Cryopreservation protective agent as well as preparation method and application thereof

Abstract

The invention belongs to the field of freezing, and particularly relates to a cryoprotectant as well as a preparation method and application thereof. The cryopreservation protective agent comprises a permeable cryopreservation protective agent, a non-permeable cryopreservation protective agent, an antioxidant and equilibrium liquid; the zwitterionic betaine with good biocompatibility maintains osmotic pressure balance inside and outside tissues through free permeation; pVP and ice crystals form strong binding force, ice crystal recrystallization in the rewarming process is inhibited, meanwhile, protein is stabilized, and the integrity of the muscle structure is protected; d-panthenol reduces oxidative damage of muscles by inhibiting oxidative stress of the muscles in the cryopreservation process, and the activity and structure of protein in the muscles are protected.

Description

Technical Field

[0001] The invention belongs to the field of freezing, and in particular relates to a cryoprotectant and a preparation method and application thereof. Background Art

[0002] Muscle is an important tissue in the body. It provides power for biological movement through the interaction of the skeletal muscle system and is of great significance to the survival of organisms. However, the prevalence of muscle diseases is increasing worldwide, such as congenital muscle disease, inflammatory muscle disease, metabolic muscle disease, etc. Among them, the prevalence of neuromuscular diseases is at least similar to the prevalence of Parkinson's disease worldwide (about 200 people are sick in 100,000 people), and is twice the prevalence of multiple sclerosis in Europe. Muscle biopsy is a necessary means for clinical diagnosis of muscle diseases, but muscles will become stiff a few hours after leaving the body, that is, rigor mortis, which affects the accuracy of biopsy. The long-term preservation technology of muscle tissue helps to realize the collection and effective preservation of muscle tissue, which is not only conducive to the in vivo research, diagnosis or allogeneic transplantation treatment of muscle diseases, but also can effectively extend the shelf life of meat products, maintain their nutritional value, facilitate storage and transportation, ensure the stability of supply, and promote the market expansion of frozen meat products.

[0003] Tissue and organ preservation technology plays an irreplaceable role in many fields such as medical treatment, scientific research, and biotechnology. Storing tissues and organs under deep cryogenic conditions can achieve metabolic stagnation and inhibit ischemic damage, and can achieve the purpose of long-term preservation without deterioration.

[0004] However, during the cryopreservation process, the formation of ice crystals is the main cause of freezing damage to organs and tissues. In particular, ice crystals formed inside tissues not only cause serious cell damage, but also destroy the structure inside the tissue. In addition, ice crystal formation can cause changes in the physical and chemical properties of lipids and proteins in tissues. When lipids are over-oxidized, their control over the flow of the solution will be greatly weakened, thereby aggravating the damage caused by changes in osmotic pressure. On the other hand, the destruction of protein structure can cause it to denature or aggregate, and the activity of some key enzymes such as proteases will also be significantly reduced. The synergistic effect of these injuries may eventually lead to the death of cells and tissues. Therefore, avoiding the formation of ice crystals or reducing the damage caused by them is a key goal in the development of cryopreservation technology. By improving freezing technology and using efficient cryoprotectants, new cryopreservation methods can be developed to minimize ice crystal damage during the freezing process. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a cryoprotectant and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A cryoprotectant comprises a permeable cryoprotectant, a non-permeable cryoprotectant, an antioxidant and a balancing solution; the mass proportion of the permeable cryoprotectant is 3-7%; the mass proportion of the non-permeable cryoprotectant is 3-7%; the mass proportion of the antioxidant is 0.2-1%.

[0008] The osmotic cryoprotectant is natural zwitterion betaine.

[0009] The non-permeable cryoprotectant is one or a mixture of polyvinyl pyrrolidone PVP, polyethylene glycol PEG, hydroxyethyl starch PEI and polyvinyl alcohol PVA.

[0010] The balancing solution is one of PBS buffer, DPBS buffer and physiological saline; preferably PBS buffer; the pH value of the balancing solution is 7.0-7.4.

[0011] Preferably, the mass proportion of the osmotic cryoprotectant is 5-7%; the mass proportion of the non-osmotic cryoprotectant is 3-5%; Preferably, the mass proportion of the osmotic cryoprotectant is 7%; the mass proportion of the non-osmotic cryoprotectant is 5%;

[0012] Preferably, the antioxidant accounts for 0.6-1% by mass, more preferably 0.8%;

[0013] Preferably, the permeable cryoprotectant is natural zwitterionic betaine; preferably, the non-permeable cryoprotectant is polyvinylpyrrolidone PVP; preferably, the antioxidant is D-panthenol.

