Mesenchymal stem cell culture medium with limited chemical components and application thereof

Abstract

The invention belongs to the field of cell culture, and discloses a mesenchymal stem cell culture medium with limited chemical components, which can be used for cell culture and exosome harvesting. The culture medium consists of a basic culture medium and an additive component, wherein the additive component is selected from one or more of a growth factor substance, a binding protein substance, an adherent factor substance, a cell factor substance, a hormone substance, a trace element-containing substance, a biogenic amine substance, an amino acid substance and a nucleoside substance. The culture medium is free of serum, human source and animal source components, the contained components are completely known, the motility rate, proliferation and cell quality of the mesenchymal stem cells in cell culture can be remarkably improved, and therefore the technical problem that in the prior art, an existing mesenchymal stem cell limited component culture medium is poor in culture effect is solved.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and particularly to a chemically defined mesenchymal stem cell culture medium and its application. Background Art

[0002] Mesenchymal stem cells (MSCs) are pluripotent cells that retain the potential to differentiate into mesodermal lineages, particularly adipocytes, chondrocytes, and osteocytes, and have received extensive attention. Mesenchymal stem cells are considered to be the stem cell products closest to clinical application. Since the isolation and extraction of mesenchymal stem cells from umbilical cord tissue do not cause any damage, and the mesenchymal stem cells in the umbilical cord are numerous, of high quality, and pure, they have become the most clinically applicable and preservable stem cells at present.

[0003] Most of the culture media used for in vitro culture of mesenchymal stem cells need to be supplemented with serum, which poses a risk of contaminating exogenous viruses and pathogenic factors during cell culture. Moreover, due to the large number of unknown components in serum and the inconsistency of bioactive factors between different batches of serum, the reproducibility of products and experimental results is poor, and the residual serum is also likely to cause allergic reactions of the inoculator to the serum, bringing huge challenges to clinical research. At present, it has been proven that human serum and platelet lysate (HPL) can support the expansion of human MSCs in vitro, and human platelet lysate has been used as the standard culture medium for clinical application of mesenchymal stem cells. However, batch differences in the preparation of HPL can affect the phenotype and function of MSCs, and at the same time, there is a risk of introducing contaminants such as viruses in human-derived components. In addition, the resources of human platelets are limited. Therefore, developing a complete chemically defined culture medium for human mesenchymal stem cells is crucial for both basic biological research and clinical application.

[0004] Chemically defined cell culture media have obvious advantages: First, safety. Biological experiments often require tracing the source. The chemical components are all from chemical synthesis or recombinant synthesis, without serum, animal-derived or human-derived components, which can minimize the risk of introducing foreign factors. Second, more consistent performance. Through a defined formula, the quality of each batch of products can be guaranteed, and the performance is more consistent. Third, it is easy to purify and downstream process. The purification process of some serum-containing culture media is relatively complex. Fourth, it improves the stability of the product. The determination of the components brings convenience to the storage, transportation conditions, and shelf life of the culture medium, and improves the stability of the product. The advantages in many aspects make serum-free and chemically defined culture media become the mainstream culture media in the future.

[0005] Currently, a variety of serum-free / xeno-free (SF / XF) and chemically defined (CD) media are commercially available for the culture and expansion of MSCs, but the effectiveness of these media remains questionable. For example, studies have shown that primary MSCs cultured poorly in StemPro MSC SFM or mTeSR medium require the addition of human AB serum. Other studies have reported that MesenCult-XF does not effectively support the isolation, expansion, phenotype, and differentiation potential of mesenchymal stem cells. Therefore, there is an urgent need to develop a defined serum-free medium suitable for both MSC culture and exosome harvesting. Summary of the invention

[0006] The purpose of the present invention is to provide a novel mesenchymal stem cell defined component culture medium, which is serum-free, human-derived, and animal-derived, and the components contained are completely known. It can significantly improve the viability, proliferation, and cell quality of mesenchymal stem cells in cell culture, thereby solving the technical problem that the existing mesenchymal stem cell defined component culture medium in the prior art has poor effect when used for culture.

