Vitrification cryoprotectants, methods of making and using the same

Abstract

The application belongs to the field of biochemistry and biomaterials, and particularly relates to a vitrification cryoprotectant and a preparation method and application thereof. The vitrification cryoprotectant comprises an antifreeze and a diluent; the antifreeze is betaine and proline. The application first explores the potential of the vitrification cryoprotectant, designs and optimizes the vitrification cryopreservation method, and successfully applies the vitrification cryopreservation method to the cryopreservation of ligaments and cartilages. The new vitrification cryoprotectant and the cryopreservation method used in the application are a long-term, efficient and low-toxicity vitrification method. The vitrification cryopreservation method of cells and tissues based on the betaine and proline vitrification cryoprotectant has not been reported, which expands the future research direction of vitrification cryopreservation and has urgent practical significance and application value for the development of cryobiology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry and biomaterials, and particularly relates to a vitrification cryoprotectant as well as a preparation method and application thereof. BACKGROUND

[0002] Long-term preservation of cells and tissues is of great significance in the fields of organ transplantation, genetic engineering, drug development, and disease treatment. Among them, the ultra-low temperature cryopreservation technology is a widely used method. This technology significantly reduces the molecular motion in biological tissues and almost completely stops all metabolic activities of cells by storing them in an environment below -140℃, thereby achieving long-term preservation of cells and tissues. Traditional ultra-low temperature cryopreservation technology usually cools biological samples to -196℃ in a slow cooling (1℃ / min) manner. During this process, the mechanical stress and osmotic stress generated by the formation of ice crystals in and outside the cells can cause various damages, including dehydration, protein denaturation, and oxidative stress, leading to cell structure damage and loss of tissue function. The addition of cryoprotectants, such as the most common dimethyl sulfoxide (DMSO) and glycerol, helps to reduce the formation of ice crystals and maintain the viability of biological tissues. They can bind to water molecules to lower the freezing point of the solution and increase the viscosity of the solution to slow down the growth rate of ice crystals, thereby reducing the damage caused by ice crystals. However, the recrystallization of ice crystals during the rewarming process and the toxicity of the protective agent limit its widespread application.

[0003] In recent years, the development of vitrification cryoprotectants has attracted widespread attention. Vitrification is a process that rapidly cools water molecules so that they cannot arrange into ice crystal structures, forming an amorphous state (i.e., a glass-like solid state). This method can effectively prevent the formation of ice crystals, thereby reducing physical damage to cells and better maintaining the structural integrity and biological function of cells and tissues. To achieve vitrification, it is necessary to ensure that the cooling and rewarming rates exceed the critical cooling rate and critical rewarming rate through rapid cooling and rewarming. In the process of vitrification cryopreservation, although the use of high concentrations of osmotic protective agents can reduce these critical rates, it also brings higher toxicity problems. In addition, the protective agent loading and unloading steps of vitrification cryopreservation are complicated, which often leads to osmotic imbalance of cells and tissues, causing osmotic damage. Therefore, finding a cryoprotectant with good biocompatibility and vitrification performance has become an important direction of current research. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provide a vitrification cryoprotectant as well as a preparation method and application thereof.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0006] A vitrification cryoprotectant, comprising an antifreezing agent and a diluent; the antifreezing agent is one or a combination of betaine or proline.

[0007] The diluent is a buffer; preferably, the diluent is one of PBS buffer, DPBS buffer, DMEM buffer or normal saline; preferably, the diluent is PBS buffer with pH of 7.0-7.4.

[0008] The molar concentration of the antifreezing agent in the diluent is greater than or equal to 5.8 mol / L.

[0009] The application further comprises a preparation method of the vitrification cryoprotectant, comprising the following steps: dissolving the antifreezing agent in the diluent.

[0010] The application further comprises an application of the vitrification cryoprotectant, which is applied to the preservation of tissues or cells; preferably, the tissues are ligaments and cartilages.

[0011] Specifically, the method comprises the following steps: 1) vitrification cryopreservation; and 2) thawing recovery.

