A method for preparing mesenchymal stem cell apoptotic vesicles

Abstract

This invention relates to the field of biomedical technology, and more particularly to a method for preparing mesenchymal stem cell apoptotic vesicles. The method primarily employs serum-free culture medium for 3D microsphere culture of stem cells, followed by induction of apoptosis into apoptotic vesicles using an apoptosis induction system, resulting in highly efficient apoptotic vesicles. This method significantly enhances the bidirectional immunomodulatory effect of stem cell-derived apoptotic vesicles, greatly expanding the application range and therapeutic efficacy of these vesicles. The serum-free culture medium avoids the safety risks and batch-to-batch variations associated with traditional culture media, and significantly improves the proliferation capacity, passage stability, and immunomodulatory effect of stem cells, enabling stable and efficient acquisition of apoptotic vesicles.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing apoptotic vesicles of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are widely distributed in connective tissues and organ stroma throughout the body. They are pluripotent stem cells possessing core characteristics such as self-renewal, multi-lineage differentiation, and immune regulation. Their low immunogenicity and immunomodulatory capabilities make them an important tool in regenerative medicine. Current research on MSCs for disease treatment includes autoimmune diseases, refractory diseases such as advanced liver, kidney, and heart diseases, inflammatory diseases, and anti-aging. Apoptotic vesicles (apoVs) are bilayer lipid structures with unique biological and functional characteristics produced during apoptosis. They are relatively large in size and high in density, and their generation process is simple, controllable, and source-specific. During in vivo treatment with stem cells, the apoptotic vesicles they secrete play a crucial role in immune regulation and tissue repair.

[0003] Mesenchymal stem cell (MSC) apoptotic vesicles are extracellular vesicles with therapeutic potential generated during MSC apoptosis. Current non-clinical studies indicate that MSC apoptotic vesicles have significant therapeutic potential in hair regeneration, liver diseases, and reproductive-related disorders. However, the preparation of MSC apoptotic vesicles currently faces challenges, including high post-transplantation safety risks and insufficient regulatory efficacy. Currently, the preparation of MSC apoptotic vesicles involves the addition of animal-derived serum, such as fetal bovine serum (FBS). However, FBS, being an animal-derived substance, introduces several adverse factors into MSC production, such as significant batch-to-batch variations, foreign protein contamination, viral contamination risks, and unstable sources. The need for specially designed processes to remove foreign substances severely hinders MSC clinical research and the translation and application of derived vesicles. Furthermore, the regulatory efficacy of MSC apoptotic vesicles remains limited, determined by factors such as culture medium composition, culture method, and extraction method. Summary of the Invention

[0004] To address the aforementioned problems, in a first aspect, the present invention provides a serum-free culture medium for mesenchymal stem cells 3-D microspheres, comprising an aqueous solution of 90%–95% (v / v) α-MEM basal medium and 5%–10% (v / v) mixed additives; the components and concentrations of the mixed additives are as follows: deoxyribonucleic acid 40–45 mg / L, ribonucleic acid 35–40 mg / L, hydrocortisone 8–10 mg / L, tetrahydroberberine 0.5–1 μg / mL, lemon exosomes 0.1–0.5 μg / mL, bFGF 10–30 ng / mL, PDGF 10–30 ng / mL, stem cell growth factor 10–30 ng / mL, TGF-α 10–30 ng / mL, and melatonin 2–5 nM.