[0007] The technical solutions provided by the present invention are as follows: A serum-free defined component mesenchymal stem cell culture medium, comprising a basal culture medium and additional components. The basal culture medium is selected from RCD288 (3D FloTrix ® The MSC chemical composition is limited to one or more of serum-free basal medium (RCD288-PYJ), αMEM, IMDM, and DMEM / F12, preferably RCD288. The supplementary ingredients are selected from one or more of growth factors, binding proteins, adhesion factors, cytokines, hormones, trace element-containing substances, biogenic amines, amino acids, and nucleosides.

[0008] Among them, growth factors can usually bind to specific receptors on the target cell membrane, thereby affecting cell mitosis, protein synthesis and differentiation. They are biological supplementary factors necessary to maintain cell growth, proliferation, division and differentiation in vitro. For example, transforming growth factor can maintain the self-renewal ability and differentiation potential of stem cells. Growth factor substances are selected from one or more of recombinant human epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, transforming growth factor β1, transforming growth factor β3, hepatocyte growth factor, vascular endothelial growth factor, connective tissue growth factor, and stem cell growth factor.

[0009] The binding protein can promote cell growth and also acts as a chelating agent for some harmful trace elements. The binding protein substance is selected from one or more of transferrin and human serum albumin.

[0010] Among them, the attachment factor is the factor for cells to attach to the bottom of the culture dish, and the attachment factor-like substances are selected from fibronectin. Among them, hormones are very important in aspects such as cell growth, metabolism, and signal transduction. For example, insulin plays an important role in the synthesis and metabolism of mammalian cells and can promote cell proliferation. The hormone-like substances are selected from one or more of insulin, dexamethasone, hydrocortisone, and progesterone.

[0011] Among them, trace elements such as selenium are the most common, and participate in the action processes of glutathione peroxidase and superoxide dismutase, and can eliminate the damage of peroxidase and oxygen free radicals to cells. The trace element-containing substances are selected from sodium selenite.

[0012] Among them, biogenic amines play an important role in stimulating cell growth, and the biogenic amine-like substances are selected from one or more of ethanolamine, putrescine, and serotonin.

[0013] Among them, amino acids play a role in many metabolic activities and can improve cell growth and activity. The amino acid-like substances are selected from one or more of taurine and non-essential amino acids.

[0014] Among them, nucleosides are the material basis for the formation of DNA and participate in the synthesis and repair processes of DNA. The nucleoside compounds are selected from one or more of deoxyadenosine, deoxycytidine, and deoxyguanosine.

[0015] Preferably, the components and contents of the additives in the serum-free defined-component mesenchymal stem cell medium of the present invention can be: recombinant human epidermal growth factor 5-20 μg / L, basic fibroblast growth factor 5-20 μg / L, transferrin 5-25 mg / L, insulin 9-20 mg / L, sodium selenite 5-8 μg / L, and ethanolamine 1-4 mg / L.

[0016] Preferably, the components and contents of the additives in the serum-free defined-component mesenchymal stem cell medium of the present invention can also be: recombinant human epidermal growth factor 5-20 μg / L, basic fibroblast growth factor 5-20 μg / L, platelet-derived growth factor 1-10 μg / L, insulin-like growth factor 2-20 μg / L, transforming growth factor 0-10 μg / L, hepatocyte growth factor 2-20 μg / L, stem cell growth factor 2-20 μg / L, transferrin 5-25 mg / L, human serum albumin 1-10 g / L, fibronectin 1-10 mg / L, insulin 9-20 mg / L, dexamethasone 2-80 μg / L, hydrocortisone 0.05-0.5 mg / L, sodium selenite 5-8 μg / L, ethanolamine 1-4 mg / L, taurine 0.5-5 mg / L. More preferably, the added components and contents of the medium are: human serum albumin / 1.25 g / L, fibronectin 5 mg / L, recombinant human epidermal growth factor 10 μg / L, basic fibroblast growth factor 10 μg / L, platelet-derived growth factor / 2 μg / L, insulin-like growth factor / 4 μg / L, transforming growth factor 2 μg / L, hepatocyte growth factor 4 μg / L, stem cell growth factor 4 μg / L, transferrin 5.5 mg / L, insulin 10 mg / L, dexamethasone 4 μg / L, hydrocortisone 150 μg / L, sodium selenite 6.7 μg / L, ethanolamine 2 mg / L, taurine 1.25 mg / L.

