen gas and then lyophilized to obtain a crude polypeptide product. (8) The polypeptide was purified by HPLC to transfer salts or desalt. HPLC: tR = 4.8 min (purification column model: Kromasil 100-5C18, 4.6 mm x 250 mm, gradient elution: 0.1% TFA in acetonitrile and 0.1% TFA in water, 1:99 for 0 min, 1:4 for 20 min). The purified solution was lyophilized to obtain the final product L-Thr-L-Arg(TR). The yield was approximately 80%. Mass spectrometry identification: 276.2 [M+H] + is.

[0143] Comparative Example 2: Cryopreservation solution 3#: Contains 10% (v / v) DMSO and 15% (v / v) fetal bovine serum per mL, with the remainder being α-MEM medium (USA, Invitrogen, C12571500BT).

[0144] Using the above cryopreservation solution, human umbilical cord mesenchymal stem cells were cryopreserved according to the method in Table 3. Specifically, the cryopreservation method for human umbilical cord stem cells was performed by digesting human umbilical cord mesenchymal stem cells in a culture dish with 25% pancreatin for 2 minutes, adding an equal volume of culture medium (10% FBS + α-MEM medium), gently tapping until all the stem cells had settled, transferring the cells to a 1.5 ml centrifuge tube, centrifuging at 1000 rpm for 5 minutes, discarding the supernatant, separating the cells from the medium, adding 10 μL of freezing solution to the bottom of the centrifuge tube, gently tapping to disperse the stem cell clumps, and placing the 10 μL of freezing solution containing the stem cells on a freezing carrier sheet, which was then placed in liquid nitrogen (-196°C) for cryopreservation (microdroplet method). To thaw, the freezing carrier sheet containing the cells and freezing solution was placed directly in α-MEM medium at 37°C. After thawing, the viability was observed by trypan blue staining, and the cell number was counted using the JIMBIO-FIL instrument, where viability = number of live cells / total number of cells (see Table 3).

[0145] [Table 7]

[0146] When using the cryopreservation solution of the present invention to cryopreserve human umbilical cord mesenchymal stem cells, the survival rate of the stem cells can reach 92.4% without the use of DMSO (Application Example 9), and even without the addition of DMSO or serum, the survival rate can reach 77.1% (Application Example 13). This freezing reagent not only achieves the same effectiveness as conventional freezing solutions for stem cells, but also has a much higher cryopreservation recovery rate than the currently commonly used cryopreservation solution containing 10% DMSO (Comparative Example 5), demonstrating that the cryopreservation effect based on PVA is significantly superior to that of Comparative Example 6, which does not contain PVA.

[0147] Example 3: Cryopreservation of whole ovarian organs and ovarian tissue sections Cryopreservation solution J: 10 mL of ethylene glycol, 17 g of sucrose (0.5 mol L−1) in a total volume of 100 mL. -1 ), 2.0 g of PVA and the remaining amount of DPBS.

[0148] Cryopreservation solution K: In a total volume of 100 mL, 10 mL of ethylene glycol, 20 mL of serum, and 17 g of sucrose (0.5 mol L−1) were added. -1 ), 1.0 g of PVA, and the remaining amount of DPBS.

[0149] Freezing solution L: 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L) -1 ), 4.0 g of poly-L-arginine (degree of polymerization 8), 1.0 g of PVA, and the remaining amount of DPBS.

[0150] Comparative Example 3: Each mL of cryopreservation solution contained 15% (v / v) DMSO, 15% (v / v) ethylene glycol, 20% (v / v) serum, 0.5 M sucrose, and the remainder DPBS.

[0151] Freezing equilibration solution a: Heat 2.0 g of PVA in an 80°C water bath and dissolve in 50 mL of DPBS using magnetic stirring. Once the PVA is completely dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix evenly, adjust the pH, and make up the remaining volume to 100 mL. Prepare for use.

[0152] Freezing equilibration solution b: Add 7.5 mL of ethylene glycol to 72.5 mL of DPBS, mix evenly, and add 20 mL of serum at the time of use.

