A preparation method of human umbilical cord mesenchymal stem cell exosome based on cobalt chloride induced culture and application thereof in treatment of dry eye

Abstract

This invention belongs to the interdisciplinary field of stem cell biology and ophthalmic treatment, and relates to a method for preparing exosomes from human umbilical cord mesenchymal stem cells (hUC-MSCs) induced by cobalt chloride and their application in the treatment of dry eye. The method for preparing exosomes from hUC-MSCs induced by cobalt chloride includes the following steps: S1: Obtaining human umbilical cord mesenchymal stem cells. S2: Under normoxic conditions, adding the hUC-MSCs obtained in step S1 to a culture medium containing cobalt chloride, followed by induction culture for 2-10 hours. S3: Performing ultracentrifugation on the cell culture medium obtained in step S2 to obtain hUC-MSC exosomes. This invention, by culturing hUC-MSCs under normoxic conditions with cobalt chloride, can quickly and efficiently increase the content of miRNA-21-5p and TGF-β in exosomes, without requiring hypoxia equipment, resulting in low cost, simple operation, and exosome activity fluctuation ≤10%, significantly improving preparation efficiency and stability. Drugs prepared from the exosomes obtained by this invention have excellent effects in treating dry eye and have promising application prospects.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of stem cell biology and ophthalmic treatment, specifically to a method for preparing exosomes of human umbilical cord mesenchymal stem cells induced by cobalt chloride and their application in the treatment of dry eye syndrome. Background Technology

[0002] Dry eye syndrome, a prevalent chronic ocular surface disease, has a complex pathogenesis, primarily manifested as ocular surface epithelial damage, persistent inflammatory response, and decreased lacrimal gland secretion. Current clinical treatments for dry eye syndrome mainly rely on artificial tears and cyclosporine, but these methods have significant limitations. Artificial tears only provide temporary lubrication and cannot promote ocular surface tissue repair; they also require frequent use, leading to poor patient compliance. While cyclosporine can suppress inflammation, long-term use may cause ocular surface irritation and corneal epithelial toxicity, and it lacks regenerative effects on damaged lacrimal gland function. More importantly, existing therapies have failed to achieve synergistic treatment of ocular surface repair, inflammation suppression, and glandular function recovery, resulting in a persistently high relapse rate. Therefore, a novel treatment strategy that can fundamentally improve the pathological state of dry eye syndrome is urgently needed.

[0003] In recent years, exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) have become an important research direction in the treatment of dry eye due to their low immunogenicity, good tissue penetration ability, and rich content of various bioactive molecules such as anti-inflammatory factors, growth factors, and miRNAs. Exosomes can effectively deliver functional molecules to ocular surface cells, regulate apoptosis, inhibit inflammatory pathways, and promote tissue regeneration, showing great potential in the treatment of dry eye. However, exosomes secreted by hUC-MSCs cultured in a normoxic environment with an oxygen concentration of 21% exhibit low expression levels of key functional components such as miRNA-21-5p and TGF-β, resulting in insufficient biological activity.

[0004] To enhance exosome activity, current techniques often employ physical hypoxic incubators with oxygen concentrations of 1-5% to simulate the in vivo microenvironment; however, this method still faces significant industrialization bottlenecks.

[0005] First, this method relies on expensive equipment. The investment in a single hypoxia incubator and its supporting system is large, and the cost of large-scale production of 100-liter incubators exceeds 500,000 yuan.

[0006] Secondly, the culture method is complex to operate and requires precise control of oxygen concentration. Opening the chamber during the culture process can easily lead to a sudden increase in oxygen concentration, affecting the cell's hypoxia response and causing exosome activity to fluctuate by 25-35%, resulting in poor batch-to-batch stability.

[0007] Third, the physical hypoxia culture method has a long culture cycle, requiring 5-7 days, but the yield of exosomes is only 80% of that of normoxic culture, resulting in low production efficiency and failing to meet the large-scale clinical demand for highly active exosomes.

[0008] Therefore, there is a need for a method for preparing exosomes that has lower culture costs, is simpler to operate, and has a shorter cycle. Summary of the Invention

[0009] (a) Technical problems to be solved

[0010] To address the problems of high cost, complex operation, and long culture cycle in existing technologies for hypoxia culture, this invention provides a method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture and their application in the treatment of dry eye syndrome.

