A method for inducing autologous mesenchymal stem cells into islet β-like cells

Abstract

The present invention belongs to the field of biotechnology, relates to the field of induced cell differentiation technology, and specifically relates to a method for inducing autologous mesenchymal stem cells into islet β-like cells. First, the present invention extracts exosomes from the pancreatic tissue of neonatal calves, adds a mixture of β-nerve growth factor and vitamin B3 to prepare an islet β-like cell inducer, and then places mesenchymal stem cells in the islet β-like cell inducer for culture to induce the acquisition of islet β-like cells. The present invention can obtain islet β-like cells at low cost, environmentally friendly and efficiently, and the induced islet β-like cells are highly sensitive to sugar concentration in vivo and in vitro, can significantly reduce the blood sugar of diabetes and significantly prolong its survival period, and have no obvious toxic and side effects on diabetic animal models and normal human bodies, and have important potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to the field of induced cell differentiation technology, and particularly relates to a method for inducing autologous mesenchymal stem cells into islet β-like cells. Background Art

[0002] Diabetes is a metabolic disease characterized by hyperglycemia. The World Health Organization estimates that more than 230 million people globally suffer from diabetes, and this figure is likely to more than double by 2030. Hyperglycemia is caused by absolute or relative deficiency of insulin secretion. Long-term hyperglycemia can lead to lesions in various tissues, especially chronic damage and dysfunction of the eyes, kidneys, heart, blood vessels, and nerves. Currently, the main treatment methods for diabetes include diet control, oral hypoglycemic drugs, and insulin injection. Although they all have certain therapeutic effects, none of them can cure the disease. Since diabetes is induced by relatively or absolutely insufficient insulin secretion from the islets, resulting in hyperglycemia, islet transplantation may be the only way to cure diabetes, especially type I diabetes with absolute insulin deficiency; using islet cell transplantation is expected to replace damaged islet cells and then secrete appropriate amounts of insulin according to blood glucose changes. However, the donor source for human islet cell transplantation is extremely difficult.

[0003] One source of islet transplantation is to isolate islets from cadaveric pancreases and then transplant them into the liver of diabetic patients or intervene in the pancreas. Its advantage is that it may be more sensitive to blood glucose response and has a better therapeutic effect on type I diabetes. However, its disadvantage is that the source is extremely difficult, and due to HLA (human leukocyte antigen) incompatibility and strong antigenicity, the transplantation is not suitable for long-term survival or requires long-term use of anti-rejection drugs. Another source is to induce insulin-secreting cells using stem cell technology, but some technologies need to introduce regulatory genes and at the same time introduce viruses, and the technology is complex. Currently, the most commonly used method in experiments is to use cytokines (such as activin A, epidermal growth factor, β-nerve growth factor, etc.) as inducers to induce mesenchymal stem cells into islet β-like cells. Although this method has a certain hypoglycemic effect on diabetic animal models, at least 3 - 5×10 7 islet β cells are required for one-time transplantation in adults. To prepare such a large number of islet β-like cells, the price of the required inducers is extremely expensive.

[0004] As is well known, adult bone marrow and adipose tissue contain abundant mesenchymal stem cells, and the materials are easily obtained. Therefore, if mesenchymal stem cells can be induced to efficiently differentiate into islet β-like cells and a sufficient amount of islet cells for adult transplantation can be obtained, it will surely have important clinical value. Summary of the Invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a method for inducing autologous mesenchymal stem cells into islet β-like cells, and successfully induces adult bone marrow and adipose mesenchymal stem cells into islet β-like cells in vitro. On the one hand, mesenchymal stem cells are derived from autologous, and are not likely to be rejected after transplantation and can survive for a long time, which is one of the ideal cell sources; on the other hand, it can obtain sufficient islet β-like cells for adult transplantation at low cost, environmentally friendly and efficiently.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a method for inducing autologous mesenchymal stem cells into islet β-like cells, comprising the following steps:

