A method for preparing agglomerated stem cell apoptotic vesicles

Abstract

The present application relates to the field of vesicle preparation method, and specifically discloses a preparation method of agglomeration state stem cell apoptosis vesicle, selects P3-P10 generation mesenchymal stem cells obtained by culture, and when the cell culture reaches 70-85% of the fusion degree, agglomeration state stem cell induction solution is added for culture, and the agglomeration state stem cells are formed after 10-15 days of culture; the agglomeration state stem cells are induced by adding stellacyanin with a concentration of 500-700 nM for 18-26 h, and after the induction is completed, the agglomeration state stem cell apoptosis vesicle is obtained by stepwise centrifugation under the conditions of 600-1000 g first centrifugation, 1500-2500 g second centrifugation and 14000-18000 g third centrifugation; the agglomeration state stem cell induction solution is a-MEM culture solution containing 8-12% FBS and 30-70 μM VC. The agglomeration state stem cell apoptosis vesicle prepared by the present application has increased immunoregulatory capacity.

Description

Technical Field

[0001] This invention relates to the field of vesicle preparation methods, and specifically to a method for preparing apoptotic vesicles of condensed stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are widely distributed throughout connective tissues and organ stroma, with the highest abundance in bone marrow. They can also be isolated from fetal umbilical cord blood, and are found in the placenta, amniotic fluid, subendothelial layer of the umbilical vein, peripheral blood, and various tissues such as the liver, fat, muscle, and skin. MSCs possess high proliferative, self-renewal, and multi-lineage differentiation potential. They can differentiate into various tissues and cells, are easy to isolate, culture, and expand, and readily accept and express exogenous genes. During long-term in vitro culture, they maintain their multi-lineage differentiation potential and exhibit a remarkably stable genetic background.

[0003] Apoptosis is a major form of programmed cell death, playing a crucial role in maintaining tissue and organ homeostasis. During apoptosis, cells shrink, chromatin condenses, and the cell membrane swells, ultimately producing a bilayer lipid structure encapsulating the cellular contents—apoptotic vesicles (apoVs). ApoVs are rich in many functional proteins related to cellular behavior and the metabolism, transport, and regulation of various diseases. In terms of molecular identification, apoVs carry general EV markers such as CD9, CD63, and CD81, as well as apoptosis-specific markers such as C1q, PS, and Fas.

[0004] Every day, billions of cells in the human body undergo apoptosis, and the resulting large number of apoVs play a crucial role in maintaining tissue and organ homeostasis. In terms of immune regulation, studies have shown that apoVs modulate the immune system through helper antigen presentation; for example, dendritic cell-derived apoVs carry the major histocompatibility complex (MHC) to activate CD4+ T cells. Conversely, normal cell-derived apoVs utilize Fas ligands to induce apoptosis in multiple myeloma cells via the Fas pathway, thereby inhibiting tumor development. Regarding tissue homeostasis, apoVs induced by hepatectomy can stimulate neutrophils to adopt a non-inflammatory regenerative phenotype, contributing to liver regeneration.

[0005] Currently established methods for preparing apoptotic vesicles include those using adherent cells and suspension cells. However, existing techniques for preparing apoptotic vesicles based on normally cultured cells and tissues result in stem cell apoptotic vesicles with low immunomodulatory capabilities, hindering the application scope and effectiveness of apoptotic vesicles. Summary of the Invention

[0006] To develop a method for preparing apoptotic stem cell vesicles that enhances the immune regulation capacity of stem cells, this invention provides a method for preparing apoptotic stem cell vesicles in agglutinated state. This invention improves the immune regulation capacity of apoptotic stem cell vesicles by inducing the generation of agglutinated stem cells and then extracting the vesicles.

[0007] This invention provides a method for preparing apoptotic vesicles of condensed stem cells.

[0008] Includes the following steps:

[0009] Select P3-P10 generation mesenchymal stem cells obtained through culture. When the cells reach a confluence of 70-85%, add agglutinin-inducing medium and culture for 10-15 days to form agglutinin-inducing stem cells.

