A kit for promoting mesenchymal stem cells to produce exosomes

Abstract

The present invention provides a kit for promoting the production of extracellular vesicles by human adipose mesenchymal stem cells and a method for promoting the production of extracellular vesicles by human adipose mesenchymal stem cells in vitro. Specifically, the present invention provides a kit containing B-300K solution, and a method for incubating and culturing human adipose mesenchymal stem cells with B-300K solution to produce extracellular vesicles is screened out. The B-300K solution is an ultrafiltrate obtained by ultracentrifugation, filtration and ultrafiltration of αMEM medium containing platelet lysate. Using the kit and method of the present invention, the yield of the produced extracellular vesicles is significantly increased, and it has no effect on the activity of the host cells of the extracellular vesicles.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically, relates to a kit for promoting human adipose mesenchymal stem cells to produce exosomes and a method for increasing the yield of extracellular exosomes. Background Art

[0002] Extracellular vesicles (EVs) refer to substances secreted by cells with a phospholipid bilayer vesicle structure. According to size and generation mechanism, they can be further divided into exosomes and microvesicles. Among them, exosomes are actively secreted by cells and are an important pathway for intercellular signal transduction. Cells secrete them extracellularly through the lysosomal pathway, when multivesicular bodies fuse with the cell plasma membrane. Their main characteristics include: 1) size ranging from 30 to 150 nm, 2) having a phospholipid bilayer, showing a cup-shaped vesicle structure under transmission electron microscopy, and 3) having tetraspanin proteins such as CD63, CD9, CD81, and markers such as TSG101 and Alix.

[0003] During cell culture, some serum-derived factors are usually used as additives to the culture medium, especially human platelet lysate. The advantage of human platelet lysate is that it is a human-derived substance, and its clinical use can reduce the immunogenicity and infection risk of animal origin. There are literature reports that after ultrafiltration, most of the exosomes in platelet lysate can be removed, and the surface markers, cell proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells cultured with this culture medium still maintain their superiority.

[0004] In summary, the present invention needs to develop a method for efficiently increasing the production of exosomes by human adipose mesenchymal stem cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for promoting human mesenchymal stem cells to produce exosomes and a kit for producing human mesenchymal stem cell exosomes.

[0006] In the first aspect of the present invention, a method for increasing the yield of extracellular exosomes is provided, including the steps of:

[0007] (a) Providing a stem cell;

[0008] (b) Under suitable culture conditions, pre-culturing the stem cell with a first culture medium to obtain a pre-cultured stem cell with a confluence of 60 - 90%;

[0009] wherein, the first culture medium is an αMEM medium containing platelet lysate;

[0010] (c) Incubate and culture the cultured stem cells with a second culture medium for 24 to 48 hours to obtain an incubation culture medium, which contains the incubated and cultured stem cells and extracellular vesicles produced by the incubated and cultured stem cells;

[0011] Wherein, the second culture medium is an ultrafiltrate obtained by subjecting an αMEM medium containing platelet lysate to ultracentrifugation, filtration, and ultrafiltration; and

[0012] (d) Isolate extracellular vesicles from the incubation culture medium obtained in the previous step.

[0013] In another preferred example, the stem cells are stem cells that have been passaged in vitro for 2 to 6 generations (P2 to P6), preferably stem cells that have been passaged in vitro for 4 generations (P4).

[0014] In another preferred example, the stem cells are mesenchymal stem cells.

[0015] In another preferred example, the mesenchymal stem cells are derived from mammals, preferably from humans.

[0016] In another preferred example, the mesenchymal stem cells are derived from human tissues, such as adipose tissue, bone marrow, and placenta.

[0017] In another preferred example, the stem cells are adipose-derived mesenchymal stem cells.

[0018] In another preferred example, in step (b), the confluence of the stem cells is 60 - 90%, preferably 70 - 90%, more preferably 75 - 80%.

[0019] In another preferred example, the concentration of platelet lysate in the first culture medium is 2% to 15%, preferably 5% to 10%, and most preferably 5%, where the percentage is by volume.

[0020] In another preferred example, the concentration of platelet lysate in the second culture medium is 2% to 15%, preferably 5% to 10%, and most preferably 5%, where the percentage is by volume.

[0021] In another preferred example, the rotational speed of the ultracentrifugation is 100,000 to 120,000 g, preferably 100,000 g; more preferably 120,000 g.

[0022] In another preferred example, the time of ultracentrifugation is 6 to 24 hours.

[0023] In another preferred example, the filtration is performed using a filter with a pore size of 0.22 μm to 0.45 μm, preferably 0.22 μm.

[0024] In another preferred example, the ultrafiltration is carried out using an ultrafiltration tube with a pore size of 100 - 300 KD, preferably 300 KD.

