Umbilical cord mesenchymal stem cell cryopreservation liquid and cryopreservation method

Abstract

The invention discloses an umbilical cord mesenchymal stem cell cryopreservation solution and a cryopreservation method. The umbilical cord mesenchymal stem cell cryopreservation solution is prepared from the following components: 10 to 20v / v percent of glycerol, 10 to 15v / v percent of polyethylene glycol, 30 to 100g / L of hydroxyethyl starch, 60 to 100g / L of trehalose, 1 to 5g / L of glutathione, 1 to 7g / L of sodium pyruvate, 10 to 20v / v percent of compound amino acid injection and the balance of dextran 40 sodium chloride injection. The cryopreservation liquid does not contain animal-derived serum, avoids the risk of animal-derived pathogenic microorganisms, is high in safety, can replace DMSO (dimethylsulfoxide), avoids the influence of DMSO on the activity of resuscitated hUC-MSCs, can achieve a better dehydration effect before freezing, and can be used for preparing the cryopreservation liquid for the resuscitated hUC-MSCs by complementation and unique molecular structures and functions in the freezing process through different mechanisms. The cryopreservation liquid has the advantages that the cryopreservation liquid can be used for cryopreservation at-80 DEG C directly, gradient cooling is not needed, the operation is simple, and a better cryopreservation liquid formula is screened for clinical application of hUC-MSCs (human UC-MSCs).

Description

Technical Field

[0001] The present invention relates to the technical field of cell preservation, and in particular to a cryopreservation solution and a cryopreservation method for umbilical cord mesenchymal stem cells. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are mesenchymal stem cells derived from the Wharton's jelly of the fetal umbilical cord. Due to their advantages such as easy collection, wide source, strong proliferation ability and low immunogenicity, they have gradually become the most promising seed cells in the fields of regenerative medicine and cell therapy.

[0003] In order to preserve the biological activity of cells for a long time, the cells need to be cryopreserved. At present, the most commonly used technology for cell cryopreservation is liquid nitrogen cryopreservation, which is to store cells in -196℃ liquid nitrogen at low temperature, so that the cells are temporarily out of the growth state and their cell characteristics are preserved. Traditional cryoprotectants often contain animal serum and dimethyl sulfoxide (DMSO), which may induce immune responses or cytotoxicity, affect cell survival and function, and are not suitable for clinical use. In addition, although the gradient cooling process can reduce cell damage to a certain extent, the operation is complicated and time-consuming. Therefore, the development of an hUC-MSCs cryopreservation solution and a cryopreservation method that is free of animal serum, DMSO, and does not require gradient cooling has important practical value and market potential, laying the foundation for further clinical applications. Summary of the invention

[0004] The object of the present invention is to provide an umbilical cord mesenchymal stem cell cryopreservation solution and a cryopreservation method. The cell cryopreservation solution of the present invention contains no animal serum, no DMSO and can realize the cryopreservation of umbilical cord mesenchymal stem cells without gradient cooling, thereby solving the problems of immune response and cytotoxicity caused by traditional cryopreservation solutions, and also solving the problem of complicated gradient cooling operation, and improving the cryopreservation effect of hUC-MSCs.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0006] The first aspect of the present invention provides an umbilical cord mesenchymal stem cell cryopreservation solution, wherein the umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components:

[0007] Glycerol 10-20v / v%, polyethylene glycol 10-15v / v%, hydroxyethyl starch 30-100g / L, trehalose 60-100g / L, glutathione 1-5g / L, sodium pyruvate 1-7g / L, compound amino acid injection 10-20v / v%, the remainder is dextran 40 sodium chloride injection.

[0008] Preferably, the umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components:

[0009] Glycerol 15v / v%, polyethylene glycol 10v / v%, hydroxyethyl starch 60g / L, trehalose 80g / L, glutathione 5g / L, sodium pyruvate 3g / L, compound amino acid injection 10v / v%, the balance is dextran 40 sodium chloride injection.

[0010] Preferably, the content of dextran 40 in the dextran 40 sodium chloride injection is 0.04-0.08 g / mL, and the content of sodium chloride is 0.08-0.10 g / mL.

