A cryopreserved umbilical cord mesenchymal stem cell solution and a cryopreservation method

Abstract

The application discloses a kind of umbilical cord mesenchymal stem cell cryopreservation solution and cryopreservation method, the umbilical cord mesenchymal stem cell cryopreservation solution includes 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 balance is dextran 40 sodium chloride injection;The cryopreservation solution of the application does not contain animal-derived serum, avoids the risk of animal-derived pathogenic microorganism, high safety, and can replace DMSO, avoid the influence of DMSO on the activity of hUC-MSCs after resuscitation, and can achieve better dehydration effect before freezing, through different mechanisms in the freezing process Complementary and unique molecular structure and function, reduce the ice crystal formation of cell in the freezing process, reduce cell damage, can be directly placed in-80 ℃ cryopreservation, without gradient cooling, simple operation, for hUC-MSCs be applied to clinical screening better cryopreservation solution formula.

Description

Technical Field

[0001] This invention relates to the field of cell preservation technology, specifically to a cryopreservation solution and method for umbilical cord mesenchymal stem cells. Background Technology

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

[0003] To preserve the biological activity of cells long-term, cryopreservation is necessary. Currently, the most commonly used technique for cell cryopreservation is liquid nitrogen cryopreservation, which involves placing cells in liquid nitrogen at -196°C to temporarily remove them from their growth state while preserving their cellular characteristics. Traditional cryoprotectants often contain animal-derived serum and dimethyl sulfoxide (DMSO), which may trigger immune responses or cytotoxicity, affecting cell viability and function, making them unsuitable for clinical use. Furthermore, while gradient cooling can reduce cell damage to some extent, it is complex and time-consuming. Therefore, developing a cryopreservation solution and method for hUC-MSCs that is free of animal-derived serum and DMSO and does not require gradient cooling has significant practical value and market potential, laying the foundation for further clinical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a cryopreservation solution and method for umbilical cord mesenchymal stem cells. The cell cryopreservation solution of this invention is free of animal-derived serum and DMSO and does not require gradient cooling to achieve cryopreservation of umbilical cord mesenchymal stem cells. It solves the problems of immune reactions and cytotoxicity caused by traditional cryopreservation solutions, and also solves the problem of complicated gradient cooling operation. Moreover, it improves the cryopreservation effect of hUC-MSCs.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0006] The first aspect of this invention provides a cryopreservation solution for umbilical cord mesenchymal stem cells, the cryopreservation solution for umbilical cord mesenchymal stem cells comprising the following components:

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

[0010] Preferably, 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.

[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 the application of the above-mentioned 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 cryopreserving umbilical cord mesenchymal stem cells, the method comprising:

[0014] Umbilical cord mesenchymal stem cells were seeded into the umbilical cord mesenchymal stem cell cryopreservation solution for cryopreservation.

[0015] Preferably, the cryopreservation concentration of the umbilical cord mesenchymal stem cells is (0.5–2.0) × 10⁻⁶. 7 cells / mL.

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

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

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

[0019] (2) The present invention uses a composite cryoprotectant that does not contain animal-derived serum, which avoids the risk of introducing animal-derived pathogens and immune responses from the body. It is highly safe and can maintain the activity and function of hUC-MSCs cells for a long time, thus improving the cryopreservation effect.

[0020] (3) This invention does not require the use of gradient cooling boxes and programmed cooling, and is simple to operate. Frozen cells can be directly placed in a -80℃ freezer, and after 24 hours, the cells can be transferred to a -196℃ liquid nitrogen tank for long-term storage. The survival rate of the revived hUC-MSCs can reach more than 95%, and it does not affect the cell proliferation ability and has good stability. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0022] Figure 1 In Example 2 of this invention, the growth status of hUC-MSCs after thawing one year after being cryopreserved in various cryopreservation solutions was observed using an inverted microscope.

[0023] Figure 2 This is the growth curve of hUC-MSCs after 1 year of cryopreservation in various cryopreservation solutions as determined by the CCK-8 assay in Example 2 of this invention.

[0024] Figure 3 This invention provides a flow cytometry analysis of the expression of cell surface markers in experimental group 5 after one year of cryopreservation of hUC-MSCs in Example 2 of this invention.

[0025] Figure 4 This invention provides an example of using Oil Red O staining to analyze the adipogenic capacity of frozen hUC-MSCs in experimental group 5 after 1 year.