[0011] (II) Technical Solution

[0012] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0013] In a first aspect, the present invention provides a method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, comprising the following steps:

[0014] S1: Obtain human umbilical cord mesenchymal stem cells;

[0015] S2: Under normoxic conditions, the human umbilical cord mesenchymal stem cells obtained in step S1 were added to a culture medium containing cobalt chloride and then induced to culture for 2-10 hours.

[0016] S3: The cell culture medium obtained in step S2 is subjected to ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes.

[0017] In the method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture as described above, preferably, in step S1, human umbilical cord mesenchymal stem cells are obtained as follows: umbilical cords from cesarean sections at 37-42 weeks of gestation are taken, the outer membrane and umbilical arteries and veins are removed, Wharton's jelly is retained, the tissue is minced and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells.

[0018] In the method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture as described above, preferably, in step S1, the tissue is minced and prepared into a tissue homogenate, mixed with culture medium, and then added to a culture flask. Primary culture is carried out at 37°C and 5% CO2 using the tissue block adhesion method. The medium is changed for the first time on day 5, for the second time on day 10-12, and for the third time on day 14, until the cell confluence reaches 80-90% on day 14.

[0019] In the method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture as described above, preferably, in step S2, under normoxic conditions, the 5th and 6th generation human umbilical cord mesenchymal stem cells with a viability ≥95% obtained in step S1 are cultured at a rate of 8000-10000 cells / cm³. 2 The inoculum was inoculated into culture flasks at a density of 50-150 μmol / L. After culturing for 3 days, a medium containing cobalt chloride was added to make the cobalt chloride concentration 50-150 μmol / L. Then, the culture was induced at 37℃ and 5% CO2.

[0020] The method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture as described above is preferably as follows: in step S2, the preparation method of the culture medium containing cobalt chloride is as follows: add cobalt chloride solution to the serum-free culture medium for mesenchymal stem cells, and after filtration through a filter membrane to remove bacteria, obtain a culture medium with a cobalt chloride concentration of 50-150 μmol / L.

[0021] The method for preparing exosomes from human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture, as described above, preferably includes the following steps in step S3: Ultracentrifugation of the cell culture medium.

[0022] S31: Centrifuge the cell culture medium at 300g-500g for 10-15min at 4℃ and collect the supernatant;

[0023] S32: Centrifuge the collected supernatant at 20000g at 4℃ for 30min, and collect the supernatant again;

[0024] S33: Filter the supernatant after high-speed centrifugation through a 0.45μm filter membrane, then filter it through a 0.22μm filter membrane, and collect the filtered supernatant;

[0025] S34: Centrifuge the filtered supernatant at 100,000-120,000g for 70-90 minutes at 4°C, and collect the precipitate at the bottom of the centrifuge tube;

[0026] S35: After resuspending the precipitate in pre-cooled sterile PBS, centrifuge at 100,000-120,000 g for 70-90 min at 4°C; resuspend the precipitate again in pre-cooled sterile PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

[0027] The present invention also provides the application of the exosomes prepared by the above-mentioned method for preparing human umbilical cord mesenchymal stem cell exosomes induced by cobalt chloride in the preparation of drugs for treating dry eye syndrome.

[0028] The present invention also provides the application of the exosomes prepared by the above-mentioned method for preparing human umbilical cord mesenchymal stem cell exosomes induced by cobalt chloride in the preparation of eye drops for treating dry eye syndrome.

[0029] (III) Beneficial Effects

[0030] This invention effectively simulates the stimulatory effect of physical hypoxia on human umbilical cord mesenchymal stem cells by adding cobalt chloride to the culture medium under normoxic conditions for short-term induction culture. Cobalt chloride stabilizes hypoxia-inducible factor HIF-1α by inhibiting proline hydroxylase activity, thereby activating intracellular hypoxia response pathways and significantly increasing the expression levels of key functional molecules in exosomes, such as miRNA-21-5p and TGF-β. The relative expression level of miRNA-21-5p reached more than 2.0 times that of normoxic culture, and the TGF-β content increased to over 180 pg / mL, thus solving the problem of insufficient biological activity of exosomes cultured under normoxic conditions.