[0011] Preferably, the umbilical cord mesenchymal stem cell cryopreservation solution is obtained by mixing the components and then filtering and sterilizing them.

[0012] A second aspect of the present invention provides a use of the above umbilical cord mesenchymal stem cell cryopreservation solution in the cryopreservation of umbilical cord mesenchymal stem cells.

[0013] A third aspect of the present invention provides a method for cryopreservation of umbilical cord mesenchymal stem cells, the method comprising:

[0014] The umbilical cord mesenchymal stem cells are inoculated into the umbilical cord mesenchymal stem cell freezing solution for cryopreservation.

[0015] Preferably, the cryopreservation concentration of the umbilical cord mesenchymal stem cells is (0.5-2.0)×10 7 cells / mL.

[0016] Preferably, the umbilical cord mesenchymal stem cells are P5 passage human umbilical cord mesenchymal stem cells.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least:

[0018] (1) The present invention uses a composite cryoprotectant that does not contain DMSO. Through its unique molecular structure and function, it reduces ice crystal formation during cell freezing, reduces cell damage, and avoids the potential toxicity risk of DMSO in clinical practice.

[0019] (2) The present invention uses a composite cryoprotectant that does not contain animal-derived serum, thereby avoiding the risk of introducing animal-derived pathogenic microorganisms and the body's immune response, having high safety, and being able to maintain the activity and function of hUC-MSCs cells for a long time, thereby improving the cryopreservation effect.

[0020] (3) The present invention does not require the use of a gradient cooling box and programmed cooling, and is simple to operate. The frozen cells can be directly placed in a -80°C refrigerator, and after 24 hours, the cells can be transferred to a -196°C liquid nitrogen tank for long-term storage. The survival rate of hUC-MSCs after resuscitation can reach more than 95%, and the ability of cell proliferation is not affected, and the stability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0022] Figure 1 In Example 2 of the present invention, an inverted microscope was used to observe the growth status of hUC-MSCs after cell recovery after being cryopreserved in each cryopreservation solution for one year;

[0023] Figure 2 The cell growth curve of hUC-MSCs after one year of cryopreservation in various cryopreservation solutions detected by CCK-8 method in Example 2 of the present invention;

[0024] Figure 3 The expression of cell surface markers in hUC-MSCs frozen in Experimental Group 5 for one year in Example 2 of the present invention was analyzed by flow cytometry;

[0025] Figure 4 The Oil Red O staining method in Example 2 of the present invention was used to analyze the adipogenic capacity of hUC-MSCs in Experimental Group 5 after being frozen for one year;

[0026] Figure 5 The alizarin red staining method in Example 2 of the present invention was used to analyze the osteogenic capacity of hUC-MSCs in Experimental Group 5 after being frozen for one year;

[0027] Figure 6 The Alcian blue staining method in Example 2 of the present invention was used to analyze the chondrogenic ability of hUC-MSCs in Experimental Group 5 after being frozen for one year. DETAILED DESCRIPTION

[0028] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0029] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0030] The present invention provides an umbilical cord mesenchymal stem cell cryopreservation solution, wherein the umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components:

[0031] Glycerol 10-20v / v%, polyethylene glycol 10-15v / v%, hydroxyethyl starch 30-100g / L, trehalose 60-100g / L, glutathione 1-5g / L, sodium pyruvate 1-7g / L, compound amino acid injection 10-20v / v%, the remainder is dextran 40 sodium chloride injection.

