Human mesenchymal stem cell supernate cytokine freeze-dried powder as well as preparation method and application thereof

Abstract

The invention provides human mesenchymal stem cell supernate cytokine freeze-dried powder as well as a preparation method and application thereof, and relates to the field of human mesenchymal stem cells. Comprising the following steps: collecting and treating umbilical cord tissues, and carrying out subculture on the umbilical cord tissues in a culture medium to obtain human mesenchymal stem cells; collecting a human mesenchymal stem cell culture solution between generations P2-P5; the preparation method comprises the following steps: adding a proper amount of sodium hyaluronate, performing centrifugation, taking a part of filtrate, and adding glycerol, sodium hyaluronate, butanediol, dipotassium glycyrrhizinate, vitamin C and hydroxypropyl tetrahydropyrantriol to prepare a freeze-dried powder solvent; adding mannitol, trehalose and glucose into the residual filtrate, and uniformly mixing to prepare mixed supernate; and mixing the supernate, and freeze-drying for 20 hours at the vacuum degree of 1Pa and the temperature of-70 DEG C to prepare freeze-dried powder. The prepared freeze-dried powder preparation can be stored for two weeks at the normal temperature and can be stably stored for 6 months or longer at the temperature of 4 DEG C, in the storage process, the activity of cell factors is high, and the storage time is 2-3 times of the stable storage time of traditional freeze-dried powder.

Description

Technical Field

[0001] The present invention relates to the field of human mesenchymal stem cells, and in particular to a human mesenchymal stem cell supernatant cytokine freeze-dried powder, a preparation method and an application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are cells with self-replication and differentiation potential. Under certain conditions, they can be induced to differentiate into a variety of human tissue cells, such as fat, bone, cartilage, tendon, ligament, nerve, myocardium, endothelium, etc. Mesenchymal stem cells are widely present in the interstitial tissues of the human body, such as bone marrow, fat, umbilical cord, placenta, etc.

[0003] Mesenchymal stem cells can secrete hundreds of cell active factors, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), platelet-derived factor (PDGF-BB), various interleukins (LL-1-19), etc. These cytokines can directly or indirectly participate in cell signaling, immune regulation, promote metabolism, accelerate wound healing, cell repair, slow down cell aging, etc.

[0004] However, traditional cytokine preparation methods and storage conditions are harsh, and they are not easy to transport over long distances. They also have low cytokine concentrations and are difficult to preserve cell activity. Therefore, a freeze-dried powder technology that can be easily used is needed. Summary of the invention

[0005] The purpose of the present invention is to provide a human mesenchymal stem cell supernatant cytokine freeze-dried powder. In order to solve the problems of low cytokine concentration and harsh storage conditions in the existing mesenchymal stem cell supernatant, the freeze-dried powder of the present invention can be stored at room temperature, is convenient for transportation, and prolongs the shelf life of cytokine activity.

[0006] Another object of the present invention is to provide a method for preparing human mesenchymal stem cell supernatant cytokine freeze-dried powder. The preparation method is simple and the prepared freeze-dried powder can be stored at room temperature.

[0007] The third purpose of the present invention is to provide an application of human mesenchymal stem cell supernatant cytokine freeze-dried powder, which uses human mesenchymal stem cell supernatant as a solvent and mixes it with freeze-dried powder to quickly activate cytokines. After being applied to the skin surface, it can be quickly absorbed and has multiple effects such as anti-wrinkle, anti-aging, antioxidant, anti-allergic, and whitening.

[0008] The present invention solves the technical problem by adopting the following technical solutions.

[0009] On the one hand, an embodiment of the present invention provides a method for preparing a lyophilized powder of cytokines from a supernatant of human mesenchymal stem cells, comprising the following steps:

[0010] S1, collecting and processing umbilical cord tissue, and subculturing human mesenchymal stem cells from the umbilical cord tissue in a culture medium;

[0011] S2, collecting the culture medium of human mesenchymal stem cells between generations P2 and P5 in step S1;

[0012] S3, centrifuging the human mesenchymal stem cell culture medium of step S2, taking part of the filtrate, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent;

[0013] S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing well to prepare a mixed supernatant;

[0014] S5, freeze-drying the mixed supernatant in step S4 at a vacuum degree of 1 Pa and a temperature of -70°C for 20 hours to prepare a freeze-dried powder.

