阅读说明：本技术 一种提高干细胞外泌体产量的培养液 (Culture solution for increasing yield of exosomes of stem cells ) 是由 申重阳 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种提高干细胞外泌体产量的细胞培养液,属于细胞培养技术领域。该培养液包括以下组分：组分1：基础培养液；组分2：血清和/或血清替代品；组分3：炎症因子；组分4：活性多肽；组分5：细胞信号通路抑制剂。本发明提供的培养液能够促进脐带间充质干细胞囊泡的形成,通过激活SIRT1基因活性,进而提高脐带间充质干细胞外泌体的产量。该培养液中,白介素-6、磷酸化MARCKS Peptide和Quercetin D5对提高脐带间充质干细胞外泌体产量发挥了协同增效的作用。本发明提供的培养液原料易得,制备方法简单,成本低廉,在制备高产量的外泌体中具有良好的应用前景。(The invention discloses a cell culture solution for improving the yield of exosomes of stem cells, and belongs to the technical field of cell culture. The culture solution comprises the following components: component 1: a basal medium; and (2) component: serum and/or serum replacement; and (3) component: an inflammatory factor; and (4) component: an active polypeptide; and (5) component: inhibitors of cellular signaling pathways. The culture solution provided by the invention can promote the formation of the umbilical cord mesenchymal stem cell vesicle, and further improve the yield of the umbilical cord mesenchymal stem cell exosome by activating the activity of the SIRT1 gene. In the culture solution, interleukin-6, phosphorylated MARCKS Peptide and Quercetin D5 play a synergistic role in improving the yield of the exosomes of the umbilical cord mesenchymal stem cells. The culture solution provided by the invention has the advantages of easily available raw materials, simple preparation method, low cost and good application prospect in preparation of high-yield exosomes.)

1. A culture solution, which is characterized in that: the culture solution comprises the following components:

component 1: a basal medium;

and (2) component: serum and/or serum replacement;

and (3) component: an inflammatory factor;

and (4) component: an active polypeptide;

and (5) component: inhibitors of cellular signaling pathways.

2. The culture solution according to claim 1, wherein: the culture solution comprises the following components:

component 1: a basal medium;

and (2) component: serum and serum replacement;

and (3) component: an inflammatory factor;

and (4) component: an active polypeptide;

and (5) component: inhibitors of cellular signaling pathways;

wherein, the volume fraction of the serum is 1 to 5 percent, the volume fraction of the serum substitute is 5 to 15 percent, the concentration of the inflammatory factor is 1 to 100pg/mL, the concentration of the active polypeptide is 1 to 20mM, and the concentration of the cell signaling pathway inhibitor is 1 to 10 MuM.

3. The culture solution according to claim 2, wherein: in the culture solution, the volume fraction of serum is 2%, the volume fraction of a serum substitute is 10%, the concentration of an inflammatory factor is 10pg/mL, the concentration of active polypeptide is 10mM, and the concentration of a cell signaling pathway inhibitor is 5 μ M.

4. The culture solution according to any one of claims 1 to 3, wherein: in the component 1, the basic culture solution is a serum-free culture medium for culturing stem cells; preferably, the stem cell is a mesenchymal stem cell, more preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell;

and/or in the component 2, the serum is fetal bovine serum, and the serum substitute is Ultroser G.

5. The culture solution according to claim 4, wherein: in the component 1, the basic culture solution is DMEM/F12.

6. The culture solution according to any one of claims 1 to 3, wherein: in the component 3, the inflammatory factor is interleukin-6; and/or, in component 4, the active polypeptide is phosphorylated MARCKS Peptide; and/or, in component 5, the cell signaling pathway inhibitor is a SIRT1 activator, and the SIRT1 activator is preferably Quercetin D5.

7. A method for preparing the culture solution according to any one of claims 1 to 6, wherein: the method comprises the following steps: and (3) uniformly mixing the components 1-5 to obtain the composition.

8. A method of preparing a stem cell exosome, comprising: the method comprises the following steps: culturing stem cells using the culture solution according to any one of claims 1 to 6, and then extracting exosomes.

9. The method of claim 8, wherein: the stem cell is a mesenchymal stem cell, preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell;

and/or the method for extracting the exosomes is differential ultracentrifugation.