[0014] The present invention also includes a method for preparing the cryoprotectant, comprising the following steps: dissolving a permeable cryoprotectant, a non-permeable cryoprotectant and an antioxidant in a balanced solution to prepare the cryoprotectant.

[0015] The present invention also includes an application of the cryoprotectant for cryopreservation of cells, tissues or organs; preferably, the tissue is muscle.

[0016] The cryopreservation of muscle specifically comprises the following steps: (1) removing the fat tissue around the isolated muscle, immersing it in a cryoprotectant, and incubating it in a 4°C refrigerator for 1-30 min; preferably 30 min; (2) cryopreserving the muscle containing the cryoprotectant in a -80°C refrigerator or a -196°C liquid nitrogen tank by rapid cooling; preferably, the storage method is a -196°C liquid nitrogen tank.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) The present invention avoids the use of traditional cryoprotectants with high toxicity and low cryopreservation efficiency. By analyzing the freezing point depression ability of betaine and four non-permeable macromolecules, a cryoprotectant formula with excellent biocompatibility is optimized and developed. Among them, the zwitterionic betaine with good biocompatibility maintains the osmotic pressure balance inside and outside the tissue through free permeation; the non-permeable cryoprotectant forms a strong binding force with ice crystals, inhibits the recrystallization of ice crystals during the rewarming process, and stabilizes the protein, protecting the integrity of the muscle structure; the antioxidant inhibits the oxidative stress of the muscle during the cryopreservation process, reduces its oxidative damage, and protects the activity and structure of the protein in the muscle.

[0019] 2) The present invention optimizes the cryopreservation method of the cryoprotectant and cryopreserves the muscle. By evaluating the protein content and enzyme activity in the muscle, it is finally concluded that cryopreservation at -196°C causes the least damage to the muscle, and cryopreservation in a -196°C liquid nitrogen environment is more conducive to muscle preservation.

[0020] 3) After freezing the muscle for one month, a systematic evaluation was conducted on its cryopreservation effect. The protein content, protein structural stability, superoxide dismutase activity, catalase activity, malondialdehyde content, myofibril structure, protein thermal stability, and mechanical properties were evaluated. The protein content, myofibril structure, antioxidant capacity, lipid oxidation degree, thermal stability, biomechanical properties, and histological structure of the muscle tissue after one month of freezing were not significantly different from those of normal tissue, and were significantly better than the commercial cryoprotectant sorbitol.

[0021] In summary, the present invention avoids the use of traditional cryoprotectants with high toxicity and low cryopreservation efficiency. By analyzing the protein content and enzyme activity of muscles after cryopreservation of permeable small molecule betaine and four non-permeable macromolecules, a cryoprotectant formula with excellent biocompatibility is optimized and developed. In addition, the present invention explores the cryopreservation effect of muscles under different deep-low temperature freezing conditions for the first time, and uses the cryoprotectant to apply to frozen muscles, so that the frozen muscles still maintain excellent tissue structure integrity, enzyme activity, protein structure stability, protein thermal stability, antioxidant capacity and mechanical properties one month after freezing. This cryopreservation strategy provides an efficient and reliable method for long-term preservation of tissues and organs. It not only helps to preserve important biological materials in the medical field, but also provides a solid foundation for possible transplantation and research in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the method for freezing muscle using the cryoprotectant of the present invention;

[0023] Figure 2 This is a graph showing the results of the cryoprotectant penetration time in an embodiment of the present invention;

[0024] Figure 3 This is a graph showing the results of histological staining tests in muscles using different cryopreservation methods in the embodiments of the present invention;

[0025] Figure 4 The graphs are the test results of protein content and enzyme activity in muscles using different freezing methods in the embodiments of the present invention;

[0026] Figure 5 This is a graph showing the test results of muscle biomechanical properties using different cryopreservation methods in the embodiments of the present invention;

[0027] Figure 6 The results of muscle protein content and enzyme activity after cryopreservation with different non-permeable cryoprotectants in the embodiments of the present invention are shown in FIG.

[0028] Figure 7 The results of muscle protein content and enzyme activity after cryopreservation with different concentrations of cryoprotectants in the embodiment of the present invention are shown in FIG.

