Culture method and application of umbilical cord mesenchymal stem cells

Abstract

The invention discloses a culture method and application of umbilical cord mesenchymal stem cells, mainly relates to the technical field of mesenchymal stem cells, and aims at solving the technical problem of obtaining umbilical cord mesenchymal stem cell exosomes with higher purity through culture and improving the cell survival rate after resuscitation. Compared with the prior art, the purification auxiliary agent is added in the culture method of the umbilical cord mesenchymal stem cells, the purification auxiliary agent is one or more of polyethylene glycol, sodium chloride and heparin sodium, the precipitation efficiency and separation purity of exosomes are improved, and residues of impurities such as impure protein are reduced; furthermore, an antioxidant is added, the antioxidant is one of ascorbyl palmitate, alpha-tocopherol and glutathione, the survival rate of the exosome after recovery is increased by reducing oxidative damage of the exosome in the treatment process, and finally the obtained high-purity and high-activity exosome is applied to eye drops for treating xerophthalmia.

Description

Technical Field

[0001] This invention relates to the field of mesenchymal stem cell technology, and more particularly to a method for culturing umbilical cord mesenchymal stem cells and its application. Background Technology

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs) are pluripotent stem cells derived from umbilical cord tissue, primarily found in Wharton's jelly and perivascular tissue. Their culture methods mainly include tissue block adhesion and enzymatic digestion. In the tissue block adhesion method, umbilical cord tissue is cut into small pieces and seeded in a culture dish, where cells migrate out of the tissue block and adhere to the culture dish for growth. In the enzymatic digestion method, cells are released from the tissue using digestive agents such as collagenase or trypsin before further culture. Current culture conditions typically include 37°C, 5% CO2, and saturated humidity. Culture media include serum-containing media, serum-free media, and mixed media, which usually contain specific growth factors and hormones to promote cell differentiation in specific directions. Choosing an appropriate culture medium is crucial for maintaining the multi-lineage differentiation potential and proliferative capacity of cells.

[0003] Dry eye syndrome is a common ocular surface disease caused by insufficient tear secretion or tear film instability. Symptoms include dry eyes, foreign body sensation, and blurred vision. Umbilical cord mesenchymal stem cells (UCMSCs) have shown significant potential and research progress in the treatment of dry eye syndrome. Treatment methods for dry eye syndrome mainly include drug therapy and surgical treatment. Umbilical cord mesenchymal stem cells possess excellent immunomodulatory and anti-inflammatory properties. By inhibiting the expression of inflammatory factors, adding them to eye drops can reduce ocular surface inflammation. They can also promote the repair and regeneration of corneal epithelial cells, improve corneal damage, and further alleviate dry eye syndrome.

[0004] CN120078715A discloses an eye drop containing exosomes, its preparation method, and its application. The eye drop contains lyophilized exosome powder and is mainly used to treat eye diseases such as dry eye, conjunctivitis, and keratitis. It can also be used for the prevention of eye diseases and the relief of eye fatigue. The addition of Houttuynia cordata extract and mesenchymal stem cell exosomes enhances the anti-inflammatory ability of the eye drop. Furthermore, the lyophilization technology maintains the stability of the active ingredients and extends its shelf life.

[0005] CN117165630A discloses a design method and preparation method of SPARC gene-modified mesenchymal stem cells and their application. SPARC, as an important extracellular matrix protein, participates in processes such as cell adhesion, migration and proliferation, and plays an important role in the repair and stability of corneal epithelial cells. This invention uses SPARC gene-modified mesenchymal stem cells to treat dry eye disease, and has a stronger therapeutic effect. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to obtain umbilical cord mesenchymal stem cell exosomes with higher purity by culturing them, and to improve the cell survival rate after resuscitation so that they can be applied to the treatment of dry eye syndrome.

[0007] To achieve the above objectives, the present invention provides a method for culturing umbilical cord mesenchymal stem cells and its application.