10. Use of the culture fluid of any one of claims 1 to 6 for the preparation of a stem cell exosome.

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a culture solution for improving the yield of a stem cell exosome.

Background

The exosome is derived from a vesicle of a late stage endocytosis body (also called a multivesicular body, MVB), wherein a cell endocytosis vesicle membrane is inwards sunken to form the multivesicular body containing a plurality of vesicles, and the multivesicular body is fused with a cell membrane and released into an extracellular matrix to form a membrane vesicle with the diameter of about 30-100 nm. Exosomes were first discovered by Johnstone et al in studying the process of reticulocyte conversion to mature red blood cells. Later studies found that not only reticulocytes were able to release such vesicles, but almost all types of living cells were able to secrete exosomes.

The exosome contains various bioactive substances such as DNA fragments, mRNA, small RNA, functional proteins, transcription factors and the like, and the membrane structure of the exosome also expresses various antigens and antibody molecules, so that various biological effects can be generated, and the exosome has wide application prospect in clinical treatment of diseases. The composition of exosomes is related to their cell source, and exosomes from different cell sources contain different specific components.

The stem cells are cells from embryonic, fetal or adult bodies and have the capacity of self-renewal and proliferation and differentiation without limitation under certain conditions, and are cells with the characteristics of self-replication capacity and multidirectional differentiation potential. Since the success of the first bone marrow cell transplantation treatment in 1968, regenerative medicine represented by cell therapy has been applied to the treatment of many diseases. Stem cell therapy has shown great potential in the field of cardiovascular diseases, especially in the treatment of myocardial infarction. Studies have shown that stem cell therapy can promote the survival of cardiomyocytes, thereby improving myocardial contractility to treat cardiovascular disease, especially myocardial infarction. However, cell therapy has certain limitations, and its low success rate of transplantation, risk of immunological rejection and tumorigenicity, etc. limit its clinical application. Recent studies have shown that stem cells can participate in the repair of damaged myocardial tissue through the paracrine mechanism of exosomes, in addition to inducing differentiation into cardiomyocytes to repair infarcted tissue. Therefore, exosomes are expected to replace stem cell therapy as a new approach to the treatment of cardiovascular diseases, especially myocardial infarction.

The human umbilical cord mesenchymal stem cells are separated from waste tissues of newborn babies, namely umbilical cords, and are stem cells with multidirectional differentiation capacity and self-renewal capacity. The umbilical cord has the advantages of easily available materials, difficult pollution, no relation to moral, ethical and legal problems and the like, and is widely used for extracting the human umbilical cord mesenchymal stem cells. Meanwhile, the in-vitro amplification and multidirectional differentiation capacity of the human umbilical cord mesenchymal stem cells is stronger than that of stem cells derived from other tissues. Researches show that the exosome derived from the human umbilical cord mesenchymal stem cells can inhibit the proliferation of peripheral blood lymphocytes, reduce the levels of cytokines IL-4 and INF-y secreted by the exosome, inhibit the growth of tumor cells in a blood system and promote the apoptosis of the tumor cells, so that the exosome derived from the human umbilical cord mesenchymal stem cells has good immunosuppressive action and tumor inhibition action, and has wide application prospect in medicaments clinically used for resisting inflammation and tumors.

At present, methods for extracting exosomes mainly comprise a density gradient centrifugation method, an organic solvent method, an immunoaffinity chromatography method, an immunomagnetic bead method, a liquid chromatography separation method, a differential ultracentrifugation method and the like. Among them, the extraction of exosomes by density gradient centrifugation is considered to be a relatively classical method, but the operation is complicated, the time consumption is long, and the clinical application is inconvenient, and some researches show that exosomes are suspended in a 30% sucrose/heavy water cushion for purification, however, the density gradient centrifugation belongs to the equal density centrifugation range, has no density gradient difference, is easy to mix with impurities with other densities, and whether heavy water has influence on human bodies cannot be evaluated. The organic solvent method is a relatively original method, although the operation is simple and the cost is low, the specificity is not high, and not only can high-abundance proteins be precipitated, but also partial low-abundance proteins can be precipitated together. The immunoaffinity chromatography has strong specificity and high efficiency, but the operation process is relatively complicated. The immunomagnetic bead method and the liquid chromatography separation method have higher cost and cannot be popularized. The differential ultracentrifugation method is a method for extracting exosomes according to the principle that centrifugal forces required by sedimentation of various substances in a sample are different, although the differential ultracentrifugation method is slightly long in time consumption, unknown potential impurity pollution does not exist, the purity is relatively high, the cost is relatively low, a large amount of exosomes can be extracted, the basic requirements of experiments are met, and the differential ultracentrifugation method is high in repeatability in practical application and can be used as a reasonable separation method.