[0029] Figure 8 The results of muscle protein content and enzyme activity after freezing with different antioxidants added in the embodiment of the present invention;

[0030] Fig. 9 The results of muscle protein content and enzyme activity after freezing with different concentrations of antioxidants added in the embodiment of the present invention;

[0031] Fig.10 This is a diagram showing the histological test results of frozen muscle in an embodiment of the present invention;

[0032] Fig.11 This is a graph showing the test results of protein content and antioxidant capacity of frozen muscle in an embodiment of the present invention;

[0033] Fig.12 This is a graph showing the test results of the muscle protein structure stability after cryopreservation in an embodiment of the present invention;

[0034] Fig.13 This is a graph showing the results of a test on thermal stability of muscle protein after cryopreservation in an embodiment of the present invention;

[0035] Fig.14 This is a graph showing the biomechanical properties of frozen muscle in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0037] Example

[0038] 1. Optimization of cryopreservation procedures;

[0039] The preparation method of a cryoprotectant comprises the following steps: dissolving a permeable cryoprotectant betaine Bet, a non-permeable cryoprotectant polyvinylpyrrolidone PVP (molecular weight 10000) and an antioxidant D-panthenol in a PBS buffer to prepare a BPD solution; wherein the concentration of betaine Bet in the PBS buffer is 7%, the concentration of polyvinylpyrrolidone PVP in the PBS buffer is 5%, and the concentration of D-panthenol in the PBS buffer is 0.8%.

[0040] The fat tissue around the isolated muscle was removed, and the BPD solution was immersed in the cryoprotectant and added to the cryotubes for incubation at 4°C for 1-30 min. The penetration capacity of the protective agent was evaluated by a freezing point osmometer. Figure 2 The results showed that the cryoprotectant and muscle could reach equilibrium after 30 minutes of co-incubation, so the co-incubation time was selected as 30 minutes.

[0041] The muscles were added to cryopreservation tubes containing PBS buffer and directly placed in a -80°C refrigerator and a liquid nitrogen tank for freezing and storage for one day, one week, and one month. After the freezing was completed, the sample tubes were placed in a 37°C water bath to thaw. Hematoxylin and eosin (H&E) staining, biuret protein content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content detection kits were used to evaluate the muscle structural integrity, muscle protein content, enzyme activity, and lipid oxidation degree at different freezing times and temperatures ( Figure 3 and Figure 4 ), and the experiment was conducted according to the instructions of the kit. The results showed that when the freezing time was 1 day, the structural integrity of the muscle fibers did not change significantly, and the protein content and malondialdehyde content did not change much. However, when the freezing time was extended to 1 week, the integrity of the tissue structure was severely damaged, the muscle bundle structure became looser, and compared with fresh muscle, the frozen muscle fibers showed obvious contraction, and the protein content and superoxide dismutase activity were greatly reduced, resulting in muscle water loss, increased tissue hardness, and increased Young's modulus ( Figure 5 ). In addition, compared with freezing in a -80℃ freezer, freezing in a -196℃ liquid nitrogen tank results in less damage to the muscle structure, less protein loss, higher enzyme activity, and lower lipid oxidation. Therefore, -196℃ conditions are more conducive to the cryopreservation of muscle.

[0042] Figure 1The schematic diagram of the cryopreservation method of muscle cold by cryoprotectant is shown. In summary, the muscle cryopreservation procedure is as follows: (1) remove the fat tissue around the isolated muscle, immerse it in cryoprotectant, and incubate it in a 4℃ refrigerator for 30 minutes to reach equilibrium; (2) quickly immerse the cryopreservation tube in liquid nitrogen to quickly cool it down, and store it in a -196℃ liquid nitrogen tank. After the cryopreservation is completed, put the sample tube in a 37℃ water bath to rewarm it, and wash the muscle with PBS buffer solution before use.

[0043] 2. Optimization of cryoprotectant formulation

[0044] 2.1. The protein content and enzyme activity of muscles protected by different cryoprotectants were evaluated using the biuret protein content and superoxide dismutase (SOD) activity detection kit.

[0045] The cryoprotectants of different components were prepared in the following manner: 5% betaine Bet was dissolved in PBS buffer with 5% polyvinyl pyrrolidone PVP (molecular weight 10,000), 5% polyethylene glycol PEG (molecular weight 10,000), 5% hydroxyethyl starch (molecular weight 10,000) PE I and 5% polyvinyl alcohol PVA (molecular weight 10,000) to prepare cryoprotectants with a concentration of 10%, and PBS was used as a control. Figure 6 It can be seen that the betaine and PVP group (BP) showed the highest protein content and enzyme activity. Therefore, the mixture of betaine and PVP was selected for subsequent tests.