[0005] In the serum-free culture medium of this invention, α-MEM basal medium serves as the basic system, providing essential amino acids, vitamins, and inorganic salts for the growth of mesenchymal stem cells, ensuring the cells' basic metabolic needs. The mixed additive aqueous solution is the core innovation of this invention; its components work synergistically and are precisely designed to address the characteristics and needs of mesenchymal stem cells. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as nucleic acid substances, not only provide raw materials for nucleic acid synthesis and participate in the transmission and expression regulation of cellular genetic information, but also help maintain normal cellular physiological functions and genome stability. Specifically, the DNA is one or more combinations of 2'-deoxyadenosine, deoxycytidine hydrochloride, deoxyguanosine, and thymine; the RNA is one or more combinations of adenosine, cytosine, guanosine, and uridine. The rational combination and concentration control of DNA and RNA can effectively promote the synthesis of DNA and RNA within cells, providing a sufficient material basis for the rapid proliferation and self-renewal of stem cells. Hydrocortisone, a glucocorticoid, regulates cellular metabolism, proliferation, and differentiation, particularly impacting the immunomodulatory function of mesenchymal stem cells (MSCs) and enhancing their anti-inflammatory capabilities. Tetrahydroberberine, a natural alkaloid, may play a role in this culture medium by regulating cell signaling pathways, inhibiting inflammatory responses, or promoting the maintenance of specific functional phenotypes. Its specific mechanism may be related to influencing the activity of certain intracellular enzymes or receptor expression. The addition of lemon exosomes is a unique feature of this invention. Exosomes, as important mediators of intercellular communication, may carry specific bioactive molecules, such as lipids, proteins, and microRNAs, which can be taken up by MSCs, thereby affecting their proliferation, differentiation, and paracrine functions. It may make a unique contribution, especially in enhancing immunomodulatory efficacy, particularly in promoting the formation of 3D microspheres during 3D culture. The combined use of multiple growth factors, including bFGF, PDGF, stem cell growth factor, and TGF-α, activates downstream signaling cascades through their specific receptor binding, synergistically promoting the in vitro proliferation activity of MSCs, maintaining the stem cell characteristics of cells, and delaying cellular senescence. The addition of melatonin, besides its antioxidant effects and protection of cells from oxidative stress damage, may also participate in regulating cellular rhythms and the expression of immune-related genes, further enhancing the immunomodulatory function of mesenchymal stem cells. Through the scientific ratio and synergistic effect of the above components, the serum-free culture medium of this invention can effectively overcome the drawbacks of traditional serum-containing culture media, while significantly improving the immunomodulatory efficacy of mesenchymal stem cells.

[0006] In a second aspect, the present invention provides a method for preparing mesenchymal stem cell apoptotic vesicles using the above-mentioned serum-free culture medium, comprising the following steps:

[0007] 1) Prepare serum-free culture medium for mesenchymal stem cells 3-D microspheres according to the following ratio. The serum-free culture medium includes 90%~95% (v / v) α-MEM basal medium and 5%~10% (v / v) aqueous solution of mixed additives. The components and concentrations of the mixed additives are as follows: deoxyribonucleic acid 40~45 mg / L, ribonucleic acid 35~40 mg / L, hydrocortisone 8~10 mg / L, tetrahydroberberine 0.8~1.5 μg / mL, lemon exosomes 0.5~1 μg / mL, bFGF 10~30 ng / mL, PDGF 10~30 ng / mL, stem cell growth factor 10~30 ng / mL, TGF-α 10~30 ng / mL, and melatonin 2~5 nM. After preparation, filter and sterilize for later use.

[0008] 2) Take umbilical cord tissue, perform Wharton jelly separation, cut the Wharton jelly into small pieces, and add it to serum-free culture medium containing mesenchymal stem cells obtained in step 1) for cell culture.

[0009] 3) On the 5th day after culture, half of the medium of the cells cultured in step 2) was changed, and then the medium was changed every 3 days. When the cell confluence reached 80%, the cells were passaged and cryopreserved.

[0010] 4) Add the serum-free medium containing mesenchymal stem cells 3-D microspheres prepared in step 1) to a rotary bottle, and add stem cell microcarriers. Take the frozen cells from step 3), thaw them, and add them to the rotary bottle. Stir for 5 minutes every 90 minutes at a stirring speed of 40 rpm. After 24 hours, switch to continuous stirring at a stirring speed of 20-40 rpm. Check the sugar content. When the sugar content is lower than 3 g / L, change 1 / 2 to 2 / 3 of the medium. After culturing for 1-4 days, proceed to the next culture step.

[0011] 5) Continue culturing the mesenchymal stem cell microspheres from step 4) in a carbon dioxide atmosphere incubator. After 5-6 days of growth, replace the serum-free medium of the mesenchymal stem cell 3-D microspheres with α-MEM basal medium and add astrocytosin induction solution. Continue culturing for 1-2 days, then collect the cell suspension, centrifuge once, and collect the supernatant. Centrifuge the supernatant a second time, collect the precipitate, resuspend the precipitate with PBS buffer, and centrifuge a third time. Collect the centrifuged precipitate to obtain the mesenchymal stem cell apoptotic vesicles.