[0032] In the present invention, glycerol as an osmotic protective agent and polyethylene glycol, hydroxyethyl starch and trehalose as non-osmotic protective agents protect the inside and outside of cells from damage by ice crystals during cryopreservation, and the use of a combined protective agent for cryopreservation improves the cryopreservation efficiency. Glycerol can increase the viscosity of the solution, reduce the formation of ice crystals and form a cell membrane to protect it; polyethylene glycol can weaken the water crystallization process, reduce damage to cell structure and function, and improve the cell recovery rate after thawing; hydroxyethyl starch, as a cell sedimentation stabilizer, can prevent or delay the sedimentation of cells during cryopreservation, prevent cells from squeezing each other and affecting the cell cryopreservation effect; the protective effect of trehalose is the interaction between the lipid membrane, maintaining the stability of proteins during cryopreservation and recovery, and can form a vitrified matrix to inhibit the formation of ice crystals in cells. At the same time, it can be used in combination with glycerol to help Trehalose penetrates the cell membrane to improve its efficacy; glutathione, as an antioxidant, can reduce the effects of reactive oxygen and cold shock damage, and improve the ability to preserve cell function after thawing; sodium pyruvate, as a cell nutrient, can replenish part of the energy consumed during cell metabolism, help maintain the stability of the cell membrane during cryopreservation, and reduce the effects of oxidative damage on the cell membrane; compound amino acid injection (18AA) is a compound preparation, a sterile aqueous solution prepared from 18 kinds of amino acids and sorbitol; dextran 40 sodium chloride injection can stabilize cell osmotic pressure and maintain cell morphology and activity. Dextran and compound amino acid injection are both clinical drugs that meet national standards, can ensure cell quality, and are directly used for clinical administration.

[0033] In one embodiment, the umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components:

[0034] Glycerol 15v / v%, polyethylene glycol 10v / v%, hydroxyethyl starch 60g / L, trehalose 80g / L, glutathione 5g / L, sodium pyruvate 3g / L, compound amino acid injection 10v / v%, the balance is dextran 40 sodium chloride injection.

[0035] In some embodiments, the content of dextran 40 in the dextran 40 sodium chloride injection is 0.04-0.08 g / mL, and the content of sodium chloride is 0.08-0.10 g / mL.

[0036] In one embodiment, the umbilical cord mesenchymal stem cell cryopreservation solution is obtained by mixing the components and then filtering and sterilizing them.

[0037] Another embodiment of the present invention provides a use of the above umbilical cord mesenchymal stem cell cryopreservation solution in the cryopreservation of umbilical cord mesenchymal stem cells.

[0038] Another embodiment of the present invention provides a method for cryopreservation of umbilical cord mesenchymal stem cells, the method for cryopreservation of umbilical cord mesenchymal stem cells comprising:

[0039] The umbilical cord mesenchymal stem cells are inoculated into the umbilical cord mesenchymal stem cell freezing solution for cryopreservation.

[0040] In some embodiments, the cryopreservation concentration of the umbilical cord mesenchymal stem cells is (0.5-2.0)×10 7 cells / mL.

[0041] In one embodiment, the umbilical cord mesenchymal stem cells are P5 passage human umbilical cord mesenchymal stem cells.

[0042] The technical solution of the present invention is further described in detail below through specific embodiments.

[0043] The raw materials used in the following examples are as follows:

[0044] Propylene glycol: injection grade, purchased from Zhejiang Suichang Huikang Pharmaceutical Co., Ltd. (National Medicine Standard No. H33020622);

[0045] Polyethylene glycol: pharmaceutical grade, purchased from Shaanxi Panlong Yihai Pharmaceutical Co., Ltd.;

[0046] Hydroxyethyl starch: pharmaceutical grade hydroxyethyl starch 130 / 0.4, purchased from Shandong Weigao Pharmaceutical Co., Ltd.;

[0047] Trehalose: injection grade, purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd. (Cat. No.: 6138-23-4);

[0048] Glutathione: purchased from Sigma (Cat. No. 1294820);

[0049] Sodium pyruvate: purchased from Sigma (catalog number: P8574);

[0050] Compound amino acid injection (18AA): purchased from Bikang Pharmaceutical Jiangsu Co., Ltd. (National Medicine Standard No. H20066340);

[0051] Dextran 40 sodium chloride injection, with a dextran 40 content of 30 g / 500 mL and a sodium chloride content of 4.5 g / 500 mL, was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. (National Medicine Standard No. H51020331).

