Cryopreservation liquid for mesenchymal stem cells and use method of cryopreservation liquid

Abstract

The invention relates to the field of cryopreservation of cells, and provides a cryopreservation solution for mesenchymal stem cells in order to solve the problem that the survival rate of the cells is obviously reduced after the use amount of a cryoprotectant in the existing cryopreservation solution is reduced, and the cryopreservation solution for the mesenchymal stem cells comprises the following components: glycerol, silk short fibers and a buffer solution, the glycerol accounts for 4wt%-6wt% of the cryopreservation solution, the silk short fibers account for 15wt%-20wt% of the cryopreservation solution, and the balance is the buffer solution; sericin is reserved on the outer surfaces of the silk short fibers; the sericin accounts for 15 to 35 weight percent of the silk short fibers. After the silk short fibers with sericin are added into the cryopreservation liquid, 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 in a low-temperature environment, the solute is directly the sericin, and by means of separation of the solute, the content of the sericin in the freezing process can be reduced. Meanwhile, sericin can be used for protecting the cells, the suspension capacity of fibers in the freezing process is improved, and aggregation is reduced.

Description

Technical Field

[0001] The present invention relates to the field of cryopreservation of cells, and more particularly, to a cryopreservation solution for mesenchymal stem cells and a method for using the same. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells with the ability of self-renewal and differentiation into multiple cell types. Due to their broad 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. Currently, the preservation of cells mainly relies on cryoprotectants, such as dimethyl sulfoxide or glycerol, which slow down the rate of biochemical reactions at low temperatures, thereby preventing the formation of destructive ice crystals in the cells and maintaining the integrity of the cell structure. However, cryoprotectants will cause irreversible damage to cells at high concentrations, and at the same time, the use amount of cryoprotectants will also be limited for some cells that are more sensitive to cryoprotectants. Under the limitation of the use amount of cryoprotectants, 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 solution with a high cell survival rate while reducing the use amount of cryoprotectants. Summary of the Invention

[0004] The purpose of the present invention is to provide a cryopreservation solution for mesenchymal stem cells, which solves the problem that the cell survival rate is significantly reduced after reducing the use amount of cryoprotectants in the existing cryopreservation solution.

[0005] Another purpose of the present invention is to provide a method for using a cryopreservation solution for mesenchymal stem cells, which can quickly separate cells from short silk fibers at a lower centrifugation speed, reducing the damage to cells caused by high-speed centrifugation and long-time dilution.

[0006] The embodiments of the present invention are achieved by the following technical solutions:

[0007] A cryopreservation solution for mesenchymal stem cells, comprising components: glycerol, short silk fibers, and a buffer solution; the glycerol accounts for 4wt%-6wt% of the cryopreservation solution, the short silk fibers account for 15wt%-20wt% of the cryopreservation solution, and the balance is the buffer solution; sericin protein remains on the outer surface of the short silk fibers; the sericin protein accounts for 15wt%-35wt% of the short silk fibers.

[0008] The preservation of mesenchymal stem cells is required for their applications in scientific research, industry, and clinical settings. After preservation, it is not only convenient for transportation but also enables the cells to be in a nearly stationary state at low temperatures. When they are needed in the future, the cells can be revived, and the research and application of them are no longer restricted by time. For example, in scientific research, after freezing mesenchymal stem cells from the same source, repeated experiments can be conducted at different times and locations; another example is in the treatment of certain urgent diseases, where pre-frozen mesenchymal stem cells can be quickly thawed and used. Although mesenchymal stem cells can be preserved by freezing, there are still some drawbacks during the freezing process, such as low cell survival rate after thawing, poor preservation of cell functions, and a long time for cell function recovery, etc.

[0009] In order to reduce the damage caused by ice crystals to cells during freezing and improve the cell survival rate, 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 operation of diluting the cryoprotectant is required during subsequent thawing, and the operation is more cumbersome. Based on this, the applicant hopes to improve the cell survival rate as much as possible while reducing the amount of cryoprotectant used, and thus has improved the cryopreservation solution.

