A method for culturing exosomes of UCMSCs in a simulated insulin-deficient microenvironment

Abstract

The present application discloses a method for culturing UCMSCs exosomes in a simulated insulin-deficient microenvironment, which relates to the technical field of exosomes. The specific steps include: S1, establishing an insulin-deficient microenvironment; S2, inoculating UCMSCs into the established simulated microenvironment; the culture temperature is 37 °C, the pH value is 7.4, the culture medium is changed every 2-3 days, inducing UCMSCs to secrete and release exosomes, culturing for 48-72 hours, and culturing to the 3-5th generation in the culture medium simulating the insulin secretion-deficient microenvironment; collecting the culture supernatant; S3, centrifuging the culture supernatant multiple times and purifying the exosomes; the high-glucose environment can simulate the blood glucose level in diabetic patients. This environment stimulates cells to produce a stress response, thereby changing the mode and content of exosome secretion. By adding these factors, a diabetic environment is simulated, so that the exosomes carry molecules related to the treatment of diabetes.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and particularly relates to a method for culturing UCMSCs exosomes by simulating an insulin-deficient microenvironment. Background Art

[0002] UCMSCs are derived from neonatal umbilical cord tissue and are a type of stem cell with self-renewal ability and multi-directional differentiation potential. Compared with mesenchymal stem cells from other sources, the acquisition process of UCMSCs is relatively simple and harmless to the donor. UCMSCs have low immunogenicity and are not easily prone to immune rejection reactions after allogeneic transplantation, making them highly safe in clinical applications. UCMSCs have the ability to differentiate across germ layers and can be induced to differentiate into various types of cells in vitro, such as neurons, muscle, endothelium, liver, pancreas, bone, cartilage, etc. In in vitro culture, UCMSCs exhibit strong proliferation ability, providing a basis for large-scale application and standardized production.

[0003] As an information transmission carrier between cells, exosomes have significantly lower immunogenicity than intact cells. This means that during allogeneic transplantation, the risk of exosomes causing immune rejection reactions is relatively low, thereby improving their safety in clinical applications. Exosomes extracted from umbilical cord mesenchymal stem cells inherit many excellent characteristics of stem cells, such as promoting tissue repair, regeneration, and immunomodulation. These functions enable exosomes to show good effects in the treatment of various diseases. For example, exosomes can promote cartilage regeneration, improve liver function, and reduce kidney injury, etc. Exosomes extracted from umbilical cord mesenchymal stem cells have no adverse effects on the kidneys and liver, and do not cause systemic allergic reactions, hemolysis, pyrogenic reactions, etc. These evidences all support exosomes as a safe therapeutic medium.

[0004] In recent years, there have been quite a few progresses in clinical research on the treatment of diabetes with exosomes. For example, some studies have shown that exosomes derived from human umbilical cord mesenchymal stem cells can improve glucose tolerance in diabetic rats, enhance insulin sensitivity, and significantly reduce blood glucose levels. In addition, there are also studies exploring the application of exosomes in the treatment of diabetic complications, such as promoting the repair of diabetic retinopathy and reducing kidney injury in diabetic nephropathy.

[0005] Currently, there is temporarily no method in the existing technology to specifically prepare exosomes dedicated to the treatment of diabetes by simulating an insulin-deficient microenvironment. Summary of the Invention

[0006] By providing a method for culturing UCMSCs exosomes by simulating an insulin-deficient microenvironment in the embodiments of the present application, the problem in the existing technology that there is no method to specifically prepare exosomes dedicated to the treatment of diabetes is solved, and a new method for preparing exosomes for the treatment of diabetes is realized.

[0007] An embodiment of the present application provides a method for culturing UCMSCs exosomes in a simulated insulin-deficient microenvironment, and the specific steps include:

[0008] S1. Establish an insulin-deficient microenvironment;

[0009] S2. Inoculate UCMSCs into the established simulated microenvironment;

[0010] The culture temperature is 37 °C, the pH value is 7.4, the culture medium is changed every 2 - 3 days to induce UCMSCs to secrete and release exosomes, and the cells are cultured for 48 - 72 hours and cultured to the 3rd - 5th generation in the culture medium simulating the insulin-secretion defect microenvironment; collect the culture supernatant;

[0011] S3. Centrifuge the culture supernatant multiple times and purify the exosomes;

[0012] Furthermore, an induction factor and a chitosan hydrogel are added to the serum-free culture medium.