[0012] The above method is applicable to the culture and extraction of mesenchymal stem cells from human, bovine, sheep, canine, feline, mouse, rat, or rabbit umbilical cords. The Wharton gel separation method effectively obtains high-purity mesenchymal stem cells, avoiding the potential contamination of other cell types in traditional whole tissue block culture. The chopped Wharton gel is directly seeded into a culture dish containing the serum-free medium of this invention. Utilizing the precise nutrient environment and growth signals provided by the medium, mesenchymal stem cells migrate from the tissue block and adhere to the culture dish. The initial half-volume medium change strategy (on day 5 after culture) removes impurities and dead cells released from the tissue block while retaining some of the active factors in the conditioned medium, which is beneficial for early cell adaptation and proliferation. Subsequent routine medium changes every 3 days ensure an adequate supply of nutrients and timely removal of metabolic waste, maintaining a stable culture microenvironment. When cell confluence reaches 80%, passage is performed. Controlling this critical point ensures optimal cell growth, avoids contact inhibition and functional alterations caused by excessive confluence, and yields a sufficient number of cells for cryopreservation and subsequent experiments. Cryopreservation preserves early-stage cells in good condition for later thawing, ensuring the stability and consistency of experimental materials. The thawed cells are then cultured and passaged again in the serum-free medium of this invention for further purification and expansion of mesenchymal stem cells. The resulting cells exhibit typical mesenchymal stem cell morphology, surface marker expression, and multi-lineage differentiation potential. Furthermore, the use of serum-free medium throughout the process effectively avoids issues such as foreign protein contamination.

[0013] As the number of passages increases, the biological characteristics of mesenchymal stem cells (MSCs) may change, such as gradually weakening proliferative capacity, reducing differentiation potential, or exhibiting a certain degree of aging. Therefore, in the culture method of this invention, the maximum passage number is explicitly limited to 30 generations. This ensures that the cultured MSCs maintain their core biological functions during passage, such as self-renewal capacity, multi-lineage differentiation potential, and crucial immunomodulatory functions, thereby guaranteeing the reliability and stability of subsequent experimental results, as well as the safety and efficacy in potential clinical applications. By strictly controlling the number of passages, the alteration of cell characteristics due to excessive passage is avoided, which could affect their application value. Preferably, a cell culture medium from the second to fifth generation is used. Second to fifth generation cells are typically in the logarithmic growth phase, with good cell condition and high viability. They respond more uniformly and sensitively to apoptosis induction, generating more and higher-quality apoptotic vesicles, effectively avoiding problems such as low apoptotic vesicle yield and poor activity caused by cell aging or poor condition.

[0014] Furthermore, regarding specific culture conditions, the volume concentration of carbon dioxide in the carbon dioxide atmosphere was set to 5%, and the culture temperature was controlled at 37℃. This environment simulates the physiological conditions in vivo, which is conducive to maintaining the stability of the metabolic activities and physiological state of cells during the induction of apoptosis, ensuring that the apoptosis process proceeds in an orderly manner. The induction culture time is 24~48h, which is an optimized time obtained through multiple experiments. This ensures that cells can fully undergo apoptosis and release apoptotic vesicles, while avoiding insufficient induction due to too short a culture time, or excessive degradation of apoptotic vesicles due to too long a culture time.

[0015] Furthermore, centrifugation is a crucial step in obtaining apoptotic vesicles. This method employs a three-step centrifugation process. The first centrifugation uses a centrifugal force of 800g and a centrifugation time of 10min, which effectively removes intact cells and larger cell debris from the cell culture medium. The second centrifugation increases the centrifugal force to 16000g and the centrifugation time to 5min, which can precipitate apoptotic vesicles with larger particle sizes. Subsequently, the precipitate is resuspended with PBS buffer to wash away residual impurities and culture medium components. A third centrifugation is then performed at a centrifugation force of 16000g and a centrifugation time of 10min to purify the apoptotic vesicles. The final collected centrifuged precipitate is a high-purity mesenchymal stem cell apoptotic vesicle.