[0015] In some embodiments of the present invention, in the mixed supernatant, the mass percentage of mannitol is 5%, the mass percentage of trehalose is 2%, the mass percentage of glucose is 2%, and the concentration of cytokine protein is 1 mg / ml.

[0016] In some embodiments of the present invention, in the above-mentioned freeze-dried powder solvent, the mass percentage of glycerol is 5%, the mass percentage of sodium hyaluronate is 0.3%, the mass percentage of butylene glycol is 1%, the mass percentage of dipotassium glycyrrhizinate is 0.15%, the mass percentage of vitamin C is 0.02%, and the mass percentage of hydroxypropyl tetrahydropyrantriol is 3%.

[0017] In some embodiments of the present invention, in the above step S3, the centrifugation conditions are: centrifugation at 4°C and 800g for 15 minutes.

[0018] In some embodiments of the present invention, in step S3, the volume of the partial filtrate is 20-25% of the total filtrate volume.

[0019] In some embodiments of the present invention, the freeze-dried powder solvent and the mixed supernatant are further sterilized by filtering using a 0.22 μm cell sieve.

[0020] In some embodiments of the present invention, in step S1, collecting, processing and subculturing umbilical cord tissue comprises the following steps:

[0021] S11, clean the remaining blood stains on the umbilical cord tissue with PBS solution, soak the umbilical cord tissue with 75% alcohol for 2 minutes, soak the umbilical cord tissue with a mixture of PBS, 1% penicillin and streptomycin for 2 minutes, and then clean the umbilical cord tissue with PBS;

[0022] S12, placing the umbilical cord tissue from step S11 in a sterile culture dish, cutting it into small pieces of 2-3 cm, cutting it open longitudinally along the umbilical cord, carefully peeling off the amniotic membrane, removing the artery and vein, peeling off the inner layer of Wharton's jelly tissue, and then transferring the Wharton's jelly tissue into a new culture dish and cutting it into minced meat;

[0023] S13, wash the Wharton's jelly tissue with a mixture of PBS, 1% penicillin and streptomycin, remove the supernatant after centrifugation, transfer the Wharton's jelly tissue to a culture bottle and evenly distribute it, and incubate it in a 37°C, 5% carbon dioxide concentration incubator overnight;

[0024] S14, add serum-free human mesenchymal stem cell complete culture medium to immerse the Wharton's jelly tissue block, and continue to culture in an incubator at 37°C and 5% carbon dioxide concentration overnight;

[0025] S15, the culture medium was fully replaced on the third day, and then half of the culture medium was replaced every other day. When the cell confluence reached 80-90%, the tissue blocks were removed, digested with 1% trypsin, and then subcultured; the operation was repeated until the fifth generation was reached.

[0026] In the present invention, when treating umbilical cord tissue, the PBS solution used is phosphate buffered saline, which is composed of Na2HPO4, KH2PO4, NaCl and KCl, and can provide a relatively stable ion environment and pH buffering capacity for umbilical cord tissue. Its pH value is between 7.2-7.4, which is isotonic with human blood.

[0027] On the other hand, an embodiment of the present invention provides a human mesenchymal stem cell supernatant cytokine freeze-dried powder, which is prepared by the above method.

[0028] In a third aspect, an embodiment of the present invention provides an application of human mesenchymal stem cell supernatant cytokine freeze-dried powder, wherein the freeze-dried powder solvent prepared in step S3 and the freeze-dried powder prepared in step S5 are evenly mixed and applied to the skin surface.

[0029] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0030] The preparation method provided by the present invention further concentrates cytokines through freeze-drying technology, and the freeze-dried powder preparation prepared can be stored for two weeks at room temperature and can be stably stored for 6 months or longer at 4°C. During the storage process, the cytokine activity is high, and the storage time is 2-3 times the stable storage time of traditional freeze-dried powder. The freeze-dried powder preparation that can be stored for a long time is easy to transport.

[0031] Compared with the traditional freeze-dried powder solvent, which uses physiological saline as a matrix, which is not conducive to the activation of cytokines, the present invention uses part of the supernatant as a solvent matrix, which can fully activate the activity of cytokines. After the freeze-dried powder is mixed with the solvent and re-dissolved, it can be quickly absorbed when applied to the face, and has multiple effects such as anti-wrinkle, anti-aging, anti-oxidation, anti-allergy, and whitening. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0033] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0034] The embodiment of the present invention provides a method for preparing a lyophilized powder of human mesenchymal stem cell supernatant cytokine, comprising the following steps:

[0035] S1, collecting and processing umbilical cord tissue, and subculturing the umbilical cord tissue in a culture medium to produce human mesenchymal stem cells; during the culture process, a serum-free mesenchymal stem cell complete culture medium is used for culture, and the obtained freeze-dried powder can avoid the irritation of serum-derived components to the skin.

