Cryopreservation method for improving survival rate of mesenchymal stem cells

Abstract

The invention relates to the field of cell cryopreservation, and provides a cryopreservation method for improving the survival rate of mesenchymal stem cells in order to solve the problem that the survival rate of the cells is obviously reduced after the usage amount of a cryoprotectant is reduced in the prior art, the cryopreservation method for improving the survival rate of the mesenchymal stem cells comprises the following steps: S100, resuspending the mesenchymal stem cells through a cryopreservation solution to obtain a cell suspension; the cryopreservation liquid comprises a permeable cryoprotectant and silk short fibers; sericin is reserved on the outer surface of the silk short fiber raw material; s200, performing freezing operation on the cell suspension to realize cell preservation; in the freezing operation process, the sericin protein is partially or completely separated out. After the silk short fibers with sericin are added into the cell suspension, the fibers can be used for protecting cells and providing attachment sites of the cells, a silk fibroin structure can also be used for promoting separation of solute, the solute is directly sericin, the osmotic pressure difference inside and outside the cells in the freezing process can be reduced through separation of the solute, and the freezing speed of the cells is increased. The survival rate of the cells is improved.

Description

Technical Field

[0001] The present invention relates to the field of cell cryopreservation, and in particular to a cryopreservation method for improving the survival rate of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells that have the ability to self-renew and differentiate into a variety of cell types. Due to their wide therapeutic potential and clinical application prospects, the long-term preservation of MSCs has become very important. Cryopreservation technology is one of the key means to achieve this goal. At present, the preservation of cells mainly relies on cryoprotectants, such as dimethyl sulfoxide or glycerol, to slow down the rate of biochemical reactions at low temperatures, thereby preventing the formation of destructive ice crystals in the water in the cells and maintaining the integrity of the cell structure. However, cryoprotectants can cause irreversible damage to cells at high concentrations, and some cells that are more sensitive to cryoprotectants will also limit the amount of cryoprotectants used. Under the limitation of the amount of cryoprotectant used, the degree to which it can lower the freezing point is limited, and thus the survival rate of cells will also be limited.

[0003] There is an urgent need for a cryopreservation technology that can reduce the amount of cryoprotectant used while maintaining a high cell survival rate. Summary of the invention

[0004] The purpose of the present invention is to provide a cryopreservation method for improving the survival rate of mesenchymal stem cells, so as to solve the problem that the survival rate of cells is significantly reduced after reducing the amount of cryoprotectant used in the prior art.

[0005] The embodiments of the present invention are implemented by the following technical solutions:

[0006] A cryopreservation method for improving the survival rate of mesenchymal stem cells comprises the following steps:

[0007] S100, resuspending the mesenchymal stem cells in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: a permeable cryoprotectant and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material;

[0008] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0009] Mesenchymal stem cells need to be preserved for scientific research, industrial and clinical applications. Preservation not only makes them easier to transport, but also keeps them in an almost static state at low temperatures. When they are needed in the future, the cells can be revived, and their research and application are no longer subject to time constraints. For example, in scientific research, after freezing mesenchymal stem cells from the same source, repeated experiments can be carried out at different times and places; for example, when treating certain urgent diseases, pre-frozen mesenchymal stem cells can be quickly thawed and put into use. Although mesenchymal stem cells can be preserved by freezing, there are also some disadvantages in the freezing process, such as low cell survival rate after thawing, poor preservation of cell function, and long cell function recovery time.

[0010] In order to reduce the damage caused by ice crystals to cells during the freezing process and improve the survival rate of cells, the prior art often uses cryoprotectants to lower the freezing point of the solution and reduce the formation of ice crystals. However, high concentrations of cryoprotectants can cause irreversible damage to cells. At the same time, the more cryoprotectants are used, the more times the cryoprotectants need to be diluted during subsequent thawing, and the operation is more cumbersome. Based on this, the applicant hopes to increase the survival rate of cells as much as possible while reducing the amount of cryoprotectants used, and thus improves the cryopreservation method.