[0052] Example 1

[0053] This example is a method for separating and culturing hUC-MSCs. The specific operation method is as follows:

[0054] 1) Working in a clean bench, take fresh full-term healthy fetal umbilical cord and add PBS containing 1% double antibody to wash the umbilical cord sample;

[0055] 2) Select an umbilical cord with good brightness, no damage and no edema, and cut it into 2-3 cm small pieces with stainless steel curved tissue scissors; use tissue scissors to throw the umbilical cord longitudinally, remove the umbilical vein and umbilical artery; cut the umbilical cord to 1mm 3 Small tissue pieces should be cut into foamy minced meat;

[0056] 3) Add 3 mL of serum-free culture medium, spread the tissue blocks evenly on the bottom of the culture dish, and culture in a 37°C, 5% CO2 incubator;

[0057] 4) Closely observe the cell growth and change the medium every 3-4 days. After 7 days of primary culture, if obvious cell clones are formed, discard the tissue blocks in time. When the number of cell clones increases, the number of cell clones in each culture dish reaches more than 7, and the cell fusion degree reaches 80-90%, the seed cells are harvested.

[0058] Example 2

[0059] 1. The specific operation methods for the culture and cryopreservation of hUC-MSCs are as follows:

[0060] 1) Seed cells were cultured at 1.0 x 10 4 / cm 2 Density seeding, according to the growth and proliferation status of cells, generally after 3 days of culture, the degree of confluence can reach 80%-90%, and then subculture is performed to P5;

[0061] 2) Harvest P5 cells, add 0.05% Trypsin-EDTA to each dish for digestion at room temperature, observe under a microscope, and when the intercellular gap increases and the cells become round, add complete medium to each dish to terminate the digestion; collect the digested cells into a 50 ml centrifuge tube, and centrifuge at 4°C, 600g for 10 min;

[0062] 3) Add fresh complete medium to each tube to resuspend the cells, mix thoroughly and then aspirate 50 μl of the cell suspension to determine the cell number and cell viability;

[0063] 4) Calculate the required amount of cells according to the experiment and divide into 8 15 mL centrifuge tubes. Centrifuge the cell suspension at 4°C, 600 g for 10 min.

[0064] 5) Resuspend the cell pellet with different freezing solutions (see Table 1). The cell density of each group is 1.0x10 7 / mL concentration cryopreservation, each cryopreservation tube was filled with 1mL of cell suspension, and hUC-MSCs were directly placed in a -80℃ refrigerator for cryopreservation without gradient cooling. After 24 hours, the cryopreservation tube was transferred to a liquid nitrogen tank (-196℃) for long-term storage. The cells of each group were revived at 3 months, 6 months, and 1 year for testing and culture.

[0065] Table 1 Grouping, ingredients and proportions of various freezing solutions

[0066]

[0067] Note: The balance in the experimental group is dextran 40 sodium chloride injection.

[0068] 2. Detection of total cell number and cell viability after resuscitation of hUC-MSCs frozen in different cryopreservation solutions in Table 1. The specific operation method is as follows:

[0069] 1) Take out hUC-MSCs of different cryopreservation concentrations from liquid nitrogen, thaw in a 37°C water bath, shake the cryopreservation tubes repeatedly at a constant speed to thaw them as quickly as possible, and quickly place them in an ultra-clean workbench;

[0070] 2) Transfer the cell suspension into a 15 mL centrifuge tube, resuspend it in the existing freezing medium: DMEM = 1:9, mix thoroughly, centrifuge at 600 g for 10 min, resuspend it in fresh complete medium, draw 50 μl of the cell suspension, and determine the total number of cells and cell viability;

[0071] 3) Count the cells using a hemocytometer, mix the cell sample thoroughly, take 20 μl of the cell suspension, then take 20 μl of 0.08% trypan blue solution and mix thoroughly, observe under a microscope, live cells are not stained, and dead cells are stained blue;

[0072] 4) Count the number of dead cells and live cells in 4 large squares under an optical microscope, and calculate the total number of cells, cell viability and cell recovery rate. The calculation formula is as follows:

[0073]

[0074] 5) Repeat the counting three times and take the average value.

[0075] The cell viability and cell recovery rate after resuscitation of hUC-MSCs cryopreserved in different cryopreservation solutions are shown in Table 2 ;

[0076] Table 2 Cell viability and cell recovery rate of hUC-MSCs after cryopreservation in different cryopreservation solutions

[0077]

[0078] From Table 2, we can see that:

[0079] As the cryopreservation time of hUC-MSCs increased, the cell viability and cell recovery rate of the cryopreservation solution in each group decreased. Among them, experimental group 5 performed best in maintaining cell viability and improving cell recovery rate, and the cell viability and cell recovery rate were significantly higher than those of the control group and other experimental groups.