[0010] During the freezing process, when a part of the cell suspension freezes, the concentration of the solution in the unfrozen part outside the cells increases, which easily leads to dehydration inside the cells and thus affects the cell survival rate. After the cryoprotectant reduces the freezing point, at the same temperature, the proportion of the unfrozen solution part increases, and thus the amount of cell dehydration can be reduced. However, as can be seen from the above, the applicant hopes to reduce the usage amount of the cryoprotectant. Therefore, short silk fibers are added to the cryopreservation solution in the present invention. The surface or inside of the short silk fibers can serve as heterogeneous nucleation sites, providing a place for solute molecules to attach and aggregate. Their charged functional groups and physical adsorption effects can also increase the precipitation of solutes. As the temperature decreases, the silk fibroin molecules in the short silk fibers will undergo a certain degree of structural reorganization, and part of the β-sheet structure will transform into a more disordered amorphous state. This structural change leads to the exposure of more active sites, thereby enhancing the adsorption ability of the short silk fibers to solutes. At low temperatures, stronger hydrogen bond networks can be formed between the silk fibroin rich in polar groups and specific solutes, improving the adsorption efficiency. Simply put, the present invention hopes that through the short silk fibers, the solutes in the solution are more likely to precipitate at low temperatures. Thus, during the freezing process, the concentration of the solution in the unfrozen part is lower than that of the cell suspension without adding short silk fibers, the dehydration of the cells is weakened, and thus the usage amount of the cryoprotectant can be reduced to a certain extent. At the same time, conventionally, silk will undergo a degumming process during production to remove the sericin protein remaining on its surface. After the present invention uses short silk fibers without removing the sericin protein, not only can the raw material price be reduced, but also the sericin protein on the fiber surface can be directly utilized to achieve the function of solute precipitation during the aforementioned freezing process, thereby reducing the osmotic pressure of the solution in the unfrozen part.

[0011] Glycerol and dimethyl sulfoxide are two common cryoprotectants. The reason for the present invention to choose glycerol is also that the solubility of sericin protein at room temperature is poor. Glycerol can be used as a cosolvent, which will neither affect the state of silk fibroin nor improve the solubility of sericin protein on the premise of ensuring its precipitation during the freezing process, so as to utilize the characteristics of sericin protein to reduce the damage of freezing operation to cells.

[0012] After the present invention improves the components of the cryopreservation solution and the freezing operation, the usage amount of the cryoprotectant can be appropriately reduced, the influence of the cryopreservation agent on cells can be reduced, and the thawing operation during cell retrieval can be simplified.

[0013] The usage amount of the short silk fibers affects the precipitation amount of sericin protein and also affects its protective effect on cells. However, if the usage amount is too high, it may lead to a reduction in the amount of cells preserved and water absorption by the cells during the freezing process. The cell structure may be damaged, and at the same time, ice crystals are more likely to form in the intracellular liquid. Therefore, it is necessary to control both the usage amount of the short silk fibers and the number of cells simultaneously. At the same time, if the usage amount of the short silk fibers is too high, it will also lead to an increase in the proportion of sericin protein in the cell suspension, thereby increasing the viscosity of the cell suspension and making it inconvenient for subsequent subpackaging operations.

[0014] Preferably, the length of the short silk fibers is 2 mm - 5 mm.

[0015] During the experiment, the applicant found that the length of the fibers affects the survival rate of cells after thawing, and through experiments, the optimal length range of the fibers was obtained as 2 - 5 mm. The applicant conjectures that the reason why the fiber length affects the cell survival rate is that if the length of the short silk fibers is too short, it will provide more nucleation sites for the formation of ice crystals and promote the formation of ice crystals. At the same time, the protection of cells by too short fibers is limited. In addition, due to the conventional specifications of cell cryotubes, the maximum length of the short silk fibers in the present invention is also limited for use in actual operations.

[0016] Preferably, the buffer solution includes: a main solution and polyethylene glycol. The main solution includes: one or more of PBS solution, HEPES solution, and TBS solution. The dosage of polyethylene glycol is 1 vt% - 3 vt%.