The Chinese patent application with the application number of 201710790115.9 discloses a process for obtaining umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: inoculating the umbilical cord mesenchymal stem cells into a bioreactor containing a Cultispher-G microcarrier and a culture solution for amplification culture, removing the umbilical cord mesenchymal stem cells after the culture is finished, and purifying to obtain the umbilical cord mesenchymal stem cell exosome; the culture solution is prepared by adding recombinant interferon gamma, a serum substitute, penicillin and streptomycin into MSCGM-CD mesenchymal stem cell culture medium. However, the process does not disclose the yield of the obtained exosomes, nor does the process give any hint how the components in the cell culture medium exert a synergistic effect on the increase of the yield of exosomes. In actual production, the development of a culture system capable of obviously improving the yield of the exosomes of the umbilical cord mesenchymal stem cells is of great significance.

Disclosure of Invention

The invention aims to provide a stem cell culture solution, which can promote the formation of umbilical cord mesenchymal stem cell vesicles and obviously improve the yield of exosomes.

Another object of the present invention is to provide a method for preparing exosomes with high yield by using the above stem cell culture solution.

The invention provides a culture solution, which comprises the following components:

component 1: a basal medium;

and (2) component: serum and/or serum replacement;

and (3) component: an inflammatory factor;

and (4) component: an active polypeptide;

and (5) component: inhibitors of cellular signaling pathways.

Further, the culture solution comprises the following components:

component 1: a basal medium;

and (2) component: serum and serum replacement;

and (3) component: an inflammatory factor;

and (4) component: an active polypeptide;

and (5) component: inhibitors of cellular signaling pathways;

wherein, the volume fraction of the serum is 1 to 5 percent, the volume fraction of the serum substitute is 5 to 15 percent, the concentration of the inflammatory factor is 1 to 100pg/mL, the concentration of the active polypeptide is 1 to 20mM, and the concentration of the cell signaling pathway inhibitor is 1 to 10 MuM.

Further, in the culture solution, the volume fraction of serum is 2%, the volume fraction of serum substitute is 10%, the concentration of inflammatory factor is 10pg/mL, the concentration of active polypeptide is 10mM, and the concentration of cell signaling pathway inhibitor is 5. mu.M.

Further, in the component 1, the basic culture solution is a serum-free culture medium for culturing stem cells; preferably, the stem cell is a mesenchymal stem cell, more preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell;

and/or in the component 2, the serum is fetal bovine serum, and the serum substitute is Ultroser G.

Further, in the component 1, the basic culture solution was DMEM/F12.

Further, in the component 3, the inflammatory factor is interleukin-6; and/or, in component 4, the active polypeptide is phosphorylated MARCKS Peptide; and/or, in component 5, the cell signaling pathway inhibitor is a SIRT1 activator.

Further, in the component 5, the SIRT1 activator is Quercetin D5.

The invention also provides a method for preparing the culture solution, which comprises the following steps: and (3) uniformly mixing the components 1-5 to obtain the composition.

The invention also provides a method for preparing a stem cell exosome, which comprises the following steps: the stem cells were cultured using the above-mentioned culture solution, and then exosomes were extracted.

Further, the stem cell is a mesenchymal stem cell, preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell;

and/or the method for extracting the exosomes is differential ultracentrifugation.

The invention also provides application of the culture solution in preparation of the stem cell exosomes.

Definitions of terms used in connection with the present invention: the initial definitions provided for by terms herein apply to that term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

Peptides are compounds in which two or more amino acids are linked by peptide bonds, play important physiological roles in the human body, and exert physiological functions. The polypeptide having activity is called active polypeptide, also called bioactive peptide or bioactive polypeptide.