[0011] During the freezing process, when the cell suspension is partially frozen, the solution concentration of the unfrozen part outside the cell increases, which easily leads to intracellular dehydration, thereby affecting the survival rate of the cell. After the cryoprotectant lowers the freezing point, at the same temperature, the proportion of the unfrozen solution increases, thereby reducing the amount of cell dehydration, but as can be seen from the above, the applicant expects to reduce the amount of cryoprotectant used, thus, the present invention adds silk staple fibers to the cell suspension, and the surface or interior of the silk staple fibers can be used as heterogeneous nucleation points to provide a place for solute molecules to attach and aggregate, and its charged functional groups and physical adsorption can also increase the precipitation of solutes. As the temperature decreases, the silk protein molecules in the silk staple fibers will undergo a certain degree of structural reorganization, and some β-folded structures will be transformed into a more disordered amorphous state. This structural change causes more active sites to be exposed, thereby enhancing the adsorption capacity of silk staple fibers to solutes. At low temperatures, a stronger hydrogen bond network can be formed between the silk protein rich in polar groups and specific solutes to improve adsorption efficiency. In simple terms, the present invention hopes to make the solutes in the solution more easily precipitated at low temperatures by using silk staple fibers, so that during the freezing process, the concentration of the solution in the unfrozen portion is lower than that of the cell suspension without the addition of silk staple fibers, and the dehydration of the cells is weakened, thereby reducing the amount of cryoprotectant used to a certain extent. At the same time, conventional silk production processes will undergo a degumming process to remove the sericin remaining on its surface. The present invention uses silk staple fibers without the sericin removed, which can not only reduce the price of raw materials but also directly utilize the sericin on the fiber surface to achieve the solute precipitation function in the aforementioned freezing process, thereby reducing the osmotic pressure of the unfrozen portion of the solution.

[0012] Preferably, the freezing operation in S200 includes the following steps:

[0013] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25℃~20℃ to 0~-5℃, with a cooling rate of 2-3℃ / min, and keep warm for 45-60min;

[0014] A200, the freezing environment continues to cool down to -20~-30℃, the cell suspension is stirred during the cooling period, and the cooling rate is 1-2℃ / min;

[0015] A300, the freezing environment is cooled to -40°C to -80°C and then the cell suspension is kept warm, and the cooling rate is 5-8°C / min;

[0016] During the operation of A200, sericin is partially or completely precipitated.

[0017] The precipitation of solutes will essentially increase the freezing point of the solution to a certain extent, thereby reducing the amount of unfrozen solution under the same temperature conditions. Therefore, after adding silk staple fibers, whether the cell survival rate is increased or decreased is controllable. It is at least necessary to ensure that the amount of solute precipitated per unit volume of solution by silk staple fibers is greater than the increase in solute per unit volume of solution after the freezing point is increased. Therefore, the applicant verified the results under different operating conditions through experiments, and then clarified the freezing operation process and parameters to improve the cell survival rate by adding silk staple fibers.

[0018] During the freezing operation, if the temperature drops rapidly in the initial stage, in addition to affecting the cells, it will also affect the structural changes of the silk staple fibers, affect the effect of promoting the precipitation of solutes, etc. Therefore, the freezing operation of the present invention should not only consider the impact on the cells, but also the impact on the performance of the silk staple fibers. During the A100 operation, by controlling the ambient temperature, cooling speed and heat preservation, the outflow of water in the cells can be reduced, the adaptability of the cells to the low temperature environment can be improved, the impact of the freezing operation on the compatibility of the silk staple fibers and cells can be reduced, the support of the silk staple fibers to the cells can be improved, and the excessive reduction of the toughness or structural damage of the silk staple fibers can be avoided, which affects the protective effect of the silk staple fibers on the cells. During the A200 operation, ice crystals begin to form, and slow cooling can reduce the formation of ice crystals and inhibit the growth of ice crystals. In this process, sericin begins to precipitate to balance the concentration changes of the extracellular solution. After the sericin is precipitated at low temperature, it can also form a highly viscous body, further protecting the cells and reducing the mechanical damage to the cells. In order to increase the uniformity of the concentration of the solution system, the cell suspension is stirred during the cooling period. Stirring can also increase the uniform dispersion of silk staple fibers and ice bodies and prevent single ice bodies from being too large to a certain extent, thereby avoiding excessive dehydration of cells during A200, and reducing the damage of ice crystal formation to the silk structure and cell structure. Since the ice bodies and silk staple fibers in the solution are evenly dispersed during the stirring process, and the movement of silk staple fibers is hindered by the hindrance of the ice bodies, the concentration of the unfrozen solution is also high at this time. In addition, the highly viscous sericin enhances the suspension ability of silk staple fibers. Therefore, during the operation of A300, the silk staple fibers can be evenly dispersed in the solution system without additional operation, and the protection effect on cells is better. At this time, the ambient temperature is lowered again to further reduce cell activity and completely freeze the cell suspension.