[0013] Furthermore, the induction factor includes:

[0014] High-glucose environment: 25 mM glucose;

[0015] Abnormal lipid metabolism: 1 mM palmitic acid;

[0016] Insulin resistance simulation: 0.005 mM leptin;

[0017] Oxidative stress: The oxygen condition of the culture environment is 25%;

[0018] Hormone: 0.01 mM inflammatory factor, and the inflammatory factor is one or a combination of interleukin 1-β and TNF-α.

[0019] Furthermore, the preparation method of the chitosan hydrogel includes:

[0020] Add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 part by weight of sodium chloride to 100 parts by weight of water and mix evenly at room temperature.

[0021] Furthermore, centrifuging the culture supernatant multiple times and purifying the exosomes specifically includes:

[0022] Collect the culture supernatant;

[0023] Primary centrifugation: Centrifuge the filtered supernatant at 300 × g for 10 minutes to remove cells and large particulate matter;

[0024] Secondary centrifugation: Centrifuge the supernatant at 2000 × g for 20 minutes to remove cell debris;

[0025] Ultracentrifugation: Centrifuge the supernatant at 100,000×g for 70 minutes, and collect the precipitate;

[0026] Washing: Resuspend the precipitate with PBS, and centrifuge again at 100,000×g for 70 minutes. Remove the supernatant, and the remaining precipitate is the purified exosomes.

[0027] Furthermore, the temperature for preparing the chitosan hydrogel is 50°C, and the pH is 10.5.

[0028] Furthermore, prepare the chitosan hydrogel into double-layer chitosan hydrogel particles, specifically:

[0029] Adjust the pH of 100 parts by weight of water to 10.5 with sodium hydroxide; heat up to 50°C, add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 parts by weight of sodium chloride, and mix evenly;

[0030] Dry and grind the above hydrogel into particles passing through a 200-mesh sieve, then place it back into 100 parts by weight of water and add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 parts by weight of sodium chloride, stir and mix evenly, and dry and grind into particles passing through a 100-mesh sieve to obtain double-layer chitosan hydrogel particles.

[0031] Furthermore, dry and grind the hydrogel into particles passing through a 200-mesh sieve, then place it back into 100 parts by weight of water, and microparticles are also added to the water.

[0032] Furthermore, the microparticles include any one or a combination of hyaluronic acid microparticles, silica particles, and silk fibroin particles.

[0033] Furthermore, the microparticles are micron-sized particles.

[0034] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0035] First, the high - glucose environment can simulate the blood - glucose level in diabetic patients. This environment stimulates cells to produce a stress response, thereby changing the pattern and content of the exosomes they secrete. By adding these factors to simulate the diabetic environment, molecules related to diabetes treatment are carried in the exosomes. Chitosan hydrogel is a biocompatible material with good water absorption and water retention. In cell culture, as part of the extracellular matrix, it provides support and protection for cells. At the same time, chitosan hydrogel can also affect the growth and secretion behavior of cells through its unique physicochemical properties; it promotes the adhesion growth and proliferation of UCMSCs, and at the same time improves the secretion efficiency and purity of exosomes. Chitosan hydrogel can also serve as a carrier for exosomes, facilitating the collection and purification of exosomes; it improves the survival rate and activity of UCMSCs in the simulated microenvironment, thereby increasing the yield and quality of exosomes. At the same time, the easy preparation and biocompatibility of chitosan hydrogel make it an ideal material in cell culture and exosome research.

[0036] Second, the increase in cross - linking degree makes the structure of the hydrogel more compact and stable, better isolating and protecting the cells in culture, reducing the interference of external impurities. During centrifugation and purification, it is easier to remove impurities, thereby improving the purity of exosomes. Increasing the cross - linking degree of chitosan hydrogel provides a more stable microenvironment for cells, thus promoting cell growth and proliferation, increasing the number of cells and exosomes; improving the purity and quantity of exosomes, and the stable microenvironment helps to reduce variables in the experiment, improving the reliability and repeatability of experimental results.