Phosphorylated MARCKS Peptide is a phosphorylated polypeptide consisting of a myristoylated alanine C kinase rich substrate protein (MARCKS) basic effector domain fragment, phosphorylation of MARCKS Peptide (151-175) reverses its inhibitory effect on phospholipase C (plc) catalyzed hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP 2). Phosphorylated MARCKS Peptide (151-175) polypeptides induce bending of the endoplasmic reticulum, increasing membrane curvature, and thus promote synthesis and secretion of secretory vesicles. The polypeptide sequence is as follows: Lys-Lys-Lys-Lys-Lys-Arg-Phe- { pSer } -Phe-Lys-Lys- { pSer } -Phe-Lys-Leu-Ser-Gly-Phe- { pSer } -Phe-Lys-Lys-Asn-Lys-Lys-Lys.

Quercetin D5, Quercetin D5, is a deuterated compound of a natural flavonoid of Quercetin, and can activate SIRT1 gene activity.

Compared with the prior art, the culture solution provided by the invention has the following beneficial effects:

the culture solution provided by the invention can promote the formation of the umbilical cord mesenchymal stem cell vesicle, and further improve the yield of the umbilical cord mesenchymal stem cell exosome by activating the activity of the SIRT1 gene. In the culture solution provided by the invention, interleukin-6, phosphorylated MARCKS Peptide and Quercetin D5 play a synergistic role in improving the yield of exosomes of umbilical cord mesenchymal stem cells.

The culture solution provided by the invention has the advantages of easily available raw materials, simple preparation method, low cost and good application prospect in preparation of high-yield exosomes.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the growth state of umbilical cord mesenchymal stem cells at passage 0 (left panel) and passage 6 (right panel).

FIG. 2 shows the results of mesenchymal stem cell flow analysis of the 6 th generation cells;

FIG. 3 is a transmission electron micrograph of exosomes isolated using the culture solution prepared in example 1.

FIG. 4 is a transmission electron micrograph of exosomes isolated using the culture solution prepared in comparative example 1.

FIG. 5 is a transmission electron micrograph of exosomes isolated using the culture solution prepared in comparative example 2.

FIG. 6 is a transmission electron micrograph of exosomes isolated using the culture solution prepared in comparative example 3.

FIG. 7 Total protein concentration in exosomes isolated using the culture solutions prepared in example 1 and comparative examples 1, 2, 3.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

In the following examples and comparative examples:

basal medium was DMEM/F12 from Gibco, model 11330.

The serum replacement was Ultroser G, purchased from PALL, model 15950-017.

Fetal bovine serum was purchased from Gibco, model 10270.

Interleukin-6 (IL-6) was purchased from Perotech and was available in the model number of 200-06.

Phosphorylated MARCKS Peptide, i.e.: MARCKS Peptide (151-175), Phosphorylated. Phosphorylated MARCKS Peptide was purchased from MCE and was designated HY-P1834.

Quercetin D5, Quercetin D5, was purchased from MCE under model 18085S.

Example 1: preparation of umbilical cord mesenchymal stem cell culture solution

The preparation steps of 1L umbilical cord mesenchymal stem cell culture solution are as follows:

adding 100mL of serum substitute and 20mL of fetal bovine serum into 880mL of DMEM/F12 basal medium, adding 10ng of interleukin-6, 10mmol of phosphorylated MARCKS Peptide and 5 mu mol of Quercetin D5 after the color of the culture medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

Example 2: preparation of umbilical cord mesenchymal stem cell culture solution

The preparation steps of 1L umbilical cord mesenchymal stem cell culture solution are as follows:

adding 110mL of serum substitute and 10mL of fetal bovine serum into 880mL of DMEM/F12 basal medium, adding 1ng of interleukin-6, 1mmol of phosphorylated MARCKS Peptide and 1 mu mol of Quercetin D5 after the color of the culture medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

Example 3: preparation of umbilical cord mesenchymal stem cell culture solution

The preparation steps of 1L umbilical cord mesenchymal stem cell culture solution are as follows:

adding 70mL of serum substitute and 50mL of fetal bovine serum into 880mL of DMEM/F12 basal medium, adding 100ng of interleukin-6, 20mmol of phosphorylated MARCKS Peptide and 10 mu mol of Quercetin D5 after the color of the culture medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

The preparation of control culture medium is as follows.