[0017] Preferably, the components and contents of the additives in the serum-free defined-component mesenchymal stem cell culture medium of the present invention can also be: platelet-derived growth factor 1-10 μg / L, insulin-like growth factor 2-20 μg / L, transforming growth factor-β1 0-10 μg / L, hepatocyte growth factor 2-20 μg / L, vascular endothelial growth factor 0-20 μg / L, connective tissue growth factor 0-10 μg / L, stem cell growth factor 2-20 μg / L, recombinant human epidermal growth factor 5-20 μg / L, basic fibroblast growth factor 5-20 μg / L, transforming growth factor-β3 0-10 μg / L, transferrin 5-25 mg / L, human serum albumin 1-10 g / L, fibronectin 1-10 mg / L, insulin 9-20 mg / L, dexamethasone 2-80 μg / L, hydrocortisone 0.05-0.5 mg / L, progesterone 0-6 μg / L, sodium selenite 5-8 μg / L, putrescine 0-10 mg / L, serotonin 0-10 mg / L, ethanolamine 1-4 mg / L, interleukin 0-20 μg / L, taurine 0.5-5 mg / L, non-essential amino acids 0-1% (V / V), deoxyadenosine 0-20 g / L, deoxycytidine 0-20 g / L, deoxyguanosine 0-20 g / L. More preferably, the added components and contents of the culture medium are: human serum albumin 1.25 g / L, fibronectin 5 mg / L, recombinant human epidermal growth factor 10 μg / L, basic fibroblast growth factor 10 μg / L, platelet-derived growth factor 2 μg / L, insulin-like growth factor 4 μg / L, vascular endothelial growth factor 4 μg / L, transforming growth factor-β1 2 μg / L, hepatocyte growth factor 4 μg / L, stem cell growth factor 4 μg / L, transforming growth factor-β3 2 μg / L, connective tissue growth factor 2 μg / L, transferrin 5.5 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, dexamethasone 4 μg / L, hydrocortisone 150 μg / L, putrescine 5.3 mg / L, taurine 1.25 mg / L, deoxyadenosine 10 g / L, deoxycytidine 11 g / L, deoxyguanosine 10 g / L, serotonin 5.5 mg / L, non-essential amino acids 1% (V / V), sodium selenite 6.7 μg / L, ethanolamine 2 mg / L, interleukin 1 μg / L.

[0018] Advantages of the present invention

[0019] (1) The defined-component culture medium of the present invention is a stem cell proliferation and maintenance culture medium with completely defined chemical components, serum-free or serum substitute (platelet lysate), and no heterologous components.

[0020] (2) The culture medium of the present invention has the characteristics of high cell proliferation multiple, good cell growth morphology, good maintenance of cell stemness, delaying aging, etc. At the same time, it supports the preparation and production of extracellular vesicles of cells, and can reduce the interference of exogenous particles.

[0021] (3) The culture medium of the present invention can be widely applied to fields such as the research and development of cell therapy drugs and industrial production, and can help biopharmaceutical customers accelerate the drug development process and clinical approval. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the following drawings are provided for the description of the present invention: Figure 1 It is the cell morphology diagram of Example 3.

[0023] Figure 2 It is the proliferation multiple of cell culture in Example 4.

[0024] Figure 3 It is the cell morphology diagram of Example 4.

[0025] Figure 4 It is the cell outgrowth situation of primary isolation of umbilical cord mesenchymal stem cells on Day 12 in Example 5.