[0046] 2.2. The optimal ratio of betaine and PVP was optimized. The ratios of PBS buffer with mass concentrations of 3%, 5%, and 7% betaine and 3%, 5%, and 7% PVP were studied. The results are as follows: Figure 7 The results showed that the combination of betaine with a mass concentration of 5%-7% betaine and PVP with a mass concentration of 3%-5% PBS buffer showed the best protection for muscle protein content and enzyme activity, especially the combination of 7% betaine and 5% PVP showed the best protection for muscle protein content and enzyme activity. Therefore, the optimal ratio of 7% betaine and 5% PVP was selected for subsequent testing, namely the 7B5P group.

[0047] 2.3. The effects of different antioxidants on muscle protection were evaluated. Fresh muscle, PBS buffer, 7% betaine + 5% PVP (BP), 7% betaine + 5% PVP + 1% L-carnitine PBS buffer (BPL), 7% betaine + 5% PVP + 1% D-panthenol PBS buffer (BPD), 7% betaine + 5% PVP + 1% ascorbyl palmitate PBS buffer (BPA), 7% betaine + 5% PVP + 1% quercetin PBS buffer (BPQ), 7% betaine + 5% PVP + 1% catechol PBS buffer (BPC), 7% betaine + 5% PVP + 1% glutathione PBS buffer (BPG), 7% betaine + 5% PVP + 1% rosarinic acid PBS buffer (BPR) were tested as cryoprotectants to preserve muscle. The results are as follows Figure 8 It can be seen that betaine + PVP + D-panthenol PBS buffer (BPD) showed the highest protein content and enzyme activity. Therefore, the mixture of betaine + PVP + D-panthenol was selected for subsequent testing.

[0048] 2.4. The concentration of D-panthenol was optimized. The ratios of D-panthenol with mass concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1% mixed with PBS buffer with mass concentrations of 7% betaine + 5% PVP were studied. The results are as follows: Fig. 9 The results showed that the combination of 0.6%-1% D-panthenol and 7% betaine + 5% PVP in PBS buffer showed the best protection for muscle protein content and enzyme activity, especially the combination of 0.8% D-panthenol and 7% betaine + 5% PVP showed the best protection for muscle protein content and enzyme activity, which was consistent with the fresh group. Therefore, the optimal ratio of 7% betaine + 5% PVP and 0.8% D-panthenol was selected for subsequent testing, namely the BPD group.

[0049] 3. Evaluation of the cryopreservation effect of the cryoprotectant on muscle

[0050] 3.1. After thawing the frozen muscles for one week and one month, the following tests were performed

[0051] (1) Histomorphological test: The structural integrity of the muscle was evaluated using an H&E staining kit. The muscle was immersed in a paraformaldehyde solution at 4°C for 24 h to fix the tissue structure. It was then dehydrated in 40% sucrose for 12 h and embedded in the tissue embedding medium OCT for 3 days. The tissue was then sectioned using a freezing microtome with a thickness of 10 μm. H&E staining analysis was then performed according to the instructions to evaluate the structural integrity of the muscle.

[0052] (2) Protein content and antioxidant capacity test: The protein content, antioxidant enzyme activity and lipid antioxidant capacity of frozen muscle were tested using the biuret protein content detection kit, superoxide dismutase (SOD) activity detection kit, catalase (CAT) activity detection kit and malondialdehyde (MDA) content detection kit, respectively.

[0053] Protein content detection by biuret method: Weigh 0.4 g of muscle, mince it, add 2 mL of extract solution, grind it thoroughly on ice to a homogenate, and test its protein content according to the instructions.

[0054] Superoxide dismutase (SOD) activity detection: Weigh 0.6 g of muscle, mince it, add 0.5 mL of extract solution, grind it thoroughly on ice to homogenate, and test its superoxide dismutase activity according to the instructions.

[0055] Catalase (CAT) activity detection: Weigh 0.2 g of muscle, mince and add 1 mL of extract, grind thoroughly on ice to homogenate, centrifuge and take the supernatant to react with the catalase activity detection kit to obtain its content.

[0056] Malondialdehyde (MDA) content detection: weigh 0.4g muscle, add 1mL extract solution after the muscle is crushed, grind it fully on ice to homogenate, take the supernatant after centrifugation and react with the malondialdehyde content detection kit to obtain its content.

[0057] (3) Protein structure test:

[0058] The myofibril structure of frozen muscle was tested using an ELISA reader and SDS-PAGE gel electrophoresis.