[0016] The technical effects of this invention are as follows: 1. This invention provides a serum-free culture medium with clearly defined components and no animal-derived contamination. By adding small molecules such as ribonucleic acid, hydrocortisone, deoxyribonucleic acid, growth factors, and tetrahydroberberine, the proliferation capacity and passage stability of mesenchymal stem cells are significantly improved while ensuring safety, solving the problems of large batch-to-batch variations and pathogen contamination risks in traditional serum-containing culture media. 2. This invention reduces cell damage during stem cell culture through the synergistic antioxidant effect of lemon exosomes and melatonin. The addition of stem cell growth factors and tetrahydroberberine enhances the proliferation capacity and bidirectional immunomodulatory ability of mesenchymal stem cells. Experimental results show that mesenchymal stem cells cultured using the culture medium of this invention can more effectively inhibit the proliferation of TH1 and TH17 pro-inflammatory cells and promote the differentiation of TH2 and Treg anti-inflammatory cells compared to traditional fetal bovine serum culture medium. Furthermore, after co-culturing with macrophages, they can secrete higher levels of the anti-inflammatory factor IL-10, exhibiting stronger immunomodulatory function. 3. In this invention, apoptotic vesicles of stem cells are prepared using a serum-free culture system consisting of 3-D microspheres and a high-lemon exosome content. This system has the advantage of mimicking the in vivo developmental microenvironment of stem cells, and lemon exosomes can maintain the stemness of mesenchymal stem cells and enhance their immunomodulatory capabilities. Experiments have confirmed that the apoptotic vesicles prepared in this invention exhibit typical morphology and structure, and possess the same bidirectional immunomodulatory and anti-inflammatory functions as stem cells, providing a new approach and a superior preparation method for cell-free therapies based on apoptotic vesicles. Attached Figure Description

[0017] Figure 1 This is a primary photograph of umbilical cord mesenchymal stem cells from Example 1.

[0018] Figure 2 The curves for detecting cell viability at different generations are shown in Example 1.

[0019] Figure 3 The bar chart shows the cellular immune regulation capacity detection for Example 1 and Comparative Example 1.

[0020] Figure 4 The bar chart shows the detection of anti-inflammatory factors after co-culturing cells with macrophages and LPS in Example 1 and Comparative Example 1.

[0021] Figure 5 This is a biological transmission electron microscope image of the apoptotic vesicles obtained in this invention.

[0022] Figure 6 The detection curve of the apoptotic vesicles obtained in this invention was obtained using a nanoparticle tracking analyzer.

[0023] Figure 7 The bar chart shows the cellular immune regulation capacity detection for Example 1 and Comparative Example 1.

[0024] Figure 8 The bar chart shows the detection of anti-inflammatory factors after co-culturing cells with macrophages and LPS in Example 1 and Comparative Example 1. Detailed Implementation

[0025] The present invention will be described below with reference to examples. These examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0026] I. Serum-free culture medium for 3-D microspheres

[0027] 1. Serum-free culture medium containing 3-D microspheres a

[0028] The solution consists of an aqueous solution containing 95% (v / v) α-MEM basal medium and 5% (v / v) mixed additives. The components and concentrations of the mixed additives are as follows: deoxyribonucleic acid 45 mg / L, ribonucleic acid 35 mg / L, hydrocortisone 8 mg / L, tetrahydroberberine 1 μg / mL, lemon exosomes 0.5 μg / mL, bFGF 30 ng / mL, PDGF 30 ng / mL, stem cell growth factor 30 ng / mL, TGF-α 10 ng / mL, and melatonin 2 nM.

[0029] 2. 3-D microsphere serum-free culture medium b

[0030] The solution consisted of an aqueous solution containing 90% (v / v) α-MEM basal medium and 10% (v / v) mixed additives. The components and concentrations of the mixed additives were as follows: deoxyribonucleic acid 42 mg / L, ribonucleic acid 37 mg / L, hydrocortisone 9 mg / L, tetrahydroberberine 0.8 μg / mL, lemon exosomes 0.3 μg / mL, bFGF 20 ng / mL, PDGF 200 ng / mL, stem cell growth factor 20 ng / mL, TGF-α 20 ng / mL, and melatonin 3.5 nM.