[0019] Preferably, the permeable cryoprotectant comprises glycerol.

[0020] Glycerol and dimethyl sulfoxide are two common cryoprotectants. The reason why glycerol is selected in the present invention is that sericin has poor solubility at room temperature. Glycerol can be used as a cosolvent, which will not affect the state of silk fibroin, and can improve the solubility of sericin while ensuring that it can be precipitated during the freezing process, so as to utilize the characteristics of sericin to reduce the damage to cells caused by the freezing operation.

[0021] Preferably, the osmotic cryoprotectant accounts for 4 wt%-6 wt% of the cryopreservation solution.

[0022] After improving the cryopreservation liquid components and the freezing operation, the present invention can appropriately reduce the amount of cryoprotectant used, reduce the influence of the cryopreservation agent on cells and simplify the thawing operation when taking cells.

[0023] Preferably, the silk staple fibers account for 15wt%-20wt% of the cryopreservation solution, and the number of cells in the cell suspension is 3×10 6 -5×10 6 cells / ml.

[0024] The amount of silk staple fibers affects the amount of solutes precipitated in A200 and its protective effect on cells. However, if the amount is too high, it may lead to a reduction in the amount of cell preservation and water absorption by cells during the freezing process. The cell structure may be damaged, and ice crystals are more likely to form in the intracellular fluid. Therefore, it is necessary to control the amount of silk staple fibers and the number of cells at the same time. At the same time, if the amount of silk staple fibers is too high, the proportion of sericin in the cell suspension will increase, thereby increasing the viscosity of the cell suspension, which is not convenient for subsequent packaging operations. In addition, too high a cell density will also increase the possibility of mechanical damage.

[0025] Preferably, the length of the silk staple fibers is 2-5 mm.

[0026] The applicant discovered during the experiment that the length of the fiber affects the survival rate of cells after thawing, and obtained through experiments that the optimal length range of the fiber is 2-5 mm. The applicant speculates that the reason why the fiber length affects the cell survival rate is that if the length of the silk staple fiber is too short, it will provide more nucleation sites for the formation of ice crystals, promoting the formation of ice crystals. At the same time, too short fibers have limited protective effects on cells. In addition, due to the conventional specification restrictions of cell cold storage tubes, the present invention also limits the maximum length of the silk staple fiber for use in actual operations.

[0027] Preferably, the stirring rate in the A200 is 10-20 r / min.

[0028] The applicant found that the stirring rate significantly affects the survival rate of cells. The applicant speculates that the reason is that a stirring rate that is too low will affect the dispersion of silk staple fibers and hinder the growth of ice crystals, while a stirring rate that is too high will easily damage the cells.

[0029] Preferably, the operation of S100 further includes: before using the cryopreservation solution, adding protease to partially hydrolyze sericin; the silk staple fibers account for 18wt%-20wt% of the cryopreservation solution.

[0030] When the applicant used silk staple fibers completely freed of sericin for cell protection and to provide cell attachment sites, the optimal amount of silk staple fibers was found to be D1, which was higher than that of silk staple fibers with retained sericin. Therefore, the applicant considered whether the amount of silk staple fibers could be further increased to improve its protective effect on cells while ensuring the function of sericin in the present invention. The applicant also conducted an experiment using only sericin powder instead of silk staple fibers, and found that the optimal amount of sericin was less than the corresponding sericin when D1 silk staple fibers were used. Therefore, the applicant considered whether the technical solution of the present invention could be further improved while still using raw materials with retained sericin on the outer surface.