[0010] Aggregated stem cells were induced with 500-700 nM astrocytoxin for 18-26 h. After induction, the cells were centrifuged at 600-1000 g for the first time, 1500-2500 g for the second time, and 14000-18000 g for the third time to obtain apoptotic vesicles of aggregated stem cells.

[0011] The induction medium for condensed stem cells is 8-12% FBS in α-MEM culture medium containing 30-70 μM of vitamin C.

[0012] Furthermore, the agglutinated stem cell induction medium is an a-MEM culture medium containing 10% FBS and 50 μM of VC.

[0013] Furthermore, the specific procedures for the first, second, and third centrifugations are as follows: the induced cell suspension is collected and centrifuged at 600-1000g for 10-15 minutes, and the supernatant is collected. The collected supernatant is centrifuged at 1500-2500g for 5-10 minutes, and the precipitate is collected. The collected precipitate is resuspended and centrifuged at 14000-18000g for 10-15 minutes, and the precipitate is collected. This precipitate is the apoptotic vesicle of the condensed stem cells.

[0014] Furthermore, the parameters for the first, second, and third centrifugations were: 800g for 10 minutes, 16000g for 5 minutes, and 16000g for 10 minutes, respectively.

[0015] Furthermore, the astrocytosin induction solution was added, and the induction culture conditions were 5% carbon dioxide and cultured at 37°C for 24 hours.

[0016] Furthermore, the concentration of the astrocytoxin is 600 nM.

[0017] The present invention also provides agglutinated stem cell apoptotic vesicles prepared by the preparation method described above.

[0018] Furthermore, the proportion of TH1, TH2, and Treg cells in the apoptotic vesicles of the condensed stem cells is increased.

[0019] Furthermore, the apoptotic vesicles of the condensed stem cells exhibit increased immunomodulatory capacity.

[0020] The present invention also provides the application of the aforementioned apoptotic vesicles of condensed stem cells in the preparation of products that enhance immunity, wherein the products are drugs or health products.

[0021] Furthermore, the drug is made from the active ingredient, apoptotic stem cell vesicles, and other pharmaceutical excipients.

[0022] Furthermore, the pharmaceutical excipients are any one or a combination of several of the following: solvents, solubilizers, cosolvents, preservatives, flavoring agents, colorants, suspending agents, emulsifiers, wetting agents, foaming agents, defoamers, fillers, absorbents, diluents, binders, disintegrants, lubricants, flow aids, coating materials, plasticizers, osmotic regulators, antioxidants, osmotic pressure regulators, integrators, penetration enhancers, thickeners, humectants, propellants, and sustained-release materials.

[0023] Furthermore, the health product is a stem cell capsule containing apoptotic vesicles of condensed stem cells.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. A method for preparing apoptotic vesicles that enhance the immune regulation of stem cell apoptotic vesicles, wherein the apoptotic vesicles possess anti-inflammatory and immunomodulatory capabilities. The method disclosed in this invention for extracting and preparing apoptotic vesicles that enhance the immune regulation of stem cell apoptotic vesicles includes mesenchymal stem cell culture, induction of condensed stem cell tissue, induction of apoptotic stem cell tissue, collection, and preparation of apoptotic vesicles.

[0026] 2. This invention improves the immunomodulatory capacity of apoptotic stem cell vesicles by inducing the generation of aggregated stem cells and then extracting vesicles.

[0027] It can be used to treat immune diseases, skin regeneration, and other conditions. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 A morphological photograph of mesenchymal stem cells when their fusion rate reaches 80%.

[0030] Figure 2 This is a morphological photograph of agglutinated stem cells.

[0031] Figure 3 Electron micrograph of apoptotic vesicles of condensed stem cells.

[0032] Figure 4 The particle size of apoptotic vesicles of condensed stem cells prepared in Example 1 was measured.

[0033] Figure 5 The effect of different concentrations of condensed stem cell induction solution on the proportion of TH1 cells was investigated. 30 μM and 70 μM represent the proportion of TH1 cells induced by condensed stem cell induction solutions containing 30 μM and 70 μM VC, respectively.

[0034] Figure 6 The effect of different concentrations of condensed stem cell induction solution on the proportion of TH2 cells was investigated. 30 μM and 70 μM represent the proportion of TH2 cells induced by condensed stem cell induction solutions containing 30 μM and 70 μM VC, respectively.