[0008] The method for culturing umbilical cord mesenchymal stem cells includes the following steps, in parts by weight:

[0009] (1) Cut 0.8-1.2 parts of umbilical cord tissue into small pieces, peel off Wharton's jelly and inoculate it into a culture dish; add 5-10 parts of serum-free culture medium and incubate in a 37℃, 5% CO2 incubator; change half of the medium on the second day, and then change the full medium every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0010] (2) Take 0.8-1.2 parts of the culture supernatant of umbilical cord mesenchymal stem cells cultured to P3-5 passage, add 0.8-1.2 parts of purification aid aqueous solution, and place at 0-5℃ for 12 hours; centrifuge at high speed for 0.5-1.5 hours, add 0.8-1.2 parts of sterile phosphate buffered saline, centrifuge at high speed for 0.5-1.5 hours, collect the precipitate, resuspend it with 0.8-1.2 parts of sterile phosphate buffered saline, and place it in a cryovial for freezing at -80℃;

[0011] Alternatively, take 0.8-1.2 parts of the culture supernatant of umbilical cord mesenchymal stem cells cultured to passage P3-5, add 0.8-1.2 parts of purification aid aqueous solution and 0.002-0.004 parts of antioxidant, and incubate at 0-5℃ for 12 hours; centrifuge at high speed for 0.5-1.5 hours, add 0.8-1.2 parts of sterile phosphate buffered saline, centrifuge at high speed for 0.5-1.5 hours, collect the precipitate, resuspend it in 0.8-1.2 parts of sterile phosphate buffered saline, and store it in cryovials at -80℃.

[0012] In step (1), the chopping process involves chopping the material to a size of 1-3mm. 3 .

[0013] The petri dish used in step (1) has a size of 70mm, 90mm, or 100mm.

[0014] The purification aid aqueous solution in step (2) is a 10-14 wt% purification aid aqueous solution; the purification aid is at least one of polyethylene glycol, sodium chloride, and sodium heparin.

[0015] The antioxidant in step (2) is one of ascorbyl palmitate, α-tocopherol, and glutathione.

[0016] The application of the umbilical cord mesenchymal stem cells is as follows: umbilical cord mesenchymal stem cells are used in eye drops to treat dry eye syndrome.

[0017] This invention addresses the problem of insufficient purity and low cell survival rate of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells) exosomes in the treatment of dry eye by optimizing initial culture conditions of umbilical cord tissue, using serum-free culture medium, and controlling the frequency of medium changes and passage timing to provide a suitable environment for stem cell growth and obtain high-quality culture supernatant. Subsequent centrifugation and resuspension processes enhance the separation and purification of exosomes. This invention also incorporates purification aids, one or more of polyethylene glycol, sodium chloride, and sodium heparin, to improve the precipitation efficiency and separation purity of exosomes and reduce the residue of impurities such as proteins. Furthermore, by adding antioxidants, one of ascorbate palmitate, α-tocopherol, and glutathione, oxidative damage to exosomes during processing is reduced, improving their survival rate after resuscitation. Finally, the obtained high-purity, highly active exosomes are applied to eye drops for the treatment of dry eye.

[0018] The beneficial effects of this invention are:

[0019] Compared with existing technologies, this invention adds a purification aid to the umbilical cord mesenchymal stem cell culture method. The purification aid is one or more of polyethylene glycol, sodium chloride, and sodium heparin, which improves the precipitation efficiency and separation purity of exosomes and reduces the residue of impurities such as extraneous proteins. Furthermore, by adding an antioxidant, which is one of ascorbate palmitate, α-tocopherol, and glutathione, the oxidative damage to exosomes during processing is reduced, thereby improving their survival rate after resuscitation. Finally, the obtained high-purity, high-activity exosomes are applied to eye drops for the treatment of dry eye syndrome. Detailed Implementation

[0020] The parameters of the specific chemical substances used in the examples are from the following sources:

[0021] Serum-free culture medium: manufactured by Youkang Biotechnology (Beijing) Co., Ltd., product number NC0103.

[0022] Sodium heparin: Manufacturer is Hubei Xindesheng Materials Technology Co., Ltd., product number DS-002.