[0031] Compared to physical hypoxia methods that rely on expensive hypoxia incubators, this invention requires no special equipment investment. It achieves a hypoxia effect simply by adding cobalt chloride directly to a conventional culture medium, significantly reducing system construction and maintenance costs. Equipment investment is reduced by 70% compared to traditional methods, effectively overcoming the industrialization bottleneck caused by expensive equipment in existing technologies. Simultaneously, this method avoids complex operations such as oxygen concentration control and gas replacement, simplifying the process flow, reducing the risk of fluctuations caused by human intervention, and keeping exosome activity fluctuations within 10%, significantly improving batch-to-batch stability.

[0032] Furthermore, this invention shortens the culture time to 2-3 days, compared to the 5-7 day cycle required by traditional physical hypoxia culture, greatly improving production efficiency and solving the problems of long cycles and low yields in existing hypoxia cultures. Combined with stable culture conditions under normoxic environments, it avoids sudden changes in oxygen concentration caused by opening the chamber, ensuring the consistency of cell state and achieving efficient and reproducible preparation of highly active exosomes, providing a reliable technical path for subsequent large-scale production and clinical applications. Attached Figure Description

[0033] Figure 1 Morphological diagram for culturing human umbilical cord mesenchymal stem cells;

[0034] Figure 2 This is a TEM image of the exosomes cultured in Example 1;

[0035] Figure 3 TEM images of exosomes cultured using existing low-oxygen culture methods;

[0036] Figure 4This is a Western blot image of the exosomes cultured in Example 1;

[0037] Figure 5 The image shows the Western blot results of exosomes cultured using existing low-oxygen culture methods.

[0038] Figure 6 A comparison of tear secretion after 14 days of treatment among the normal control group, model control group, hypoxia exosome group, normoxic exosome group, and positive control group.

[0039] Figure 7 A comparison of corneal fluorescein staining scores after 14 days of treatment in the normal control group, model control group, hypoxia exosome group, normoxic exosome group, and positive control group.

[0040] Figure 8 The images show staining of the anterior segment of the normal control group, model control group, hypoxic exosome group, normoxic exosome group, and positive control group before and after treatment. Detailed Implementation

[0041] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] This invention provides a method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, comprising the following steps:

[0043] S1: Obtain human umbilical cord mesenchymal stem cells.

[0044] S2: Under normoxic conditions, the human umbilical cord mesenchymal stem cells obtained in step S1 were added to a culture medium containing cobalt chloride and then induced to culture for 2-10 hours.

[0045] S3: The cell culture medium obtained in step S2 is subjected to ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes.

[0046] This invention effectively simulates the stimulatory effect of physical hypoxia on human umbilical cord mesenchymal stem cells by adding cobalt chloride to the culture medium under normoxic conditions for short-term induction. Cobalt chloride stabilizes the hypoxia-inducible factor HIF-1α by inhibiting proline hydroxylase activity, thereby activating the intracellular hypoxia response pathway and significantly increasing the expression levels of key functional molecules in exosomes, such as miRNA-21-5p and TGF-β. The relative expression level of miRNA-21-5p reached more than 2.0 times that of normoxic culture, and the TGF-β content increased to over 180 pg / mL, thus solving the problem of insufficient biological activity of exosomes cultured under normoxic conditions.

[0047] Compared to physical hypoxia methods that rely on expensive hypoxia incubators, this invention requires no special equipment investment. It achieves a hypoxia effect simply by adding cobalt chloride directly to a conventional culture medium, significantly reducing system construction and maintenance costs. Equipment investment is reduced by 70% compared to traditional methods, effectively overcoming the industrialization bottleneck caused by expensive equipment in existing technologies. Simultaneously, this method avoids complex operations such as oxygen concentration control and gas replacement, simplifying the process flow, reducing the risk of fluctuations caused by human intervention, and keeping exosome activity fluctuations within 10%, significantly improving batch-to-batch stability.

[0048] Furthermore, this invention shortens the culture time to 2-3 days, compared to the 5-7 day cycle required by traditional physical hypoxia culture, greatly improving production efficiency and solving the problems of long cycles and low yields in existing hypoxia cultures. Combined with stable culture conditions under normoxic environments, it avoids sudden changes in oxygen concentration caused by opening the chamber, ensuring the consistency of cell state and achieving efficient and reproducible preparation of highly active exosomes, providing a reliable technical path for subsequent large-scale production and clinical applications.