[0036] S2, collecting the culture medium of human mesenchymal stem cells between generations P2 and P5 in step S1;

[0037] S3, centrifuging the human mesenchymal stem cell culture medium of step S2, taking part of the filtrate, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizinate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent; the lyophilized powder solvent contains the supernatant of the human mesenchymal stem cell culture medium, and the supernatant is used as a matrix. When the lyophilized powder is used, the lyophilized powder can be quickly revived and the cytokines in the lyophilized powder can be activated. Compared with the traditional solvent mainly composed of physiological saline,

[0038] S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing and preparing a mixed supernatant; preparing freeze-dried powder with the supernatant, which can fully preserve the cytokines in the supernatant; mannitol and glucose are freeze-drying protective agents and fillers respectively; trehalose is a stabilizer and antioxidant; adding mannitol to the supernatant can protect the protein from denaturation during the freeze-drying process, and trehalose can prevent the freeze-dried powder from agglomerating, which helps to keep the freeze-dried powder in a good state.

[0039] S5, freeze-drying the mixed supernatant in step S4 at a vacuum degree of 1 Pa and a temperature of -70°C for 20 hours to prepare a freeze-dried powder.

[0040] In some embodiments of the present invention, in the above-mentioned mixed supernatant, the mass percentage of mannitol is 5%, the mass percentage of trehalose is 2%, and the mass percentage of glucose is 2%. The concentration of cytokine protein is 1 mg / ml. The concentration of cytokine protein in the supernatant is adjusted to facilitate the control of the uniformity of the lyophilized powder product. Adding the above-mentioned mass percentages of mannitol, trehalose and glucose to the mixed supernatant can protect the cytokines during the freeze-drying process and avoid cytokine inactivation. During long-term storage, the cytokines are still active.

[0041] In some embodiments of the present invention, the lyophilized powder solvent contains 5% by mass of glycerol, 0.3% by mass of sodium hyaluronate, 1% by mass of butylene glycol, 0.15% by mass of dipotassium glycyrrhizinate, 0.02% by mass of vitamin C, and 3% by mass of hydroxypropyl tetrahydropyrantriol. Glycerol is a protective agent; sodium hyaluronate provides moisturizing effect for skin care and is also a stabilizer for the active ingredients of lyophilized powder; butylene glycol is a stabilizer and antibacterial agent; dipotassium glycyrrhizinate has anti-inflammatory and skin tolerance enhancing effects; vitamin C is an antioxidant that can promote the synthesis of collagen and has the effect of removing spots; hydroxypropyl tetrahydropyrantriol can promote collagen production, enhance skin barrier function, and improve skin texture.

[0042] In some embodiments of the present invention, in the above step S3, the centrifugation conditions are: centrifugation at 4°C and 800g for 15 minutes.

[0043] In some embodiments of the present invention, in step S3, the volume of the partial filtrate is 20-25% of the total filtrate volume.

[0044] In some embodiments of the present invention, the freeze-dried powder solvent and the mixed supernatant are further sterilized by filtering using a 0.22 μm cell sieve.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] The umbilical cord used was donated voluntarily by the mother in accordance with ethical and moral standards. The mother's HBV antibody antigen, HCV antibody, anti-HIV antibody, anti-syphilis Treponema pallidum antibody, Epstein-Barr virus and other test results were all negative, and she had no previous history of tumors or family history.

[0048] S11, clean the remaining blood stains on the umbilical cord tissue with PBS solution, soak the umbilical cord tissue with 75% alcohol for 2 minutes, soak the umbilical cord tissue with a mixture of PBS, 1% penicillin and streptomycin for 2 minutes, and then clean the umbilical cord tissue with PBS;

[0049] S12, placing the umbilical cord tissue from step S11 in a sterile culture dish, cutting it into small pieces of 2-3 cm, cutting it open longitudinally along the umbilical cord, carefully peeling off the amniotic membrane, removing the artery and vein, peeling off the inner layer of Wharton's jelly tissue, and then transferring the Wharton's jelly tissue into a new culture dish and cutting it into minced meat;