Comparative example 1:

the culture solution of this comparative example is different from the culture solution of example 1 only in that: interleukin-6 was not added. Specifically, the preparation procedure of the culture solution of this comparative example was as follows:

adding 100mL of serum substitute and 20mL of fetal bovine serum into 880mL of DMEM/F12 basal medium, adding 10mmol of phosphorylated MARCKS Peptide and 5 mu mol of Quercetin D5 after the color of the culture medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

Comparative example 2:

the culture solution of this comparative example is different from the culture solution of example 1 only in that: no phosphorylated MARCKS Peptide was added. Specifically, the preparation procedure of the culture solution of this comparative example was as follows:

adding 100mL of serum substitute and 20mL of fetal calf serum into 880mL of DMEM/F12 basal medium, adding 10ng of interleukin-6 and 5 mu mol of Quercetin D5 after the color of the medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

Comparative example 3:

the culture solution of this comparative example is different from the culture solution of example 1 only in that: quercetin D5 was not added. Specifically, the preparation procedure of the culture solution of this comparative example was as follows:

adding 100mL of serum substitute and 20mL of fetal bovine serum into 880mL of DMEM/F12 basal medium, adding 10ng of interleukin-6 and phosphorylated MARCKS Peptide after the color of the culture medium is stable, and fully mixing to obtain the product, and storing at 4 ℃.

The following test examples demonstrate the advantageous effects of the present invention.

Test example 1: subculturing umbilical cord mesenchymal stem cells

(1) Test method

Culturing the primary umbilical cord mesenchymal stem cells by using the culture solution prepared in example 1 for adherence, wherein the cells grow rapidly, the cell morphology is normal, when the cell confluence degree reaches 85%, the adherence umbilical cord mesenchymal stem cells are digested by using 0.25% trypsin (sigma 110M 7362V)/0.02% EDTA digestive juice, and the cells are collected by centrifugation according to the following steps of 1: 6, subculturing, and analyzing the growth state of the cells by taking pictures by using an inverted microscope.

Digesting 6 th generation adherent umbilical cord mesenchymal stem cells by using Accutase enzyme (eBioscience, 00-4555) to prepare a single cell suspension, centrifuging at 1000rpm for 5min, discarding supernatant, and placing 1 × 10 in each flow tube⁶The umbilical cord mesenchymal stem cells and 50 μ l of the antibody, the cell staining protocol is: two flow tubes were added (eBioscience, anti-CD90 FITC, anti-CD45 PE-cy, anti-HLA-DR APC, anti-CD34 PE) and (eBioscience, anti-CD73 FITC, anti-CD19 PE, anti-CD11b APC, anti-CD105 PE-cy) to each tube, incubated at room temperature for 30 minutes, washed 2 times with PBS buffer, resuspended in 400. mu.l PBS, and positive cell detection was performedIndices are CD105, CD73 and CD 90; negative indicators include CD45, CD34, CD11b, CD19, HLA-DR. The streaming results were analyzed using flowjo software.

(2) Test results

As can be seen from FIG. 1, during the in vitro culture of umbilical cord mesenchymal stem cells, the morphology of the 0 th generation cells and the 6 th generation cells is normal. Flow cytometry analysis results show (figure 2), the 6 th generation umbilical cord mesenchymal stem cell marker is expressed normally, wherein the expression amounts of positive indexes CD105, CD73 and CD90 are all more than 95%, the expression amounts of negative indexes CD45, CD34, CD11b, CD19 and HLA-DR are all less than 0.5%, and the identification standard of umbilical cord mesenchymal stem cells is met.