[0023] Example 1

[0024] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0025] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0026] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, add 0.12g of polyethylene glycol 8000 to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0027] Example 2

[0028] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0029] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0030] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, add 0.1g of polyethylene glycol 8000 and 0.02g of sodium chloride to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0031] Example 3

[0032] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0033] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0034] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, add 0.1g of polyethylene glycol 8000 and 0.02g of heparin sodium to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0035] Example 4

[0036] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0037] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0038] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, add 0.1g of polyethylene glycol 8000, 0.016g of sodium chloride and 0.004g of sodium heparin to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryopreservation tube and freeze at -80℃.

[0039] Example 5

[0040] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0041] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0042] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, add 0.1g of polyethylene glycol 8000, 0.016g of sodium chloride and 0.004g of sodium heparin to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution and 0.003g of ascorbate palmitate antioxidant, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0043] Example 6

[0044] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0045] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0046] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, take 0.1g of polyethylene glycol 8000, 0.016g of sodium chloride, 0.004g of sodium heparin, add to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution and 0.003g of α-tocopherol antioxidant, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0047] Example 7

[0048] A method for culturing umbilical cord mesenchymal stem cells includes the following steps:

[0049] (1) Cut 1g of umbilical cord tissue into pieces with a size of 2mm. 3 Peel Wharton's gum and inoculate it into a 90 mm culture dish; add 6 mL of serum-free medium and incubate at 37°C in a 5% CO2 incubator; perform a half-volume medium change on the second day, and then perform a full-volume medium change every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells.

[0050] (2) Take 1g of culture supernatant of umbilical cord mesenchymal stem cells cultured to P4 generation, take 0.1g of polyethylene glycol 8000, 0.016g of sodium chloride, 0.004g of sodium heparin, add to 1g of water to obtain a purification aid aqueous solution, add 1g of 12wt% purification aid aqueous solution and 0.003g of glutathione antioxidant, place at 4℃ for 12 hours; centrifuge at 110000×g for 1 hour, add 1mL of sterile phosphate buffered saline, centrifuge at 110000×g for 1 hour, collect the precipitate, resuspend in 1mL of sterile phosphate buffered saline, place in cryovial and freeze at -80℃.

[0051] Test Example 1

[0052] The umbilical cord mesenchymal stem cells obtained by the culture methods in Examples 1-7 were applied to eye drops. The umbilical cord mesenchymal stem cells obtained by the culture methods in Examples 1-7 were added to physiological saline as test samples for the eye drops in Examples 1-7, with a concentration of 0.3 wt%.

[0053] Forty adult male SD rats weighing 300-310g were selected as experimental mice to construct a dry eye mouse model. 0.2% benzalkonium chloride solution was instilled once a day, 5μL per eye, for 14 days, thus creating dry eye mice.

[0054] Mice with dry eye syndrome were randomly divided into 8 groups, corresponding to groups 1-7 of Examples and a control group. Groups 1-7 were instilled with the test samples of the eye drops from Examples 1-7, while the control group was instilled with only physiological saline. The instillation was performed once daily, 5 μL per eye each time, for 7 days. Tear secretion in the mice was recorded using the tear film assay before administration on day 1 and after administration on day 7. The difference in the wetted length of the tear film before and after administration was also recorded, and the average values ​​were summarized in Table 1.

[0055] Table 1

[0056]

[0057]