[0050] S13, wash the Wharton's jelly tissue with a mixture of PBS, 1% penicillin and streptomycin, remove the supernatant after centrifugation, transfer the Wharton's jelly tissue to a culture bottle and evenly distribute it, and incubate it in a 37°C, 5% carbon dioxide concentration incubator overnight;

[0051] S14, add serum-free human mesenchymal stem cell complete culture medium to immerse the Wharton's jelly tissue block, and continue to culture in an incubator at 37°C and 5% carbon dioxide concentration overnight;

[0052] S15, the culture medium was fully replaced on the third day, and then half of the culture medium was replaced every other day. When the cell confluence reached 80-90%, the tissue blocks were removed, digested with 1% trypsin, and then subcultured; the operation was repeated until the fifth generation was reached.

[0053] S2, collecting the culture medium of human mesenchymal stem cells between the P2-P5 generations;

[0054] S3, centrifuging the collected human mesenchymal stem cell culture medium at 4°C and 800g for 15 min, taking 25% of the total filtrate volume, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent; the final mass percentage of each substance in the lyophilized powder solvent is as follows: 5% by mass of glycerol, 0.3% by mass of sodium hyaluronate, 1% by mass of butylene glycol, 0.15% by mass of dipotassium glycyrrhizate, 0.02% by mass of vitamin C, and 3% by mass of hydroxypropyl tetrahydropyrantriol.

[0055] S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing and preparing a mixed supernatant; the final concentration of each substance is: 5% by mass of mannitol, 2% by mass of trehalose, and 2% by mass of glucose.

[0056] The above-mentioned lyophilized powder solvent and mixed supernatant were sterilized by filtration using a 0.22 μm cell sieve.

[0057] S5, in a sterile dust-free environment, the mixed supernatant in step S4 is dispensed into 7 ml vials at 3 ml / vial, half-stoppered, freeze-dried at a vacuum degree of 1 Pa and a temperature of -70°C for 20 h, and capped to obtain a lyophilized powder.

[0058] When in use, mix the prepared freeze-dried powder solvent and freeze-dried powder evenly, and apply it on the skin surface that needs to be treated.

[0059] Example 2

[0060] The difference from Example 1 is that in this example, in step S3, 20% of the total filtrate volume is used to prepare a freeze-dried powder solvent. The remaining steps are the same as those of Example 1.

[0061] S1, culturing human mesenchymal stem cells using the same method as in Example 1;

[0062] S2, collecting the culture medium of human mesenchymal stem cells between the P2-P5 generations;

[0063] S3, centrifuging the collected human mesenchymal stem cell culture medium at 4°C and 800g for 15 min, taking 20% ​​of the total filtrate volume, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent; the final mass percentage of each substance in the lyophilized powder solvent is as follows: 5% by mass of glycerol, 0.3% by mass of sodium hyaluronate, 1% by mass of butylene glycol, 0.15% by mass of dipotassium glycyrrhizate, 0.02% by mass of vitamin C, and 3% by mass of hydroxypropyl tetrahydropyrantriol.

[0064] S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing and preparing a mixed supernatant; the final concentration of each substance is: 5% by mass of mannitol, 2% by mass of trehalose, and 2% by mass of glucose.

[0065] The above-mentioned lyophilized powder solvent and mixed supernatant were sterilized by filtration using a 0.22 μm cell sieve.

[0066] S5, in a sterile dust-free environment, the mixed supernatant in step S4 is dispensed into 7 ml vials at 3 ml / vial, half-stoppered, freeze-dried at a vacuum degree of 1 Pa and a temperature of -70°C for 20 h, and capped to obtain a lyophilized powder.

[0067] Example 3

[0068] The difference from Example 1 is that in this example, in step S3, 23% of the total filtrate volume is used to prepare a freeze-dried powder solvent. The remaining steps are the same as those of Example 1.

[0069] S1, culturing human mesenchymal stem cells using the same method as in Example 1;

[0070] S2, collecting the culture medium of human mesenchymal stem cells between the P2-P5 generations;

[0071] S3, centrifuging the collected human mesenchymal stem cell culture medium at 4°C and 800g for 15 min, taking 23% of the total filtrate volume, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent; the final mass percentage of each substance in the lyophilized powder solvent is as follows: 5% by mass of glycerol, 0.3% by mass of sodium hyaluronate, 1% by mass of butylene glycol, 0.15% by mass of dipotassium glycyrrhizate, 0.02% by mass of vitamin C, and 3% by mass of hydroxypropyl tetrahydropyrantriol.