[0008] S1. Cut the pancreas of a newborn calf into tissue blocks, culture them adherently in a serum-free medium for 2-4 days, after removing the tissue blocks, continue to culture the adherent cells for 3-5 days, collect the supernatant of the medium, centrifuge the supernatant at low temperature, and then freeze and ultra-centrifuge to collect the precipitate, and suspend it with normal saline to obtain exosomes of the pancreas of a newborn calf;

[0009] S2. Add β-nerve growth factor and vitamin B3 to the exosomes of the pancreas of a newborn calf, and mix evenly to obtain an islet β-like cell inducer;

[0010] S3. After culturing the mesenchymal stem cells to the third generation, add the islet β-like cell inducer and culture and induce for at least 3 weeks to induce autologous mesenchymal stem cells into islet β-like cells.

[0011] The method of the present invention can differentiate 100% of mesenchymal stem cells into islet β-like cells, and can obtain sufficient islet β-like cells for adult transplantation at low cost, environmentally friendly and efficiently (autologous bone marrow mesenchymal stem cells have the characteristics of convenient material collection and strong proliferation ability, and cell proliferation is obvious during the induction process, and any amount of cells can be obtained); at the same time, the induced islet β-like cells are highly sensitive to sugar concentration, can significantly reduce the blood sugar of diabetes and significantly prolong its survival period, and have no obvious toxic and side effects on diabetic animal models and normal human bodies, and can completely replace islet β cells.

[0012] Preferably, the sources of the mesenchymal stem cells include but are not limited to umbilical cord, adipose, and bone marrow.

[0013] Preferably, in S1, the exosomes of the pancreas of a newborn calf are suspended with normal saline to a concentration of 1-10×10 12 / mL.

[0014] Preferably, in S2, the concentration of the exosomes of the pancreas of a newborn calf in the inducer is 1x10 10 -10x10 10, the concentration of β-nerve growth factor in the inducer is 90-110 μg / L, and the concentration of vitamin B3 in the inducer is 9-12 μg / mL.

[0015] Preferably, after adding the islet β-like cell inducer in S3, the final concentration of neonatal calf pancreatic exosomes is controlled to be 1x10 10 -10x10 10 / mL, the final concentration of β-nerve growth factor is 90-110 μg / L, and the final concentration of vitamin B3 is 9-12 μg / mL.

[0016] Preferably, after culturing the mesenchymal stem cells to the third generation in S3, the concentration is adjusted to 1-10x10 4 / mL, and then the inducer is added.

[0017] Preferably, the culture and induction conditions in S3 are 37 °C and 5% CO2.

[0018] Preferably, the size of the tissue block in S1 is 0.8-1.2 mm.

[0019] The second aspect of the present invention provides an inducer for inducing autologous mesenchymal stem cells into islet β-like cells, and the inducer includes neonatal calf pancreatic exosomes, β-nerve growth factor and vitamin B3.

[0020] Preferably, the preparation method of the neonatal calf pancreatic exosomes is: cutting the neonatal calf pancreas into tissue blocks, culturing adherently in a serum-free medium for 2-4 days, removing the tissue blocks and continuing to culture the adherent cells for 3-5 days, collecting the culture medium supernatant, centrifuging the supernatant at low temperature, and then freeze-ultracentrifuging to collect the precipitate, which is obtained after suspension with physiological saline.

[0021] Preferably, the concentration of the neonatal calf pancreatic exosomes in the inducer is 1x10 10 -10x10 10 , the concentration of β-nerve growth factor in the inducer is 90-110 μg / L, and the concentration of vitamin B3 in the inducer is 9-12 μg / mL.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention discloses a method for inducing autologous mesenchymal stem cells into islet β-like cells. First, exosomes are extracted from neonatal calf pancreatic tissue, and β-nerve growth factor and vitamin B3 are added to prepare an islet β-like cell inducer. Then, the mesenchymal stem cells are cultured in the islet β-like cell inducer, and thus islet β-like cells are successfully induced. The present invention has the following advantages:

[0024] (1) The exosomes of the present invention are extracted from the discarded pancreas of neonatal calves, and then β-nerve growth factor and vitamin B3 are added to prepare an inducer, which is inexpensive and environmentally friendly;