[0012] The specific steps of step 1) are as follows: sequentially loading the tissues or cells in the vitrification cryoprotectant with gradient concentrations; then transferring the tissues or cells to a cryopreservation tube containing the vitrification cryoprotectant for cooling, preferably from 4℃ to -150℃; preferably, the cooling rate is greater than the critical cooling rate of the vitrification cryoprotectant; preferably, the cooling rate is greater than or equal to 2℃ / min; and rapidly transferring the cooled cryopreservation tube to a liquid nitrogen tank at -196℃ for preservation.

[0013] The gradient concentrations in step 1) are sequentially increased by a factor of two to the molar concentration of the vitrification cryoprotectant in the tissues or cells, which is greater than or equal to 5.8 mol / L; preferably, the vitrification cryoprotectant is 1.475 mol / L, 2.95 mol / L and 5.9 mol / L, respectively.

[0014] The specific steps of step 2) are as follows: taking the cryopreservation tube out of the liquid nitrogen tank, rapidly placing it in a 37℃ water bath for 5-10 min, then sequentially transferring the tissues or cells to the vitrification cryoprotectant with gradient concentrations for recovery, and then transferring them to a washing solution for incubation; preferably, the washing solution is PBS buffer.

[0015] The gradient concentrations in step 2) are sequentially decreased by a factor of two, preferably, the vitrification cryoprotectant is 2.95 mol / L and 1.475 mol / L, respectively.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] 1) The application uses natural zwitterionic betaine or proline as a vitrification cryoprotectant. Specifically, betaine is a commonly used osmotic pressure regulator that can penetrate the cell membrane, regulate the osmotic pressure inside and outside the cell, and is a biocompatible molecule. Proline is a natural osmotic protectant and antioxidant with good biocompatibility.

[0018] When its concentration is greater than 5.8 mol / L, vitrification can be achieved and the de-vitrification does not occur during the rewarming process. The use of betaine or proline as a vitrification cryoprotectant reduces the damage to cells and tissues caused by chemical reagents during the cryopreservation process, and avoids the formation of ice crystals during the preservation process and the damage to cells and tissues.

[0019] 2) The application measures the physicochemical properties of the vitrification cryoprotectant, and designs and optimizes the vitrification cryopreservation method accordingly. The gradient loading method is used to alleviate the dehydration of the tissue caused by the high concentration of the protectant during the process, and the temperature is lowered at a rate greater than the critical cooling rate of the vitrification cryoprotectant to achieve stable vitrification.

[0020] 3) The application uses the above-mentioned vitrification cryopreservation method to vitrify and freeze ligaments in liquid nitrogen for a month. Through experiments, it is found that the biomechanical properties of the ligaments after cryopreservation remain stable, the hydroxyproline content and glycosaminoglycan content are basically unchanged, and the tissue structure is normal. Compared with the traditional DMSO slow freezing preservation effect, the application has better effect and higher safety.

[0021] 4) The application uses the above-mentioned vitrification cryopreservation method to vitrify and freeze cartilage in liquid nitrogen for a month. Through experiments, it is found that the cartilage after cryopreservation has complete tissue structure and stable cell activity.

[0022] In summary, in order to solve the problem of high toxicity and poor biocompatibility of the vitrification cryopreservation agent in the prior art, the application selects natural zwitterionic betaine or proline as the main component of the protectant. Betaine and proline are natural osmotic protectants with good ability to reduce the freezing point of water and adjust the osmotic pressure, and have been applied to the slow freezing preservation of various cell lines. The application first explores the potential of betaine and proline as vitrification cryoprotectants, designs and optimizes the vitrification cryopreservation method, and successfully applies it to the cryopreservation of ligaments and cartilage. The new vitrification cryoprotectant and cryopreservation method used in the application is a long-term, efficient and low-toxicity vitrification method. The vitrification cryopreservation method based on betaine or proline has not been reported, which expands the research direction of future vitrification cryopreservation and has urgent practical significance and application value for the development of cryobiology. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Schematic diagram of the method for vitrification cryopreservation of ligament according to the present application;

[0024] Figure 2 Macroscopic schematic diagram of the process of liquid nitrogen cooling and water bath rewarming with different cryoprotectants in Example 1 of the present application;

[0025] Figure 3 Graph of concentration change during the process of loading and unloading of vitrification cryoprotectant in Example 2;

[0026] Figure 4 Graph of the results of biomechanical performance test of ligament after cryopreservation in Example 4;