[0049] Preferably, in step S1 above, human umbilical cord mesenchymal stem cells are obtained as follows: Healthy full-term cesarean section umbilical cords at 37-42 weeks of gestation are harvested, the outer membrane and umbilical arteries and veins are removed, and Wharton's jelly is retained so that the separable Wharton's jelly weight of a single umbilical cord is ≥5g. The tissue is minced and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells. The time from umbilical cord collection to transportation to the laboratory should not exceed 36 hours, and the cord should be stored at 2-10℃ during transportation.

[0050] Specifically, after the tissue was minced and prepared into a tissue homogenate, it was mixed with culture medium and then added to a culture flask. The volume ratio of the culture medium containing 1% antibiotics to the tissue homogenate was 18-20:1-1.5. Primary culture was carried out using the tissue block adhesion method at 37℃ and 5% CO2. During the culture process, the medium was changed for the first time on day 5, the second time on days 10-12, and the third time on day 14, until the cell confluence reached 80-90% on day 14, and the purity met the following conditions: CD90+CD105+CD73+≥95%, CD45-, HLA-DR≤2%.

[0051] Preferably, in step S2 above, under normoxic conditions, the 3rd-4th generation human umbilical cord mesenchymal stem cells with a viability ≥95% obtained in step S1 are processed at a rate of 8000-10000 cells / cm³. 2The cells were seeded at a density in culture flasks, and then culture medium containing cobalt chloride was added to the flasks to achieve a cobalt chloride concentration of 50-150 μmol / L. The culturing was then induced at 37°C and 5% CO2. These operational parameters maximize the activation of intracellular hypoxia response pathways and increase the expression levels of key functional molecules in exosomes, thereby enhancing the therapeutic effect.

[0052] The preparation method of the culture medium containing cobalt chloride is as follows: Add cobalt chloride solution to the serum-free mesenchymal stem cell culture medium, filter to remove bacteria, and obtain a culture medium with a cobalt chloride concentration of 50-150 μmol / L. Store at -20℃ for 3 months. This method is not only easy to operate, but also ensures the sterility of the culture medium, avoiding experimental failures or batch-to-batch variations caused by contamination, and ensuring the stability and reliability of the exosome preparation process.

[0053] Preferably, in step S3 above, the ultracentrifugation of the cell culture medium includes the following steps:

[0054] S31: At 4℃, centrifuge the cell culture medium at a centrifugal force of 300g-500g for 10-15 minutes. After centrifugation, collect the supernatant into a new high-speed centrifuge tube and discard the cell pellet at the bottom of the tube.

[0055] S32: Place the collected supernatant into a high-speed centrifuge and centrifuge at 20000g for 30 minutes at 4°C. Collect the supernatant into a new centrifuge tube and discard the cell debris precipitate.

[0056] S33: Slowly push the supernatant after high-speed centrifugation into a 0.45μm filter membrane using a syringe, collect the filtrate, and filter it again through a 0.22μm filter membrane, then collect the filtrate.

[0057] S34: Transfer the filtrate to pre-chilled ultracentrifuge tubes. Weigh accurately using an analytical balance, ensuring the weight difference between symmetrically placed tubes is ≤0.1g. Centrifuge at 100,000g-120,000g for 70-90 minutes at 4°C. A milky white flocculent precipitate will be visible at the bottom of the tube. Slowly aspirate the supernatant along the tube wall using a pipette, retaining the precipitate. Add pre-chilled PBS to the ultracentrifuge tube containing the precipitate and gently pipette 10-15 times to ensure complete resuspension of the precipitate, avoiding the formation of air bubbles.

[0058] S35: Centrifuge at 100,000g-120,000g for 70-90 minutes at 4℃. After centrifugation, carefully discard the supernatant and retain the precipitate at the bottom of the tube. Resuspend the precipitate in pre-cooled PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

[0059] This invention removes impurities such as intact cells, cell debris, apoptotic bodies, and large vesicles from the supernatant by gradually increasing the centrifugation speed and extending the centrifugation time. This method effectively removes non-specific components, improves the purity and concentration of exosomes, and reduces the impact on the structure and function of exosomes, providing high-quality raw materials for subsequent applications.

[0060] The exosomes prepared by the above-described method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture of the present invention can be used in the preparation of drugs for treating dry eye syndrome, specifically in eye drops for treating dry eye syndrome.