[0031] 3. Serum-free culture medium containing 3-D microspheres

[0032] The solution consisted of an aqueous solution containing 93% (v / v) α-MEM basal medium and 7% (v / v) mixed additives. The components and concentrations of the mixed additives were as follows: 40 mg / L deoxyribonucleic acid, 40 mg / L ribonucleic acid, 10 mg / L hydrocortisone, 0.5 μg / mL tetrahydroberberine, 0.1 μg / mL lemon exosomes, 10 ng / mL bFGF, 10 ng / mL PDGF, 10 ng / mL stem cell growth factor, 30 ng / mL TGF-α, and 5 nM melatonin.

[0033] II. Cultured Mesenchymal Stem Cell Microspheres

[0034] The cultivation method includes the following steps:

[0035] 1) Prepare serum-free culture medium, filter and sterilize before use;

[0036] 2) Take umbilical cord tissue, perform Wharton gel separation, cut the Wharton gel into small pieces and add it to a culture dish containing the serum-free culture medium obtained in step 1) for cell culture;

[0037] 3) On the 5th day after culture, change half of the medium for the cells cultured in step 2), and then change the medium every 3 days. When the cell confluence is 80%, passage and cryopreserve.

[0038] 4) Add the serum-free culture medium for mesenchymal stem cell 3-D microspheres prepared in step 1) to a rotary bottle, and add stem cell microcarriers. Take the frozen cells from step 3), thaw them, and add them to the rotary bottle. Stir for 5 minutes every 90 minutes at a stirring speed of 40 rpm. After 24 hours, switch to continuous stirring at a stirring speed of 20-40 rpm. Detect the sugar content and determine the medium replacement time based on the sugar consumption. After culturing for 1-4 days, mesenchymal stem cell microspheres are obtained.

[0039] Example 1

[0040] Human umbilical cord mesenchymal stem cells were cultured using the above method, with 3-D microsphere serum-free medium a as the culture medium, and passaged to 30 generations.

[0041] Example 2

[0042] Human umbilical cord mesenchymal stem cells were cultured using the above method, with 3-D microsphere serum-free medium b as the culture medium, and passaged to 25 generations.

[0043] Example 3

[0044] Bovine umbilical cord mesenchymal stem cells were cultured using the above method, with 3-D microsphere serum-free medium c as the culture medium, and passaged to 30 generations.

[0045] Comparative Example 1

[0046] Fetal bovine serum culture medium was used as a control for the experiment. In this control, the culture medium was prepared according to the following volume ratio: 90% α-MEM basal medium and 10% fetal bovine serum. The prepared fetal bovine serum-containing culture medium was stored at a temperature of 2~8℃.

[0047] The following steps were taken to culture mesenchymal stem cells: the prepared culture medium containing fetal bovine serum was filtered and sterilized, human umbilical cord was obtained, Wharton gel was separated, the Wharton gel was cut into small pieces and added to a culture dish, and culture medium containing fetal bovine serum was added; half of the medium was changed on the 5th day after culture; the medium was changed every 3 days, and the cells were passaged when the confluence reached 80%.

[0048] III. Obtaining Apoptotic Vesicles from Mesenchymal Stem Cells

[0049] The mesenchymal stem cell microspheres obtained in Examples 1-3 and the mesenchymal stem cells obtained in Comparative Example 1 were cultured separately in a carbon dioxide atmosphere incubator for 5-6 days. The culture medium was then replaced with α-MEM basal medium, and astrocytosin induction medium was added. After culturing for another 1-2 days, the cell suspension was collected, centrifuged once, and the supernatant was collected. The supernatant was then centrifuged a second time, and the precipitate was collected. The precipitate was resuspended using PBS buffer, and then centrifuged a third time. The centrifuged precipitate was collected, yielding the mesenchymal stem cell apoptotic vesicles. The carbon dioxide concentration was 5% (v / v), the culture temperature was 37°C, and the culture time was 26 hours. The centrifugation force for the first centrifugation was 800g, and the centrifugation time was 10 minutes. The centrifugation force for the second centrifugation was 16000g, and the centrifugation time was 5 minutes. The centrifugation force for the third centrifugation was 16000g, and the centrifugation time was 10 minutes.