[0026] Figure 5 It is the comparison of the senescence situation of mesenchymal stem cells P6 in Example 6.

[0027] Figure 6 It is the situation of harvesting exosomes from cell culture in Example 7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The following describes in detail the mesenchymal stem cell culture medium and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1: Influence of Different Component Formulations on Two-Dimensional Culture of MSCs The composition and concentration of a clinical-grade human mesenchymal stem cell serum-free complete culture medium provided by the present invention are shown in Table 1. The following components are dissolved, mixed according to their respective characteristics, made up to volume with the basal medium, filtered and sterilized with a 0.22 μm filter membrane, and stored at 4°C.

[0030] Table 1. Components and Concentrations of Culture Media A, B, and C are as follows:

[0031] Human umbilical cord mesenchymal stem cells P4 were resuscitated and seeded (seeding amount 7.7×10^4) in a 6-well plate, cultured for 4 days, and the cell number, proliferation multiple, cell viability, and cell diameter were detected. The experimental results showed that the cells in group C proliferated 13.11 times, which was significantly better than other groups (see Table 2). Group C was preferred, but group B was a more cost-effective combination.

[0032] Table 2. Cell Culture Conditions on the 4th Day of Two-Dimensional Culture in Example 1

[0033] Example 2: Effect of adding Component C to different basal media on two-dimensional culture of MSCs Component C and Component B were added to different basal media respectively to test the effect of different basal media on the two-dimensional culture of MSCs. Human umbilical cord mesenchymal stem cells at passage P4 were resuscitated and inoculated (inoculation amount: 7.7×10^4 cells) in a 6-well plate, cultured for 4 days, and the cell number, proliferation multiple, cell viability and cell diameter were detected. The experimental results showed the effect of different basal media on cell proliferation: the cells in the RCD288-PYJ basal medium proliferated 12.42 and 9.22 times respectively, which was significantly better than other groups (see Tables 3 and 4), and the RCD288-PYJ basal medium was preferred.

[0034] Table 3 Cell culture conditions on the 4th day of two-dimensional culture in Example 2 - Group C

[0035] Table 4 Cell culture conditions on the 4th day of two-dimensional culture in Example 2 - Group B

[0036] Example 3: Detection of continuous passage and proliferation ability of cells Experimental method: When culturing mesenchymal stem cells with the defined-component medium described in the present invention, it is not necessary to pre-coat the bottom of the culture dish. Add 15 mL of the defined-component medium into a T75 cell culture flask, and inoculate umbilical cord mesenchymal stem cells at a density of 8000 cells / cm 2 for continuous passage culture. When the confluence of umbilical cord mesenchymal stem cells reaches about 85% (about 72 - 96 h), subculture can be carried out. The subculture is still inoculated in a T75 culture flask at a density of 8000 cells / cm 2 and continuously passaged to passage P10, and the cell morphology and cell number of each passage are recorded.

[0037] Table 5 Cell proliferation during continuous passage of two-dimensional culture in Example 3

[0038] According to the above experimental method for culturing umbilical cord mesenchymal stem cells, continuous passage was carried out. The results showed that under the culture conditions of the defined-component medium prepared in Example 1 - Group C, umbilical cord mesenchymal stem cells maintained good proliferation (as shown in Table 5) and still maintained nearly 6-fold proliferation after continuous passage to passage P10. At the same time, they adhered well and all showed a spindle-shaped fibroblast-like cell morphology, growing in a vortex or radial shape as a whole (such as Figure 1 ).

[0039] Example 4: Comparison of Proliferation Ability with Existing Commercially Available Products The defined component medium of the present invention and three commercially available serum-free media in the control group (as shown in Table 6) were used to continuously passage and culture the umbilical cord mesenchymal stem cells for three generations using the experimental method of umbilical cord mesenchymal stem cells described in Example 3, and three independent repeated experiments were set up.