[0027] Figure 5 Schematic diagram of the microscope for ligament tissue morphology after cryopreservation in Example 4;

[0028] Figure 6 Graph of the results of protein subenthalpy change, hydroxyproline content and glycosaminoglycan content test of ligament after cryopreservation in Example 4;

[0029] Figure 7 Schematic diagram of the microscope for H&E staining of cartilage after cryopreservation in Example 6;

[0030] Figure 8 Graph of cell survival rate of cartilage after cryopreservation in Example 6. DETAILED DESCRIPTION

[0031] In order to enable the persons skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below with reference to the accompanying drawings and the best mode.

[0032] Example 1: The method for preparing the vitrification cryoprotectant comprises the following steps: dissolving the antifreezing agent in the diluent to obtain. Figure 1 Schematic diagram of the method for vitrification cryopreservation of ligament according to the present application;

[0033] Specifically, the following steps are included: preparation of the vitrification cryoprotectant and selection of the concentration;

[0034] Preparation of the vitrification cryoprotectant: 0.48 mol, 0.53 mol, 0.58 mol and 0.63 mol of betaine were respectively dissolved in 100 mL of PBS buffer (pH 7.0-7.4), and after being fully stirred, they were used as the vitrification cryoprotectants A1, B1, C1 and D1;

[0035] 0.48mol, 0.53mol, 0.58mol, 0.63mol proline respectively were dissolved in 100mL PBS buffer (pH 7.0-7.4), and used as glassing freezing protectant A2, B2, C2, D2 after fully stirring.

[0036] As shown in Figure 2 The above glassing freezing protectant solution was added into 1.5mL centrifuge tube, and the freezing protectant solution with good glassing performance was selected by rapid cooling with nitrogen and water bath rewarming method, and it was found that when the concentration of betaine or proline was greater than 0.58mol (i.e. 5.8mol / L), glassing could be realized during the cooling process and no glassing was occurred during the water bath rewarming process. In order to reduce the toxicity of the protectant, glassing freezing protectants C1 and C2 were selected for subsequent experiments.

[0037] Example 2: Loading and unloading of glassing freezing protectant

[0038] The tissue used in this embodiment is ligament, specifically the lateral cross ligament of SD rats, and the gradient concentration of glassing freezing protectants E1, F1, G1 are 1.475mol / L, 2.95mol / L, 5.9mol / L (environmental concentration is slightly greater than the concentration in the tissue, so that the concentration of glassing freezing protectant in the ligament reaches 5.8mol / L) respectively, the solute is betaine, and the solvent is PBS buffer.

[0039] Similarly, proline is used as the solute to obtain E2, F2, G2, which are 1.475mol / L, 2.95mol / L, 5.9mol / L respectively.

[0040] The gradient multi-step loading method can reduce osmotic damage and avoid excessive dehydration caused by excessive osmotic pressure difference between the inside and outside of the tissue and cells.

[0041] The present application adopts a step-by-step loading and unloading scheme to gradually adjust the concentration of glassing freezing protectant in the ligament to reduce osmotic damage.

[0042] According to Example 1, the glassing freezing protectant used is glassing freezing protectant C1 or C2 of Example 1.

[0043] Figure 3 The concentration change of the glassing freezing protectant with betaine as the solute during the loading and unloading process is shown, and the specific loading process is that the ligament is placed in glassing freezing protectants E1, F1, G1 with gradient concentrations for 15min respectively, and the final glassing freezing protectant concentration in the ligament is 5.8M.

[0044] The unloading process is to sequentially transfer the ligament to the vitrification cryoprotectant F1, E1, respectively, and place it for 15 min, and then transfer it to the cleaning solution for incubation for 15 min, and finally realize the complete removal of the vitrification cryoprotectant. Among them, the cleaning solution is a PBS buffer.

[0045] The loading and unloading process of the vitrification cryoprotectant with proline as the solute is similar to betaine, which will not be repeated here.