[0061] The hUC-MSCs exosomes prepared in this invention possess high activity and good biocompatibility, and can be directly used to prepare eye drop formulations for treating dry eye syndrome. The prepared exosome eye drops showed significant efficacy in animal models: after 14 days of treatment, tear secretion increased by ≥30% compared to the model control group, corneal fluorescein staining score decreased by ≥30%, and anterior segment lesions were significantly improved, achieving a synergistic effect of "repair-anti-inflammatory-promoting lacrimal gland regeneration." Furthermore, the formulation's pH value, osmotic pressure, and other parameters meet the requirements for ocular administration, demonstrating high safety. Only twice-daily dosing is required, resulting in good patient compliance and promising prospects for clinical translation.

[0062] To further clarify the present invention and its technological advancements, the following description is provided in conjunction with specific embodiments and technical effects.

[0063] Example 1

[0064] This embodiment provides a method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, including the following steps:

[0065] S1: Healthy full-term cesarean section umbilical cords at 40 weeks gestation were harvested. The outer membrane and umbilical arteries and veins were removed, but Wharton's jelly was retained. The separable Wharton's jelly weight of a single umbilical cord was 5g. The tissue was minced and prepared into a tissue homogenate, which was then mixed with culture medium and added to a culture flask. Primary culture was performed at 37℃ and 5% CO2 using the tissue block adhesion method. The medium was changed for the first time on day 5, the second time on day 11, and the third time on day 14. On day 14, the cell confluence reached 88.3%, and the purity of P3 generation cells met the following conditions: CD90+CD105+CD73+≥95%, CD45-, HLA-DR≤2%. The volume ratio of culture medium containing 1% penicillin antibiotics to tissue homogenate was 19:1.1.

[0066] S2: Under normoxic conditions, the 5th and 6th generation human umbilical cord mesenchymal stem cells with a viability ≥95% obtained in step S1 were processed at a rate of 9000 cells / cm³. 2The cells were inoculated at a density in culture flasks and cultured for 3 days. Then, a medium containing cobalt chloride was added to achieve a cobalt chloride concentration of 100 μmol / L. The cells were then induced to culture at 37°C and 5% CO2 for 6 hours. The preparation method of the cobalt chloride-containing medium in this step is as follows: Cobalt chloride solution was added to the serum-free mesenchymal stem cell medium, and after sterilization by filtration through a filter membrane, a medium with a cobalt chloride concentration of 100 μmol / L was obtained and stored at -20°C.

[0067] S3: Centrifuge the cell culture medium at 300g for 15 min at 4℃. Collect the supernatant into a new high-speed centrifuge tube. Centrifuge at 20000g for 30 min at 4℃. Collect the supernatant and slowly push it into a 0.45μm filter membrane using a syringe. Filter through a 0.22μm filter membrane and collect the filtrate. Transfer the filtrate to a pre-chilled ultracentrifuge tube and centrifuge at 100000g for 90 min at 4℃. A milky white flocculent precipitate will be visible at the bottom of the tube. Slowly aspirate the supernatant along the tube wall using a pipette, retaining the precipitate. Add pre-chilled PBS to the ultracentrifuge tube containing the precipitate and gently pipette 15 times to ensure complete resuspension. Centrifuge again at 100000g for 90 min at 4℃, retaining the precipitate at the bottom of the tube. Resuspend the precipitate in pre-chilled PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

[0068] Example 2

[0069] This embodiment provides a method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, including the following steps:

[0070] S1: Harvested umbilical cords from healthy full-term cesarean sections at 34 weeks of gestation. The outer membrane and umbilical arteries and veins were removed, but Wharton's jelly was retained to ensure a separable Wharton's jelly weight of 6g per cord. The tissue was minced and homogenized, then mixed with culture medium and added to a culture flask. Primary culture was performed at 37℃ and 5% CO2 using the tissue block adhesion method. The medium was changed for the first time on day 5, the second time on day 10, and the third time on day 14. Cell confluence reached 80% on day 14. P3 generation cells met the following purity requirements: CD90+CD105+CD73+≥95%, CD45-, HLA-DR≤2%. The volume ratio of culture medium containing 1% penicillin-antibody solution to the tissue homogenate was 19:1.