[0025] In another preferred example, in step I, the second culture medium is prepared by the following method:

[0026] (5) Provide an αMEM medium containing 2% - 15% platelet lysate;

[0027] (ii) Ultracentrifuge the αMEM medium containing 2% - 15% platelet lysate for 6 - 24 hours to obtain the supernatant after centrifugation; wherein, the speed of the ultracentrifugation is 100,000 - 120,000 g, preferably 100,000 g; more preferably, 120,000 g;

[0028] (iii) Filter the supernatant after centrifugation using a filter with a pore size of 0.22 μm - 0.45 μm (preferably 0.22 μm) to obtain the filtered filtrate;

[0029] (iv) Ultrafilter the filtered filtrate using an ultrafiltration tube with a pore size of 100 - 300 KD (preferably 300 KD) to obtain the ultrafiltrate, that is, obtain the second culture medium.

[0030] In another preferred example, the second culture medium has the following characteristics:

[0031] (1) Contains 2% - 15% platelet lysate;

[0032] (2) Almost does not contain exosomes in the platelet lysate;

[0033] (3) Enables mesenchymal stem cells to maintain a relatively good proliferation ability;

[0034] (4) Enables mesenchymal stem cells to maintain relatively good stem cell characteristics; and

[0035] (5) Promotes mesenchymal stem cells to secrete more exosomes.

[0036] In another preferred example, "almost does not contain exosomes in the platelet lysate" means that the content of exosomes in the platelet lysate in the second culture medium is less than 10.0%, preferably 5.0%, and most preferably 2.0%.

[0037] In another preferred example, in step (c), the incubation time is 24 - 72 hours, preferably 24 - 48 hours, and most preferably 48 hours.

[0038] In another preferred example, in step (d), it includes:

[0039] (d1) Separate the incubation culture medium to obtain a cell-free supernatant, wherein the supernatant contains the extracellular vesicles.

[0040] (d2) Incubate the supernatant in step (d1) with a polyethylene glycol (PEG) solution overnight to obtain a mixture.

[0041] (d3) Separate the mixture obtained in step (d2) to obtain a precipitate, wherein the precipitate contains the extracellular vesicles.

[0042] In another preferred example, in step (d1), centrifuge the incubation culture medium to obtain a cell-free supernatant.

[0043] In another preferred example, the centrifugation conditions are 3000 - 10000 g, differential centrifugation for 10 - 40 minutes.

[0044] In another preferred example, in step (d2), the polyethylene glycol (PEG) is PEG4000 - PEG8000, preferably PEG6000.

[0045] In another preferred example, in step (d2), the final concentration of polyethylene glycol (PEG) in the polyethylene glycol (PEG) solution is 8% - 20%.

[0046] In another preferred example, in step (d3), centrifuge the mixture to obtain a precipitate, wherein the precipitate contains the extracellular vesicles.

[0047] In another preferred example, the centrifugation conditions are 3000 - 15000 g, differential centrifugation for 40 - 70 minutes.

[0048] In another preferred example, the method further includes step (e): detecting the particle concentration and the marker expression level of the extracellular vesicles obtained in step (d); detecting cell proliferation, cell viability, and cell surface markers of the cells obtained in step (c).

[0049] In another preferred example, the detection of the marker expression level includes detecting the protein expression level of the marker.

[0050] In another preferred example, the markers include extracellular vesicle-specific markers, cell-specific markers, or a combination thereof.

[0051] In another preferred example, the extracellular vesicle-specific marker is CD81.

[0052] In another preferred example, the cell-specific marker is selected from the group consisting of: CD73, CD90, CD105, CD34, CD45, HLA DR, or a combination thereof.

[0053] In a second aspect of the present invention, there is provided an extracellular exosome, which is obtained by the method described in the first aspect of the present invention.

[0054] In another preferred example, the exosome is derived from human cells.

[0055] In another preferred example, the human cells are selected from induced stem cells and mesenchymal stem cells.

[0056] In another preferred example, compared with the exosomes obtained by incubating and culturing with the "control group culture medium", the exosomes obtained by the method described in the first aspect of the present invention have one or more characteristics selected from the group consisting of:

[0057] (1) High exosome yield;

[0058] (2) High expression level of exosome-specific markers.

[0059] In another preferred example, compared with the stem cells incubated and cultured with the "control group culture medium", the stem cells incubated and cultured by the method described in claim 1 have one or more characteristics selected from the group consisting of:

[0060] (1) Strong cell proliferation ability and good cell activity;

[0061] (2) The cell-specific markers meet the standards of mesenchymal stem cell-specific markers.

[0062] In another preferred example, the exosome-specific marker is CD81.

[0063] In another preferred example, the cell-specific marker is selected from CD73, CD90, CD105, CD34, CD45, HLA DR or a combination thereof.

[0064] In another preferred example, the standards for mesenchymal stem cell-specific markers are: CD73+ / CD90+ / CD105+>95%; CD34+ / CD45+ / HLA DR+<2%.

[0065] In another preferred example, the "control group culture medium" is selected from the group consisting of: αMEM basal medium, αMEM + 5% EliteGro, solution B, B-100KD, or serum-free medium.