[0025] (2) The present invention can induce 100% of human mesenchymal stem cells (including umbilical cord, adipose, and bone marrow) into fully functional islet β-like cells, while still retaining the characteristics of stem cells; and the mesenchymal stem cells are derived from autologous, so they are not prone to rejection after transplantation and can survive for a long time, which is one of the ideal cell sources;

[0026] (3) The islet β-like cells induced by the present invention can completely replace islet β cells;

[0027] (4) The method of the present invention takes into account that the vast majority of fetal calves or neonatal bull calves in dairy farms are immediately euthanized after birth, and the pancreas is discarded as waste, while the pancreas of fetal calves and neonatal calves is rich in a complete cytokine network and exosomes that induce the differentiation of pancreatic stem cells into islets. Therefore, the present invention can make full use of the discarded pancreatic tissues of fetal calves and neonatal calves, not only realizing waste utilization, but also being beneficial to environmental protection;

[0028] (5) The islet β-like cells induced by the present invention are highly sensitive to glucose concentration, can significantly reduce the blood sugar of diabetes and significantly prolong its survival period, and have no obvious toxic and side effects on diabetic animal models and normal human bodies. Description of the Drawings

[0029] Figure 1 Electron microscopy image of isolated and purified exosomes from neonatal calf pancreas;

[0030] Figure 2 Mesenchymal stem cells cultured to the third generation (ordinary optical microscope, spindle-shaped cells), where A-D are taken in different backgrounds and the cells are spindle-shaped;

[0031] Figure 3 Flow cytometry identification structure of mesenchymal stem cells, where CD34- / HLA-DR- / , CD29+ / CD73+ / CD90+CD105+ are surface markers of mesenchymal stem cells;

[0032] Figure 4 Mesenchymal stem cells induced into islet cell clusters (A represents induction for 10 days without staining; B represents the result of dithizone staining); Note: The islet cell map shows pink after dithizone staining, indicating that the cells contain a large amount of insulin;

[0033] Figure 5 Flow cytometry identification result of induced islet β-like cells (strong positive of islet β cell markers DNER and DISP2 while still expressing mesenchymal stem cell markers CD90 and CD105);

[0034] Figure 6 Insulin secretion (mU / L) of different cells cultured in low-glucose and high-glucose media for different times; where LG: low-glucose medium; HG: high-glucose medium; Control: blank control (DMEM / F12 culture medium); MSC: uninduced mesenchymal stem cells; Routine: β-like cells induced by the conventional method; Novel: β-like cells prepared in Example 1; from left to right, they represent LG medium (10 min), HG medium (10 min), LG medium (60 min), HG medium (60 min);

[0035] Figure 7 Survival time (weeks) of diabetic rat models transplanted with different cells; where Control: blank control group without injecting any cells; MSC: transplanted mesenchymal stem cells; routineβcell: transplanted β cells induced by the conventional method; novelβcell: transplanted pancreatic islet β cells prepared in Example 1;

[0036] Figure 8 Blood glucose (mM) change curves of different cells transplanted into diabetic rat models at different times (weeks); where line A is the control group without injecting cells; line B is uninduced mesenchymal stem cells; pink line, pancreatic islet β-like cells induced by the conventional method; line C is pancreatic islet β-like cells prepared in Example 1;

[0037] Figure 9 Effect of pancreatic islet β-like cells prepared in Example 1 injected into normal human body on blood glucose (mM) and insulin (pM) secretion. Specific embodiments

[0038] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following specific embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0040] Example 1 A method for inducing autologous mesenchymal stem cells into pancreatic islet β-like cells

[0041] 1. Preparation of pancreatic islet β-like cell inducer, specifically including the following steps:

[0042] (1) In a dairy farm (the GRP laboratory of the Animal Laboratory of Inner Mongolia Medical University), the pancreas of a newborn calf was aseptically collected. In the laboratory, at 4°C, the pancreas was cut into 1-mm tissue blocks and cultured adherently in a serum-free medium (DMEM / F12 medium from Gibco, USA, and the amount of the medium should just cover the tissue blocks) for 3 - 4 days. Then the tissue blocks were removed, and the adherent cells were cultured for another 4 days. The supernatant in the culture flask was collected, centrifuged at 3000 rpm for 15 minutes at low temperature (4°C), and the supernatant was collected. Then it was ultracentrifuged at 100000 g for 60 minutes at 4°C, and the supernatant was discarded. Finally, the precipitate was resuspended in physiological saline to obtain calf pancreatic exosomes. Observation of the exosomes isolated from the pancreas of a newborn calf by electron microscopy showed that their diameter was about 30 - 150 nm( Figure 1 ). At the same time, the concentration of the obtained exosome particles was adjusted to 10 12 / mL by a nano-flow cytometer and stored at -80°C for later use;

[0043] (2) The calf pancreatic exosomes were suspended in a serum-free medium and suspended at a concentration of 1×10 10 - 10×10 10 / mL. Then, β-nerve growth factor (purchased from PEPROTECH, USA) at a concentration of 100 μg / L and vitamin B3 at a concentration of 10 μg / mL were added. After mixing evenly, an islet β-cell inducer was obtained.

[0044] 2. Induce autologous bone marrow mesenchymal stem cells into islet β-like cells, specifically as follows:

[0045] First, under aseptic conditions, 10 mL of bone marrow tissue was punctured and extracted from the femoral diaphysis marrow cavity and injected into a centrifuge tube containing sodium citrate anticoagulant. Then, an equal volume of DMEM / F12 culture medium containing 10% FBS was added, and the whole bone marrow adherent culture method was used for culture. After culturing in a 37°C, 5% CO2 incubator for 7 days, the culture medium was changed to obtain primary BM-MSC, and then the culture medium was changed every 3 days. After the cells were fused to 80% - 90%, subculture was carried out, and the bone marrow mesenchymal stem cells were cultured to the third generation with a conventional mesenchymal stem cell complete medium (DMEM / F12 medium). Then, the cell surface markers were identified by flow cytometry (positive markers CD90, CD105, CD73, negative markers CD44, CD34, HLA-DR)( Figure 2 、 3 ), and the cell concentration was adjusted to 10 4 cells / mL with a serum-free medium.

[0046] Then, the islet β-cell inducer prepared in step 1 was added to the bone marrow mesenchymal stem cells, and the final concentration of the calf pancreatic exosomes in the islet β-cell inducer was controlled to be 1×10 10-10×10 10 / mL, the final concentration of β-nerve growth factor is 100 μg / L, and the final concentration of vitamin B3 is 10 μg / mL. Then transfer it into a T175 culture flask and culture and induce for 3 weeks at 37°C and 5% CO2. As Figure 2 shown, the mesenchymal stem cells are spindle-shaped, Figure 3 and the flow cytometry identification results of Figure 4 also confirmed that the spindle-shaped cells are mesenchymal stem cells. After induced culture, the cells become round and aggregate into spherical islet cell clusters ( Figure 4 A), and further immunohistochemical staining confirmed that the cell clusters can secrete insulin, which is exactly the same as the islet cell cluster structure isolated from the pancreas, and shows red after dithizone staining ( Figure 5 B), and 100% of the mesenchymal stem cells differentiate into islet cell clusters.

[0047] Example 2 Verification of the application effect of the islet β-like cell induction method

[0048] 1. Insulin secretion of various cells at different times in low-glucose (LG medium) and high-glucose media

[0049] Equal amounts (1×10 6Put (a certain number of cells) into a 24-well culture plate, with 6 wells repeated for each type of cell. After culturing in low-glucose medium (LG medium) and high-glucose medium (HG medium) at 37°C for different times (10 min, 60 min), collect the supernatant of the medium, and quantitatively detect the insulin content in the medium by ELISA. Among them, the high-glucose medium is Gibco's DMEM medium, with the product number C11995500BT, containing 4500 mg / L Glucose; the low-glucose medium is Tianjin Haoyang's DMEM low-glucose medium (product batch number Lot20190212 - 0265, model TBD11054), containing 1000 mg / L D-Glucose. The induction of β-like cells adopts the conventional factor induction method: Take human bone marrow mesenchymal stem cells at the 3rd passage. When they are fused to 80%-90%, add 100 μg / L β-nerve growth factor, 10 mg / L vitamin B3, and 20 μg / L epidermal growth factor to the medium and induce for 14 days. Then add 10 mg / L vitamin B3, 10 μg / L basic fibroblast growth factor, and 1% insulin-transferrin-selenium to the medium and continue to induce for 14 days, for a total of 28 days of induction (for the specific method, refer to "Shan Xia, Cui Xiaolan, Shi Han, etc. Transplantation of islet-like cells at different stages of induced differentiation of human umbilical cord mesenchymal stem cells for the treatment of diabetes [J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4703 - 4708.").