[0080] 3. Observation of cell morphology and total number of growth after resuscitation of hUC-MSCs frozen in different cryopreservation solutions. The specific operation methods are as follows:

[0081] 1) hUC-MSCs revived from different cryopreservation solutions were cultured at 1.0 x 10 4 / cm 2 The density was inoculated in φ10 cm culture dishes, 8 mL of culture medium was added to each dish, and the dishes were placed in a 37°C, 5% CO2 incubator for culture;

[0082] 2) Observe the growth of hUC-MSCs after cryopreservation and resuscitation in several groups of cryopreservation solutions under an inverted microscope. The cells should grow adherently and present a long spindle shape. Generally, the degree of confluence can reach 80%-90% after 3 days of culture. Take photos and save them. The results of the photos are as follows: Figure 1 As shown;

[0083] Depend on Figure 1 It can be seen that:

[0084] Microscopic observation revealed that the cells in each group had good morphology, showing normal spindle or polygonal shapes. Among them, experimental group 5 was able to quickly adhere to the wall and begin to proliferate during the culture process, with the highest cell fusion.

[0085] 3) Add 1 mL of 0.05% Trypsin-EDTA to each culture dish and observe under a microscope. When the intercellular gap increases and the cells become shorter, add 8 mL of the culture supernatant collected previously to terminate the digestion; after mixing, take 50 μl of the cell suspension and measure the total number of cells and cell viability of each culture dish. The measurement results are shown in Table 3;

[0086] Table 3 Total cell count, viability and culture period of hUC-MSCs cryopreserved in different cryopreservation solutions after 1 year of recovery

[0087] Group Total number of cells Cell viability Cultivation cycle Experimental Group 1 <![CDATA[3.85x 10 6 ]]> 94.1％ 4 days Experimental Group 2 <![CDATA[3.37x 10 6 ]]> 92.6％ 4 days Experimental Group 3 <![CDATA[4.18x 10 6 ]]> 94.4％ 3 days Experimental Group 4 <![CDATA[3.41x 10 6 ]]> 92.8％ 4 days Experimental Group 5 <![CDATA[4.46x 10 6 ]]> 96.6％ 3 days Experimental Group 6 <![CDATA[4.12x 10 6 ]]> 94.7％ 3 days Experimental Group 7 <![CDATA[3.28x 10 6 ]]> 90.4％ 4 days Experimental Group 8 <![CDATA[3.71x 10 6 ]]> 92.5％ 4 days Experimental Group 9 <![CDATA[3.59x 10 6 ]]> 92.0％ 4 days Experimental Group 10 <![CDATA[3.87x 10 6 ]]> 93.7％ 4 days Experimental Group 11 <![CDATA[3.25x 10 6 ]]> 90.9％ 4 days Control group 1 <![CDATA[3.56x 10 6 ]]> 92.8％ 4 days

[0088] From Table 3, we can see that:

[0089] The culture period of hUC-MSCs cryopreserved in each group for 1 year after thawing was 3-4 days. Among them, experimental group 5 performed best in terms of total cell number and cell viability, which were significantly higher than those of the control group and other experimental groups, and the culture period was shorter.

[0090] 4. CCK-8 method was used to detect the cell growth curve of hUC-MSCs after recovery after being frozen in different cryopreservation solutions. The specific operation method is as follows:

[0091] 1) 2x 10 per well 4 Cell inoculation volume: Inoculate 7 24-well cell culture plates at the same time, calculate the total number of cells required, and dilute the cells to a concentration of 2 x 10 in a 15 mL centrifuge tube. 4 / mL, add 1mL to each well; at the same time, only add culture medium to one well in each plate as the daily zero control well.