[0072] S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing and preparing a mixed supernatant; the final concentration of each substance is: 5% by mass of mannitol, 2% by mass of trehalose, and 2% by mass of glucose.

[0073] The above-mentioned lyophilized powder solvent and mixed supernatant were sterilized by filtration using a 0.22 μm cell sieve.

[0074] S5, in a sterile dust-free environment, the mixed supernatant in step S4 is dispensed into 7 ml vials at 3 ml / vial, half-stoppered, freeze-dried at a vacuum degree of 1 Pa and a temperature of -70°C for 20 h, and capped to obtain a lyophilized powder.

[0075] Comparative Example 1

[0076] The difference from Example 1 is that in this comparative example, in step S4, only trehalose and glucose are added to the mixed supernatant, and mannitol is not added. The remaining steps are the same as those of Implementation 1.

[0077] Comparative Example 2

[0078] The difference from Example 1 is that in this comparative example, in step S4, only mannitol and trehalose are added to the mixed supernatant, and glucose is not added. The remaining steps are the same as those of Implementation 1.

[0079] Comparative Example 3

[0080] The difference from Example 1 is that in this comparative example, in step S4, no mannitol, glucose or trehalose is added to the mixed supernatant, and the remaining steps are the same as those of Implementation 1.

[0081] Experimental example

[0082] The lyophilized powders prepared in the above Examples 1-3 and Comparative Examples 1-3 were stored at 4°C for 0, 6 and 12 months, and representative fibroblast growth factor (FGF) and epidermal growth factor (EGF) were selected as representative indicators. The corresponding lyophilized powder solvents were mixed with the lyophilized powders, and the contents of the above cytokines were detected by ELISA kits. The results are shown in Table 1. The decomposition rates of fibroblast growth factor (FGF) and epidermal growth factor (EGF) after 6 and 12 months were calculated. The results are shown in Table 1.

[0083] As shown in Table 2.

[0084]

[0085] Table 1 Concentration of lyophilized powder of Example 1-3 after storage at 4°C

[0086]

[0087] In the above Table 1, 0 months refers to the detection of the corresponding cytokine content immediately after the freeze-dried powder is prepared. It can be concluded that the freeze-dried powders of Examples 1-3 and Comparative Examples 1-3 are stored at 4°C. As time goes by, the content of cytokines in the freeze-dried powders decreases. In the first 6 months, the content decreases more slowly. In the 6-12 month stage, the content of cytokines in the last 6 months decreases faster than in the first 6 months.

[0088] Table 2 Decomposition rate of cytokines after each freeze-dried powder was stored at 4°C

[0089]

[0090] As can be seen from Table 2, the cytokines of Examples 1-3 degraded slowly during storage, while the cytokines of Comparative Examples 1-3 degraded quickly, indicating that the lyophilized powder prepared by the method provided in this example has a long storage time at 4°C, and the cytokines still have high activity during long-term storage.

[0091] For the freeze-dried powders of Comparative Examples 1-3, only part of the protective agent or no protective agent was added during preparation. When stored at 4° C., the cytokine content decreased significantly with time. After 12 months of storage, the cytokine content decreased by 50%.

[0092] In summary, the preparation method provided by the present invention further concentrates cytokines through freeze-drying technology, and the freeze-dried powder preparation prepared can be stored for two weeks at room temperature and can be stably stored for 6 months or longer at 4°C. During the storage process, the cytokine activity is high, and the storage time is 2-3 times the stable storage time of traditional freeze-dried powder. Secondly, the freeze-dried powder preparation is easy to transport.

[0093] Compared with the traditional freeze-dried powder solvent, which uses physiological saline as a matrix, which is not conducive to the activation of cytokines, the present invention uses part of the supernatant as a solvent matrix, which can fully activate the activity of cytokines. After the freeze-dried powder is mixed with the solvent and re-dissolved, it can be quickly absorbed when applied to the face, and has multiple effects such as anti-wrinkle, anti-aging, anti-oxidation, anti-allergy, and whitening.