[0035] Figure 7 The effect of different concentrations of condensed stem cell induction solution on the proportion of TH17 cells was investigated. 30 μM and 70 μM represent the proportion of TH17 cells induced by condensed stem cell induction solutions containing 30 μM and 70 μM VC, respectively.

[0036] Figure 8 The effect of different concentrations of condensed stem cell induction solution on the proportion of Treg cells was investigated. 30 μM and 70 μM represent the proportions of Treg cells induced by condensed stem cell induction solutions containing 30 μM and 70 μM VC, respectively. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0038] Example 1: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0039] I. Mesenchymal stem cell culture

[0040] P0 generation mesenchymal stem cells were revived using a 37°C water bath, and P1 generation mesenchymal stem cells were obtained after centrifugation. Cell culture medium (aMEM culture medium containing 10% fetal bovine serum) was added, and the cells were shaken well and then placed in a cell culture incubator (5% carbon dioxide, 37°C) for culture.

[0041] After culturing for 3 days, when the cells have grown to 80% confluence, the cells are digested, centrifuged to obtain P2 generation mesenchymal stem cells, added to cell culture medium (aMEM culture medium containing 10% fetal bovine serum), shaken well and placed in a cell culture incubator (5% carbon dioxide, 37℃) for culture.

[0042] After culturing for 3 days, when the cells have grown to 80% confluence, the cells are digested, centrifuged to obtain P3 generation mesenchymal stem cells, added to cell culture medium (aMEM culture medium containing 10% fetal bovine serum), shaken well and placed in a cell culture incubator (5% carbon dioxide, 37℃) for culture.

[0043] After 3 days of culture, when the cells reached 80% confluence, they were digested, centrifuged, and P3 generation mesenchymal stem cells were obtained. Flow cytometry was used for cell identification (as shown in Table 1). The surface molecular detection results of the mesenchymal stem cells 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 2%, indicating normal surface molecular expression of the stem cells.

[0044] Table 1. Results of molecular detection on the surface of mesenchymal stem cells.

[0045]

[0046] The results are shown in Table 1, which indicate that the mesenchymal stem cells meet the requirements of the International Cell Therapy Association.

[0047] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0048] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well in a cross-hatching method and then placed in a cell culture incubator (5% carbon dioxide, 37°C).

[0049] 2. When the mesenchymal stem cell confluence reaches 80% (as shown in cell morphology photographs) Figure 1As shown, the grown cells adhered to the wall and formed a spindle shape, which meets the morphological requirements of mesenchymal stem cells. The cell culture medium was replaced with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC), and the cells were placed in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0050] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 12-day culture period; (cell morphology photos are shown below). Figure 2 As shown in the electron micrograph of apoptotic vesicles of condensed stem cells, Figure 3 As shown, the results indicate that condensed stem cells are elongated spindle-shaped, with reduced intercellular spaces and increased extracellular matrix compared to conventionally cultured stem cells, forming a three-dimensional growth structure for stem cells.

[0051] 4. Replace the agglutinated stem cell induction medium, add 3 mL of basal culture medium (aMEM culture medium) to each well, and add 600 nM of different concentrations of astrocytosin induction medium for treatment. Place the wells in an incubator (5% carbon dioxide, 37℃) and continue induction at different time points of 18 h, 20 h, 22 h, 24 h and 26 h before proceeding to the next step.

[0052] The astrocytosin induction solution is prepared by dissolving astrocytosin to the required concentration in DMSO (manufacturer: OriGen, catalog number: CP-70);

[0053] The manufacturer of Astragalus is MCE, product number: 62996-74-1.

[0054] 5. Collect the cell suspension induced in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0055] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0056] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0057] The collected precipitates were resuspended in 1 mL of PBS and the protein concentration was determined. The results are shown in Table 2. For specific detection methods, please refer to the instructions of the Yisheng BCA Protein Concentration Assay Kit.