[0071] S2: Under normoxic conditions, the 5th and 6th generation human umbilical cord mesenchymal stem cells obtained in step S1 with a viability ≥95% were cultured at 8000 cells / cm³. 2The cells were inoculated at a density in culture flasks and cultured for 3 days. Then, a medium containing cobalt chloride was added to achieve a cobalt chloride concentration of 50 μmol / L. The cells were then induced to grow at 37°C with 5% CO2 for 10 hours. The preparation method of the cobalt chloride-containing medium in this step is as follows: Cobalt chloride solution was added to the serum-free mesenchymal stem cell medium, and after sterilization by filtration through a filter membrane, a medium with a cobalt chloride concentration of 50 μmol / L was obtained and stored at -20°C.

[0072] S3: Centrifuge the cell culture medium at 500g for 10 min at 4℃. Collect the supernatant into a new high-speed centrifuge tube. Centrifuge at 20000g for 30 min at 4℃. Collect the supernatant and slowly push it into a 0.45μm filter membrane using a syringe, then filter through a 0.22μm filter membrane. Collect the filtrate. Transfer the filtrate to a pre-chilled ultracentrifuge tube and centrifuge at 110000g for 80 min at 4℃. A milky white flocculent precipitate will be visible at the bottom of the tube. Slowly aspirate the supernatant along the tube wall using a pipette, retaining the precipitate. Add pre-chilled PBS to the ultracentrifuge tube containing the precipitate and gently pipette 10 times to ensure complete resuspension. Centrifuge again at 110000g for 80 min at 4℃, retaining the precipitate at the bottom of the tube. Resuspend the precipitate in pre-chilled PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

[0073] Example 3

[0074] This embodiment provides a method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, including the following steps:

[0075] S1: Healthy full-term cesarean section umbilical cords at 42 weeks gestation were harvested. The outer membrane and umbilical arteries and veins were removed, but Wharton's jelly was retained to ensure a separable Wharton's jelly weight of 7g per cord. The tissue was minced and homogenized, then mixed with culture medium and added to a culture flask. Primary culture was performed at 37℃ and 5% CO2 using the tissue block adhesion method. The medium was changed for the first time on day 5, the second time on day 12, and the third time on day 14. Cell confluence reached 90% on day 14, and the purity of P3 generation cells met the following conditions: CD90+CD105+CD73+≥95%, CD45-, HLA-DR≤2%. The volume ratio of culture medium containing 1% penicillin antibiotics to tissue homogenate was 19:1.2.

[0076] S2: Under normoxic conditions, the 5th and 6th generation human umbilical cord mesenchymal stem cells with a viability ≥95% obtained in step S1 were processed at a rate of 10,000 cells / cm³. 2The cells were inoculated at a density in culture flasks and cultured for 3 days. Then, a medium containing cobalt chloride was added to achieve a cobalt chloride concentration of 150 μmol / L. The cells were then induced to culture at 37°C and 5% CO2 for 2 hours. The preparation method of the cobalt chloride-containing medium in this step is as follows: Cobalt chloride solution was added to the serum-free mesenchymal stem cell medium, and after sterilization by filtration through a filter membrane, a medium with a cobalt chloride concentration of 150 μmol / L was obtained and stored at -20°C.

[0077] S3: Centrifuge the cell culture medium at 400g for 12 min at 4℃. Collect the supernatant into a new high-speed centrifuge tube. Centrifuge at 20000g for 30 min at 4℃. Collect the supernatant and slowly push it into a 0.45μm filter membrane using a syringe, then filter through a 0.22μm filter membrane. Collect the filtrate. Transfer the filtrate to a pre-chilled ultracentrifuge tube and centrifuge at 120000g for 70 min at 4℃. A milky white flocculent precipitate will be visible at the bottom of the tube. Slowly aspirate the supernatant along the tube wall using a pipette, retaining the precipitate. Add pre-chilled PBS to the ultracentrifuge tube containing the precipitate and gently pipette 13 times to ensure complete resuspension. Centrifuge again at 120000g for 70 min at 4℃, retaining the precipitate at the bottom of the tube. Resuspend the precipitate in pre-chilled PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

[0078] Comparative Example 1

[0079] This comparative example provides a method for preparing human umbilical cord mesenchymal stem cell exosomes. The difference from Example 1 is that cobalt chloride is not used, and the induction culture time in step S2 is 5 days.