[0058] The treatment in the control group, which involved instilling saline solution, only provided temporary physical hydration to the ocular surface and could not repair the pathological mechanisms of dry eye, such as ocular surface epithelial damage and decreased lacrimal gland function. Therefore, the treatment and hydration effects on dry eye were minimal. In contrast, the treatments in Examples 1-7 involved instilling saline eye drops containing umbilical cord mesenchymal stem cells. Through the exosomes carrying proteins, nucleic acids, and other bioactive molecules, these drops improved symptoms by promoting ocular surface epithelial cell proliferation, regulating inflammatory responses, and stimulating lacrimal gland secretion. In the dry eye animal model of Example 1, the saline eye drops containing umbilical cord mesenchymal stem cells reduced the levels of pro-inflammatory factors such as IL-1β and IL-6, upregulated the anti-inflammatory factor IL-10, and increased epithelial growth factor (EGF) in tears to promote corneal repair. In Example 1, polyethylene glycol (PEG) was added as a purification aid in the culture method. It primarily dehydrated and concentrated exosomes through size exclusion and osmotic pressure effects. However, since exosome precipitation was promoted solely by osmotic pressure without synergistic regulation from other aids, the exosomes were prone to membrane rupture and degradation of active ingredients during purification due to a lack of protection. This resulted in low effective exosome concentrations and weak activity, offering limited improvement to tear secretion. Examples 2 and 3 introduced sodium chloride and sodium heparin, respectively, for compounding. The sodium chloride added in Example 2 increased ionic strength, neutralized the surface charge of exosome particles, and further promoted the precipitation of exosomes by PEG. This was achieved by adjusting the solution... The enhanced ionic strength of polyethylene glycol (PEG) increases the osmotic pressure gradient, accelerates exosome aggregation and precipitation, and improves precipitation efficiency. In Example 3, the added sodium heparin carries a high anionic charge, which can bind to ligands on the surface of exosomes. At the same time, it captures positively charged impurities (such as lipoproteins) through electrostatic interaction, reducing non-specific adsorption. In Example 4, a multi-component compound of PEG, sodium chloride, and sodium heparin is used. PEG provides osmotic pressure to drive exosome precipitation, sodium chloride amplifies the precipitation effect by optimizing ionic strength, and sodium heparin specifically captures impurities to achieve targeted removal. The three components work synergistically to enhance the effect of eye drops on improving ocular surface moisture in mice with dry eye syndrome. Examples 5-7 introduced different types of active protective antioxidants based on Example 4. Ascorbyl palmitate added in Example 5, as a lipid-soluble derivative of vitamin C, can be located at the lipid-water interface of the membrane, scavenging oxygen free radicals near the membrane and synergistically regenerating oxidized vitamin E, providing dual protection for the membrane structure and membrane proteins. It has both lipid solubility and antioxidant stability, can penetrate the exosome membrane to scavenge free radicals, and simultaneously inhibits membrane lipid peroxidation and oxidative degradation of water-soluble active ingredients. α-Tocopherol added in Example 6 can embed itself in the biological membrane bilayer to capture lipid peroxidation free radicals, thereby protecting membrane lipids from oxidative damage. It mainly inhibits lipid peroxidation by embedding itself in the exosome membrane structure, but its antioxidant effect on water-soluble components within the membrane is weak. Glutathione added in Example 7 is an endogenous water-soluble antioxidant. Although it can scavenge some free radicals, its short half-life and difficulty in penetrating the lipid membrane result in insufficient protection for lipid-soluble components. Therefore, the retention rate of umbilical cord mesenchymal stem cell exosome activity and the therapeutic effect on dry eye syndrome in the eye drops of Examples 6-7 are lower than those in Example 5.

[0059] Test Example 2

[0060] The P5 generation umbilical cord mesenchymal stem cells obtained by the culture methods in Examples 1-7 were cryopreserved at -80℃ for 1 month, transferred to 37℃, and thawed in a water bath for 2 hours. They were then seeded into 1 mL of serum-free culture medium and cultured in a 37℃, 5% CO2 incubator for 2 days. After ultracentrifugation for 5 min, the precipitate was collected, resuspended in PBS buffer, and cell viability was detected. Three control groups were set up for each example. The cell viability is summarized in Table 2.

[0061] Table 2

[0062]

[0063] In Example 1, polyethylene glycol (PEG) was added as a purification aid. This method promoted cell precipitation solely through osmotic pressure, which could lead to impurities disrupting the stability of the extracellular microenvironment during cryopreservation. Cell membrane integrity was also easily damaged during thawing, resulting in the lowest cell viability after thawing. Example 2 introduced sodium chloride, which increased ionic strength to neutralize the surface charge of exosomes, further promoting PEG precipitation. This ionic strength regulation maintained osmotic pressure balance, reducing cell shrinkage or swelling damage during cryopreservation and thawing, thus improving cell viability after thawing. Example 3 added sodium heparin, which specifically binds to impurity proteins through charge-electric interactions, reducing the physical compression of cells by impurities and further improving cell viability after thawing. Example 4 combined PEG, sodium chloride, and sodium heparin to create a synergistic effect of efficient precipitation, osmotic pressure stabilization, and targeted impurity removal, providing a purer and more stable cryopreservation microenvironment for cells and improving cell viability after thawing.