[0066] In another preferred example, the αMEM + 5% EliteGro is an αMEM medium containing 5% platelet lysate.

[0067] In another preferred example, the solution B is the supernatant obtained by subjecting αMEM medium containing 5% platelet lysate to ultracentrifugation at 100,000 - 120,000 g for 6 - 24 hours.

[0068] In another preferred example, the B - 100KD is the ultrafiltrate obtained by subjecting αMEM medium containing 5% platelet lysate to ultracentrifugation at 100,000 - 120,000 g for 6 - 24 hours to obtain the supernatant, filtering the supernatant through a filter with a pore size of 0.22 μm, and then ultrafiltering the filtrate through an ultrafiltration tube with a pore size of 100KD.

[0069] In another preferred example, the serum - free medium is KnockOut Serum Replacement, a serum - free medium produced by Thermo.

[0070] In the third aspect of the present invention, a kit is provided, which comprises:

[0071] (Z1) Human stem cells;

[0072] (Z2) A first culture medium and a second culture medium; wherein, the first culture medium is αMEM medium containing 2% - 15% platelet lysate, and the second culture medium is the ultrafiltrate obtained by subjecting αMEM medium containing 2% - 15% platelet lysate to ultracentrifugation, filtration and ultrafiltration; and

[0073] (Z3) Polyethylene glycol (PEG).

[0074] In another preferred example, the kit further includes (c) a label or an instruction manual, which indicates that the kit is used for producing human stem cell exosomes.

[0075] It should be understood that within the scope of the present invention, the above - mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0076] Figure 1 Shows the proliferation changes of passage 4 adipose - derived mesenchymal stem cells incubated with different culture media for 48 hours; wherein, A: After incubation with different culture media for 48 hours, cell counting is performed; B: After incubation with different culture media for 48 hours, the cell viability is detected using a CCK8 kit. The experimental results are statistically analyzed using One Way ANOVA, *P < 0.05; ns, no significant difference; Error bars, S.D.

[0077] Figure 2Shows the expression of the exosome-specific membrane protein CD81 (loaded with the same volume) in the exosomes collected after incubating adipose-derived mesenchymal stem cells of passage P4 with different culture media for 48 hours. Among them, M is Marker; EG is EliteGro.

[0078] Figure 3 Shows the particle concentrations of the exosomes harvested before and 48 hours after incubating cells with different culture media using NTA. Error bars, S.D.

[0079] Figure 4 Shows the plating form of the cells in Example 2.

[0080] Figure 5 Shows the particle concentrations of the exosomes harvested after incubating adipose-derived mesenchymal stem cells of passage P4 with B-300K solution for different times. Among them, B-300K solution is the blank control; the particle concentration secreted by the cells = total particle concentration - particle concentration of B-300K solution. Error bars, S.D.

[0081] Figure 6 Shows the apoptosis ratios of adipose-derived mesenchymal stem cells of passage P4 at different incubation time points. Among them, 24h: the apoptosis rate of passage P4 adipose MSCs after incubating with solution B for 24 hours; 48h: the apoptosis rate of passage P4 adipose MSCs after incubating with solution B for 48 hours; 72h: the apoptosis rate of passage P4 adipose MSCs after incubating with solution B for 72 hours; all groups are compared with the positive control, *P<0.05; Error bars, S.D.

[0082] Figure 7 Shows the cell apoptosis at different time points detected by Annexin V-FITC. Among them, A-D: apoptotic cells detected by AnnexinV-FITC, showing green; E-H: nuclei stained by Hochest 33342, showing blue. All groups are statistically analyzed with the positive control, *P<0.05; Error bars, S.D.; scale bar: 100μm. Detailed implementation manners

[0083] Through extensive and in-depth research and a large number of screenings, the present inventor has developed for the first time a kit for promoting human adipose mesenchymal stem cells to produce exosomes and screened out a method that can effectively increase the yield of human extracellular exosomes. Experiments show that after incubating human adipose mesenchymal stem cells with B-300K solution culture system for 48 hours, it can effectively increase the yield of cell exosomes without affecting the activity of human adipose mesenchymal stem cells, and moreover, the expression level of the specific protein CD81 of the obtained exosomes increases. On this basis, the present invention is completed.

[0084] Extracellular vesicles

[0085] There are three main types of extracellular vesicles (EVs), namely exosomes, microvesicles, and apoptotic bodies. All three main types of EVs are enclosed by a lipid bilayer and have a diameter range of 30 - 2000 nm.

[0086] The term exosomes refers to a subclass of EVs with a diameter of 50 - 100 nm derived from endosomes, which are the main components of the paracrine substances of various cell types, including mesenchymal stem cells (MSCs). MSCs exosomes are a type of EVs with a diameter in the range of 50 - 100 nm among the EVs derived from MSCs and have a complete lipid bilayer membrane structure.