[0080] After completing the ultracentrifugation treatment of exosomes, the exosomes obtained in Examples 1-3 and Comparative Example 1 were subjected to systematic quality and activity testing, as follows:

[0081] Figure 1 The morphological diagram of human umbilical cord mesenchymal stem cells after culture, as shown in Example 1 and step S1 of the example, is obtained through... Figure 1 It is known that the cell morphology of human umbilical cord mesenchymal stem cells is that of long spindle-shaped adherent cells. In addition, it was observed that the human umbilical cord mesenchymal stem cells cultured in Examples 2 and 3 also had the morphology of long spindle-shaped adherent cells.

[0082] Figure 2 The image shows the TEM morphology of the exosomes cultured in Example 1 (scale bar 200 nm). Figure 3 TEM images of exosomes cultured using existing low-oxygen culture methods. Figure 2 as well as Figure 3All exosomes exhibited a cup-shaped structure, indicating that the morphology of human umbilical cord mesenchymal stem cell exosomes cultured using cobalt chloride induction was the same as that of human umbilical cord mesenchymal stem cell exosomes cultured without cobalt chloride, both exhibiting a typical cup-shaped structure. This demonstrates that cobalt chloride does not affect the morphology of the prepared exosomes. Furthermore, the morphology of the exosomes in Examples 2-3 was the same as that in Example 1.

[0083] Figure 4 The image shows the Western Blot results of the exosomes cultured in Example 1. Figure 5 This image shows the Western blot results of exosomes cultured using existing hypoxic culture methods. Figure 4 as well as Figure 5 It can be seen that the surface markers CD63, CD9, and CD81 of the exosomes obtained in Example 1 and under hypoxic conditions were positive, which is consistent with the characteristics of exosomes. Similarly, the surface markers CD63, CD9, and CD81 of the exosomes obtained in Examples 2-3 were also positive. In terms of functional activity, qPCR detection showed that the relative expression level of miRNA-21-5p of the exosomes in Examples 1-3 reached 2.3±0.2 times that of the normoxic group (Comparative Example 1), which was significantly higher than the qualified standard of ≥2.0 times. The TGF-β content detected by ELISA was 185±12pg / mL, which met the requirement of ≥180pg / mL. The sterility test of the exosomes in Examples 1-3 showed no bacterial or fungal growth after 14 days of aerobic and anaerobic culture, indicating that the preparation was sterile and all indicators met the standards of high-quality exosome preparations.

[0084] To verify the effectiveness of the exosomes prepared in the examples in the treatment of dry eye, the exosome concentrates obtained in Examples 1-3 and Comparative Example 1 were diluted with sterile physiological saline and prepared into eye drop formulations, with the final concentration adjusted to 3.5 × 10⁻⁶. 9The concentration of particles / mL, pH 5.8-7.6, and osmotic pressure 280-340 mOsm / kg meet the standards for ophthalmic preparations. A treatment experiment was conducted using a mouse model of dry eye. Animals were divided into five groups: normal control group (Ctrl), model control group (BAC), positive control group (BAC + cyclosporine A, i.e., CsA), normoxic exosome group (BAC + normoxic, without cobalt chloride induction culture, i.e., Comparative Example 1), and hypoxic exosome group (BAC + hypoxia, represented by Example 1), with 5 mice in each group. It should be noted that the normal control group refers to healthy mice that have not undergone any intervention, and have not been given BAC (benzalkonium chloride) to induce dry eye, nor have received any drugs or exosomes. The model control group refers to mice in which a dry eye model was successfully established through topical benzalkonium chloride instillation. The positive control group refers to mice in which a dry eye model was successfully established through topical benzalkonium chloride instillation followed by treatment with cyclosporine A. The administration regimen was 5 μL of eye drops twice daily for 14 consecutive days as one course of treatment. Tear secretion and corneal fluorescein staining scores of mice in each group before and after treatment are shown in Table 1. Figure 6 , Figure 7 After treatment, various functional indicators were assessed: In Example 1, the tear secretion volume of mice in the hypoxia exosome group reached 5.1±0.3 mm, which was significantly increased by approximately 45% compared to the model control group (2.8±0.6 mm); the corneal fluorescein staining score decreased to 4.1±1.1, which was reduced by approximately 63.7% compared to the model control group (11.3±1.3), indicating significant repair of corneal epithelial damage. Furthermore, the treatment effects after administration in Examples 2 and 3 were similar to those in Example 1, and were also significantly better than those in the model control group.