[0026] Figure 5 This invention provides an example of using alizarin red staining to analyze the osteogenic capacity of frozen hUC-MSCs in experimental group 5 after 1 year.

[0027] Figure 6 This invention relates to the Alcian blue staining method used in Example 2 to analyze the chondrogenic capacity of cryopreserved hUC-MSCs in experimental group 5 after 1 year. Detailed Implementation

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

[0029] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0030] This invention provides a cryopreservation solution for umbilical cord mesenchymal stem cells, which comprises the following components:

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

[0032] In this invention, glycerol, acting as a permeable cryoprotectant, is combined with non-permeable cryoprotectants polyethylene glycol, hydroxyethyl starch, and trehalose to protect the intracellular and extracellular spaces from ice crystal damage during cryopreservation. This combined cryoprotectant approach improves cryopreservation efficiency. Glycerol increases solution viscosity, reduces ice crystal formation, and forms a protective cell membrane. Polyethylene glycol weakens the water crystallization process, reducing damage to cell structure and function and improving cell recovery after thawing. Hydroxyethyl starch acts as a cell sedimentation stabilizer, preventing or delaying cell sedimentation during cryopreservation and preventing cell compression that could affect cryopreservation results. Trehalose's protective effect stems from its interaction with lipid membranes, maintaining protein stability during cryopreservation and thawing. It forms a vitrified matrix to inhibit intracellular ice crystal formation, and its combined use with glycerol further enhances cryopreservation efficiency. Trehalose penetrates cell membranes to enhance its efficacy; glutathione, as an antioxidant, reduces the effects of reactive oxygen species and cold shock damage, improving the preservation of cell function after thawing; sodium pyruvate, as a cell nutrient, replenishes some of the energy consumed during cell metabolism, helps maintain cell membrane stability during cryopreservation, and reduces the impact of oxidative damage on cell membranes; compound amino acid injection (18AA) is a compound preparation, a sterile aqueous solution formulated with 18 amino acids and sorbitol; dextran 40 sodium chloride injection stabilizes cell osmotic pressure and maintains cell morphology and activity. Both dextran and compound amino acid injections are clinical drugs that meet national standards, ensuring cell quality and allowing for direct 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, with the remainder being dextran 40 sodium chloride injection.

[0035] In some embodiments, 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.

[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 the application of the above-mentioned 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 cryopreserving umbilical cord mesenchymal stem cells, the method comprising:

[0039] Umbilical cord mesenchymal stem cells were seeded into the umbilical cord mesenchymal stem cell cryopreservation solution for cryopreservation.

[0040] In some embodiments, the cryopreservation concentration of the umbilical cord mesenchymal stem cells is (0.5–2.0) × 10⁻⁶. 7 cells / mL.

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

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

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

[0044] Glycerol: Injection grade, purchased from Zhejiang Suichang Huikang Pharmaceutical Co., Ltd. (National Drug Approval Number 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. (item number: 6138-23-4);

[0048] Glutathione: Purchased from Sigma (product number: 1294820);

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

[0050] Compound Amino Acid Injection (18AA): Purchased from Bicon Pharmaceutical Jiangsu Co., Ltd. (National Drug Approval Number H20066340);

[0051] Dextran 40 sodium chloride injection, with a dextran 40 content of 30g / 500mL and a sodium chloride content of 4.5g / 500mL, was purchased from Sichuan Kelun Pharmaceutical Co., Ltd. (National Drug Approval Number H51020331).

[0052] Example 1

[0053] This embodiment describes the isolation and culture of hUC-MSCs. The specific operation method is as follows:

[0054] 1) Perform the operation in a clean bench, take a 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 that is bright, undamaged, and without edema. Cut it into 2-3 cm segments using stainless steel tissue scissors. Use tissue scissors to longitudinally cut the umbilical cord and remove the umbilical vein and artery. Cut the umbilical cord to 1 mm. 3 Small pieces of tissue should be cut into a foamy, minced meat-like consistency.

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

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

[0058] Example 2

[0059] 1. The specific procedures for culturing and cryopreserving hUC-MSCs are as follows:

[0060] 1) Mix the seed cells at a ratio of 1.0 x 10 4 / cm 2 Density seeding is performed, and the confluence can generally reach 80%-90% after 3 days of culture, depending on the growth and proliferation status of the cells. Then, the cells are passaged to P5.