[0037] Third, the bilayer structure better isolates and protects the cells in the culture environment, reducing the interference of external impurities. The highly cross - linked inner layer forms a more compact and stable structure, blocking the entry of macromolecules and particles, thus keeping the culture medium clean. The low - cross - linked design of the outer layer allows the penetration of nutrients and the excretion of waste, while still being able to block smaller impurities, making it easier to remove impurities during centrifugation and purification, thereby improving the purity of exosomes;

[0038] Fourth, the addition of micron - sized particles increases the connection between the outer and inner layers of the hydrogel, and makes the inner - layer hydrogel contact the cells with a lower viscosity after the hydrolysis of the outer - layer hydrogel, preventing cell discomfort and resulting in cell death; the micron - sized particles with good biocompatibility interact with the extracellular matrix, simulating the in - vivo physiological environment and providing a growth environment closer to the natural state for cells; the addition of micron - sized particles enhances the structural strength of the outer - layer hydrogel, making it more stable and better able to withstand various stresses and challenges during culture, such as temperature fluctuations, pH value changes, etc.; micron - sized particles can serve as carriers for drugs or growth factors. By controlling the pore size and surface properties of the particles, the slow release of drugs or growth factors can be achieved, thereby maintaining their biological activity for a longer time. Brief Description of the Drawings

[0039] Figure 1 It is a result diagram of the therapeutic effect of exosomes on diabetes. Detailed Description of the Invention

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs; the terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0041] Example 1

[0042] A method for culturing UCMSCs exosomes by simulating an insulin-deficient microenvironment in the present application specifically includes the following steps:

[0043] S1. Establish an insulin-deficient microenvironment;

[0044] An inducer and chitosan hydrogel are added to a serum-free medium. The inducer includes:

[0045] High-glucose environment: 25 mM glucose;

[0046] Abnormal lipid metabolism: 1 mM palmitic acid;

[0047] Insulin resistance simulation: 0.005 mM leptin;

[0048] Oxidative stress: The oxygen condition of the culture environment is 25%;

[0049] Hormone: 0.01 mM inflammatory factor; the inflammatory factor is one or a combination of interleukin 1-β and TNF-α;

[0050] The preparation method of the chitosan hydrogel includes:

[0051] 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 part by weight of sodium chloride are added to 100 parts by weight of water and mixed evenly at room temperature to obtain it;

[0052] The serum-free medium selects Procell MSC SFM;

[0053] S2. Inoculate UCMSCs into the established simulated microenvironment;

[0054] The culture temperature is 37°C, the pH value is 7.4, the medium is changed every 2 - 3 days to induce UCMSCs to secrete and release exosomes, cultured for 48 - 72 hours, and cultured to the 3rd - 5th generation in the medium simulating the insulin secretion-deficient microenvironment; collect the culture supernatant;

[0055] S3. Centrifuge the culture supernatant multiple times and purify exosomes;

[0056] Using ultracentrifugation, centrifuge the culture supernatant multiple times to isolate exosomes. Specifically:

[0057] Collect the culture supernatant;

[0058] Primary centrifugation: Centrifuge the filtered supernatant at 300×g for 10 minutes to remove cells and large particulate matter;

[0059] Secondary centrifugation: Centrifuge the supernatant at 2000×g for 20 minutes to remove cell debris;

[0060] Ultracentrifugation: Centrifuge the supernatant at 100,000×g for 70 minutes and collect the precipitate;

[0061] Washing: Resuspend the precipitate with PBS and centrifuge again at 100,000×g for 70 minutes. Remove the supernatant, and the remaining precipitate is the purified exosome.