[0050] According to the insulin secretion amounts of different cells in the medium with different sugar concentrations in Table 1, it can be seen that the sensitivity of the islet β-like cells prepared in Example 1 to sugar concentration is more than 3 times that of the islet β-like cells prepared by the conventional method. At the same time, from Figure 6 it can be seen that the islet β-like cells induced in Example 1 are highly sensitive to sugar concentration. The insulin secretion amount after 10 min in the low-glucose medium is 38.3 + 22.1 mU / L, and the secretion amount after 60 min in the low-glucose medium is 69.6 + 32.8 mU / L. The secretion amount after 10 min in the high-glucose medium is 269.4 + 131.1 mU / L, and the secretion amount after 60 min in the high-glucose medium is 933.4 + 205.3 mU / L. Compared with the blank control, the uninduced mesenchymal stem cells have no obvious response to sugar concentration. The islet β cells prepared by the method of Example 1 have a significantly higher response sensitivity to high sugar than the islet β-like cells prepared by the conventional method.

[0051] Table 1 Insulin secretion amounts (mU / mL) of different cells after culturing in low-glucose and high-glucose media for different times

[0052]

[0053] 2. Survival study of diabetic rat models after injection with different cells:

[0054] Forty diabetic rat models were prepared (Wista rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Modeling method: After feeding with a high-sugar and high-fat diet for 4 weeks, streptozotocin was intraperitoneally injected at a dose of 50 mg / kg for 3 consecutive times. A fasting blood glucose concentration higher than 16.7 mol / L indicated successful modeling). The fasting blood glucose of all the rat models exceeded 20 mM. They were randomly divided into 4 groups: Control group, MSC group, routine β cell group, and novel group. Among them, no cells were injected into the Control group; 10 6 mesenchymal stem cells per kg were injected into the tail vein of the MSC group; 10 6 islet β-like cells prepared by the routine method per kg were injected into the tail vein of the routine β cell group; 10 6 islet β-like cells prepared in Example 1 per kg were injected into the tail vein of the novel group.

[0055] As Figure 7 can be seen, after transplantation of the islet β cells prepared in Example 1 to treat the diabetic rat model, their survival time exceeded 60 weeks, while all the diabetic rats in the other cell transplantation groups died within 35 weeks. Therefore, this experiment ended at 60 weeks.

[0056] As Figure 8 can be seen, the hypoglycemic effect of the islet β-like cells prepared in Example 1 lasted for about 20 weeks. Although the blood glucose was relatively high later, the survival period was up to about 60 weeks, and the surviving diabetic rats remained in a healthy state. However, the islet β-like cells prepared by the routine method could only last within 10 weeks, and the survival period was less than 35 weeks. This shows that the islet β-like cells prepared by the present invention can not only reduce blood glucose but also repair the damage caused by hyperglycemia to the body, significantly prolonging its survival period.

[0057] 3. Safety of islet β-like cells for normal human body

[0058] The islet β cells induced by the method of Example 1 were injected around the umbilicus of the inventor Li Xin (without diabetes) at a dose of 1×10 6 cells per kg of body weight, and the fasting blood glucose and insulin were measured every day for 2 consecutive weeks.

[0059] As Figure 9As shown, curve A is the test curve of fasting blood glucose, and curve B is the test curve of fasting insulin. After the injection of islet β-like cells, there is no significant effect on the fasting insulin secretion and blood glucose of normal adults. After periumbilical injection, except for mild local pain, there are no other serious adverse reactions such as allergy and fever. It has no obvious hypoglycemic effect on normal adults, nor can it significantly increase the insulin secretion level, indicating that the islet β-like cells prepared by the method of the present invention have no obvious adverse reactions on normal adults.