[0061] 2) Harvest P5 cells. Add 0.05% Trypsin-EDTA to each culture dish for digestion at room temperature. Observe under a microscope. When the intercellular spaces increase and the cells become rounded, add complete culture medium to each dish to stop the digestion. Collect the digested cells into 50ml centrifuge tubes and centrifuge at 4℃, 600g for 10min.

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

[0063] 4) Based on the calculated cell volume required for the experiment, dispense the cells into eight 15mL centrifuge tubes and centrifuge the cell suspension at 4°C and 600g for 10 minutes.

[0064] 5) Resuspend the cell pellet in different cryopreservation solutions (see Table 1), with a cell density of 1.0 x 10⁻⁶ cells per group. 7hUC-MSCs were cryopreserved at a concentration of / mL. Each cryovial contained 1mL of cell suspension. No gradient cooling was required. The cells were directly placed in a -80°C freezer for 24 hours. After 24 hours, the cryovials were transferred to a liquid nitrogen tank (-196°C) for long-term storage. Cells from each group were revived at 3 months, 6 months, and 1 year for testing and culture.

[0065] Table 1. Grouping, components, and proportions of each cryopreservation solution formulation

[0066]

[0067] Note: The remaining amount in the experimental group was dextran 40 sodium chloride injection.

[0068] 2. The total cell count and cell viability of hUC-MSCs after thawing in different cryopreservation solutions (Table 1) are determined using the following specific procedures:

[0069] 1) Remove hUC-MSCs of different cryopreservation concentrations from liquid nitrogen, thaw them in a 37°C water bath, shake the cryopreservation tubes repeatedly at a uniform speed to thaw them as quickly as possible, and then place them in a clean bench.

[0070] 2) Transfer the cell suspension to a 15 mL centrifuge tube, resuspend it according to the existing cryopreservation solution: DMEM = 1:9, mix thoroughly, centrifuge at 600 g for 10 min, resuspend it with fresh complete culture medium, take 50 μl of cell suspension, and determine the total number of cells and cell viability.

[0071] 3) Use a hemocytometer to count cells. Mix the cell sample thoroughly. Take 20 μl of cell suspension and then take 20 μl of 0.08% trypan blue solution. Mix thoroughly and observe under a microscope. Live cells will not be stained, while dead cells will be stained blue.

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

[0073]

[0074] 5) Repeat the count 3 times and take the average.

[0075] Table 2 shows the cell viability and cell recovery rate of hUC-MSCs after thawing from cryopreservation in different cryopreservation solutions.

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

[0077]

[0078] As shown in Table 2:

[0079] As the cryopreservation time of hUC-MSCs increased, the cell viability and cell recovery rate of the cryopreservation solutions in all groups decreased. Among them, experimental group 5 showed the best performance in maintaining cell viability and improving cell recovery rate, with both cell viability and cell recovery rate significantly higher than the control group and other experimental groups.

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

[0081] 1) hUC-MSCs thawed from different cryopreservation solutions were divided into groups of 1.0 x 10⁻⁶. 4 / cm 2 The culture medium was inoculated into φ10cm petri dishes at the specified density, with 8mL of culture medium added to each petri dish. The dishes were then incubated at 37℃ in a 5% CO2 incubator.

[0082] 2) Observe the growth of hUC-MSCs after cryopreservation and thawing in several groups under an inverted microscope. The cells should adhere to the wall and exhibit an elongated spindle shape. Generally, the confluence can reach 80%-90% after 3 days of culture. Take pictures and save them. The picture results are as follows: Figure 1 As shown;

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

[0084] Microscopic observation revealed that the cells in all groups had good morphology, exhibiting normal spindle or polygonal shapes. Among them, experimental group 5 was able to rapidly adhere to the culture vessel and begin to proliferate during the culture process, showing the highest degree of cell confluence.

[0085] 3) Add 1 mL of 0.05% Trypsin-EDTA to each culture dish and observe under a microscope. When the intercellular spaces increase and the cells become shorter, add 8 mL of the previously collected culture supernatant to stop the digestion. After mixing, take 50 μl of cell suspension and measure the total number of cells and cell viability of each culture dish. The 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] 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] As shown in Table 3:

[0089] After cryopreservation in each group for one year, the culture period for passage to culture dishes was 3-4 days. Among them, experimental group 5 showed the best performance in terms of total cell count and cell viability maintenance, with both the total cell count and cell viability significantly higher than the control group and other experimental groups, and the culture period was shorter.