[0087] Platelet lysate

[0088] Platelets are formed by the adhesion of small cytoplasmic fragments of megakaryocytes to each other. The platelet lysate described in the present invention is a product obtained by directly subjecting human - derived platelet cells to continuous repeated freeze - thaw lysis to release a large amount of activated growth factors. Therefore, human platelet lysate is a cell culture medium additive derived from human platelets, free of xenogeneic sources and animal serum. It contains all the growth factors and proteins required for cell growth.

[0089] The method of the present invention

[0090] The present invention provides a method for increasing the yield of human stem cell exosomes in vitro. Specifically, it includes the steps:

[0091] (1) Cultivate passage P2 - P6 adipose - derived mesenchymal stem cells in αMEM medium containing 5% platelet lysate, with a cell seeding density of 10000 - 15000 cells / cm 2 , until the cells reach 60% - 90% confluence.

[0092] (2) Prepare B - 300K solution, which is αMEM medium containing 5% platelet lysate. Ultra - centrifuge it at 100000 - 120000 g for 6 - 24 hours, obtain the supernatant, filter the supernatant through a filter with a pore size of 0.22 - 0.45 μm, and then ultra - filter the filtrate through an ultra - filtration tube with a pore size of 100 - 300 KD to harvest the obtained ultra - filtrate.

[0093] (3) Incubate the cells in step (1) with B-300K solution for 24 to 48 hours, and then collect the conditioned medium.

[0094] (4) The conditioned medium obtained in step (3) is subjected to differential centrifugation at 3000 to 10000 g to obtain the supernatant.

[0095] (5) The supernatant obtained in step (4) is co-incubated overnight with PEG4000 - 8000 with a final concentration of 8% - 20%, and then subjected to differential centrifugation at 3000 to 150000 g to obtain the precipitate, which is the extracellular vesicles derived from human stem cells.

[0096] In another preferred example, the mesenchymal stem cells are mesenchymal stem cells derived from human tissues, and the cells are selected from the P2, P3, P4, P5, and P6 generations of primary cells after subculture.

[0097] In another preferred example, the mesenchymal stem cells are adipose mesenchymal stem cells derived from human adipose tissue, and the cells are selected from the P4 generation.

[0098] In another preferred example, the seeding density of the mesenchymal stem cells is 10000 cells / cm 2 。

[0099] In another preferred example, the final concentration of the platelet lysate used is 2%, 5%, or 10%.

[0100] In another preferred example, the optimal final concentration of the platelet lysate used is 5%, and the relatively optimal concentration is 10%.

[0101] In another preferred example, the confluence of the mesenchymal stem cells reaches any confluence in the range of 70% - 90%.

[0102] In another preferred example, the optimal confluence of the mesenchymal stem cells is 80%, and the relatively optimal confluence is 70%.

[0103] In another preferred example, the B-300K solution is an αMEM medium containing platelet lysate, and the optimal concentration of the platelet lysate is 5%, and the relatively optimal concentration is 10%.

[0104] In another preferred example, the B-300K solution is prepared as follows: an αMEM medium containing 5% platelet lysate is ultracentrifuged at 120000 g for 6 hours to obtain the supernatant, the supernatant is filtered through a filter with a pore size of 0.22 μm to obtain the filtrate, and the filtrate is ultrafiltered through an ultrafiltration tube with a pore size of 300 KD to obtain the ultrafiltrate.

[0105] In another preferred example, in step (4), the conditioned medium obtained in step (3) is centrifuged at 3000 g for 15 minutes.

[0106] In another preferred example, in step (4), the conditioned medium obtained in step (3) is centrifuged at 10,000 g for 30 minutes.

[0107] In another preferred example, in step (5), the supernatant obtained in step (4) is co-incubated overnight with solution C, and solution C contains PEG4000 - 8000 with a final concentration of 8% - 20%.

[0108] In another preferred example, solution C contains PEG6000 with a final concentration of 12%.

[0109] In another preferred example, in step (5), after the supernatant obtained in step (4) is co-incubated overnight with solution C, differential centrifugation is carried out at 3000 - 15,000 g.

[0110] In another preferred example, in step (5), after the supernatant obtained in step (4) is co-incubated overnight with solution C, it is centrifuged at 3000 g for 1 hour.

[0111] In another preferred example, in step (5), after the supernatant obtained in step (4) is co-incubated overnight with solution C, it is centrifuged at 120,000 g for 70 minutes.

[0112] In another preferred example, the method further includes the steps: (6) detecting cell viability and detecting the protein expression level of the marker.

[0113] In another preferred example, in step (6), the monitoring includes using the western blot method to detect the extracellular vesicle-specific protein markers derived from human cells: CD9 and CD81. The expression level of this marker is positively correlated with the extracellular vesicle-specific protein.

[0114] In another preferred example, in step (6), the monitoring includes using the NTA method to detect the concentration of human extracellular vesicles.

[0115] In the method screened by the present invention, it has a significant effect on improving the yield of extracellular vesicles and specific markers, and has no effect on the viability and specific markers of cells.