[0058] Example 2: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0059] I. Mesenchymal stem cell culture

[0060] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0061] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0062] 1. The obtained P4 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0063] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0064] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0065] 4. Replace the induction medium for agglutinated stem cells, add 3 mL of basal culture medium to each well, add 300 nM astrocytoxin induction medium for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 24 h before proceeding to the next step.

[0066] 5. Collect the cell suspension induced in step 4, centrifuge at 600g for 15 minutes, and collect the supernatant.

[0067] 6. After centrifuging the supernatant collected in step 5 at 15000g, collect the precipitate;

[0068] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 14000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0069] The collected apoptotic vesicles of condensed stem cells were resuspended in 1 mL of PBS and their particle size was analyzed. The results are as follows: Figure 4 As shown, the results indicate that the diameter of apoptotic vesicles in condensed stem cells is between 100 and 200 nm.

[0070] Example 3: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0071] I. Mesenchymal stem cell culture

[0072] P5 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0073] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0074] 1. The obtained P5 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0075] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0076] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0077] 4. Replace the induction medium for agglutinated stem cells, add 3 mL of basal culture medium to each well, add 400 nM astrocytoxin induction medium for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 24 h before proceeding to the next step.

[0078] 5. Collect the cell suspension induced in step 4, centrifuge at 1000g for 15 minutes, and collect the supernatant.

[0079] 6. After centrifuging the supernatant collected in step 5 at 25000g for 5 minutes, collect the precipitate;

[0080] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 18000g for 8 minutes and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0081] Example 4: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0082] I. Mesenchymal stem cell culture

[0083] P6 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0084] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0085] 1. The obtained P6 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0086] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0087] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0088] 4. Replace the induction medium for agglutinated stem cells, add 3 mL of basal culture medium to each well, add 500 nM astrocytosine for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 24 h before proceeding to the next step.

[0089] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 900g for 15 minutes, and collect the supernatant.

[0090] 6. After centrifuging the supernatant collected in step 5 at 20000g for 6 minutes, collect the precipitate;

[0091] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 15000g for 8 minutes and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0092] Example 5: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0093] I. Mesenchymal stem cell culture

[0094] P7 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0095] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0096] 1. The obtained P7 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0097] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0098] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0099] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 700 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 24 h before proceeding to the next step.

[0100] 5. Collect the cell suspension induced by astrocytosine in step 4, centrifuge at 700g for 15 minutes, and collect the supernatant.

[0101] 6. After centrifuging the supernatant collected in step 5 at 18000g for 6 minutes, collect the precipitate;

[0102] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 8 minutes and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0103] Example 6: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0104] I. Mesenchymal stem cell culture

[0105] P3 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0106] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0107] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0108] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0109] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0110] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 600 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 26 h before proceeding to the next step.

[0111] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0112] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0113] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0114] Example 7: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0115] I. Mesenchymal stem cell culture

[0116] P10 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0117] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0118] 1. The obtained P10 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well in a cross-hatching method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0119] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0120] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0121] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 600 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 24 h before proceeding to the next step.

[0122] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0123] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0124] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0125] Example 8: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0126] I. Mesenchymal stem cell culture

[0127] P3 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0128] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0129] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0130] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0131] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0132] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 600 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 22 h before proceeding to the next step.

[0133] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0134] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0135] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0136] Example 9: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0137] I. Mesenchymal stem cell culture

[0138] P3 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0139] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0140] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0141] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0142] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0143] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 600 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 20 h before proceeding to the next step.

[0144] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0145] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0146] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0147] Example 10: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0148] I. Mesenchymal stem cell culture

[0149] P3 generation mesenchymal stem cells were cultured according to the culture method in Example 1.

[0150] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0151] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well by the cross method and then placed in a cell culture incubator (5% carbon dioxide, 37℃).

[0152] 2. When the mesenchymal stem cells reach 80% confluence, replace the cell culture medium with agglutinated stem cell induction medium (10% FBS a-MEM culture medium containing 50uM VC) and place the cells in a cell culture incubator for 24 hours (5% carbon dioxide, 37℃).

[0153] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 15-day culture period.

[0154] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add 600 nM astrocytoxin for treatment, and place in an incubator (5% carbon dioxide, 37℃) to continue induction for 18 h before proceeding to the next step.