[0153] Comparative Example 3: Freezing equilibration solution 1#: Contains 7.5% (v / v) DMSO, 7.5% (v / v) ethylene glycol, and 20% (v / v) fetal bovine serum per mL, with the remainder being DPBS.

[0154] Cryopreservation solution 1#: 15% (v / v) DMSO, 15% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, 0.5 mol L per mL -1 of sucrose, the remainder being DPBS.

[0155] Melt 1#: Melt I (1.0 mol L -1 of sucrose, 20% serum, and the remainder is DPBS), melting solution II (0.5 mol L -1 of sucrose, 20% serum, and the balance is DPBS), melting solution III (0.25 mol L -1 sucrose, 20% serum, the remainder is DPBS), and thawing solution IV (20% serum, the remainder is DPBS).

[0156] Melt 2#: Melt I (1.0 mol L -1 of sucrose, 20 mg mL -1 of PVA, the balance being DPBS), melting solution II (0.5 mol L -1 of sucrose, 20 mg mL -1 of PVA, the balance being DPBS), melting solution III (0.25 mol L -1of sucrose, 20 mg mL -1 PVA, the remaining volume is DPBS), melting solution IV (20 mg mL -1 PVA, the remainder is DPBS).

[0157] Using the above-mentioned cryopreservation solution and the comparative freezing equilibration solution and cryopreservation solution, cryopreservation is carried out according to the methods in Tables 4 and 5 on intact ovarian organs from mice aged 3 days or less and ovarian tissue sections from sexually mature mice, respectively.

[0158] First, the intact ovarian organ or ovarian tissue slice was placed in an equilibration solution and allowed to equilibrate at room temperature for 25 minutes. Then, it was placed in the prepared cryopreservation solution for 15 minutes. The intact ovarian organ or ovarian tissue slice was then placed in a freezing carrier and stored in liquid nitrogen. After thawing, the intact ovarian organ or ovarian tissue slice was placed in a culture medium (10% FBS + α-MEM) and then placed in a 37°C, 5% CO2 incubator to recover for 2 hours. It was then fixed in 4% paraformaldehyde, embedded in paraffin, and stained with HE staining for morphology. The results are shown in Figures 1-10. Figure 1 shows a section of a fresh, unfrozen ovarian organ, and Figure 6 shows a section of a fresh, unfrozen ovarian tissue.

[0159] [Table 8]

[0160] [Table 9]

[0161] As can be seen from Figures 1-5, compared with Comparative Example 7 without the addition of amino acid bionic ice-control material and the use of fresh ovarian organs that have not been frozen, the primordial follicle structure of Examples 14-16 is relatively complete, the mesenchymal structure is relatively complete, the cytoplasm is homogenized, there are relatively more light stainings, the cell nuclei are shrunken, and there are relatively less dark stainings; the vascular wall structure is complete, there is little collapse of the vascular lumen, the endothelial cytoplasm is homogenized, there are relatively more light stainings, the cell nuclei are shrunken, and there are relatively less dark stainings. As can be seen from the above, the cryopreservation effect of the ovarian organs of Examples 14-16 is better.

[0162] As can be seen from Figures 6-10, compared to Comparative Example 8 and fresh ovarian tissue that had not been frozen, the methods of Examples 17-19 produced relatively complete antral follicle structures, relatively complete mesenchymal structures, homogenized cytoplasm, relatively more lightly stained cytoplasm, shrunken cell nuclei, and relatively less darkly stained cytoplasm. As can be seen from the above, the cryopreservation solution for cryopreserving ovarian tissue according to the present invention has superior effects to the prior art.