[0064] Examples 5-7, based on Example 4, added antioxidants to further block the free radical burst caused by ice crystals at -80℃ during cryopreservation and the oxidative stress damage caused by lipid peroxidation exacerbated by the 37℃ temperature rise during thawing. Among them, the ascorbate palmitate added in Example 5 has both lipid solubility and antioxidant stability, can penetrate the exosome membrane to scavenge free radicals, inhibit membrane lipid peroxidation and oxidative degradation of water-soluble active ingredients, and provides comprehensive protection for cell activity, resulting in the highest survival rate of revived cells. The α-tocopherol added in Example 6 focuses on protecting the cell membrane lipid structure and reducing lipid peroxidation, but its antioxidant protection effect on intracellular water-soluble components is slightly weaker. The glutathione added in Example 7, as an endogenous antioxidant, can penetrate the cell membrane to scavenge intracellular free radicals, protect organelle structures such as mitochondria, and inhibit oxidative damage caused by ice crystal formation.

[0065] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for culturing umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps, in parts by weight: (1) Cut 0.8-1.2 parts of umbilical cord tissue into small pieces, peel off Wharton's jelly and inoculate it into a culture dish; add 5-10 parts of serum-free culture medium and incubate in a 37℃, 5% CO2 incubator; change half of the medium on the second day, and then change the full medium every 3 days thereafter. When the cell confluence reaches 80-90%, passage the cells. (2) Take 0.8-1.2 parts of the culture supernatant of umbilical cord mesenchymal stem cells cultured to P3-5 passage, add 0.8-1.2 parts of purification aid aqueous solution, and place at 0-5℃ for 12 hours; centrifuge at high speed for 0.5-1.5 hours, add 0.8-1.2 parts of sterile phosphate buffered saline, centrifuge at high speed for 0.5-1.5 hours, collect the precipitate, resuspend it with 0.8-1.2 parts of sterile phosphate buffered saline, and place it in a cryovial for freezing at -80℃; Alternatively, take 0.8-1.2 parts of the culture supernatant of umbilical cord mesenchymal stem cells cultured to passage P3-5, add 0.8-1.2 parts of purification aid aqueous solution and 0.002-0.004 parts of antioxidant, and incubate at 0-5℃ for 12 hours; centrifuge at ultraspeed for 0.5-1.5 hours, add 0.8-1.2 parts of sterile phosphate buffered saline, centrifuge at ultraspeed for 0.5-1.5 hours, collect the precipitate, resuspend it in 0.8-1.2 parts of sterile phosphate buffered saline, and store it in cryovials at -80℃; The purification aid is one or more of polyethylene glycol, sodium chloride, and sodium heparin.

2. The method for culturing umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, In step (1), the chopping process involves chopping the material to a size of 1-3mm. 3 .

3. The method for culturing umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The petri dish used in step (1) has a size of 70mm, 90mm, or 100mm.

4. The method for culturing umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The purification aid aqueous solution in step (2) is a 10-14 wt% purification aid aqueous solution.

5. The method for culturing umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The purification aid is at least one of polyethylene glycol, sodium chloride, and sodium heparin.

6. The method for culturing umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, The antioxidant in step (2) is one of ascorbyl palmitate, α-tocopherol, and glutathione.

7. An umbilical cord mesenchymal stem cell, characterized in that, It is cultured by the culture method described in any one of claims 1-6.

8. An application of umbilical cord mesenchymal stem cells as described in claim 7, characterized in that, The umbilical cord mesenchymal stem cells were used in eye drops to treat dry eye syndrome.

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