Test example 2: exosomes were isolated and morphometric

(1) Test method

Exosomes were isolated using differential ultracentrifugation. The specific operation is as follows:

referring to the method of test example 1, 4 th-generation umbilical cord mesenchymal stem cells were cultured using the culture solutions prepared in example 1 and comparative examples 1, 2, and 3, respectively, and the supernatant was collected after 48 hours of culture. Placing the supernatant in a 50ml centrifuge tube, centrifuging at 4 deg.C and 2000g for 30 min, removing cell debris, placing the centrifuged supernatant in an ultracentrifuge tube, centrifuging at 4 deg.C and 1000g for 70 min, and retaining the supernatant to obtain exosome concentrate 1. Transferring the exosome concentrated solution 1 into a new ultracentrifuge tube, centrifuging at 4 ℃ and 100000g for 70 minutes, then retaining the precipitate, diluting the precipitate with PBS, centrifuging at 4 ℃ and 100000g for 70 minutes, then retaining the supernatant to obtain an exosome concentrated solution 2, filtering the exosome concentrated solution 2 through a low-adsorption sterile filter membrane (Pall, 0.22 mu m), and storing at-80 ℃ after the PBS is resuspended to obtain the exosome.

25. mu.L of the exosome was dropped on a copper mesh and left to stand for 2 minutes, and then stained with 20g/L of an aqueous uranium acetate solution (1%, pH 4.0) for 2 minutes, and after drying naturally, the exosome was observed and photographed using a transmission electron microscope.

(2) Test results

As can be seen from fig. 3 to 6, exosomes isolated from the culture solutions prepared in example 1 and comparative examples 1, 2 and 3 showed typical characteristics, and were round or oval vesicles with distinct membrane structures, which were individually distributed and aggregated into vesicle clusters, and low electron density substances were observed in the vesicles.

Test example 3: determination of Total protein concentration in isolated exosomes

(1) Test method

Separately, exosomes separated from the culture solution prepared in test example 2 by using example 1 and comparative examples 1, 2 and 3 were added with protein lysate RAPA (containing 1% PMSF), and the mixture was vigorously shaken on a vortex shaker for 30 seconds and left to stand at 4 ℃ for 30 minutes to lyse proteins sufficiently. The supernatant was transferred to a new 1.5mL EP tube by centrifugation at 12000g for 15 minutes at 4 ℃ and 5 Xloading reagent buffer was added and boiled for 7 minutes to obtain an exosome protein sample. And (3) adding 10 mu L of exosome protein sample into 200 mu L of BCA protein quantitative working solution, standing at 37 ℃ for 30 minutes, detecting the total protein amount in exosomes by using an enzyme-linked immunosorbent assay, and calculating the total protein concentration in exosomes.

(2) Test results

As can be seen from fig. 7, the total protein concentration in exosomes isolated after umbilical cord mesenchymal stem cells were cultured using the culture solutions prepared in example 1 of the present invention was significantly higher than that in exosomes isolated after umbilical cord mesenchymal stem cells were cultured using the culture solutions prepared in comparative examples 1, 2, and 3. Furthermore, the total protein concentration in the exosomes isolated after the umbilical cord mesenchymal stem cells were cultured with the culture solution prepared in example 1 of the present invention was even higher than the sum of the total protein concentrations in the exosomes isolated after the umbilical cord mesenchymal stem cells were cultured with the culture solutions prepared in comparative examples 1, 2, 3.

The experimental results show that the exosome yield obtained by separating after umbilical cord mesenchymal stem cells are cultured by using the culture solution prepared in the embodiment 1 of the invention is obviously improved; in addition, in the culture solution prepared in the embodiment 1 of the invention, interleukin-6, phosphorylated MARCKS Peptide and Quercetin D5 play a synergistic role in improving the yield of the exosomes of the umbilical cord mesenchymal stem cells.

In conclusion, the invention provides a cell culture solution for improving the yield of the exosomes of the stem cells. The experimental result shows that the culture solution provided by the invention can promote the formation of the umbilical cord mesenchymal stem cell vesicle, and further improve the yield of the umbilical cord mesenchymal stem cell exosome by activating the activity of the SIRT1 gene. In the culture solution provided by the invention, interleukin-6, phosphorylated MARCKS Peptide and Quercetin D5 play a synergistic role in improving the yield of exosomes of umbilical cord mesenchymal stem cells. The culture solution provided by the invention has the advantages of easily available raw materials, simple preparation method, low cost and good application prospect in preparation of high-yield exosomes.