[0092] 2) After all inoculations are completed, place in a 37°C, 5% CO2 incubator for culture;

[0093] 3) Take a 24-well plate on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day respectively, and select a control well, add 100 μL CCK-8 reagent to each well, and place it in a 37°C, 5% CO2 incubator for 4 hours;

[0094] 4) Preheat the microplate reader for at least 15 minutes in advance, gently shake the 24-well plate to mix, take 150 μL and transfer to a 96-well plate, repeat 3 times for each sample, and measure the absorbance value (OD value) at 450 nm; collect data from days 1 to 7 to draw a cell growth curve, see Figure 2 ;

[0095] Depend on Figure 2 It can be seen that:

[0096] Different cryopreservation solutions had a significant effect on the cell proliferation ability of hUC-MSCs after recovery. The growth curves of hUC-MSCs frozen in each group of cryopreservation solutions for one year showed a trend of first increasing and then gradually decreasing. Among them, the cells in experimental group 5 were able to adapt to the culture environment quickly after recovery, quickly entered the logarithmic growth phase and maintained a high proliferation rate, and finally reached a high cell density, indicating that the cryopreservation solution performed well in maintaining cell proliferation ability, caused less damage to cells, and could better protect the growth potential of cells.

[0097] 5. Flow cytometry was used to analyze the expression of surface markers of hUC-MSCs after resuscitation after cryopreservation in different cryopreservation solutions. The specific operation method is as follows:

[0098] hUC-MSCs frozen in different freezing solutions for 3 months, 6 months and 1 year were taken, digested and washed after subculturing, resuspended in PBS, and transferred to flow cytometry tubes. Each tube contained 1x 10 6hUC-MSCs were labeled with CD73, CD90, CD44, CD105, CD34 and CD45, and the results of flow cytometric analysis are shown in Table 4. The expression of cell surface markers in hUC-MSCs in group 5 frozen for 1 year after flow cytometry analysis is shown in Table 4. Figure 3 As shown;

[0099] Table 4 Expression of cell surface markers in hUC-MSCs cryopreserved in different cryopreservation solutions after thawing for 1 year

[0100]

[0101]

[0102] From Table 4, we can see that:

[0103] After the cryopreservation solution of each group was revived, the positive expression rates of CD73, CD90, CD44 and CD105 of hUC-MSCs were all above 95%, while the negative references CD34 and CD45 were below 2%. Among them, experimental group 5 performed well in maintaining the expression levels of positive and negative surface markers, with a positive expression rate of more than 99%, while the negative reference was as low as below 0.4%, indicating that hUC-MSCs have strong specific recognition ability and can stably express various surface markers.

[0104] 6. Detection of the three-lineage differentiation potential of hUC-MSCs after resuscitation by freezing in different cryopreservation solutions, analysis of the adipogenic ability of stem cells by Oil Red O staining, analysis of the osteogenic ability of stem cells by Alizarin Red staining, and analysis of the chondrogenic ability of stem cells by Alcian Blue staining. The specific operation methods are as follows:

[0105] 1) Adipogenic differentiation: The adipogenic differentiation medium was DMEM containing 10-6 mol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L isobutylxanthine, 200 μmol / L indomethacin and 10% fetal bovine serum. hUC-MSCs frozen for 3, 6 and 12 months in different cryopreservation solutions were taken and incubated at 1.0×10 4 / cm 2 The cells were inoculated in a 12-well plate and replaced with adipose-induced differentiation medium when the cell confluence was 70%-80%. The medium was changed every 4 days and cultured for about 14 days (oil droplets were seen in the cells under the microscope). The cells were observed under the microscope using Oil Red O staining. The results of the Oil Red O staining analysis of the cells in Group 5 after cryopreservation of hUC-MSCs for 1 year were as follows: Figure 4 As shown;

[0106] 2) Osteogenic differentiation: The osteogenic differentiation medium was DMEM containing 10 mol / L dexamethasone, 10 mmol / L β-glycerol phosphate, 0.05 mmol / L 2-phosphate ascorbic acid and 10% fetal bovine serum. hUC-MSCs frozen in different freezing solutions for 3, 6 and 12 months were cultured at 1.0×10 4 / cm 2 The cells were seeded in 12-well plates, and the osteogenic differentiation medium was replaced when the cell confluence reached 70%-80%. The medium was changed every 4 days. The cells were cultured for about 21 days and observed under a microscope using Alizarin red staining. The results of the analysis of the cells in Group 5 after cryopreservation of hUC-MSCs for 1 year were as follows: Figure 5 As shown;