[0062] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0063] The hyperglycemic environment can simulate the blood glucose level in diabetic patients. This environment stimulates cells to produce a stress response, thereby changing the pattern and content of exosomes they secrete. Palmitic acid is a saturated fatty acid. Excessive presence will lead to abnormal lipid metabolism, causing exosomes to carry molecular markers or signaling molecules related to glucose metabolism and insulin resistance; adding palmitic acid to the culture medium can simulate the diabetic environment, making exosomes rich in molecules related to lipid metabolism, inflammation, and oxidative stress; leptin is a hormone secreted by adipocytes and is involved in regulating appetite and energy metabolism. By adding leptin and adjusting its concentration, an environment of insulin resistance can be simulated, resulting in exosomes carrying molecules related to insulin resistance, energy metabolism, and inflammation. Factors such as hyperglycemia and abnormal lipid metabolism can lead to oxidative stress, which is an important pathological mechanism of diabetes and various chronic diseases. By increasing the oxygen concentration in the culture environment, an oxidative stress state can be simulated; making exosomes rich in antioxidant enzymes, inflammatory factors, and molecules related to injury repair; inflammatory factors play an important role in the occurrence and development of diabetes and its complications. By adding these factors to simulate the diabetic-related inflammatory environment, exosomes carry molecules related to inflammation, immune regulation, and tissue repair;

[0064] Chitosan hydrogel is a biocompatible material with good water absorption and water retention properties. In cell culture, as part of the extracellular matrix, it provides support and protection for cells. At the same time, chitosan hydrogel can also affect the growth and secretion behavior of cells through its unique physicochemical properties; it promotes the adhesion growth and proliferation of UCMSCs, and at the same time improves the secretion efficiency and purity of exosomes. Chitosan hydrogel can also serve as a carrier for exosomes, facilitating the collection and purification of exosomes; it improves the survival rate and activity of UCMSCs in the simulated microenvironment, thus increasing the yield and quality of exosomes. At the same time, the easy preparation and biocompatibility of chitosan hydrogel make it an ideal material in cell culture and exosome research;

[0065] Chitosan hydrogel has a three-dimensional porous structure, providing a growth environment similar to the extracellular matrix (ECM) for cells; this environment helps the adhesion and proliferation of UCMSCs, enabling cells to grow and differentiate better in the simulated insulin-deficient microenvironment. The three-dimensional structure also promotes cell-cell interaction and signal transduction;

[0066] Chitosan hydrogel adsorbs and stabilizes inductive factors, ensuring a certain concentration gradient of inductive factors in the culture environment, thus more precisely simulating the in vivo environment of diabetic patients; chitosan hydrogel also has a sustained-release effect, slowly releasing the adsorbed inductive factors, enabling cells to be continuously stimulated by inductive factors for a long time, which is conducive to studying the responses and changes of cells under continuous stress;

[0067] Chitosan hydrogel has a certain buffering capacity, helping to maintain the pH value of the culture environment within an appropriate range, reducing the impact of pH fluctuations on cell growth and exosome secretion; chitosan hydrogel absorbs harmful substances and metabolic wastes generated during the culture process, keeping the culture environment clean and stable, which is beneficial to the healthy growth of cells; the three-dimensional structure and biocompatibility of chitosan hydrogel help to promote the secretion of exosomes by UCMSCs, increasing the yield and collection efficiency of exosomes; the separation of chitosan hydrogel from the culture supernatant is relatively easy, simplifying the purification process of exosomes and improving the purification efficiency and purity.

[0068] After 8 weeks of feeding with a high-fat diet (HFD) in SD rats, streptozotocin (STZ, 25 mg / kg) was intraperitoneally administered to induce type 2 diabetic rats. Tail vein blood was collected continuously for 3 days after STZ injection, and blood glucose was monitored using a blood glucose meter. A blood glucose value ≥ 16.7 mmol / L was considered successful in establishing the model. The type 2 diabetic rats were randomly divided into a model group of 10 rats, an experimental group of 10 rats, and a control group of 10 rats. Another 10 rats fed normally during the same period were used as the normal group.

[0069] After 7 days of STZ injection, the experimental group was injected with 5 mg / rat of exosomes via the tail vein every day, and the model group was infused with an equal amount of PBS. The control group was given acarbose by gavage (100 mg / (kg·d)), once a day for 10 days; blood glucose was monitored after 10 days of administration; the results were as Figure 1 ;

[0070] Example 2

[0071] In the above example, by simulating the diabetic microenvironment and chitosan hydrogel, exosomes have better effects for diabetes. To improve the survival rate of cells in the microenvironment and increase the extraction amount of exosomes, further improvements were made on the basis of Example 1.