[0017] The main use of the main solution is to maintain the pH stability and osmotic pressure of cells during freezing and thawing, protect cells from damage caused by pH changes, and ensure the ion balance inside and outside cells. In addition to adjusting the osmotic pressure inside and outside cells, polyethylene glycol can also improve the uniform distribution of glycerol and has a certain cryoprotective effect. The main purpose of adding polyethylene glycol in the present invention is to improve the dispersibility of short silk fibers, reduce the aggregation of short silk fibers during use, improve the dissolution efficiency of sericin before freezing, and facilitate the removal of short silk fibers during dilution operation.

[0018] Preferably, a method for using a cryopreservation solution includes the following steps:

[0019] S100. The mesenchymal stem cells are resuspended with the cryopreservation solution to obtain a cell suspension;

[0020] S200. The cell suspension is cryopreserved to preserve the cells; during the cryopreservation operation, part or all of the sericin precipitates;

[0021] The dilution operation after thawing when taking cells includes the following steps:

[0022] A100. Transfer the thawed cell suspension to a preheated first culture medium, centrifuge to obtain a cell pellet. The first culture medium includes: a conventional culture solution, calcium chloride, and dithiothreitol. The dosage of calcium chloride is 2.5 mM - 3.5 mM, and the dosage of dithiothreitol is 0.5 mM - 1.5 mM;

[0023] A200. Transfer the cell pellet to a preheated second culture medium, centrifuge to obtain a cell pellet;

[0024] After resuspending the cell pellet, a cell suspension with completed dilution is obtained.

[0025] The main purpose of the dilution operation is to remove glycerol, reduce its toxicity to cells, and restore the osmotic pressure balance inside and outside the cells, enabling the cell environment to gradually transition to normal culture conditions. Since the amount of glycerol used in the cryopreservation solution provided by the present invention is small, the dilution operation only needs to be carried out twice. When the cells are just thawed, they are relatively fragile, so the centrifugation speed should not be too fast to avoid damaging the cells. However, to completely separate the cells from the short silk fibers, a higher centrifugal force or a longer centrifugation time is required. A longer centrifugation time will increase the influence time of the solution on the cells and reduce the cell survival rate. Therefore, the present invention additionally adds calcium chloride and dithiothreitol to the first culture medium, causing protein structure changes and reducing the binding force between the short silk fibers and the cells, so as to reduce the dilution and separation time. Although using protease to degrade fibrin can also separate the cells from the short silk fibers, the degradation time is relatively long, the influence time of the solution on the cells increases, and the final cell survival rate is reduced. Therefore, the applicant selects the technical solution of protein denaturation to reduce the adhesion between the protein and the cells by changing the protein structure. In addition, after treatment with calcium chloride and dithiothreitol, the fiber structure becomes more loose and is more likely to float to the supernatant, facilitating the removal of the fibers. Dithiothreitol can also reduce the damage to cells caused by excessive oxidation. During resuspension, a conventional culture medium can be used, and the conventional culture medium can be M16 culture medium.

[0026] Preferably, the centrifugation speed in steps A100 and A200 is 800 rpm - 1000 rpm.

[0027] Preferably, the second culture medium includes: a conventional culture medium and glutathione, and the dosage of the glutathione is 4 mg / L - 6 mg / L.

[0028] Glutathione can scavenge free radicals, reduce the stress damage of cells, and also has a detoxification function, helping cells to recover normal physiological functions faster. In addition, glutathione can also maintain the structure and function of proteins.

[0029] Preferably, the freezing operation in S200 includes the following steps:

[0030] B100. After placing the cell suspension in a freezing environment, the freezing environment is cooled from 25°C to 20°C to 0°C to -5°C, and the cooling rate is 2 - 3°C / min, and keep warm for 45 - 60 min;

[0031] B200. The freezing environment continues to cool to -20°C to -30°C, and the cell suspension is stirred during the cooling process, and the cooling rate is 1 - 2°C / min;

[0032] B300, cool the cell suspension to -40°C to -80°C and then store it under insulation. The cooling rate is 5 - 8°C / min;

[0033] Among them, during the operation of B200, sericin partially or completely precipitates.