[0116] The kit of the present invention

[0117] The present invention also provides a kit for producing human stem cell extracellular vesicles. Specifically, the kit includes:

[0118] (Z1) Human stem cells;

[0119] (Z2) The first culture medium and the second culture medium; wherein, the first culture medium is an αMEM medium containing 2% - 15% (preferably 5%) platelet lysate, and the second culture medium is the ultrafiltrate obtained by ultracentrifugation, filtration, and ultrafiltration of an αMEM medium containing 2% - 15% platelet lysate; and

[0120] (Z3) Polyethylene glycol (PEG) 4000 - 8000.

[0121] In another preferred embodiment, the kit further comprises (c) a label or an instruction manual, which indicates that the kit is used for the production of human stem cell exosomes.

[0122] In another preferred embodiment, the human stem cells are mesenchymal stem cells, and the mesenchymal stem cells include but are not limited to adipose mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.

[0123] In another preferred embodiment, the second culture medium is prepared by the following method:

[0124] (i) Provide an αMEM medium containing 2% - 15% platelet lysate;

[0125] (ii) Ultracentrifuge the αMEM medium containing platelet lysate for 6 - 24 hours to obtain the supernatant after centrifugation; wherein, the rotational speed of the ultracentrifugation is 100000 - 120000g, preferably 100000g; more preferably, 120000g;

[0126] (iii) Filter the supernatant after centrifugation using a filter with a pore size of 0.22μm - 0.45μm (preferably 0.22μm) to obtain the filtered filtrate;

[0127] (iv) Ultrafilter the filtered filtrate using an ultrafiltration tube with a pore size of 100 - 300KD (preferably 300KD) to obtain the ultrafiltrate, that is, obtain the second culture medium.

[0128] In another preferred embodiment, the second culture medium has the following characteristics:

[0129] (1) Contains 2% - 15% platelet lysate;

[0130] (2) Contains fewer exosomes in the platelet lysate;

[0131] (3) Enables mesenchymal stem cells to maintain a relatively excellent proliferation ability;

[0132] (4) Enables mesenchymal stem cells to maintain relatively excellent stem cell characteristics; and

[0133] (5) Promote mesenchymal stem cells to secrete more exosomes.

[0134] The human stem cell exosomes produced using the kit of the present invention have the following characteristics:

[0135] (1) High exosome yield;

[0136] (2) High expression level of exosome-specific markers, and the exosome-specific markers include CD81.

[0137] In addition, during the exosome production process, the human stem cells used for exosome production have the following characteristics after incubation and culture:

[0138] (1) Strong cell proliferation ability and good cell activity;

[0139] (2) The cell-specific markers meet the mesenchymal stem cell-specific marker standards (CD73+ / CD90+ / CD105+>95%; CD34+ / CD45+ / HLA DR+<2%).

[0140] The main advantages of the present invention include:

[0141] (1) The present invention provides a kit for producing human stem cell exosomes, and the kit contains B-300K solution. The particle yield of exosomes produced by the cells cultured using this kit is 1.6 - 2.0×10 10 times that of αMEM medium containing 5% platelet lysate.

[0142] (2) The expression level of the specific protein CD81 of the exosomes produced by culturing cells using the kit of the present invention is not affected, but instead has the highest expression level.

[0143] (3) The present invention verifies from the protein level, cell proliferation and cell activity that the B-300K solution has no effect on the activity of the cultured cells, does not produce toxic and side effects on the cells, and does not affect the expression level of the exosome-specific protein CD81.

[0144] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following embodiments can adopt the conventional methods in the art, such as referring to "Molecular Cloning: A Laboratory Manual" (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the supplier. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels without special instructions.

[0145] Experimental Materials and Methods

[0146] Experimental Materials

[0147] (1) Human adipose mesenchymal stem cells

[0148] Human adipose mesenchymal stem cells are mesenchymal stem cells derived from human adipose tissue and are a type of pluripotent stem cells. They possess all the common characteristics of stem cells, namely self-renewal and multi-directional differentiation ability.

[0149] (2) Culture media used in the present invention

[0150] αMEM: A medium containing non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin, ascorbic acid, ribonucleosides, and deoxyribonucleosides, and free of proteins, lipids, and growth factors.

[0151] αMEM + 5% EliteGro: An αMEM medium containing 5% platelet lysate.

[0152] Solution B: The supernatant obtained by ultracentrifuging the αMEM medium containing 5% platelet lysate at 100,000 - 120,000 g for 6 - 24 hours.

[0153] B-100KD: The αMEM medium containing 5% platelet lysate is ultracentrifuged at 100,000 - 120,000 g for 6 - 24 hours to obtain the supernatant, which is then filtered through a 0.22 μm pore size filter, and the filtrate is further ultrafiltered through a 100KD pore size ultrafiltration tube to harvest the ultrafiltrate.

[0154] B-300KD (B-300K solution): The αMEM medium containing 5% platelet lysate is ultracentrifuged at 100,000 - 120,000 g for 6 - 24 hours to obtain the supernatant, which is then filtered through a 0.22 μm pore size filter, and the filtrate is further ultrafiltered through a 300KD pore size ultrafiltration tube to harvest the ultrafiltrate.