[0090] 4. The CCK-8 assay was used to detect the cell growth curves of hUC-MSCs after thawing from cryopreservation in different cryopreservation solutions. The specific operation method is as follows:

[0091] 1) According to 2 x 10 per hole 4 Cell seeding volume: Inoculate seven 24-well cell culture plates simultaneously, calculate the required total number of cells, and dilute the cells to a concentration of 2 x 10⁻⁶ cells / mL in 15 mL centrifuge tubes. 4 Add 1 mL of culture medium per well; at the same time, add only one well in each plate as a daily zeroing control well.

[0092] 2) After all inoculations are completed, the incubator is placed at 37°C and incubated with 5% CO2.

[0093] 3) Take one 24-well plate on days 1, 2, 3, 4, 5, 6 and 7 respectively, and select one control well at the same time. Add 100 μL of CCK-8 reagent to each well and incubate at 37℃ in a 5% CO2 incubator for 4 hours.

[0094] 4) Preheat the microplate reader for at least 15 minutes. Gently shake the 24-well plate to mix, then transfer 150 μL to a 96-well plate. Repeat each sample three times. Measure the absorbance (OD) at 450 nm. Collect data from days 1-7 to plot the cell growth curve. See [link to relevant documentation]. Figure 2 ;

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

[0096] Different cryopreservation solutions significantly affected the cell proliferation capacity of hUC-MSCs after thawing. The growth curves of hUC-MSCs cryopreserved in each group for one year all showed an initial increase followed by a gradual decrease. Among them, cells in experimental group 5 adapted to the culture environment more quickly after thawing, rapidly entered the logarithmic growth phase, maintained a high proliferation rate, and ultimately achieved a high cell density. This indicates that the cryopreservation solution performed excellently in maintaining cell proliferation capacity, caused minimal cell damage, and effectively protected the cells' growth potential.

[0097] 5. Flow cytometry analysis of the expression of surface markers in hUC-MSCs after thawing from cryopreservation in different cryopreservation solutions. The specific operation method is as follows:

[0098] hUC-MSCs were cryopreserved for 3 months, 6 months, and 1 year using different cryopreservation solutions. After passage, they were digested, washed, resuspended in PBS, and transferred to flow cytometry tubes, 100 μl per tube containing 1 x 10⁻⁶ MSCs. 6Table 4 shows the results of flow cytometry analysis of hUC-MSCs labeled with CD73, CD90, CD44, CD105, CD34, and CD45. The expression of cell surface markers in group 5 of the experimental group after one year of cryopreservation is also shown in the table. Figure 3 As shown;

[0099] Table 4. Expression of cell surface markers in hUC-MSCs thawed one year after cryopreservation in different cryopreservation solutions.

[0100]

[0101]

[0102] As shown in Table 4:

[0103] After thawing from the cryopreserved solutions, the positive expression rates of CD73, CD90, CD44, and CD105 in hUC-MSCs were all above 95%, while the negative control CD34 and CD45 were below 2%. Experimental group 5 demonstrated excellent performance in maintaining the expression levels of both positive and negative surface markers, with a positive expression rate exceeding 99% and a negative control rate as low as 0.4%, indicating that hUC-MSCs have strong specific recognition capabilities and can stably express various surface markers.

[0104] 6. Detection of the three-lineage differentiation potential of hUC-MSCs after thawing from cryopreservation in different solutions: Oil Red O staining was used to analyze the adipogenic capacity of stem cells, Alizarin Red staining was used to analyze the osteogenic capacity of stem cells, and Alcian Blue staining was used to analyze the chondrogenic capacity of stem cells. Specific operating procedures are as follows:

[0105] 1) Adipogenic differentiation induction: The adipogenic differentiation induction medium was DMEM medium 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 months, 6 months, and 12 months in different cryopreservation solutions were cultured at a concentration of 1.0 × 10⁻⁶ mol / L. 4 / cm 2 Cells were seeded in 12-well plates and replaced with adipogenic differentiation medium when cell confluence reached 70%-80%. The medium was changed every 4 days. After approximately 14 days of culture (when oil droplets were observed in the cells under a microscope), cells were stained with Oil Red O and observed under a microscope. The results of Oil Red O staining analysis of the experimental group 5 hUC-MSCs after 1 year of cryopreservation are shown below. Figure 4 As shown;