[0034] The precipitation of solutes will essentially increase the freezing point of the solution to a certain extent, and then reduce the amount of the unfrozen part of the solution under the same temperature condition. Therefore, after adding short silk fibers, whether the survival rate of the cells increases or decreases is controllable. It is at least necessary to ensure that the amount of solutes precipitated in the solution per unit volume by the short silk fibers is greater than the increase in the amount of solutes in the solution per unit volume after the freezing point is increased. Therefore, the applicant verifies the results under different operating conditions through experiments, and then clarifies the freezing operation process and parameters for improving the survival rate of cells by adding short silk fibers.

[0035] During the freezing operation process, if the temperature drops rapidly in the initial stage, it will not only affect the cells, but also affect the structural changes of the short silk fibers and their effect of promoting solute precipitation. Therefore, the freezing operation of the present invention needs to consider not only the impact on the cells, but also the impact on the performance of the short silk fibers. During the operation of A100, by controlling the environmental temperature, cooling rate and insulation, the outflow of intracellular water can be reduced, the adaptability of the cells to the low-temperature environment can be improved, the impact of the freezing operation on the cell compatibility of the short silk fibers can be reduced, the supporting effect of the short silk fibers on the cells can be improved, and the over-reduction of the toughness or structural damage of the short silk fibers can be avoided, which may affect the protective effect of the short silk fibers on the cells. During the operation of A200, ice crystals begin to form. Slow cooling can reduce the formation of ice crystals and inhibit the growth of ice crystals. During this process, sericin begins to precipitate to balance the concentration change of the extracellular solution. After sericin precipitates at low temperature, it can also form a highly viscous state to further protect the cells and reduce the mechanical damage suffered by the cells. In order to increase the uniformity of the solution system concentration, the cell suspension is stirred during the cooling period. Stirring can also increase the uniform dispersion of the short silk fibers and the ice crystals and a certain degree of hindrance to the excessive growth of single ice crystals, thereby avoiding excessive dehydration of the cells during A200 and reducing the damage of ice crystal formation to the silk structure and cell structure. Since the ice crystals and short silk fibers in the solution are evenly dispersed during stirring, and the movement of the short silk fibers is blocked by the ice crystals, the concentration of the unfrozen solution is relatively high at this time, plus the highly viscous sericin, the suspension ability of the short silk fibers is enhanced. Therefore, during the operation of A300, the short silk fibers can be evenly dispersed in the solution system without additional operation, and the protective effect on the cells is better. At this time, the environmental temperature is further reduced to further reduce the cell activity and make the cell suspension completely frozen.

[0036] Preferably, the stirring rate in A200 is 10 - 20 r / min.

[0037] The applicant found that the stirring rate significantly affects the cell viability. The applicant conjectured that the reason is that too low a stirring rate will affect the dispersion effect of the short silk fibers and the effect of hindering ice crystal growth, but too high a stirring rate is likely to cause damage to the cells.

[0038] Preferably, the operation of S100 further includes: before using the cryopreservation solution, adding protease first to partially hydrolyze sericin; the short silk fibers account for 18wt%-20wt% of the cryopreservation solution.

[0039] When the applicant used short silk fibers with completely removed sericin for cell protection and providing cell attachment sites, the best dosage of the short silk fibers was found to be D1 parts in the experiment, which is higher than that when using short silk fibers with remaining sericin. Thus, the applicant wondered whether, on the premise of ensuring the function of sericin in the present invention, the dosage of the short silk fibers could be further increased to improve its protective effect on cells. The applicant also conducted an experiment using only sericin powder without using short silk fibers and found that the best dosage of sericin is less than that corresponding to using D1 parts of short silk fibers. Therefore, the applicant wondered whether the technical solution of the present invention could be further improved on the premise of still using raw materials with sericin remaining on the outer surface.

[0040] 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 then not easily precipitated during the freezing process. At this time, the dosage of the short silk fibers can be increased to the best, while the amount of sericin maintaining the protein structure does not exceed the best dosage. Finally, under the verification of the experiment, it was proved that the improved solution of the applicant can improve the viability of mesenchymal stem cells after cryopreservation. The protease can be α-chymotrypsin, papain or cocoonase.

[0041] Preferably, the dosage of the protease is 0.08 - 0.15mg / ml, the enzymolysis temperature is 20 - 25°C, and the enzymolysis time is 1 - 3h.