[0155] KnockOut Serum Replacement: A serum-free medium produced by Thermo

[0156] Experimental methods

[0157] (1) Culture of human adipose-derived mesenchymal stem cells

[0158] The passage 4 human adipose-derived mesenchymal stem cells were seeded at a density of 1×10 4 cells / cm 2 When the cells grew to 80%, the αMEM medium containing 5% platelet lysate that had been used to culture the cells was discarded. The control group culture medium and B-300K solution were added respectively, and the cells were cultured at 37 °C and 5% CO2 for 48 hours.

[0159] (2) Isolation of extracellular vesicles derived from human adipose-derived mesenchymal stem cells

[0160] The cell culture supernatant was collected, centrifuged at 3000g for 15 min, the supernatant was aspirated to remove the precipitate; then centrifuged at 10,000g for 0.5 h, the supernatant was aspirated to remove cell debris. 24% PEG6000 was added to the centrifuged medium and incubated overnight at 4 °C; then centrifuged at 3200g for 1 h at 4 °C, the supernatant was discarded, and the precipitate was resuspended with PBS; centrifuged at 120,000g for 70 min, the supernatant was discarded, and the precipitate collected at the bottom of the centrifuge tube was resuspended with PBS.

[0161] (3) Measurement of cell proliferation and viability

[0162] The cells were digested with trypsin containing 0.25% EDTA, and cell counting was performed using trypan blue.

[0163] The CCK8 kit was used to test the metabolic values of the cells after culturing in different culture media for 48 hours.

[0164] The mitochondrial membrane potential and cell apoptosis detection kit was added to each well plate for fluorescence color development. Apoptotic cells showed green fluorescence. The cell nuclei showed blue fluorescence.

[0165] (4) Flow cytometry for detecting the expression of cell surface markers

[0166] The cell suspension was collected, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded (leaving about 100 μl of liquid). Antibodies (CD73, CD90, CD105, CD34, CD45, HLA-DR) were added and incubated for 20 minutes in the dark. 1 ml / tube of PBS was added, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 500 μl of PBS was added, and the sample was used for flow cytometry.

[0167] (5) Western blot for detecting the expression of extracellular vesicle-specific proteins

[0168] Lyse exosomes using NP-40 with protease inhibitor added. Detect the protein concentration of exosomes using a BCA kit. Add 4×loading buffer to the remaining samples and boil them at 95°C for 5 min. Load the samples onto a precast protein gel in the same volume, run at 80 V for 0.5 h, and then run at 120 V for 1 h. After running the gel, transfer to a PVDF membrane at a constant current of 250 mA for 1.5 h. After membrane transfer, block for 0.5 h. Add the primary antibody (CD81) (1:1000) to the blocking solution and incubate overnight at 4°C. Wash the membrane with TBST, 10 min each time, wash 3 times, use the corresponding secondary antibody (1:3000) and incubate at room temperature for 3 h. Wash the membrane with TBST, 10 min each time, wash 3 times, and develop with HRP.

[0169] (6) NTA detection of the particle concentration and particle size of extracellular exosomes

[0170] Wash the sample cell of the ZetaView nanoparticle tracking analyzer with deionized water. Calibrate the instrument using polystyrene microspheres. Wash the sample cell with PBS. Dilute the sample with PBS and detect it.

[0171] Example 1: Screening of incubation culture media

[0172] 1.1: Effects of different culture media on the proliferation and viability of passage 4 adipose mesenchymal stem cells

[0173] After incubating cells with different culture media for 48 hours, detect the cell viability using CCK8 and perform cell counting using trypan blue. The experimental results are as Figure 1 shown.

[0174] The results showed that after incubating cells with different culture media for 48 hours, it was found that αMEM + 5% EliteGro, solution B, and B-300KD were most suitable for cell proliferation, while αMEM, B-100KD, and KnockOut Serum Replacement were not suitable for cell proliferation.

[0175] 1.2: Effects of different culture media on the surface markers of passage 4 adipose mesenchymal stem cells

[0176] After incubating cells with different culture media for 48 hours, it was found that αMEM, αMEM + 5% EliteGro, Solution B, B-300KD, and KnockOut Serum Replacement had relatively stable effects on the surface markers of P4 working bank cells, with CD73+ > 95%, CD90+ > 95%, CD105+ > 95%, HLA DR+ < 2%, CD34+ < 2%, and CD45+ < 2%; the expression of cell surface markers by B-100KD was unstable, with both HLA DR+ and CD34+ greater than 2%. The experimental results are shown in Table 1.

[0177] Table 1 shows the expression of cell surface markers after incubating passage 4 adipose-derived mesenchymal stem cells with different culture media for 48 hours. These include CD73+, CD90+, CD105+, HLA DR+, CD34+, and CD45+. According to the detection requirements for mesenchymal surface markers, CD73+ > 95%, CD90+ > 95%, CD105+ > 95%, HLA DR+ < 2%, CD34+ < 2%, and CD45+ < 2%.