[0031] The technical solution conceived by the applicant is as follows: after adding protease to the cell suspension, sericin can be partially hydrolyzed to convert it into amino acids, which are not easy to precipitate during the freezing process. At this time, the amount of silk staple fibers can be increased to the optimum, while the amount of sericin that maintains the protein structure will not exceed the optimum amount. Finally, under the verification of the experiment, it was proved that the applicant's improved solution can improve the survival rate of mesenchymal stem cells after cryopreservation. The protease can be α-chymotrypsin, papain or cocoon enzyme.

[0032] Preferably, the dosage of protease is 0.08-0.15 mg / ml, the enzymolysis temperature is 20-25° C., and the enzymolysis time is 1-3 h.

[0033] The unit ml refers to the volume of the cell suspension. Due to the presence of cells in the cell suspension, it is difficult to increase the enzymatic hydrolysis rate by increasing the enzymatic hydrolysis temperature in the present invention, and thus the enzymatic hydrolysis time is correspondingly increased to achieve the purpose of hydrolyzing the required amount of sericin.

[0034] Preferably, when the cell suspension is used again, it is thawed in a constant temperature water bath at a temperature of 35-38°C.

[0035] The present invention has at least the following beneficial effects:

[0036] The present invention not only protects cells and provides attachment sites for cells by adding silk staple fibers containing sericin to the cell suspension, but also promotes the precipitation of solutes by utilizing the structure of silk fibroin under low temperature, and the solute is directly sericin. The precipitation of solutes can reduce the osmotic pressure difference between the inside and outside of cells during freezing, thereby improving the survival rate of cells. At the same time, sericin can also be used to protect cells and increase the suspension ability of fibers during freezing, thereby reducing aggregation. DETAILED DESCRIPTION

[0037] In order to make the purpose, method scheme and advantages of the embodiments of the present invention clearer, the method scheme in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0038] Example 1: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0039] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 4 wt% of the cryopreservation solution; the silk staple fibers account for 15 wt% of the cryopreservation solution; the number of cells in the cell suspension is 3×10 6 cells / ml. The length of silk staple fiber is 2mm.

[0040] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0041] The freezing operation in S200 includes the following steps:

[0042] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25°C to 0°C at a cooling rate of 2°C / min and keep warm for 45 minutes;

[0043] A200, the freezing environment continues to cool down to -20°C, the cell suspension is stirred during the cooling period, and the cooling rate is 1°C / min; the stirring rate in A200 is 10r / min;

[0044] A300, the freezing environment is cooled to -40°C and then the cell suspension is kept warm, and the cooling rate is 5°C / min;

[0045] During the operation of A200, sericin is partially or completely precipitated.

[0046] Example 2: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0047] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 6wt% of the cryopreservation solution; the silk staple fibers account for 20wt% of the cryopreservation solution; the number of cells in the cell suspension is 5×10 6 cells / ml. The length of silk staple fiber is 5mm.

[0048] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0049] The freezing operation in S200 includes the following steps:

[0050] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 20°C to -5°C at a cooling rate of 3°C / min and keep warm for 60min;

[0051] A200, the freezing environment continues to cool down to -30°C, the cell suspension is stirred during the cooling period, and the cooling rate is 2°C / min; the stirring rate in A200 is 20r / min;

[0052] A300, the freezing environment is cooled to -80°C and then the cell suspension is kept warm, and the cooling rate is 8°C / min;

[0053] During the operation of A200, sericin is partially or completely precipitated.

[0054] Example 3: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0055] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 5wt% of the cryopreservation solution; the silk staple fibers account for 18wt% of the cryopreservation solution; the number of cells in the cell suspension is 4×10 6 cells / ml. The length of silk staple fiber is 3mm.

[0056] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0057] The freezing operation in S200 includes the following steps:

[0058] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25°C to -5°C at a cooling rate of 2.5°C / min and keep warm for 60 minutes;

[0059] A200, the freezing environment continues to cool down to -25°C, the cell suspension is stirred during the cooling period, and the cooling rate is 1.5°C / min; the stirring rate in A200 is 15r / min;

[0060] A300, the freezing environment is cooled to -80°C and then the cell suspension is kept warm, and the cooling rate is 6°C / min;

[0061] During the operation of A200, sericin is partially or completely precipitated.