[0042] In 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 enzymolysis rate by increasing the enzymolysis temperature in the present invention. Therefore, the enzymolysis time is correspondingly increased to achieve the purpose of the required amount of sericin hydrolysis.

[0043] The present invention has at least the following beneficial effects:

[0044] After adding silk short fibers with sericin protein to the cryopreservation solution, the present invention can not only use the fibers to protect cells and provide cell attachment sites, but also utilize the sericin protein structure to promote the precipitation of solutes under low-temperature conditions. The solute is directly sericin protein. By the precipitation of solutes, the osmotic pressure difference inside and outside cells during freezing can be reduced, improving cell survival rate. At the same time, sericin protein can be used to protect cells and increase the suspension ability of fibers during freezing, reducing aggregation. When the present invention is diluted for the first time, calcium chloride and dithiothreitol are added, which can not only separate silk short fibers from cells at a low centrifugation speed, but also make the fiber structure looser, easier to float to the supernatant for removal. In addition, it can reduce the damage to cells caused by excessive oxidation. Detailed implementation manners

[0045] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention.

[0046] Embodiment 1: A cryopreservation solution for mesenchymal stem cells includes components: glycerol, silk short fibers and buffer solution; the glycerol accounts for 4 wt% of the cryopreservation solution, the silk short fibers account for 15 wt% of the cryopreservation solution, and the balance is the buffer solution; sericin protein remains on the outer surface of the silk short fibers; the sericin protein accounts for 15 wt% of the silk short fibers. The length of the silk short fibers is 2 mm. The buffer solution includes: PBS solution and polyethylene glycol, and the dosage of the polyethylene glycol is 1 vt%.

[0047] Embodiment 2: A cryopreservation solution for mesenchymal stem cells includes components: glycerol, silk short fibers and buffer solution; the glycerol accounts for 6 wt% of the cryopreservation solution, the silk short fibers account for 20 wt% of the cryopreservation solution, and the balance is the buffer solution; sericin protein remains on the outer surface of the silk short fibers; the sericin protein accounts for 35 wt% of the silk short fibers. The length of the silk short fibers is 5 mm. The buffer solution includes: PBS solution and polyethylene glycol, and the dosage of the polyethylene glycol is 1 vt% - 3 vt%.

[0048] Embodiment 3: A cryopreservation solution for mesenchymal stem cells includes components: glycerol, silk short fibers and buffer solution; the glycerol accounts for 5 wt% of the cryopreservation solution, the silk short fibers account for 18 wt% of the cryopreservation solution, and the balance is the buffer solution; sericin protein remains on the outer surface of the silk short fibers; the sericin protein accounts for 25 wt% of the silk short fibers. The length of the silk short fibers is 3 mm. The buffer solution includes: PBS solution and polyethylene glycol, and the dosage of the polyethylene glycol is 1 vt% - 3 vt%.

[0049] Example 4: A method for using a cryopreservation solution for mesenchymal stem cells, comprising the following steps:

[0050] S100, umbilical cord mesenchymal stem cells were resuspended in cryopreservation solution to obtain a cell suspension; the number of cells in the cell suspension was 4×10 6 cells / ml;

[0051] S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin is partially or completely precipitated.

[0052] The freezing operation in S200 includes the following steps:

[0053] B100, 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 45min;

[0054] B200, 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 B200 is 10r / min;

[0055] B300, the freezing environment is cooled to -40°C and then the cell suspension is kept warm at a cooling rate of 5°C / min;

[0056] Wherein, during the operation of B200, sericin is partially or completely precipitated;

[0057] When sampling, take out the 2 ml cold storage tube containing the cell suspension and thaw it in a constant temperature water bath at 37°C. During the water bath, shake the cold storage tube;

[0058] The dilution operation after thawing when taking cells for use includes the following steps:

[0059] A100, transferring the thawed cell suspension to 5 ml of the first culture medium preheated to 37° C., and obtaining a cell precipitate after centrifugation, wherein the first culture medium comprises: M16 culture medium, calcium chloride and dithiothreitol, wherein the amount of the calcium chloride is 2.5 mM, and the amount of the dithiothreitol is 0.5 mM; and the centrifugation speed is 800 rpm;

[0060] A200, transferring the cell pellet to 5 ml of the second culture medium preheated to 37°C, and obtaining the cell pellet after centrifugation; the second culture medium comprises: M16 culture medium and glutathione, wherein the amount of glutathione is 4 mg / L; the centrifugation speed is 800 rpm;

[0061] A300, after resuspending the cell pellet with M16 culture medium, a diluted cell suspension was obtained.