[0046] Example 3: Cryopreservation experiment of ligament

[0047] The vitrification cryopreservation method of ligament with betaine as the solute: sequentially place the ligament in the equilibration solutions E1, F1, G1 as described in Example 2 for 15 min respectively; then transfer the above ligament to a 1.8 mL cryovial containing 1 mL of the vitrification cryoprotectant (vitrification cryoprotectant C1, concentration 5.8 mol / l); transfer the cryovial containing the ligament to the programmed cooling instrument with the program set in advance for cooling, and cool from 4℃ to -150℃ at a speed of 2℃ / min; quickly transfer the cooled cryovial containing the ligament to a liquid nitrogen tank at -196℃ for storage.

[0048] The vitrification cryopreservation method of ligament with proline as the solute: sequentially place the ligament in the equilibration solutions E2, F2, G2 as described in Example 2 for 15 min respectively; then transfer the above ligament to a 1.8 mL cryovial containing 1 mL of the vitrification cryoprotectant (vitrification cryoprotectant C2, concentration 5.8 mol / l); transfer the cryovial containing the ligament to the programmed cooling instrument with the program set in advance for cooling, and cool from 4℃ to -150℃ at a speed of 2℃ / min; quickly transfer the cooled cryovial containing the ligament to a liquid nitrogen tank at -196℃ for storage.

[0049] Comparative Example 1: Traditional cryopreservation method of ligament: immerse the ligament in 10% DMSO (10 mL DMSO dissolved in 90 mL PBS buffer) for 15 min, then transfer it to a 1.8 mL cryovial containing 1 mL of traditional cryoprotectant (10 mL DMSO dissolved in 90 mL PBS buffer, thoroughly stirred and used as traditional cryoprotectant); then put it into a liquid nitrogen tank for storage.

[0050] Comparative Example 2: Cryopreservation of ligament in water: place the ligament in a 1.8 mL cryovial containing 1 mL of distilled water; then put it into a liquid nitrogen tank for storage.

[0051] Thawing steps of ligament with betaine as the solute:

[0052] The cryopreservation tube containing the ligament was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for 5-10 min. The ligament was then transferred into the vitrification cryoprotectant F1 (2.95 mol / L) and E1 (1.475 mol / L) for 15 min, respectively, and then into the washing solution for 15 min to obtain the ligament Bet.

[0053] The thawing step of the ligament with proline as the solute:

[0054] The cryopreservation tube containing the ligament was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for 5-10 min. The ligament was then transferred into the vitrification cryoprotectant F2 (2.95 mol / L) and E2 (1.475 mol / L) for 15 min, respectively, and then into the washing solution for 15 min to obtain the ligament Pro.

[0055] The washing solution is PBS buffer.

[0056] Example 4: Detection and analysis of the results of ligament cryopreservation

[0057] 1. The ligaments cryopreserved for 7 days and 30 days and then thawed were subjected to the following functional tests

[0058] (1) Biomechanical property test: a mechanical tester was used to evaluate the biomechanical properties of the ligament. The ligament Bet and Pro obtained in Example 3 and the ligaments of Comparative Examples 1 and 2 were cut into samples with a length of 6 mm using a scalpel, and 2 mm on both sides were fixed on two pieces of sandpaper using cyanoacrylate. The average cross-sectional area of the ligament was 2 mm 2 . The sandpaper was clamped with an aluminum clamp of the testing instrument to firmly fix the ligament. Uniaxial tensile test was performed at a constant rate of 2 mm / min until the ligament broke. The stress-strain data of the tensile test were analyzed to calculate the ultimate stress and ultimate strain of the sample. The slope of the linear region of the stress-strain curve was calculated to obtain the Young's modulus of the sample.

[0059] (2) Histomorphological test: the thawed ligament was fixed with paraformaldehyde for 24 h, dehydrated with 40% sucrose at 4°C for 12 h, embedded with OCT for 24 h, and then cut into sections with a thickness of 10 μm using a freezing microtome. The tissue sections were stained according to the instructions of the hematoxylin and eosin (H&E) staining and Masson staining kits to evaluate the morphology of the cryopreserved tissue.

[0060] (3) Protein thermal denaturation detection: the cryopreserved ligament was analyzed by differential scanning calorimetry. 10 mg of the cryopreserved ligament sample was accurately weighed, and the sample was subjected to calorimetry by differential scanning calorimeter at a heating rate of 10 ℃ / min from 20 ℃ to 100 ℃, and the enthalpy change (ΔH) of protein thermal denaturation was obtained by integrating thermal denaturation.