[0062] Example 4: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0063] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 5wt% of the cryopreservation solution; the silk staple fibers account for 20wt% of the cryopreservation solution; the number of cells in the cell suspension is 4×10 6 cells / ml. The length of silk staple fiber is 3mm.

[0064] The operation of S100 also includes: before using the cryopreservation solution, adding papain to partially hydrolyze the sericin, wherein the amount of papain used is 0.08 mg / ml, the enzymolysis temperature is 20° C., and the enzymolysis time is 1 hour.

[0065] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0066] The freezing operation in S200 includes the following steps:

[0067] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25°C to -5°C at a cooling rate of 2.5°C / min and keep warm for 60 minutes;

[0068] A200, the freezing environment continues to cool down to -25°C, the cell suspension is stirred during the cooling period, and the cooling rate is 1.5°C / min; the stirring rate in A200 is 15r / min;

[0069] A300, the freezing environment is cooled to -80°C and then the cell suspension is kept warm, and the cooling rate is 6°C / min;

[0070] During the operation of A200, sericin is partially or completely precipitated.

[0071] Example 5: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0072] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 5wt% of the cryopreservation solution; the silk staple fibers account for 20wt% of the cryopreservation solution; the number of cells in the cell suspension is 4×10 6 cells / ml. The length of silk staple fiber is 3mm.

[0073] The operation of S100 also includes: before using the cryopreservation solution, adding papain to partially hydrolyze the sericin, wherein the amount of papain used is 0.15 mg / ml, the enzymolysis temperature is 25° C., and the enzymolysis time is 3 hours.

[0074] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0075] The freezing operation in S200 includes the following steps:

[0076] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25°C to -5°C at a cooling rate of 2.5°C / min and keep warm for 60 minutes;

[0077] A200, the freezing environment continues to cool down to -25°C, the cell suspension is stirred during the cooling period, and the cooling rate is 1.5°C / min; the stirring rate in A200 is 15r / min;

[0078] A300, the freezing environment is cooled to -80°C and then the cell suspension is kept warm, and the cooling rate is 6°C / min;

[0079] During the operation of A200, sericin is partially or completely precipitated.

[0080] Example 6: A cryopreservation method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0081] S100, umbilical cord mesenchymal stem cells are resuspended in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: M16 nutrient solution, glycerol and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; glycerol accounts for 5wt% of the cryopreservation solution; the silk staple fibers account for 20wt% of the cryopreservation solution; the number of cells in the cell suspension is 4×106 cells / ml. The length of silk staple fiber is 3mm.

[0082] The operation of S100 also includes: before using the cryopreservation solution, adding papain to partially hydrolyze the sericin, wherein the amount of papain used is 0.1 mg / ml, the enzymolysis temperature is 25° C., and the enzymolysis time is 2 hours.

[0083] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0084] The freezing operation in S200 includes the following steps:

[0085] A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25°C to -5°C at a cooling rate of 2.5°C / min and keep warm for 60 minutes;

[0086] A200, the freezing environment continues to cool down to -25°C, the cell suspension is stirred during the cooling period, and the cooling rate is 1.5°C / min; the stirring rate in A200 is 15r / min;

[0087] A300, the freezing environment is cooled to -80°C and then the cell suspension is kept warm, and the cooling rate is 6°C / min;

[0088] During the operation of A200, sericin is partially or completely precipitated.

[0089] Comparative Example 1: The difference from Example 6 is that: silk staple fibers with sericin removed are used.

[0090] Comparative Example 2: The difference from Example 6 is that there is no stirring operation in A200.

[0091] Comparative Example 3: The difference from Example 6 is that dimethyl sulfoxide is used as the permeable cryoprotectant.

[0092] Comparative Example 4: The difference from Example 6 is that the freezing preservation solution does not contain silk staple fibers.

[0093] Comparative Example 5: The difference from Example 6 is that the cryopreservation solution does not contain silk staple fibers and glycerol accounts for 10 wt % of the cryopreservation solution.

[0094] Comparative Example 6: The difference from Example 6 is that the silk staple fibers account for 18 wt % of the cryopreservation solution.