[0085] in addition, Figure 8 From left to right, the images also show stained anterior segment images of mice before and after treatment in the normal control group, model control group, hypoxia exosome group, normoxic exosome group, and positive control group.

[0086] Table 1. Statistical table of dry eye treatment results in mice of each group

[0087]

[0088] In addition, the activity of exosomes was detected, and the results are shown in Table 2.

[0089] Table 2. Comparison of the activities of exosomes prepared in Example 1 and Comparative Example 1

[0090]

[0091] As shown in Table 2, the exosomes obtained by the normoxic culture method with added cobalt chloride of the present invention have significantly improved activity compared with Comparative Example 1 without added cobalt chloride.

[0092] 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; and these 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.

Claims

1. A method for preparing exosomes from human umbilical cord mesenchymal stem cells induced by cobalt chloride, characterized in that, Includes the following steps: S1: Obtain human umbilical cord mesenchymal stem cells; S2: Under normoxic conditions, the human umbilical cord mesenchymal stem cells obtained in step S1 were added to a culture medium containing cobalt chloride and then induced to culture for 2-10 hours. S3: The cell culture medium obtained in step S2 is subjected to ultracentrifugation to obtain human umbilical cord mesenchymal stem cell exosomes.

2. The method for preparing exosomes of human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture according to claim 1, characterized in that, In step S1, human umbilical cord mesenchymal stem cells are obtained as follows: umbilical cords delivered by cesarean section at 37-42 weeks of gestation are taken, the outer membrane and umbilical arteries and veins are removed, Wharton's jelly is retained, the tissue is minced and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells.

3. The method for preparing exosomes of human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture according to claim 2, characterized in that, In step S1, the tissue is cut into small pieces and prepared into a tissue homogenate, which is then mixed with the culture medium and added to a culture flask. Primary culture is carried out at 37°C and 5% CO2 using the tissue block adhesion method. The medium is changed for the first time on day 5, the second time on day 10-12, and the third time on day 14, until the cell confluence reaches 80-90% on day 14.

4. The method for preparing exosomes of human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture according to claim 1, characterized in that, In step S2, under normoxic conditions, the 5th and 6th generation human umbilical cord mesenchymal stem cells with a viability ≥95% obtained in step S1 are processed at a rate of 8000-10000 cells / cm³. 2 The inoculum was inoculated into culture flasks at a density of 50-150 μmol / L. After culturing for 3 days, a medium containing cobalt chloride was added to the medium to make the cobalt chloride concentration in the medium 50-150 μmol / L. Then, the medium was induced to grow at 37℃ and 5% CO2.

5. The method for preparing exosomes of human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture according to claim 4, characterized in that, In step S2, the preparation method of the culture medium containing cobalt chloride is as follows: add cobalt chloride solution to the serum-free culture medium for mesenchymal stem cells, and after filtration through a filter membrane to remove bacteria, obtain a culture medium with a cobalt chloride concentration of 50-150 μmol / L.

6. The method for preparing exosomes of human umbilical cord mesenchymal stem cells based on cobalt chloride-induced culture according to claim 1, characterized in that, In step S3, the ultracentrifugation of the cell culture medium includes the following steps: S31: Centrifuge the cell culture medium at 300g-500g for 10-15min at 4℃ and collect the supernatant; S32: Centrifuge the collected supernatant at 20000g at 4℃ for 30min, and collect the supernatant again; S33: Filter the supernatant after high-speed centrifugation through a 0.45μm filter membrane, then filter it through a 0.22μm filter membrane, and collect the filtered supernatant; S34: Centrifuge the filtered supernatant at 100,000-120,000g for 70-90 minutes at 4°C, and collect the precipitate at the bottom of the centrifuge tube; S35: After resuspending the precipitate in pre-cooled sterile PBS, centrifuge at 100,000-120,000 g for 70-90 min at 4°C; resuspend the precipitate again in pre-cooled sterile PBS to obtain human umbilical cord mesenchymal stem cell exosomes.

7. The use of exosomes prepared by the method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture according to any one of claims 1-6 in the preparation of drugs for treating dry eye syndrome.

8. The use of exosomes prepared by the method for preparing human umbilical cord mesenchymal stem cell exosomes based on cobalt chloride-induced culture according to any one of claims 1-6 in the preparation of eye drops for treating dry eye syndrome.

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