[0072] Increase the cross-linking degree of the hydrogel to increase the degradation time of the hydrogel, specifically:

[0073] Adjust the pH of 100 parts by weight of water to 10.5 with sodium hydroxide; heat to 50 °C, add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose and 0.5 part by weight of sodium chloride, and mix evenly to obtain;

[0074] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0075] The increase in the cross-linking degree makes the structure of the hydrogel more compact and stable, better isolates and protects the cells in culture, reduces the interference of external impurities, and can more easily remove impurities during centrifugation and purification, thereby improving the purity of exosomes. Increasing the cross-linking degree of chitosan hydrogel provides a more stable microenvironment for cells, thereby promoting cell growth and proliferation, increasing the number of cells and exosomes; improving the purity and quantity of exosomes, and the stable microenvironment helps to reduce variables in the experiment and improve the reliability and repeatability of experimental results.

[0076] The protein concentration of exosomes in the sample was determined by the BCA method, and the number of exosome particles with a particle size range of 20 - 150 nm in the sample was statistically analyzed using a nano-flow cytometer to calculate the purity of exosomes. The results are shown in Table 1:

[0077]

[0078]

[0079] Table 1

[0080] Example 3

[0081] In Example 2, by increasing the cross-linking degree of chitosan hydrogel, the number and purity of exosomes were increased. To further optimize the cell culture environment, further improvements were made on the basis of Example 2.

[0082] Prepare double-layer hydrogel particles, specifically as follows:

[0083] Adjust the pH of 100 parts by weight of water to 10.5 with sodium hydroxide; heat to 50 °C, add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 part by weight of sodium chloride, and mix evenly.

[0084] Dry and grind the above hydrogel into particles and pass through a 200-mesh sieve, then re-place it in 100 parts by weight of water and add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose, and 0.5 part by weight of sodium chloride, stir and mix evenly, dry and grind into particles and pass through a 100-mesh sieve to obtain double-layer chitosan hydrogel particles.

[0085] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0086] The double-layer structure better isolates and protects cells in the culture environment, reduces the interference of external impurities. The inner layer with a high degree of cross-linking forms a more compact and stable structure, blocking the entry of macromolecular substances and particles, thereby keeping the culture medium clean. The design of the outer layer with a low degree of cross-linking enables the penetration of nutrients and the excretion of waste, while still being able to block smaller impurities, making it easier to remove impurities during centrifugation and purification, thus improving the purity of exosomes;

[0087] The double-layer chitosan hydrogel particles provide a more stable and suitable microenvironment, which helps the growth and proliferation of cells. The high degree of cross-linking of the inner layer provides a stronger support and protection effect, enabling cells to better maintain their morphology and function during the culture process;

[0088] The design of the double-layer chitosan hydrogel particles optimizes the cell culture environment, improves the growth efficiency of cells and the secretion amount of exosomes; the double-layer structure enhances the stability of the hydrogel particles, enabling them to better withstand various stresses during the culture process, thus maintaining the integrity and activity of exosomes;

[0089] The protein concentration of exosomes reaches 22.8 ± 1.8 mg / ml, and the purity reaches 90.8%.

[0090] Example 4

[0091] In Example 3, by designing double-layer hydrogel particles, the number of cells and exosomes is increased; to further optimize cell culture and the function of the hydrogel, it is improved on the basis of Example 3.

[0092] In the preparation of the double-layer hydrogel particles, when preparing the outer layer, the solution also contains microparticles;

[0093] The microparticles include any one or combination of hyaluronic acid microparticles, silica particles, and silk fibroin particles;

[0094] The microparticles are micron-sized particles;

[0095] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0096] The addition of micron-sized particles increases the connection between the outer layer and the inner layer of the hydrogel, and enables the inner layer of the hydrogel to contact cells with a lower viscosity after the hydrolysis of the outer layer of the hydrogel, so as to prevent cell discomfort and cause cell death; The micron-sized particles with good biocompatibility interact with the extracellular matrix, simulate the in-vivo physiological environment, and provide a growth environment closer to the natural state for cells; The addition of micron-sized particles enhances the structural strength of the outer layer of the hydrogel, making it more stable and better able to withstand various stresses and challenges during the culture process, such as temperature fluctuations, pH value changes, etc.; Micron-sized particles can be used as carriers of drugs or growth factors. By controlling the pore size and surface properties of the particles, the slow release of drugs or growth factors can be achieved, thereby maintaining their biological activity for a longer time;