[0061] (4) Hydroxyproline (HYP) detection: an appropriate amount of the rewarmed ligament was weighed and ground, and hydrochloric acid was used for digestion at 100 ℃ for 3 h, and the PH was adjusted to neutral by sodium hydroxide. The hydroxyproline content was determined according to the kit instructions.

[0062] (5) Glycosaminoglycan (GAGs) detection: an appropriate amount of the rewarmed ligament was weighed, centrifuged at a speed of 5000 rpm for ten minutes, and the supernatant was collected. The glycosaminoglycan content was determined according to the ELISA kit instructions.

[0063] 2、Results analysis

[0064] (1) Figure 4 The biomechanical properties of the ligament after cryopreservation are shown in Table 1. The results of the test can be seen that the Young's modulus and ultimate stress of the water group (comparative example 2) decrease over time, which may be due to the damage to the collagen fiber structure caused by the formation of ice crystals during the cryopreservation process, and the decrease of the cross-linking density and triple helix content of collagen fibers. The biomechanical properties of the vitrification group are consistent with those of the fresh group, and are significantly better than those of the traditional DMSO (comparative example 1) slow cryopreservation method, indicating that the vitrification cryoprotectant and cryopreservation method of the present application can keep the original biomechanical properties of the ligament unchanged.

[0065] (2) Figure 5 The histomorphological results of the ligament after cryopreservation are shown in Table 2. Among them, the H&E staining results show that the collagen fibers of the fresh group are wavy and arranged in parallel, and there are sparse cells in the dense fiber gap, and the cell nucleus is flat; the collagen fibers of the DMSO group are obviously broken, the fiber gap is increased, and the cell density is reduced; in the water group, large-area cracking occurs between the collagen fibers, the structure is severely damaged, the cells are arranged in disorder, and part of the cell nuclei are atrophy; in contrast, the collagen fibers of the vitrification group of the present application maintain a parallel wavy appearance and are minimally damaged, in addition, the cell distribution is orderly and the structure is intact.

[0066] The Masson staining results are consistent with the H&E staining results. The collagen fiber cryopreservation effect of the vitrification group of the present application is the best, and the tissue structure after rewarming is very close to that of the fresh group. These results show that the vitrification cryoprotectant and cryopreservation method of the present application can effectively keep the morphological characteristics of the tissue stable.

[0067] (3) Figure 6The relevant function detection results (enthalpy change when the intracellular protein is heat denatured, hydroxyproline content, and glycosaminoglycan content) of the ligament after cryopreservation are shown in Table 1. It can be seen that the enthalpy change, hydroxyproline content, and glycosaminoglycan content of the vitrification group are consistent with those of the fresh group, and are obviously better than those of the DMSO group and the water group, indicating that the vitrification cryoprotective agent and the cryopreservation method of the present application can play a good protective effect on the ligament and reduce the related damage in the low-temperature preservation process.

[0068] The above results show that the betaine or proline-based vitrification cryoprotective agent can not only maintain the stable biomechanical properties of the ligament, the complete tissue structure, but also prevent the loss of important components such as glycosaminoglycan during the vitrification cryopreservation process, that is, the vitrification cryoprotective agent and the cryopreservation method thereof have excellent cryopreservation effect.

[0069] Example 5: Cryopreservation experiment of cartilage

[0070] The difference between Example 5 and Example 3 is only that the cryopreserved tissue is replaced by cartilage;

[0071] Example 6: Detection and analysis of cartilage cryopreservation results

[0072] 1. The following function detection is performed on the cartilage cryopreserved for 30 days and thawed

[0073] (1) Tissue morphology detection: the thawed cartilage is fixed with paraformaldehyde for 24 h, dehydrated with 40% sucrose at 4°C for 12 h, embedded with OCT for 24 h, and then cut into slices with a thickness of 10 μm by using a freezing microtome. The tissue slices are dyed according to the H&E staining kit instructions to evaluate the morphology of the cryopreserved tissue.

[0074] (2) Tissue cell activity detection: the thawed cartilage is digested with collagenase type II for 12 h, filtered by a 200-mesh cell filter, and then centrifuged to collect the cartilage cells. The cell survival rate is detected by using the live and dead staining method.