[0040] The results showed that after continuous passage three times under the culture conditions of the defined component medium prepared in Example 1 - Group C, the umbilical cord mesenchymal stem cells showed better and more stable proliferation ability, and the cell harvest was higher than that of the existing commercially available serum-free medium in the control group (as Figure 2 ), and the cells grew in a fibroblast-like manner after culture and passage, and at the same time, the cells were arranged homogenously and in a swirling pattern (as Figure 3 ).

[0041] Table 6 Conditions of the Medium of the Present Invention and the Control Group Medium

[0042] Example 5: Primary Isolation of Umbilical Cord Mesenchymal Stem Cells The defined component medium of the present invention and a commercially available product in the control group (as shown in Table 6) were used as media for the primary isolation of umbilical cord mesenchymal stem cells. For the primary isolation of umbilical cord mesenchymal stem cells, cells crawled out from the tissue blocks when cultured to Day 12, and the primary cells were harvested until Day 19. The cell crawling speeds and the number of tissue blocks with effectively crawled-out cells of the two media were similar (as Figure 4 ), but the defined component medium of the present invention could harvest a greater number of cells, and at the same time, the cell viability was higher (as shown in Table 7).

[0043] Table 7 Conditions of the Primary Isolation of Umbilical Cord Mesenchymal Stem Cells

[0044] Example 6: Detection of Cell Surface by Flow Cytometry The defined component medium of the present invention and three commercially available serum-free media in the control group (as shown in Table 6) were used to continuously passage and culture the umbilical cord mesenchymal stem cells to P6 using the experimental method of umbilical cord mesenchymal stem cells described in Example 3. Cell immunostaining was performed according to the antibody instruction manual, and marker analysis was carried out by flow cytometry.

[0045] Table 8 Conditions of the Phenotype Detection of Umbilical Cord Mesenchymal Stem Cells

[0046] The results showed (as shown in Table 8) that under the culture conditions in the defined-component medium prepared in Example 1, the umbilical cord mesenchymal stem cells exhibited better biological characteristics: the expression rates of the positive markers CD90 and CD73 were higher than those in the control group of the existing commercial serum-free medium, and the expression rates of the negative markers CD45, CD34, CD19, CD14, and HLA-DA were lower than those in the control group of the existing commercial serum-free medium.

[0047] Example 7: Senescence detection For the defined-component medium of the present invention and three commercial serum-free media in the control group (as shown in Table 6), using the experimental method of umbilical cord mesenchymal stem cells described in Example 3, continuous passage culture was carried out until the P6 generation, and β-galactosidase staining (Solarbio, G1580) was performed on the umbilical cord mesenchymal stem cells. According to the recommended operation in the instruction manual, 5-7 fields of view were randomly selected under an inverted optical microscope, and the number of dark blue cells (senescent cells) in the fields of view was recorded.

[0048] The results showed that under the culture conditions in the defined-component medium prepared in Example 1, in the presence of β-galactoside, the substrate did not change to dark blue, and the defined-component medium of the present invention showed a delayed cell senescence state compared with the control group (as Figure 5 )

[0049] Example 8: Cell culture to harvest exosomes For the defined-component medium of the present invention, using the umbilical cord mesenchymal stem cell culture method described in Example 3, two-dimensional culture was carried out for 72-96 h, and three-dimensional culture was carried out for 4-12 days. The cell supernatant was harvested, and nanoparticle tracking analysis (NTA) technology was used to detect the characteristics of exosome particle number, particle size, etc. in the culture supernatant. The defined-component medium of the present invention can be used for three-dimensional culture, continuously harvesting exosomes until Day 12, and the net production value of exosomes was higher than 2E+09 particles / mL every two days (as Figure 6 )

[0050] The three-dimensional culture method is as follows: 1) Prepare microcarriers: Place the microcarriers (Beijing Huakan, W01) in a culture container, and add the defined-component complete medium to make the microcarriers evenly dispersed.