[0062] Example 5: A method for using a cryopreservation solution for mesenchymal stem cells, comprising the following steps:

[0063] S100. Umbilical cord mesenchymal stem cells are resuspended with the cryopreservation solution to obtain a cell suspension; the number of cells in the cell suspension is 4×10 6 cells / ml;

[0064] S200. The cell suspension is cryopreserved through a freezing operation; during the freezing operation, sericin partially or completely precipitates.

[0065] The freezing operation in S200 includes the following steps:

[0066] B100. After placing the cell suspension in a freezing environment, the freezing environment is cooled from 20°C to -5°C at a cooling rate of 3°C / min and incubated for 60 min;

[0067] B200. The freezing environment is further cooled to -30°C, and the cell suspension is stirred during the cooling period at a cooling rate of 2°C / min; the stirring rate in B200 is 20 r / min;

[0068] B300. After the freezing environment is cooled to -80°C, the cell suspension is incubated and preserved at a cooling rate of 8°C / min;

[0069] Among them, during the operation of B200, sericin partially or completely precipitates;

[0070] When sampling, after taking out the 2-ml cryotube containing the cell suspension, it is thawed in a constant-temperature water bath at 37°C, and the cryotube is shaken during the water bath process;

[0071] The dilution operation after thawing when the cells are taken includes the following steps:

[0072] A100. Transfer the thawed cell suspension to a 5-ml first culture medium preheated to 37°C, centrifuge to obtain a cell pellet, the first culture medium includes: M16 culture solution, calcium chloride and dithiothreitol, the dosage of calcium chloride is 3.5 mM, and the dosage of dithiothreitol is 1.5 mM; the centrifugation speed is 1000 rpm;

[0073] A200. Transfer the cell pellet to a 5-ml second culture medium preheated to 37°C, centrifuge to obtain a cell pellet; the second culture medium includes: M16 culture solution and glutathione, the dosage of glutathione is 6 mg / L; the centrifugation speed is 1000 rpm;

[0074] A300. After resuspending the cell pellet with M16 culture solution, a diluted cell suspension is obtained.

[0075] Example 6: A method for using a cryopreservation solution for mesenchymal stem cells, comprising the following steps:

[0076] S100. Umbilical cord mesenchymal stem cells are resuspended with the cryopreservation solution to obtain a cell suspension; the number of cells in the cell suspension is 4×10 6 cells / ml;

[0077] S200. The cell suspension is cryopreserved through a freezing operation; during the freezing operation, sericin partially or completely precipitates.

[0078] The freezing operation in S200 includes the following steps:

[0079] B100. After placing the cell suspension in a freezing environment, the freezing environment is cooled from 25°C to -5°C at a cooling rate of 2.5°C / min and incubated for 60 min;

[0080] B200. The freezing environment is further cooled to -25°C, and the cell suspension is stirred during the cooling period at a cooling rate of 1.5°C / min; the stirring rate in B200 is 15 r / min;

[0081] B300. After the freezing environment is cooled to -80°C, the cell suspension is incubated and stored, and the cooling rate is 6°C / min;

[0082] Among them, during the operation of B200, sericin partially or completely precipitates;

[0083] When sampling, after taking out the 2-ml cryotube containing the cell suspension, it is thawed in a constant-temperature water bath at 37°C, and the cryotube is shaken during the water bath process;

[0084] The dilution operation after thawing when the cells are taken out includes the following steps:

[0085] A100. The thawed cell suspension is transferred to a 5-ml first culture medium preheated to 37°C, and after centrifugation, a cell pellet is obtained. The first culture medium includes: M16 culture solution, calcium chloride, and dithiothreitol. The dosage of calcium chloride is 3 mM, and the dosage of dithiothreitol is 1 mM; the centrifugation speed is 1000 rpm;

[0086] A200. The cell pellet is transferred to a 5-ml second culture medium preheated to 37°C, and after centrifugation, a cell pellet is obtained. The second culture medium includes: M16 culture solution and glutathione. The dosage of glutathione is 5 mg / L; the centrifugation speed is 1000 rpm;

[0087] A300. After resuspending the cell pellet with M16 culture solution, a diluted cell suspension is obtained.