[0155] 5. Collect the cell suspension induced by astrocytoxin in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0156] 6. After centrifuging the supernatant collected in step 5 at 16000g for 5 minutes, collect the precipitate;

[0157] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g for 10min and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0158] Example 11: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0159] I. Mesenchymal stem cell culture

[0160] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0161] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0162] The method is basically the same as that in Example 2, except that:

[0163] The sample was treated with 300 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 26 hours for further induction.

[0164] Example 12: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0165] I. Mesenchymal stem cell culture

[0166] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0167] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0168] The method is basically the same as that in Example 2, except that:

[0169] The sample was treated with 300 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 22 h of induction.

[0170] Example 13: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0171] I. Mesenchymal stem cell culture

[0172] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0173] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0174] The method is basically the same as that in Example 2, except that:

[0175] The sample was treated with 300 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 28 hours for further induction.

[0176] Example 14: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0177] I. Mesenchymal stem cell culture

[0178] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0179] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0180] The method is basically the same as that in Example 2, except that:

[0181] The sample was treated with 300 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 22 h of induction.

[0182] Example 15: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0183] I. Mesenchymal stem cell culture

[0184] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0185] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0186] The method is basically the same as that in Example 2, except that:

[0187] The sample was treated with 300 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 20 hours for further induction.

[0188] Example 16: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0189] I. Mesenchymal stem cell culture

[0190] P4 generation mesenchymal stem cells were cultured according to the culture method in Example 2.

[0191] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0192] The method is basically the same as that in Example 2, except that:

[0193] The sample was treated with 300 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 18 hours for further induction.

[0194] Example 17: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0195] I. Mesenchymal stem cell culture

[0196] Same as Example 3.

[0197] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0198] The basic steps are the same as in Example 3, except that:

[0199] The sample was treated with 400 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 26 hours for further induction.

[0200] Example 18: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0201] I. Mesenchymal stem cell culture

[0202] Same as Example 3.

[0203] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0204] The basic steps are the same as in Example 3, except that:

[0205] The sample was treated with 400 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 28 hours for further induction.

[0206] Example 19: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0207] I. Mesenchymal stem cell culture

[0208] Same as Example 3.

[0209] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0210] The basic steps are the same as in Example 3, except that:

[0211] The sample was treated with 400 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 22 h of induction.

[0212] Example 20: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0213] I. Mesenchymal stem cell culture

[0214] Same as Example 3.

[0215] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0216] The basic steps are the same as in Example 3, except that:

[0217] The sample was treated with 400 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 20 hours for further induction.

[0218] Example 21: A method for preparing apoptotic vesicles of congealed stem cells with enhanced immune regulation.

[0219] I. Mesenchymal stem cell culture

[0220] Same as Example 3.

[0221] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0222] The basic steps are the same as in Example 3, except that:

[0223] The sample was treated with 400 nM astrocytoxin induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 18 hours for further induction.

[0224] Example 22: A method for preparing apoptotic vesicles of condensed stem cells with enhanced immune regulation.

[0225] I. Mesenchymal stem cell culture

[0226] Same as Example 4.

[0227] II. Preparation steps of apoptotic vesicles of condensed stem cells

[0228] The basic steps are the same as in Example 4, except that:

[0229] The sample was treated with 500 nM astrocytosine induction solution and placed in an incubator (5% carbon dioxide, 37°C) for 26 hours for further induction.

[0230] Following the above pattern, astrocytosin at concentrations of 300 nM, 400 nM, 500 nM, 600 nM, and 700 nM was used for induction for 18 h, 20 h, 22 h, 24 h, and 26 h, respectively. The remaining steps were consistent with Example 1. The protein concentration results of the obtained apoptotic vesicles in condensed stem cells are shown in Table 2.

[0231] Table 2. Results of protein concentration detection in astrocytokine-induced apoptotic vesicles of condensed stem cells.