[0075] 2. Result analysis

[0076] (1) Figure 7 The H&E staining results of the cartilage after cryopreservation are shown in Table 2. Among them, the H&E staining results show that the tissue morphology of the vitrification group is consistent with that of the fresh group, with a small amount of pores and uniform cell distribution; the pores of the DMSO group are increased and the number of cells is reduced; the water group has a large number of holes and the structure is completely destroyed.

[0077] (2) Figure 8The cell survival rate in the tissue after cryopreservation of cartilage is shown in the figure. Among them, the cell survival rate is more than 80% after one month of cryopreservation by vitrification; the cell activity of the DMSO group is obviously reduced, and the survival rate is less than 60%; only a small amount of cells survive in the pure water group.

[0078] The above results show that the betaine or proline-based vitrification cryoprotectant can also be applied to the vitrification cryopreservation of cartilage and achieve ideal preservation effect, and the tissue structure is complete and the cell activity is maintained.

[0079] In summary, in order to solve the problem of high toxicity and poor biocompatibility of the vitrification cryopreservation agent in the prior art, the natural zwitterion betaine or proline is selected as the main component of the protective agent. Betaine and proline are natural osmotic pressure protective agents, which have good ability to reduce the freezing point of water and adjust the osmotic pressure, and have been applied to slow freezing preservation of various cell lines. The application first explores the potential of betaine and proline as vitrification cryoprotective agents, designs and optimizes the vitrification cryopreservation method, and successfully applies it to the cryopreservation of ligament and cartilage. The new vitrification cryoprotective agent and the cryopreservation method used in the application are a long-term, efficient and low-toxicity vitrification method. The vitrification cryopreservation method of the betaine or proline-based vitrification cryoprotective agent for cells and tissues has not been reported, which expands the research direction of future vitrification cryopreservation, and has urgent practical significance and application value for the development of cryobiology.

[0080] The above only describes the preferred embodiments of the application, and it should be pointed out that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principles of the application, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. The application of a vitrification cryoprotectant, characterized in that, It is used for the preservation of ligaments or cartilage; Includes the following steps: 1) Vitrification and cryopreservation; The ligaments or cartilage were sequentially placed in vitrified cryoprotectants of varying concentrations for loading; then the ligaments or cartilage were transferred to cryovials containing vitrified cryoprotectants for cooling, from 4°C to -150°C; the cooling rate was greater than the critical cooling rate of the vitrified cryoprotectants; the cooled cryovials were then rapidly transferred to a liquid nitrogen tank at -196°C for storage. 2) Thawing and recovery; The vitrification cryoprotectant comprises an antifreeze and a diluent; the antifreeze is one or a combination of betaine and proline; the diluent is one of PBS buffer, DPBS buffer, DMEM buffer, or physiological saline; and the molar concentration of the antifreeze in the diluent is ≥5.8 mol / L.

2. The application according to claim 1, characterized in that, The pH of the diluent is 7.0-7.

4.

3. The application according to claim 1, characterized in that, Cooling rate ≥ 2℃ / min.

4. The application according to claim 1, characterized in that, In step 1), the gradient concentration is increased sequentially until the molar concentration of the vitrification cryoprotectant in the ligament or cartilage is ≥5.8 mol / L.

5. The application according to claim 1, characterized in that, The gradient concentrations in step 1) are 1.475 mol / L, 2.95 mol / L, and 5.9 mol / L, respectively, of the vitrification cryoprotectant.

6. The application according to claim 1, characterized in that, Step 2) The specific steps are as follows: Take the cryovial out of the liquid nitrogen tank, quickly place it in a 37°C water bath for 5-10 min to rewarm, then transfer the ligament or cartilage to a gradient concentration of vitrification cryoprotectant for recovery, and then transfer it to a washing solution for incubation; the washing solution is PBS buffer.

7. The application according to claim 6, characterized in that, The gradient concentrations in step 2) decrease sequentially by a factor of two; they are 2.95 mol / L and 1.475 mol / L, respectively.

8. The application of a vitrification cryoprotectant according to any one of claims 1-7, characterized in that, The preparation of a vitrified cryoprotectant includes the following steps: dissolving the antifreeze in a diluent.

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