[0097] Hyaluronic acid is an important component of the extracellular matrix and has the functions of promoting cell growth, migration and proliferation. Therefore, hyaluronic acid microparticles can simulate the natural extracellular matrix in the cell culture environment, provide a more suitable growth environment for cells, and thus promote cells to secrete more exosomes;

[0098] Hyaluronic acid has good moisturizing and lubricating effects and can protect cells from damage by the external environment. During the cell culture process, hyaluronic acid microparticles maintain the healthy state of cells, which is beneficial to the stable secretion of exosomes;

[0099] Silica particles have high hardness and stability and play a physical support role in the outer layer of the double-layer chitosan hydrogel particles, maintaining the structural integrity of the hydrogel particles and providing a stable growth environment for cells; Although silica itself does not have direct biological activity, its surface properties (such as charge, hydrophilicity and hydrophobicity, etc.) enable cells to attach, grow and proliferate on its surface, and then regulate the secretion of exosomes. Porous silica particles have a large specific surface area and pore volume. As carriers of drugs or growth factors, during the cell culture process, these carriers can slowly release drugs or growth factors, affect the physiological activities of cells, and then regulate the secretion of exosomes;

[0100] Silk fibroin is a natural polymer material with good biocompatibility and degradability. Silk fibroin particles can provide a growth environment similar to the natural extracellular matrix for cells in the cell culture environment, which is beneficial to cell attachment, growth and proliferation; Silk fibroin particles affect the differentiation process and functional expression of cells through their unique physical and chemical properties.

[0101] Detect the concentration and purity of exosomes by using different microparticle hydrogel particles, as shown in Table 2:

[0102]

[0103] Table 2

[0104] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for culturing exosomes of UCMSCs by simulating an insulin-deficient microenvironment, characterized in that, The specific steps include: S1. Establish an insulin-deficient microenvironment; The simulated microenvironment is specifically to add an inducer and a double-layer chitosan hydrogel in a serum-free medium; The inducer includes: High-glucose environment: 25 mM glucose; Abnormal lipid metabolism: 1 mM palmitic acid; Insulin resistance simulation: 0.005 mM leptin; Oxidative stress: The oxygen condition of the culture environment is 25%; Hormone: 0.01 mM inflammatory factor, and the inflammatory factor is interleukin 1-β; The preparation method of the double-layer chitosan hydrogel includes: Adjust the pH of 100 parts by weight of water to 10.5 with sodium hydroxide; heat to 50 °C, then add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose and 0.5 part by weight of sodium chloride, and mix evenly to obtain a single-layer hydrogel; Dry and grind the above single-layer hydrogel into particles and pass through a 200-mesh sieve, then re-place it in 100 parts by weight of water and add 10 parts by weight of water-soluble chitosan, 3 parts by weight of hydroxyethyl cellulose and 0.5 part by weight of sodium chloride, stir and mix evenly, and dry and grind into particles and pass through a 100-mesh sieve to obtain double-layer chitosan hydrogel particles; S2. Inoculate UCMSCs into the established simulated microenvironment, the culture temperature is 37 °C, the pH value is 7.4, the medium is changed every 2-3 days, induce UCMSCs to secrete and release exosomes, culture for 48-72 hours, and culture in the medium simulating the insulin secretion-deficient microenvironment until the 3rd to 5th generation; collect the culture supernatant; S3. Centrifuge the culture supernatant multiple times and purify the exosomes.

2. The method for culturing UCMSCs exosomes by simulating an insulin-deficient microenvironment according to claim 1, wherein, Centrifuge the culture supernatant multiple times and purify the exosomes, specifically: Collect the culture supernatant; Primary centrifugation: Centrifuge the filtered supernatant at 300×g for 10 minutes to remove cells and large particulate matter; Secondary centrifugation: Centrifuge the supernatant at 2000×g for 20 minutes to remove cell debris; Ultracentrifugation: Centrifuge the supernatant at 100,000×g for 70 minutes to collect the precipitate; Washing: Resuspend the precipitate with PBS and centrifuge at 100,000×g for 70 minutes again to remove the supernatant, and the remaining precipitate is the purified exosome.

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