[0050] IV. Characterization and Detection

[0051] Figure 1 The image shows a primary photograph of umbilical cord mesenchymal stem cells from Example 1. As can be seen from the image, the umbilical cord mesenchymal stem cells cultured in Example 1 exhibit a typical fibroblast-like spindle-shaped morphology after adhesion. The cells are evenly and neatly arranged, have good refractive index, are in good condition, and are free from obvious contamination by other cells.

[0052] Cell surface molecular analysis was performed on cells from passages P5, P10, P15, P20, P25, and P30 in step 4) of Example 1. The results are shown in Table 1. Cell identification was performed using flow cytometry. The surface molecular analysis results of mesenchymal stem cells from passages P5, P10, P15, P20, P25, and P30 all met the requirements of the International Cell Therapy Association. CD73, CD90, and CD105 were all above 95%, while CD14, CD19, CD34, CD45, and HLA-DR were all below 1%, indicating that this serum-free culture can support long-term culture of mesenchymal stem cells and support the expression of surface molecules of mesenchymal stem cells.

[0053] Table 1. Cell surface molecular detection

[0054]

[0055] Cell viability was tested at P5, P10, P15, P20, P25, and P30 generations from step 4 of Example 1. The results are as follows: Figure 2 As shown in the figure. Cell viability was detected using the trypan blue exclusion assay. The results showed that the viability of P5, P10, P15, P20, P25, and P30 cells were all above 90%, indicating that this serum-free culture can support the proliferation activity of mesenchymal stem cells.

[0056] Cellular immune regulation capacity was tested using P5 generation cells from Example 1 and Comparative Example 1, respectively. The results are as follows: Figure 3 As shown in the figure. The results showed that co-culturing mesenchymal stem cells and immune cells with serum-free culture medium relative to fetal bovine serum could inhibit the proliferation of TH1 and TH17 cells and promote the differentiation of TH2 and Treg cells, demonstrating a very strong immunomodulatory function.

[0057] P5 and P10 passage cells from Example 1 and Comparative Example 1 were co-cultured with macrophages and LPS, and the anti-inflammatory factor (IL-10) was detected. The results are as follows: Figure 4 As shown in the figure. The results of anti-inflammatory factors of mesenchymal stem cells showed that co-culture of mesenchymal stem cells with LPS can secrete anti-inflammatory factor (IL-10), and serum-free culture medium has a stronger ability to inhibit inflammation than fetal bovine serum.

[0058] The apoptotic vesicles of stem cells obtained in this invention were examined by transmission electron microscopy (TEM), and the results are as follows: Figure 5 As shown, the results indicate that the apoptotic vesicles of stem cells exhibit a typical "cup saucer" or cup-shaped structure, consistent with the typical structural characteristics of apoptotic vesicles of stem cells.

[0059] The apoptotic vesicles of stem cells obtained in this invention were detected using a nanoparticle tracking analyzer (NTA). The detection results are as follows: Figure 6 As shown, the results indicate that the average particle size of the obtained stem cell apoptotic vesicles is approximately 100 nm, with a particle size range of 80–200 nm.

[0060] The apoptotic vesicles of stem cells obtained in this invention and the P5 generation cells from Comparative Example 1 were used to detect their cellular immune regulation capabilities. The immune regulation results are as follows: Figure 7 As shown in the figure. The results showed that co-culturing mesenchymal stem cells with immune cells in serum-free medium relative to fetal bovine serum could inhibit the proliferation of TH1 and TH17 cells and promote the differentiation of TH2 and Treg cells, demonstrating a very strong immunoregulatory function, indicating that apoptotic vesicles of stem cells inherit the immunoregulatory ability of stem cells.