[0178] Table 1 Expression of cell surface markers after incubating P4 working bank cells with different culture media for 48 hours

[0179]

[0180] 1.3: Detection of CD81 expression in exosomes isolated from each group by Western blot

[0181] Western Blot was used to detect the expression of specific markers of extracellular exosomes produced after incubating cells with culture media from each group. The experimental results are as Figure 2 shown.

[0182] The results showed that after culturing cells with αMEM + 5% EliteGro, Solution B, and B-300KD, the collected extracellular vesicles expressed more CD81, while after culturing cells with αMEM, B-100KD, and KnockOut Serum Replacement, the collected extracellular vesicles basically did not express CD81.

[0183] 1.4: Detection of the yield of extracellular vesicles after incubating cells with different culture media for 48 hours by NTA

[0184] ZetaView was used to detect the particle concentration of extracellular exosomes produced after incubating cells with culture media from each group for 48 hours. The experimental results are as Figure 3 shown.

[0185] The results showed that αMEM and B-100KD contained basically no nanoparticles when not in use. However, after incubating cells for 48 hours, the yield of exosomes secreted by the cells was also extremely low. KnockOut Serum Replacement is a chemically synthesized medium that contains a small amount of nanoparticles when not in use, but after incubating cells for 48 hours, the yield of extracellular vesicles secreted by the cells is also relatively low. αMEM + 5% EliteGro contained a large amount of nanoparticles when not in use. After incubating cells for 48 hours, although the yield of extracellular vesicles secreted by the cells was high, compared with the non-use state, the actual yield of extracellular vesicles secreted by the cells was not high. Solution B and B-300KD contained a relatively low amount of nanoparticles when not in use. After incubating cells for 48 hours, the yield of extracellular vesicles secreted by the cells was relatively high.

[0186] Example 2: Screening of Incubation Time

[0187] 2.1: Detection of exosomes secreted by cells at each time point by NTA

[0188] Seed P4 generation haMSCs at a density of 1.5×10 4 / cm 2 in a 6-well cellbind plate (the seeding pattern is as Figure 4 shown). After 80% confluence, change to Solution B-300K and incubate for 24 hours, 48 hours, and 72 hours respectively. Then collect the conditioned medium and cells. Use the Solution B-300K at each time point as the blank control, and use the total particle concentration collected at the corresponding time point as the experimental group. Detect the number of exosome particles secreted by the cells at the corresponding time point by NTA (total particle number - particle number of the Solution B-300K blank control group) (Table 2, Figure 5 ).

[0189] The results showed that after incubating with Solution B-300K for 48 hours, the number of particles secreted by the cells was the largest; after incubating for 24 hours, the number of particles secreted by the cells was the smallest.

[0190] Table 2 Number of exosome particles secreted by cells at different time points

[0191]

[0192] Note: Solution B-300K is used as the blank control; the particle concentration secreted by the cells = total particle concentration - particle concentration of Solution B-300K.

[0193] 2.2: Detection of cell viability

[0194] Use a mitochondrial membrane potential and cell apoptosis detection kit to detect cell apoptosis. Use Hochest33342 for nuclear staining, and the cell nuclei appear blue ( Figure 7);Using Annexin V-FITC staining, if the cells start to apoptose, the apoptotic cells will show green fluorescence( Figure 7 )。Use Image J to perform "analyze particles" to analyze the apoptosis ratio (Table 3 and Figure 6 )。The cells in the positive control group were treated with 10 mM H2O2 for 5 min.

[0195] Table 3 Apoptosis ratios of cells at different time points

[0196]

[0197] The results showed that compared with the cells in the positive control group, after incubation with B-300K solution for 24 hours and 48 hours, the apoptosis ratio of the cells was significantly lower than that of the positive control group, while after incubation for 72 hours, the apoptosis ratio of the cells was comparable to that of the positive control group (Table 3 and Figure 6 )。It was proved that the cell activity remained at a relatively high level after incubation with B-300K solution for 24 hours and 48 hours.

[0198] In summary, after incubating passage 4 adipose mesenchymal stem cells with B-300K solution, the proliferation ability and activity of the cells are both excellent, and the exosome production of the cells is the highest, showing a significant advantage compared with the control group. Although B-300K solution has undergone a series of ultracentrifugation, filtration and ultrafiltration steps, it has no toxic or side effects on the cells, and the cell surface markers still maintain their original characteristics.

[0199] After incubating passage 4 adipose mesenchymal stem cells with B-300K solution for 48 hours, the exosome production of the cells is the highest and the cell activity is the best.

[0200] Therefore, the method provided by the present invention can effectively improve the exosome production of mesenchymal stem cells without affecting the activity of human mesenchymal stem cells.