[0060] In summary, the present invention successfully induces adult mesenchymal stem cells into islet β-like cells in vitro by using an inducer composed of neonatal calf pancreatic exosomes, β-nerve growth factor and vitamin B3, and can obtain sufficient islet β-like cells for adult transplantation at low cost, environmentally friendly and efficiently; at the same time, the induced islet β-like cells are highly sensitive to sugar concentration, can completely replace islet β cells, can significantly reduce the blood glucose of diabetes and significantly prolong its survival period, and have no obvious toxic and side effects on diabetic animal models and normal human bodies, having important potential clinical application value.

[0061] The above has detailed the embodiments of the present invention, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. A method for inducing autologous mesenchymal stem cells into islet β-like cells, characterized in that, It includes the following steps: S1. Sterilely collect the pancreas of a newborn calf in a dairy farm. Cut the pancreas into tissue blocks of 1 mm in an environment of 4°C in the laboratory, and perform adherent culture in DMEM / F12 serum-free medium for 3 - 4 days. Remove the tissue blocks, and continue to culture the adherent cells for 4 days. Collect the supernatant in the culture flask, centrifuge at 3000 rpm for 15 minutes at 4°C, collect the supernatant, then perform cryo-ultrahigh-speed centrifugation at 100,000 g for 60 minutes at 4°C, discard the supernatant, and finally resuspend the precipitate with physiological saline to obtain exosomes of the pancreas of a newborn calf; S2. Add β-nerve growth factor and vitamin B3 to the exosomes of the pancreas of a newborn calf, and mix evenly to obtain an islet β-like cell inducer; S3. After culturing human bone marrow mesenchymal stem cells to the third generation, add the islet β-like cell inducer and culture and induce for at least 3 weeks to induce autologous mesenchymal stem cells into islet β-like cells.

2. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that In S1, newborn calf pancreatic exosomes were suspended in saline to a concentration of 1×10 12 -10×10 12 Pieces / mL.

3. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that, The concentration of neonatal calf pancreatic exosomes in S2 in the inducer is 1x10 10 -10x10 10 per mL, the concentration of β-nerve growth factor in the inducer is 90-110 μg / L, and the concentration of vitamin B3 in the inducer is 9-12 μg / mL.

4. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that, After adding islet β-like cell inducer in S3, control the final concentration of neonatal calf pancreatic exosomes to be 1x10 10 -10x10 10 per mL, the final concentration of β-nerve growth factor is 90-110 μg / L, and the final concentration of vitamin B3 is 9-12 μg / mL.

5. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that, After culturing human bone marrow mesenchymal stem cells in S3 to the third generation, adjust the concentration to 1x10 4 -10 x10 4 cells / mL, and then add the inducer.

6. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that, The conditions for culturing and inducing in S3 are 37°C and 5% CO2.

7. A method for inducing autologous mesenchymal stem cells into islet β-like cells according to claim 1, characterized in that, In S1, the size of the tissue block is 0.8 - 1.2 mm.

8. An inducer for inducing autologous mesenchymal stem cells into islet β-like cells, characterized in that, The inducer includes exosomes of the pancreas of a newborn calf, β-nerve growth factor and vitamin B3; the preparation method of the exosomes of the pancreas of a newborn calf is: sterilely collect the pancreas of a newborn calf in a dairy farm, cut the pancreas into tissue blocks of 1 mm in an environment of 4°C in the laboratory, perform adherent culture in DMEM / F12 serum-free medium for 3 - 4 days, remove the tissue blocks, continue to culture the adherent cells for 4 days, collect the supernatant in the culture flask, centrifuge at 3000 rpm for 15 minutes at 4°C, collect the supernatant, then perform cryo-ultrahigh-speed centrifugation at 100,000 g for 60 minutes at 4°C, discard the supernatant, and finally resuspend the precipitate with physiological saline to obtain exosomes of the pancreas of a newborn calf.

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