[0107] 3) Chondrogenic differentiation: Chondrogenic differentiation medium is DMEM medium containing 0.1 μmol / L dexamethasone, 50 mg / L L-ascorbic acid, 1 mmol / L sodium pyruvate, 10 μg / L transforming growth factor β, 50 g / L ITS, and 10% fetal bovine serum. hUC-MSCs frozen in different freezing solutions for 3 months, 6 months, and 12 months were taken and incubated at 1.0×10 4 / cm 2 The cells were seeded in 12-well plates and replaced with cartilage differentiation medium when the cell confluence reached 70%-80%. The medium was changed every 4 days. After 14 days of culture, the cells were observed under an Alcian blue staining microscope. The results of the Alcian blue staining analysis of the hUC-MSCs in group 5 after one year of cryopreservation were as follows: Figure 6 As shown;

[0108] Depend on Figures 4 to 6 It can be seen that:

[0109] In experimental group 5, after adipogenesis was induced by Oil Red O staining, dense red lipid droplets appeared in the cytoplasm of a large number of cells. The lipid droplets were relatively uniform in size, deeply stained and widely distributed, indicating that this group of cells had strong adipogenic differentiation ability; after osteogenesis was induced by Alizarin Red staining, a large number of dense red calcium nodules were formed in the extracellular matrix. The nodules were bright in color and connected to each other, indicating that this group of cells had strong osteogenic ability and high osteoblast activity; after cartilage was induced by Alcian Blue staining, the cell clusters showed a rich blue color, indicating that the acidic mucopolysaccharide content in the extracellular matrix was high and the chondroblasts had a strong ability to synthesize cartilage-specific matrix.

[0110] The above experimental data show that the present invention evaluates the cryopreservation effects of the cryopreservation solutions of 11 experimental groups and 1 control group from aspects such as cell morphology, cell viability, cell recovery rate, cell growth trend, passage growth status, cell culture cycle, expression of surface markers and tri-lineage differentiation ability after cryopreservation and resuscitation. Among them, the cryopreservation effect of experimental group 5 is the best.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A cryopreservation solution of umbilical cord mesenchymal stem cells, characterized in that: The umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components: Glycerol 10-20v / v%, polyethylene glycol 10-15v / v%, hydroxyethyl starch 30-100g / L, trehalose 60-100g / L, glutathione 1-5g / L, sodium pyruvate 1-7g / L, compound amino acid injection 10-20v / v%, the remainder is dextran 40 sodium chloride injection.

2. The umbilical cord mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that: The umbilical cord mesenchymal stem cell cryopreservation solution comprises the following components: Glycerol 15v / v%, polyethylene glycol 10v / v%, hydroxyethyl starch 60g / L, trehalose 80g / L, glutathione 5g / L, sodium pyruvate 3g / L, compound amino acid injection 10v / v%, the balance is dextran 40 sodium chloride injection.

3. The umbilical cord mesenchymal stem cell cryopreservation solution according to claim 1 or 2, characterized in that: The dextran 40 sodium chloride injection contains 0.04-0.08 g / mL of dextran 40 and 0.08-0.10 g / mL of sodium chloride.

4. The umbilical cord mesenchymal stem cell cryopreservation solution according to claim 1 or 2, characterized in that: The umbilical cord mesenchymal stem cell freezing solution is obtained by mixing the components and then filtering and sterilizing them.

5. Use of the umbilical cord mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 4 in cryopreservation of umbilical cord mesenchymal stem cells.

6. A method for cryopreservation of umbilical cord mesenchymal stem cells, characterized in that: The cryopreservation method of the umbilical cord mesenchymal stem cells comprises: The umbilical cord mesenchymal stem cells are inoculated into the umbilical cord mesenchymal stem cell freezing solution according to any one of claims 1 to 4 for cryopreservation.

7. The method for cryopreservation of umbilical cord mesenchymal stem cells according to claim 6, characterized in that: The cryopreservation concentration of the umbilical cord mesenchymal stem cells is (0.5-2.0)×10 7 cells / mL.

8. The method for cryopreservation of umbilical cord mesenchymal stem cells according to claim 6, characterized in that: The umbilical cord mesenchymal stem cells are P5 generation human umbilical cord mesenchymal stem cells.

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