[0059] Myofibril intrinsic fluorescence spectrum test: Weigh 0.4g muscle and mince it, add 2mL of extract, break it with a cell disruptor and centrifuge it, take out the lower layer of sediment, dilute it with HEPES buffer solution, and put it into the microplate reader for detection, with an excitation wavelength of 295nm.

[0060] SDS-PAGE gel electrophoresis test: The lower precipitate was obtained in the same way as above and diluted with HEPES buffer solution, and 20uL was taken to make gel using a one-step gel making kit. After Coomassie brilliant blue staining, the stained protein gel was destained with a decolorizing solution containing acetic acid and methanol, and the protein bands were recorded using a gel imaging analyzer.

[0061] (4) Protein thermal stability test: DSC was used to evaluate the thermal stability of muscle protein. 20 mg of muscle was weighed and placed in a crucible, which was sealed and placed in the sample chamber. The temperature was raised from 20°C to 100°C at a rate of 10°C / min. The temperature was then lowered to 20°C, and the denaturation temperature of the muscle was obtained based on the peak temperature in the heat flow curve during the heating process.

[0062] (5) Biomechanical properties test: A universal testing machine was used to perform compression tests on the muscles to evaluate their biomechanical properties. The muscles were modified into 5×10×2 mm 3 The sample was taken and the surface moisture was wiped off. The muscle was placed on the compression table and the muscle compression test was performed at a speed of 2 mm / min along the plane perpendicular to the muscle placement plane until the maximum stress reached 450 N. The slope of the linear part of the stress-strain curve was recorded as Young's modulus.

[0063] 3.2 Results Analysis

[0064] (1) Fig.10 The histomorphological analysis of muscle after cryopreservation is shown in Figure 2. The results show that after the muscle was cryopreserved for one week under the conditions of various cryopreservation protectants, the muscle fiber structure remained intact and no obvious damage occurred. However, after the cryopreservation time reached one month, the muscle preserved using the commercial cryoprotectant sorbitol group (Sorbitol) showed certain collagen fiber breakage, increased fiber gaps, partial fragmentation, and muscle fiber contraction and depolymerization. In contrast, the muscle preserved in the cryoprotectant group still showed a complete fiber structure even after one month of cryopreservation.

[0065] (2) Fig.11 The protein content and antioxidant capacity of muscle after cryopreservation are shown in the figure. The protein content results show that with the extension of cryopreservation time, the protein content of the cryoprotectant group decreased slightly, but it was still consistent with the content of the normal group. The protein content of the commercial cryoprotectant sorbitol group was only 79.2%, while the protein content of the non-protectant group was greatly reduced. In addition, the results of SOD, CTA and MDA showed that after one month of cryopreservation, the cryoprotectant group could retain 90.55% of SOD activity and 95.16% CAT activity, indicating that it has a good ability to preserve the antioxidant enzyme activity in muscle; relative to the sorbitol group, the malondialdehyde content increased significantly to 178.2%, and the malondialdehyde content of the cryoprotectant group increased slightly, indicating that its lipid oxidation degree was small. Therefore, this protective agent can protect the protein content in muscle from loss and has antioxidant capacity.

[0066] (3) Fig.12The protein structural characteristics after muscle cryopreservation are shown in Figure 2. The endogenous fluorescence spectrum results show that after one month of cryopreservation, the endogenous fluorescence spectrum peak of the cryoprotectant group remained basically unchanged, retaining the original tertiary conformation of the protein in the muscle, while the endogenous fluorescence spectrum peak of the sorbitol group was greatly reduced, indicating that its protein peptide chain was severely damaged by hydroxyl free radicals, reducing endogenous tryptophan and causing a decrease in fluorescence intensity. In addition, SDS-PAGE electrophoresis bands showed that the group without cryoprotectant showed lighter bands of myosin heavy chain, myosin light chain and actin, indicating that this phenomenon showed that the spatial conformation of protein molecules in myofibrils changed, leading to the aggregation and accelerated degradation of protein molecules; while the electrophoresis bands of myosin heavy chain, myosin light chain and actin in the cryoprotectant group were similar in color to those in the fresh group, and the actin band was significantly darker than the band after cryopreservation with sorbitol, indicating that the cryoprotectant can effectively inhibit the aggregation, oxidation and degradation of myosin and actin during the freezing process, thereby maintaining the stability of the myofibrillar protein structure.