[0095] Test: After the umbilical cord mesenchymal stem cells were stored according to the cryopreservation methods provided in Examples 1-6 and Comparative Examples 1-5, samples were taken after 20 days, and the cell viability was determined by the trypan blue exclusion test. After five groups of tests were performed for each storage method, the test data were averaged. When sampling, the cold storage tube containing the cell suspension was taken out and thawed in a constant temperature water bath at a temperature of 35-38°C. During the water bath, the cold storage tube was shaken. The test results are shown in Table 1.

[0096] Table 1

[0097] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Survival rate (%) 83.6 85.1 86.0 92.4 93.3 96.7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Survival rate (%) 69.4 77.9 74.2 62.8 92.6 87.3

[0098] It can be seen from the experimental results of Examples 1-6 that after mesenchymal stem cells are preserved by the cryopreservation method provided by the present invention, the cells have a good survival rate when they are taken out.

[0099] From the comparison between Example 3 and Examples 4-6, it can be seen that the survival rate of cells can be improved after the cell suspension is partially hydrolyzed with sericin and the amount of silk staple fibers is increased.

[0100] From the comparison between Example 6 and Comparative Example 6, it can be seen that after the cell suspension partially hydrolyzes the sericin, the optimal amount of the silk staple fibers increases.

[0101] From the comparison between Comparative Examples 1-4 and Example 6, it can be seen that the retention of sericin, the stirring operation in A200, the selection of osmotic cryoprotectants, and the addition of silk staple fibers in the cryopreservation solution can all improve the survival rate of mesenchymal stem cells after freezing.

[0102] From the comparison between Comparative Example 5 and Examples 4-6, it can be seen that after reducing the amount of cryopreservative used in the present invention, the cell survival rate is still comparable to or even higher than that of conventional technologies.

[0103] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cryopreservation method for improving the survival rate of mesenchymal stem cells, characterized in that: The following steps are involved: S100, resuspending the mesenchymal stem cells in a cryopreservation solution to obtain a cell suspension; the cryopreservation solution comprises: a permeable cryoprotectant and silk staple fibers; sericin is retained on the outer surface of the silk staple fiber raw material; S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

2. The cryopreservation method according to claim 1, characterized in that The freezing operation in S200 includes the following steps: A100, after placing the cell suspension in a freezing environment, cool the freezing environment from 25℃~20℃ to 0~-5℃, with a cooling rate of 2-3℃ / min, and keep warm for 45-60min; A200, the freezing environment continues to cool down to -20~-30℃, the cell suspension is stirred during the cooling period, and the cooling rate is 1-2℃ / min; A300, the freezing environment is cooled to -40°C to -80°C and then the cell suspension is kept warm, and the cooling rate is 5-8°C / min; During the operation of A200, sericin is partially or completely precipitated.

3. The cryopreservation method according to claim 1, characterized in that The permeating cryoprotectant includes glycerol.

4. The cryopreservation method according to claim 1, characterized in that: The permeable cryoprotectant accounts for 4 wt%-6 wt% of the cryopreservation solution.

5. The cryopreservation method according to claim 1, characterized in that: The silk staple fibers account for 15wt%-20wt% of the cryopreservation solution, and the number of cells in the cell suspension is 3×10 6 -5×10 6 cells / ml.

6. The cryopreservation method according to claim 1, characterized in that: The length of the silk staple fibers is 2-5 mm.

7. The cryopreservation method according to claim 1, characterized in that: The stirring rate in the A200 is 10-20 r / min.

8. The cryopreservation method according to any one of claims 1 to 7, characterized in that: The operation of S100 also includes: before using the cryopreservation solution, adding protease to partially hydrolyze sericin; the silk staple fibers account for 18wt%-20wt% of the cryopreservation solution.

9. The cryopreservation method according to claim 8, characterized in that: The dosage of protease is 0.08-0.15 mg / ml, the enzymolysis temperature is 20-25°C, and the enzymolysis time is 1-3 h.

10. The cryopreservation method according to claim 8, characterized in that: When the cell suspension is used again, it is thawed in a constant temperature water bath at a temperature of 35-38°C.