[0088] Example 7: The difference from Example 6 is that the short silk fibers account for 20 wt% of the cryopreservation solution, and on the basis of Example 6, the following operation is added in step S100: before the cryopreservation solution is used, papain is added first to partially hydrolyze sericin. The dosage of papain is 0.08 mg / ml, the enzymatic hydrolysis temperature is 20 °C, and the enzymatic hydrolysis time is 1 h.

[0089] Example 8: The difference from Example 6 is that the short silk fibers account for 20 wt% of the cryopreservation solution, and on the basis of Example 6, the operation of S100 further includes: before the cryopreservation solution is used, papain is added first to partially hydrolyze sericin. The dosage of papain is 0.15 mg / ml, the enzymatic hydrolysis temperature is 25 °C, and the enzymatic hydrolysis time is 3 h.

[0090] Example 9: The difference from Example 6 is that the short silk fibers account for 20 wt% of the cryopreservation solution, and on the basis of Example 6, the operation of S100 further includes: before the cryopreservation solution is used, papain is added first to partially hydrolyze sericin. The dosage of papain is 0.1 mg / ml, the enzymatic hydrolysis temperature is 25 °C, and the enzymatic hydrolysis time is 2 h.

[0091] Comparative Example 1: The difference from Example 9 is that the first culture medium only uses M16 culture solution.

[0092] Comparative Example 2: The difference from Example 9 is that dithiothreitol is not added to the first culture medium.

[0093] Comparative Example 3: The difference from Example 9 is that the second culture medium only uses M16 culture solution.

[0094] Comparative Example 4: The difference from Example 9 is that short silk fibers without sericin are used.

[0095] Comparative Example 5: The difference from Example 9 is that there is no stirring operation in A200.

[0096] Comparative Example 6: The difference from Example 9 is that the permeating cryoprotectant uses dimethyl sulfoxide.

[0097] Comparative Example 7: The difference from Example 9 is that the cryopreservation solution does not contain short silk fibers.

[0098] Comparative Example 8: The difference from Example 9 is that the cryopreservation solution does not contain short silk fibers and glycerol accounts for 10 wt% of the cryopreservation solution.

[0099] Comparative Example 9: The difference from Example 9 is that the short silk fibers account for 18 wt% of the cryopreservation solution.

[0100] Experiment 1: The cryopreservation solutions provided in Examples 1-3 were used to cryopreserve umbilical cord mesenchymal stem cells for 30 days according to the usage method provided in Example 4. After sampling, they were thawed and diluted, and finally, the trypan blue exclusion test was used to determine the cell survival rate. After five groups of tests were conducted for each cryopreservation solution, the test data were averaged. The measurement results are shown in Table 1.

[0101] Table 1

[0102] Example 1 Example 2 Example 3 Survival rate (%) 85.9 84.8 87.5

[0103] As can be seen from the results in Table 1, the best formulation ratio of the cryopreservation solution is the one provided in Example 3.

[0104] Experiment 2: The cryopreservation solution provided in Example 3 was used to cryopreserve umbilical cord mesenchymal stem cells for 30 days according to the usage methods provided in Examples 4-9 and Comparative Examples 1-9. After sampling, they were thawed and diluted, and finally, the trypan blue exclusion test was used to determine the cell survival rate. After five groups of tests were conducted for each cryopreservation solution, the test data were averaged. The measurement results are shown in Table 2.

[0105] Table 2

[0106] Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Survival rate (%) 87.5 89.4 90.1 94.0 95.6 97.2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Survival rate (%) 96.3 94.4 93.8 71.7 81.2 72.6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Survival rate (%) 64.1 95.1 92.5

[0107] As can be seen from the test results of Examples 4-9, after using the cryopreservation solution provided by the present invention in combination with the usage method, the survival rate of the cells increased significantly.