[0067] (4) Fig.13 The results show that the denaturation temperature of myosin and actin in muscle increased significantly with the increase of freezing time, and the cryoprotectant group can form a protective layer on the outer layer of liposomes, stabilize the protein structure, and reduce ice crystal damage, so that the denaturation temperature after one week and one month of freezing is not significantly different from that of the fresh group.

[0068] (5) Fig.14 The results show that the cryoprotectant group showed a Young's modulus similar to that of the fresh group, whether it was frozen for one week or one month, while the Young's modulus of the sorbitol group increased to a certain extent, and the Young's modulus of the non-protectant group increased significantly, indicating that the cryoprotectant group can better preserve the arrangement and integrity of the myofibril bundles and maintain the mechanical properties of the muscle.

[0069] The above results show that the cryoprotectant based on betaine, PVP and D-panthenol as the main components can not only have a good protective effect on the protein content, protein structure stability, enzyme activity and antioxidant capacity of the muscle, but also preserve the biomechanical properties and histological structural integrity of the muscle. Therefore, the cryoprotectant and the cryopreservation method developed by the present invention show excellent cryopreservation effect.

[0070] In summary, the present invention avoids the use of traditional cell cryoprotectants with high toxicity and low cryopreservation efficiency, as well as commercial meat cryoprotectants with high sugar, high calories and high salt content. By analyzing the protein content and enzyme activity of muscles after cryopreservation with permeable small molecule betaine and 4 non-permeable macromolecules, a cryoprotectant formula with excellent biocompatibility is optimized and developed. In addition, the present invention explores the cryopreservation effect of muscles under different deep-low temperature freezing conditions for the first time, and uses the cryoprotectant to freeze frozen muscles, so that the frozen muscles still maintain excellent tissue structural integrity, enzyme activity, protein structural stability, protein thermal stability, antioxidant capacity and mechanical properties after one month. This cryopreservation strategy provides an efficient and reliable method for long-term preservation of tissues and organs. It not only helps to preserve important biological materials in the medical field, but also provides a solid foundation for possible transplantation and research in the future.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cryoprotectant, characterized in that: It comprises a permeable cryoprotectant, a non-permeable cryoprotectant, an antioxidant and a balancing solution; the mass proportion of the permeable cryoprotectant is 3-7%; the mass proportion of the non-permeable cryoprotectant is 3-7%; the mass proportion of the antioxidant is 0.2-1%.

2. The cryoprotectant according to claim 1, characterized in that The osmotic cryoprotectant is natural zwitterion betaine.

3. The cryoprotectant according to claim 1, characterized in that The non-permeable cryoprotectant is one or a mixture of polyvinyl pyrrolidone PVP, polyethylene glycol PEG, hydroxyethyl starch PEI and polyvinyl alcohol PVA.

4. The cryoprotectant according to claim 1, characterized in that The antioxidant is one or a mixture of L-carnitine, D-panthenol, ascorbyl palmitate, quercetin, catechol, glutathione and rosmarinic acid.

5. The cryoprotectant according to claim 1, characterized in that The balancing solution is one of PBS buffer, DPBS buffer and physiological saline; preferably PBS buffer; the pH value of the balancing solution is 7.0-7.

4.

6. The cryoprotectant according to claim 1, characterized in that The mass proportion of the osmotic cryoprotectant is 5-7%; the mass proportion of the non-osmotic cryoprotectant is 3-5%; preferably, the mass proportion of the osmotic cryoprotectant is 7%; the mass proportion of the non-osmotic cryoprotectant is 5%.

7. The cryoprotectant according to claim 1, characterized in that: The antioxidant accounts for 0.6%-1% by mass, and more preferably 0.8% by mass.

8. A method for preparing the cryoprotectant according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: dissolving a permeable cryoprotectant, a non-permeable cryoprotectant and an antioxidant in a balanced solution to obtain the prepared product.

9. A use of the cryoprotectant according to any one of claims 1 to 7, characterized in that: Applicable to cryopreservation of cells, tissues or organs; preferably, the tissue is muscle.

10. The use according to claim 9, characterized in that: The cryopreservation of muscle specifically comprises the following steps: (1) removing the fat tissue around the isolated muscle, immersing it in a cryoprotectant, and incubating it in a 4°C refrigerator for 1-30 min; preferably 30 min; (2) cryopreserving the muscle containing the cryoprotectant in a -80°C refrigerator or a -196°C liquid nitrogen tank by rapid cooling; preferably, the storage method is a -196°C liquid nitrogen tank.

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