[0232]

[0233] As shown in Table 2, the protein concentration detection results of stem cell vesicles induced by different concentrations of astrocytokinin at different times showed that the astrocytokinin concentration was directly proportional to the release rate of apoptotic vesicles. As the astrocytokinin concentration increased, the release rate of apoptotic vesicles accelerated. When the concentration exceeded 600 nM, the number of vesicles reached a plateau. The effect of different induction times on the amount of vesicles released showed that after 24 hours, the number of apoptotic vesicles no longer increased or increased slowly. Therefore, the optimal induction scheme for condensed stem cells was obtained by inducing 24 hours after astrocytokinin at concentrations above 600 nM, providing a method for preparing apoptotic vesicles of condensed stem cells.

[0234] Comparative Example 1: Preparation process of conventionally cultured stem cell vesicles.

[0235] 1. The obtained P10 generation mesenchymal stem cells were seeded into 6-well plates, culture medium (10% FBS in a-MEM culture medium) was added, and the cells were shaken well by the cross method and then placed in a cell culture incubator for culture.

[0236] 2. When the mesenchymal stem cells reach 80% fusion, add astrocytosine (600 nM) for treatment and induce for 24 h;

[0237] 3. After collecting the cell suspension, centrifuge at 800g for 10 minutes and collect the supernatant;

[0238] 4. After centrifuging the supernatant collected in step 5 at 16000g, collect the precipitate;

[0239] 5. After resuspending the cells in PBS, centrifuge at 16000g and collect the precipitate, which is the stem cell apoptosis vesicle.

[0240] The collected apoptotic vesicles of stem cells were resuspended in 1 mL of PBS.

[0241] Comparative Example 2: A method for preparing apoptotic vesicles of stem cells.

[0242] 1. The obtained P3 generation mesenchymal stem cells were seeded into 24-well plates at a rate of 1E5 / well. After seeding, 3 mL of culture medium (10% FBS in α-MEM culture medium) was added to each well, and the cells were shaken well in a cross-hatching method and then placed in a cell culture incubator (5% carbon dioxide, 37°C).

[0243] 2. When the mesenchymal stem cells reach 80% fusion, the cell culture medium is replaced with different concentrations of agglutinated stem cell induction medium, and three groups of agglutinated stem cell induction medium concentrations are set up (group A, group B, and group C).

[0244] Group A: 10% FBS α-MEM culture medium containing 30 μM VC; Group B: 10% FBS α-MEM culture medium containing 50 μM VC; Group C: 10% FBS α-MEM culture medium containing 70 μM VC. Cells were cultured in a cell culture incubator for 24 hours (5% CO2, 37°C).

[0245] 3. Change the induction medium for agglutinated stem cells every 3 days. Agglutinated stem cells will form after a 12-day culture period.

[0246] 4. Replace the induction medium, add 3 mL of basic culture medium to each well, add astrocytosin (600 nM) for treatment, and place in an incubator (5% carbon dioxide, 37℃) for 24 h before proceeding to the next step.

[0247] 5. Collect the cell suspension induced in step 4, centrifuge at 800g for 10 minutes, and collect the supernatant.

[0248] 6. After centrifuging the supernatant collected in step 5 at 16000g, collect the precipitate;

[0249] 7. After resuspending the precipitate collected in step 6 with PBS, centrifuge at 16000g and collect the precipitate, which is the apoptotic vesicle of agglutinated stem cells.

[0250] The collected precipitates were resuspended in 1 mL of PBS and the protein concentration was determined. The results are shown in Table 3. For specific detection methods, please refer to the instructions of the Yisheng BCA Protein Concentration Assay Kit.

[0251] Table 3. Protein concentration detection results of apoptotic vesicles in condensed stem cells induced by different concentrations of vitamin C for 24 hours.

[0252] concentration 30μM 50 μM (Example 1) 70μM protein concentration 0.103 0.275 0.215

[0253] As shown in Table 3, the concentration of VC in the induction medium for agglutinated stem cells has a significant effect on the protein concentration of apoptotic vesicles in agglutinated stem cells induced for 24 hours. When the agglutinated stem cell induction medium is 10% FBS a-MEM culture medium containing 50 μM VC, that is, when the VC concentration is 50 μM, the protein concentration of apoptotic vesicles in agglutinated stem cells induced for 24 hours is significantly higher than that in group A (30 μM) and group C (70 μM).