[0106] 2) Osteogenic induction differentiation: The osteogenic induction differentiation medium was DMEM medium containing 10 mol / L dexamethasone, 10 mmol / L β-glycerol phosphate, 0.05 mmol / L ascorbic acid 2-phosphate, and 10% fetal bovine serum. hUC-MSCs frozen for 3 months, 6 months, and 12 months in different cryopreservation solutions were cultured at a concentration of 1.0 × 10⁻⁶. 4 / cm 2 Cells were seeded in 12-well plates. When cell confluence reached 70%-80%, the medium was replaced with osteogenic induction differentiation medium, and the medium was changed every 4 days. After approximately 21 days of culture, cells were observed under a microscope after Alizarin Red staining. The Alizarin Red staining method was used to analyze the observation results of frozen hUC-MSCs in experimental group 5 after 1 year. Figure 5 As shown;

[0107] 3) Chondrogenic differentiation induction: The chondrogenic differentiation induction medium was DMEM 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 for 3 months, 6 months, and 12 months in different cryopreservation solutions were cultured at a concentration of 1.0 × 10⁻⁶ mcg / mL. 4 / cm 2 Cells were seeded in 12-well plates and replaced with chondrocyte-inducing differentiation medium when cell confluence reached 70%-80%. The medium was changed every 4 days. After 14 days of culture, cells were observed under a microscope after Alcian blue staining. The Alcian blue staining analysis of the experimental group 5 cryopreserved hUC-MSCs after 1 year is shown below. Figure 6 As shown;

[0108] Depend on Figures 4-6 It can be known that:

[0109] In experimental group 5, after oil red O staining to induce adipogenesis, a large number of cells showed dense red lipid droplets in their cytoplasm. The droplets were relatively uniform in size, deeply stained, and widely distributed, indicating that the cells in this group had a strong adipogenic differentiation capacity. After alizarin red staining to induce osteogenic differentiation, a large number of dense red calcium nodules formed in the extracellular matrix. The nodules were brightly colored and connected to each other, indicating that the cells in this group had a vigorous osteogenic capacity and high osteoblast activity. After Alcian blue staining to induce chondrogenesis, the cell clumps showed a deep blue color, indicating that the extracellular matrix contained a high content of acidic mucopolysaccharides and that the chondrocytes had a strong ability to synthesize cartilage-specific matrix.

[0110] The experimental data above show that this invention evaluated the cryopreservation effects of cryopreservation solutions in 11 experimental groups and 1 control group based on aspects such as cell morphology, cell viability, cell recovery rate, cell growth trend, passage growth status, cell culture cycle, expression of surface markers, and three-lineage differentiation ability after cryopreservation and thawing. Among them, experimental group 5 showed the best cryopreservation effect.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A cryopreservation solution for umbilical cord mesenchymal stem cells, characterized in that, The cryopreservation solution for umbilical cord mesenchymal stem cells 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, with the remainder being dextran 40 sodium chloride injection.

2. The cryopreservation solution for umbilical cord mesenchymal stem cells according to claim 1, 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.

3. The cryopreservation solution for umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The cryopreservation solution for umbilical cord mesenchymal stem cells is obtained by mixing the components and then filtering and sterilizing them.

4. The use of the cryopreservation solution for umbilical cord mesenchymal stem cells according to any one of claims 1 to 3 in the cryopreservation of umbilical cord mesenchymal stem cells.

5. A method for cryopreserving umbilical cord mesenchymal stem cells, characterized in that, The cryopreservation method for the umbilical cord mesenchymal stem cells includes: Umbilical cord mesenchymal stem cells are seeded into the umbilical cord mesenchymal stem cell cryopreservation solution described in any one of claims 1 to 3 for cryopreservation.

6. The method for cryopreservation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that, The cryopreservation concentration of the umbilical cord mesenchymal stem cells was (0.5~2.0) × 10⁻⁶. 7 cells / mL.

7. The method for cryopreservation of umbilical cord mesenchymal stem cells according to claim 5, characterized in that, The umbilical cord mesenchymal stem cells are P5 generation human umbilical cord mesenchymal stem cells.

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