[0201] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for increasing the yield of extracellular exosomes, characterized in that, Comprising the steps: (a) Providing mesenchymal stem cells; (b) Under suitable culture conditions, pre-culturing the mesenchymal stem cells using a first culture medium to obtain pre-cultured stem cells with a confluence of 60-90%; Wherein, the first culture medium is an αMEM medium containing platelet lysate; (c) Incubating and culturing the pre-cultured mesenchymal stem cells with a second culture medium for 24-96 hours to obtain an incubation culture medium, the incubation culture medium containing the incubated and cultured mesenchymal stem cells and extracellular vesicles produced by the incubated and cultured mesenchymal stem cells; Wherein, the second culture medium is an ultrafiltrate obtained by ultracentrifuging an αMEM medium containing platelet lysate at 100,000-120,000 g for 6-24 hours, filtering the centrifuged supernatant using a filter with a pore size of 0.22 μm, and ultrafiltering the filtered filtrate using an ultrafiltration tube with a pore size of 300 KD; and (d) Separating extracellular vesicles from the incubation culture medium obtained in the previous step.

2. The method according to claim 1, characterized in that, The mesenchymal stem cells are stem cells passaged in vitro for 2 to 6 generations.

3. The method according to claim 2, wherein The mesenchymal stem cells are stem cells passaged in vitro for 4 generations.

4. The method according to claim 1, wherein The concentration of platelet lysate in the first culture medium is 2%-15%, wherein the percentage is by volume.

5. The method according to claim 4, characterized in that, The concentration of platelet lysate in the first culture medium is 5%-10%.

6. The method according to claim 5, wherein The concentration of platelet lysate in the first culture medium is 5%.

7. The method according to claim 1, characterized in that, The concentration of platelet lysate in the second culture medium is 2%-15%, wherein the percentage is by volume.

8. The method according to claim 7, wherein The concentration of platelet lysate in the second culture medium is 5%-10%.

9. The method according to claim 8, characterized in that, The concentration of platelet lysate in the second culture medium is 5%.

10. The method according to claim 1, characterized in that, The second culture medium is prepared using the following method: (i) Providing an αMEM medium containing 2%-15% platelet lysate, wherein the percentage is by volume; (ii) Ultracentrifuging the αMEM medium containing 2%-15% platelet lysate for 6-24 hours to obtain a centrifuged supernatant; wherein, the rotational speed of the ultracentrifugation is 100,000-120,000 g; (iii) Filtering the centrifuged supernatant using a filter with a pore size of 0.22 μm to obtain a filtered filtrate; (iv) Ultrafiltering the filtered filtrate using an ultrafiltration tube with a pore size of 300 KD to obtain an ultrafiltrate, i.e., obtaining the second culture medium.

11. The method according to claim 10, characterized in that, Providing an αMEM medium containing 5%-10% platelet lysate.

12. The method according to claim 11, characterized in that, Providing an αMEM medium containing 5% platelet lysate.

13. The method according to claim 1, characterized in that, The second culture medium has the following characteristics: ( 1) Containing 2%-15% of platelet lysate; (2) Almost free of extracellular vesicles in platelet lysate; (3) Enabling mesenchymal stem cells to maintain a relatively excellent proliferation ability; (4) Enabling mesenchymal stem cells to maintain relatively excellent stem cell characteristics; and (5) Promoting mesenchymal stem cells to secrete more extracellular vesicles.

14. The method according to claim 1, wherein In step (c), the incubation and culture time is 24-72 hours.

15. The method according to claim 14, wherein In step (c), the incubation time is 24 - 48 hours.

16. The method according to claim 15, characterized in that, In step (c), the incubation time is 48 hours.

17. The method according to any one of claims 1 to 16, characterized in that, In step (d), it includes: (d1) Separating the incubation culture solution to obtain a cell-free supernatant, wherein the supernatant contains the extracellular vesicles; (d2) Incubating the supernatant in step (d1) with a polyethylene glycol (PEG) solution overnight to obtain a mixture; and (d3) Separating the mixture obtained in step (d2) to obtain a precipitate, wherein the precipitate contains the extracellular vesicles.

18. A kit, characterized in that, The kit contains: (Z1) Human mesenchymal stem cells; (Z2) A first culture solution and a second culture solution; wherein, the first culture solution is an αMEM medium containing 2% - 15% platelet lysate, and the second culture solution is an ultrafiltrate obtained by ultracentrifuging the αMEM medium containing 2% - 15% platelet lysate at 100000 - 120000 g for 6 - 24 hours, filtering the centrifuged supernatant using a filter with a pore size of 0.22 μm, and ultrafiltering the filtered filtrate using an ultrafiltration tube with a pore size of 300 KD, wherein the percentage is by volume; and (Z3) Polyethylene glycol (PEG).

19. The kit according to claim 18, characterized in that, The αMEM medium containing platelet lysate is an αMEM medium containing 5% - 10% platelet lysate.

20. The kit according to claim 19, wherein The αMEM medium containing platelet lysate is an αMEM medium containing 5% platelet lysate.

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