[0254] The apoptotic vesicles of condensed stem cells obtained in Example 1 were co-cultured with PBMC immune cells and stimulated with phytohemagglutinin (manufacturer: MCE, catalog number: HY-18739) (working concentration: 5ug / mL), and the proportions of TH1, TH2, TH17 and Treg cells were detected.

[0255] The results are as follows Figure 5As shown, the immunomodulatory capacity of apoptotic stem cell vesicles in condensed state was higher than that of ordinary cultured stem cell vesicles prepared by the methods in Comparative Example 1 and Comparative Example 2.

[0256] like Figure 6 As shown, the immunomodulatory capacity of apoptotic stem cell vesicles in condensed state was higher than that of ordinary cultured stem cell vesicles prepared by the methods in Comparative Example 1 and Comparative Example 2, compared with those in Comparative Example 2.

[0257] like Figure 7 As shown, the immunomodulatory capacity of apoptotic stem cell vesicles in condensed state was lower than that of conventionally cultured stem cell vesicles prepared by the methods in Comparative Examples 1 and 2 compared to those in Comparative Examples 2.

[0258] like Figure 8 As shown, the immunomodulatory capacity of apoptotic stem cell vesicles in condensed state was lower than that in Comparative Example 1 and Comparative Example 2 compared to the conventionally cultured stem cell vesicles prepared by the same method, with a lower proportion of Treg cells.

[0259] The ratio of TH1, TH2, TH17 and Treg reflects the immune regulation capacity of stem cells, thus indicating that this scheme provides a method for preparing apoptotic vesicles of agglutinated stem cells that can enhance immune regulation capacity.

[0260] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0261] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing apoptotic vesicles of condensed stem cells, characterized in that, Includes the following steps: Select P3-P10 generation mesenchymal stem cells obtained through culture. When the cells reach a confluence of 70-85%, add agglutinin-inducing medium and culture for 10-15 days to form agglutinin-inducing stem cells. Aggregated stem cells were induced with 500-700 nM astrocytoxin for 18-26 h. After induction, the cells were centrifuged at 600-1000 g for the first time, 1500-2500 g for the second time, and 14000-18000 g for the third time to obtain apoptotic vesicles of aggregated stem cells. The induction medium for condensed stem cells is 8-12% FBS in α-MEM culture medium containing 50 μM VC.

2. The method for preparing apoptotic vesicles of condensed stem cells according to claim 1, characterized in that, The specific procedures for the first, second, and third centrifugation are as follows: After collecting the cell suspension induced by apoptosis, centrifuge at 600-1000g for 10-15 minutes and collect the supernatant. Centrifuge the collected supernatant at 1500-2500g for 5-10 minutes and collect the precipitate. After resuspending the precipitate, centrifuge at 14000-18000g for 10-15 minutes and collect the precipitate, which is the apoptotic vesicle of the condensed stem cells.

3. The method for preparing apoptotic vesicles of condensed stem cells according to claim 2, characterized in that, The parameters for the first, second, and third centrifugations were: 800g for 10 minutes, 16000g for 5 minutes, and 16000g for 10 minutes, respectively.

4. The method for preparing apoptotic vesicles of condensed stem cells according to claim 1, characterized in that, The culture conditions for inducing astrocytokinin were 5% carbon dioxide and cultured at 37°C for 24 hours.

5. The method for preparing apoptotic vesicles of condensed stem cells according to claim 1, characterized in that, The concentration of the astrocytoxin was 600 nM.

6. Aggregated stem cell apoptotic vesicles prepared by the preparation method according to any one of claims 1-5.

7. The apoptotic vesicles of condensed stem cells according to claim 6, characterized in that, The proportion of TH1, TH2 and Treg cells in the apoptotic vesicles of the condensed stem cells increased.

8. The apoptotic vesicles of condensed stem cells according to claim 7, characterized in that, The apoptotic vesicles of the condensed stem cells have increased immune regulatory capacity.

9. The use of the agglutinated stem cell apoptotic vesicles of claim 6 in the preparation of a product for regulating the proportion of immune cells, characterized in that, The regulation of immune cell proportions includes increasing the proportion of TH1 and TH2 cells and decreasing the proportion of TH17 cells.

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