[0163] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention. [Brief explanation of the drawings]

[0164] [Figure 1] This is an image of a stained section of a fresh (not frozen) ovarian organ from a 3-day-old mouse. [Figure 2] 10 shows stained images of sections of the cryopreserved intact ovarian organs of Comparative Example 7 after thawing. [Figure 3] 10 shows stained images of sections of cryopreserved intact ovarian organs of Application Example 14 after thawing. [Figure 4] 10 shows stained images of sections of cryopreserved intact ovarian organs after thawing in Application Example 15. [Figure 5] 13 is a stained image of a section of the cryopreserved ovarian organ of Application Example 16 after thawing. [Figure 6]This is an image of a stained section of fresh (not frozen) ovarian tissue from a sexually mature mouse. [Figure 7] 13 shows stained images of cryopreserved ovarian tissue sections of Comparative Example 8 after thawing. [Figure 8] 13 is a stained image of a section of ovarian tissue that has been cryopreserved in Application Example 17 and has been thawed. [Figure 9] 13 is a stained image of a section of ovarian tissue that has been cryopreserved in Application Example 18 and has been thawed. [Figure 10] 13 is a stained image of a section of ovarian tissue that has been cryopreserved in Application Example 19 and has been thawed.

Claims

1. The volume per 100 mL is calculated as follows: 0.01-50 g of bionic ice control material, 5.0-45 mL of polyol, and 0.1-1.0 mol L of water-soluble sugar. -1 , containing 0-30 mL of serum, the remaining volume being buffer solution, wherein the bionic ice-controlling material is selected from atactic PVA having a syndiotacticity of 15%-60%, a molecular weight of 10-500 kDa, and a degree of hydrolysis of more than 80%, or a combination of the atactic PVA and an amino acid ice-controlling material; The amino acid ice-controlling material is selected from one or more of polyamino acids, amino acids and peptide compounds having a degree of polymerization of ≥ 2; The peptide compound is a polypeptide, a glycopeptide derivative, or a compound represented by formula (I), 【Chemical 1】 wherein R is selected from substituted or unsubstituted alkyl groups, and the substituents are -OH, -NH 2 , -COOH, -CONH 2 where n is an integer greater than or equal to 1 and less than or equal to 1000; the polypeptide is selected from one or more of L-Thr-L-Arg, L-Thr-L-Pro, L-Arg-L-Thr, L-Pro-L-Thr, L-Thr-L-Arg-L-Thr, L-Thr-L-Pro-L-Thr and L-Ala-L-Ala-L-Thr; A DMSO-free cryopreservation solution, characterized in that the glycopeptide derivative is at least one of GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val and GDL-L-Ser.

2. The cryopreservation solution of claim 1, wherein R is a substituted or unsubstituted C1-6 alkyl group.

3. R is -CH 3 , -CH 2 CH 3 or -CH 2 CH 2 The cryopreservation solution according to claim 2, characterized in that it is COOH.

4. The content of the atactic PVA is 0.1-6.0 g; The polyol is C 2-5 is a polyol of the water-soluble saccharide is at least one of a non-reducing disaccharide, a water-soluble polysaccharide, and a glycoside; the buffer is at least one of DPBS, Hepes-buffered HTF buffer and other cell buffers; The cryopreservation solution according to any one of claims 1 to 3, wherein the serum is selected from human serum albumin or a substitute thereof for cryopreserved objects of human origin, and from fetal bovine serum or bovine serum albumin for cryopreserved objects of non-human origin.

5. The cryopreservation solution according to claim 4, wherein the polyol is any one of ethylene glycol, propylene glycol, and glycerol.

6. The cryopreservation solution according to claim 4, wherein the water-soluble saccharide is selected from the group consisting of sucrose, water-soluble cellulose, trehalose, and ficoll.

7. The cryopreservation solution according to claim 6, wherein the water-soluble cellulose is hydroxypropyl methylcellulose.

8. The cryopreservation solution according to claim 4, wherein the substitute is sodium dodecyl sulfonate.

9. A freezing preservation solution described in any one of claims 1 to 8, characterized in that the content of the bionic ice control material is 0.01-50g.

10. The cryopreservation solution of claim 9, wherein the bionic ice-controlling material is a combination of the atactic PVA and the amino acid and / or the polyamino acid.