[0061] The apoptotic vesicles of stem cells obtained in this invention and the P5 and P10 generation cells from Comparative Example 1 were co-cultured with macrophages and LPS, respectively, and the anti-inflammatory factor (IL-10) was detected. The detection results are as follows: Figure 8 As shown, co-culturing mesenchymal stem cells with macrophages and LPS can secrete anti-inflammatory factors (IL-10). Serum-free culture medium and FBS have stronger anti-inflammatory capabilities, indicating that apoptotic vesicles of stem cells inherit the anti-inflammatory ability of stem cells.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing apoptotic vesicles of mesenchymal stem cells, characterized in that, Includes the following steps: 1) Prepare serum-free culture medium for mesenchymal stem cells 3-D microspheres according to the following ratio. The serum-free culture medium includes 90%~95% (v / v) α-MEM basal medium and 5%~10% (v / v) aqueous solution of mixed additives. The components and concentrations of the mixed additives are as follows: deoxyribonucleic acid 40~45 mg / L, ribonucleic acid 35~40 mg / L, hydrocortisone 8~10 mg / L, tetrahydroberberine 0.8~1.5 μg / mL, lemon exosomes 0.5~1 μg / mL, bFGF 10~30 ng / mL, PDGF 10~30 ng / mL, stem cell growth factor 10~30 ng / mL, TGF-α 10~30 ng / mL, and melatonin 2~5 nM. After preparation, filter and sterilize for later use. 2) Take umbilical cord tissue, perform Wharton jelly separation, cut the Wharton jelly into small pieces, and add it to serum-free culture medium containing mesenchymal stem cells obtained in step 1) for cell culture. 3) Replace half of the medium on the 5th day after culture of the cells cultured in step 2), and then replace the medium every 3 days thereafter. Passage the cells according to their confluence and freeze them. 4) Add the serum-free medium containing 3-D microspheres of mesenchymal stem cells prepared in step 1) to a rotary bottle, and add stem cell microcarriers. Take the frozen cells from step 3), thaw them, and add them to the rotary bottle. Stir for 5 minutes every 90 minutes at a stirring speed of 40 rpm. After 24 hours, switch to continuous stirring at a stirring speed of 20-40 rpm. Detect the sugar content and determine the medium replacement time based on the sugar consumption. After culturing for 1-4 days, proceed to the next culture step. 5) Continue culturing the mesenchymal stem cell microspheres from step 4) in a carbon dioxide atmosphere incubator. After 5-6 days of growth, replace the serum-free medium of the mesenchymal stem cell 3-D microspheres with α-MEM basal medium and add astrocytosin induction solution. Continue culturing for 1-2 days, then collect the cell suspension, centrifuge once, and collect the supernatant. Centrifuge the supernatant a second time, collect the precipitate, resuspend the precipitate with PBS buffer, and centrifuge a third time. Collect the centrifuged precipitate to obtain the mesenchymal stem cell apoptotic vesicles.

2. The method according to claim 1, characterized in that, In step 1), the deoxyribonucleic acid is one or more of 2'-deoxyadenosine, deoxycytidine hydrochloride, deoxyguanosine, or thymine.

3. The method according to claim 1, characterized in that, In step 1), the ribonucleic acid is one or more of adenosine, cytosine, guanosine, or uridine.

4. The method according to claim 1, characterized in that, In step 2), the umbilical cord is one of the following: human umbilical cord, bovine umbilical cord, sheep umbilical cord, dog umbilical cord, cat umbilical cord, mouse umbilical cord, rat umbilical cord, or rabbit umbilical cord.

5. The method according to claim 1, characterized in that, In step 3), the cells are passaged when the cell fusion rate is 80%.

6. The method according to claim 1, characterized in that, In step 4), the criteria for judging the liquid replacement are: when the sugar content is lower than 3g / L, replace 1 / 2 to 2 / 3 of the liquid.

7. The method according to claim 1, characterized in that, In step 5), the volume concentration of carbon dioxide is 5%, the culture temperature is 37℃, and the induction culture time is 24~48h.

8. The method according to claim 1, characterized in that, In step 5), the centrifugal force for the first centrifugation is 800g and the centrifugation time is 10min; the centrifugal force for the second centrifugation is 16000g and the centrifugation time is 5min; and the centrifugal force for the third centrifugation is 16000g and the centrifugation time is 10min.

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