[0094] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing lyophilized powder of human mesenchymal stem cell supernatant cytokine, characterized in that: The steps include: S1, collecting and processing umbilical cord tissue, and subculturing human mesenchymal stem cells from the umbilical cord tissue in a culture medium; S2, collecting the culture medium of human mesenchymal stem cells between generations P2 and P5 in step S1; S3, centrifuging the human mesenchymal stem cell culture medium of step S2, taking part of the filtrate, adding glycerol, sodium hyaluronate, butylene glycol, dipotassium glycyrrhizate, vitamin C, and hydroxypropyl tetrahydropyrantriol to prepare a lyophilized powder solvent; S4, adding mannitol, trehalose and glucose to the remaining filtrate of step S3, mixing well to prepare a mixed supernatant; S5, freeze-drying the mixed supernatant in step S4 at a vacuum degree of 1 Pa and a temperature of -70°C for 20 hours to prepare a freeze-dried powder.

2. The method for preparing the human mesenchymal stem cell supernatant cytokine freeze-dried powder according to claim 1, characterized in that: In the mixed supernatant, the mass percentage of mannitol is 5%, the mass percentage of trehalose is 2%, and the mass percentage of glucose is 2%.

3. The method for preparing the human mesenchymal stem cell supernatant cytokine freeze-dried powder according to claim 1, characterized in that: The concentration of cytokine protein in the mixed supernatant is 1 mg / ml.

4. The method for preparing human mesenchymal stem cell supernatant cytokine freeze-dried powder according to claim 1, characterized in that: In the freeze-dried powder solvent, the mass percentage of glycerol is 5%, the mass percentage of sodium hyaluronate is 0.3%, the mass percentage of butylene glycol is 1%, the mass percentage of dipotassium glycyrrhizinate is 0.15%, the mass percentage of vitamin C is 0.02%, and the mass percentage of hydroxypropyl tetrahydropyrantriol is 3%.

5. The method for preparing freeze-dried cytokine powder of human mesenchymal stem cell supernatant according to claim 1, characterized in that: In step S3, the centrifugation conditions are: centrifugation at 4°C and 800g for 15 minutes.

6. The method for preparing freeze-dried cytokine powder of human mesenchymal stem cell supernatant according to claim 1, characterized in that: In the step S3, the volume of the partial filtrate is 20-25% of the volume of the total filtrate.

7. The method for preparing freeze-dried cytokine powder of human mesenchymal stem cell supernatant according to claim 1, characterized in that: The method further includes filtering and sterilizing the lyophilized powder solvent and the mixed supernatant using a 0.22 μm cell sieve.

8. The method for preparing freeze-dried cytokine powder of human mesenchymal stem cell supernatant according to claim 1, characterized in that: In step S1, collecting, processing and subculturing umbilical cord tissue includes the following steps: S11, clean the remaining blood stains on the umbilical cord tissue with PBS solution, soak the umbilical cord tissue with 75% alcohol for 2 minutes, soak the umbilical cord tissue with a mixture of PBS, 1% penicillin and streptomycin for 2 minutes, and then clean the umbilical cord tissue with PBS; S12, placing the umbilical cord tissue from step S11 in a sterile culture dish, cutting it into small pieces of 2-3 cm, cutting it open longitudinally along the umbilical cord, carefully peeling off the amniotic membrane, removing the artery and vein, peeling off the inner layer of Wharton's jelly tissue, and then transferring the Wharton's jelly tissue into a new culture dish and cutting it into minced meat; S13, wash the Wharton's jelly tissue with a mixture of PBS, 1% penicillin and streptomycin, remove the supernatant after centrifugation, transfer the Wharton's jelly tissue to a culture bottle and evenly distribute it, and incubate it in a 37°C, 5% carbon dioxide concentration incubator overnight; S14, add serum-free human mesenchymal stem cell complete culture medium to immerse the Wharton's jelly tissue block, and continue to culture in an incubator at 37°C and 5% carbon dioxide concentration overnight; S15, the culture medium was fully replaced on the third day, and then half of the culture medium was replaced every other day. When the cell confluence reached 80-90%, the tissue blocks were removed, digested with 1% trypsin, and then subcultured; the operation was repeated until the fifth generation was reached.

9. A human mesenchymal stem cell supernatant cytokine freeze-dried powder, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. A use of the human mesenchymal stem cell supernatant cytokine freeze-dried powder as claimed in claim 9, characterized in that: The freeze-dried powder solvent prepared in step S3 is evenly mixed with the freeze-dried powder prepared in step S5, and the mixture is applied on the skin surface.

Images