11. The cryopreservation solution of claim 10, characterized in that the bionic ice control material consists of 0.1-5.0 g of the atactic PVA and 8.0-35 g of the amino acid and / or 1.0-9.0 g of the polyamino acid.

12. The atactic PVA has a syndiotacticity of 50%-60%; the polyamino acid is selected from homopolymers of at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, and glycine; The cryopreservation solution according to any one of claims 1 to 11, wherein the homopolymer has a degree of polymerization of ≧2.

13. The cryopreservation solution according to any one of claims 1 to 12, characterized in that the content of the polyol is 6.0-28 mL.

14. The cryopreservation solution according to claim 13, characterized in that the serum content is 0.

15. The cryopreservation solution according to claim 13, characterized in that the content of the water-soluble sugars is 0.1-1.0 mol L-1.

16. The cryopreservation solution according to claim 13, characterized in that the pH of the cryopreservation solution is 6.5-7.

6.

17. A method for preparing a cryopreservation solution described in any one of claims 1 to 16, characterized in that it includes the steps of dissolving the bionic ice control material in a buffer solution, cooling to room temperature, adjusting the pH, and dissolving other components in the remaining buffer solution, cooling, and mixing.

18. The preparation method includes the steps of: (1) dissolving the atactic PVA in a portion of a buffer solution, cooling the solution to room temperature, and then adjusting the pH to obtain solution 1; (2) optionally dissolving the polyamino acid or the amino acid in a buffer solution, and adjusting the pH after cooling to room temperature to form solution 2; (3) dissolving the water-soluble saccharide in a portion of the buffer solution, and after all of the water-soluble saccharide is dissolved, adding other components other than serum to obtain a solution 3; (4) mixing Solution 1, optional Solution 2, and Solution 3 after they have cooled to room temperature, adjusting the pH, and adjusting the volume to a predetermined volume with a buffer to obtain the cryopreservation solution; 18. The method for preparing a cryopreservation solution according to claim 17, wherein optionally, when the cryopreservation solution contains serum, the serum is added when the cryopreservation solution is used.

19. Calculated per 100 mL, it contains 0.1-5.0 g of atactic PVA, 5.0-45 mL of polyol, 0-30 mL of serum, and the remaining amount of buffer solution. The atactic PVA is a DMSO-free frozen equilibration solution characterized by a syndiotacticity of 15%-60%, a molecular weight of 10-500 kDa, and a degree of hydrolysis of more than 80%.

20. 20. The DMSO-free freezing equilibration solution of claim 19, wherein the content of atactic PVA is 0.1-4.0 g.

21. The freezing equilibration solution according to claim 19 or 20, characterized in that, per 100 mL, the freezing equilibration solution contains 7.5-15 mL of the polyol, 10-20 mL of serum, and the remaining amount of buffer solution.

22. The freeze-equilibrating solution according to claim 21, characterized in that, per 100 mL, the freeze-equilibrating solution contains 0.5-3.5 g of the atactic PVA, 7.5-15 mL of the polyol, and the remaining amount of buffer solution.

23. The cryopreservation solution according to any one of claims 1 to 16 and the freezing equilibrium solution according to any one of claims 19 to 22, wherein the cryopreservation solution and the freezing equilibrium solution exist independently; The atactic PVA is a DMSO-free cryopreservation reagent characterized by a syndiotacticity of 15%-60%, a molecular weight of 10-500 kDa, and a degree of hydrolysis of more than 80%.

24. The DMSO-free cryopreservation reagent of claim 23, characterized in that the serum content is 0, and the freezing equilibration solution contains, per 100 mL, 0.5-2.5 g of atactic PVA, 7.5-15 mL of polyol, and the remaining amount of buffer solution.

25. Use of the cryopreservation solution according to any one of claims 1 to 16 and / or the freezing equilibrium solution according to any one of claims 19 to 22 in cryopreservation of biological tissue.

26. The biological tissue is selected from at least one of an oocyte, an embryo, a stem cell, an organ, and a tissue.

26. The use according to claim 25.

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