[0108] As can be seen from the comparison of the results of Examples 7-9 with those of Example 6, after the cell suspension partially hydrolyzes sericin and increases the dosage of short silk fibers, the survival rate of the cells can be improved.

[0109] As can be seen from the comparison of the results of Comparative Examples 1-2 with those of Example 9, the components of the first culture medium have little effect on the cell survival rate, but they will affect the final cell yield. Adding dithiothreitol can improve the cell survival rate to a certain extent.

[0110] As can be seen from the comparison of the results of Comparative Example 3 with those of Example 9, using the components of the second culture medium provided by the present invention can improve the cell survival rate.

[0111] As can be seen from the comparison of the results of Comparative Examples 4-7 with those of Example 9, the retention of sericin, the stirring operation in B200, the selection of permeating cryoprotectant, and the addition of short silk fibers to the cryopreservation solution can all improve the survival rate of mesenchymal stem cells after cryopreservation.

[0112] As can be seen from the comparison of Comparative Example 5 with Examples 4-6, after the present invention reduces the usage amount of the cryopreservation agent, the cell survival rate is still comparable to that of the conventional technology, or even higher.

[0113] It can be seen from the comparison between Comparative Example 8 and Examples 7-9 that after reducing the usage amount of the cryopreservative, the cell survival rate of the present invention is still comparable to that of the conventional technology, or even higher.

[0114] It can be seen from the comparison between Comparative Example 9 and Example 9 that after partial hydrolysis of sericin in the cell suspension, the optimal dosage of the short silk fibers increases.

[0115] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A cryopreservation solution for mesenchymal stem cells, characterized in that: The invention comprises the following components: glycerol, silk staple fibers and buffer solution; the glycerol accounts for 4wt%-6wt% of the cryopreservation solution, the silk staple fibers account for 15wt%-20wt% of the cryopreservation solution, and the remainder is the buffer solution; sericin is retained on the outer surface of the silk staple fibers; the sericin accounts for 15wt%-35wt% of the silk staple fibers.

2. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that The length of the silk staple fibers is 2 mm to 5 mm.

3. The cryopreservation solution for mesenchymal stem cells according to claim 1 or 2, characterized in that: The buffer solution comprises: a main solution and polyethylene glycol, the main solution comprises: one or more of a PBS solution, a HEPES solution and a TBS solution, and the amount of the polyethylene glycol is 1vt%-3vt%.

4. A method for using the cryopreservation solution for mesenchymal stem cells according to any one of claims 1 to 3, characterized in that: The following steps are involved: S100, resuspending the mesenchymal stem cells according to any one of claims 1 to 3 in a cryopreservation solution to obtain a cell suspension; S200, the cell suspension is frozen to preserve the cells; during the freezing operation, the sericin protein is partially or completely precipitated; The dilution operation after thawing when taking cells for use includes the following steps: A100, transferring the thawed cell suspension to a preheated first culture medium, and obtaining a cell precipitate after centrifugation, wherein the first culture medium comprises: a conventional culture medium, calcium chloride and dithiothreitol, wherein the amount of the calcium chloride is 2.5 mM-3.5 mM, and the amount of the dithiothreitol is 0.5 mM-1.5 mM; A200, transfer the cell pellet to the preheated second culture medium, and obtain the cell pellet after centrifugation; A300, after resuspending the cell pellet, a diluted cell suspension is obtained.

5. The method of use according to claim 4, characterized in that: The centrifugal speed in the A100 and A200 steps is 800 rpm-1000 rpm.

6. The method of use according to claim 4, characterized in that: The second culture medium comprises: a conventional culture solution and glutathione, wherein the amount of glutathione used is 4 mg / L-6 mg / L.

7. The method of use according to any one of claims 4 to 6, characterized in that: The freezing operation in S200 includes the following steps: B100, 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; B200, 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; B300, the freezing environment is cooled to -40°C to -80°C and then the cell suspension is kept warm, with a cooling rate of 5-8°C / min; During the operation of B200, sericin is partially or completely precipitated.

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

9. The method of use according to claim 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.

10. The method of use according to claim 9, 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.