[0051] 2) Cell seeding and culture: Add the cell suspension to the culture container and place it in an incubator for culture; the parameters of the reactor are set to a combination of variable-speed culture and constant-speed culture, for example, 40 rpm for 5 min, 1 rpm for 25 min, and continue for 1 day. After optionally supplementing the mesenchymal stem cell complete medium, the reactor is adjusted to a constant speed of 40 rpm and continue to culture for several days, for example, 1 day, 2 days, 3 days, 4 days, and the time to stop culture is determined according to the cell sampling and detection results. 3) Harvest cell supernatant: Stop stirring the bioreactor on Day 4 of culture. After the microcarriers have settled, collect the cell supernatant, and then harvest the cell supernatant every two days.

[0052] The above are only the preferred embodiments of the present invention. It should be noted that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention, without deviating from the scope defined by the claims of the present invention.

Claims

1. A serum-free defined-component mesenchymal stem cell culture medium, characterized in that, The culture medium consists of a basal medium and additive components, and the additive components are selected from one or more of growth factor substances, binding protein substances, adhesion factor substances, cytokine substances, hormone substances, trace element-containing substances, biogenic amine substances, amino acid substances, and nucleoside substances.

2. The culture medium according to claim 1, characterized in that, The basal medium is selected from one or more of RCD288 (3D FloTrix MSC chemically defined serum-free basal medium RCD288-PYJ of Beijing Huakan Biotechnology Co., Ltd.), αMEM, IMDM, and DMEM / F12, and preferably RCD288. ® ​ 3. The culture medium according to claim 1 or 2, characterized in that, The growth factor substances are selected from one or more of recombinant human epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, transforming growth factor β1, transforming growth factor β3, hepatocyte growth factor, vascular endothelial growth factor, connective tissue growth factor, and stem cell growth factor; the binding protein substances are selected from one or more of transferrin and human serum albumin; the adhesion factor substance is selected from fibronectin; the cytokine substance is selected from interleukin 2; the hormone substances are selected from one or more of insulin, dexamethasone, hydrocortisone, and progesterone; the trace element-containing substance is selected from sodium selenite; the biogenic amine substances are selected from one or more of ethanolamine, putrescine, and serotonin; the amino acid substances are selected from one or more of taurine and non-essential amino acids; the nucleoside compounds are selected from one or more of deoxyadenosine, deoxycytidine, and deoxyguanosine.

4. The culture medium according to claim 1 or 3, characterized in that, The additive components and their contents in the culture medium are: recombinant human epidermal growth factor 5 - 20 μg / L, basic fibroblast growth factor 5 - 20 μg / L, transferrin 5 - 25 mg / L, insulin 9 - 20 mg / L, sodium selenite 5 - 8 μg / L, and ethanolamine 1 - 4 mg / L.

5. The culture medium according to claim 1 or 3, characterized in that, The additive components and their contents in the culture medium are: recombinant human epidermal growth factor 5 - 20 μg / L, basic fibroblast growth factor 5 - 20 μg / L, platelet-derived growth factor 1 - 10 μg / L, insulin-like growth factor 2 - 20 μg / L, transforming growth factor 0 - 10 μg / L, hepatocyte growth factor 2 - 20 μg / L, stem cell growth factor 2 - 20 μg / L, transferrin 5 - 25 mg / L, human serum albumin 1 - 10 g / L, fibronectin 1 - 10 mg / L, insulin 9 - 20 mg / L, dexamethasone 2 - 80 μg / L, hydrocortisone 0.05 - 0.5 mg / L, sodium selenite 5 - 8 μg / L, ethanolamine 1 - 4 mg / L, and taurine 0.5 - 5 mg / L.

6. The culture medium according to claim 1 or 3, characterized in that, The added components and their contents in the medium are as follows: platelet-derived growth factor 1 - 10 μg / L, insulin-like growth factor 2 - 20 μg / L, transforming growth factor β1 0 - 10 μg / L, hepatocyte growth factor 2 - 20 μg / L, vascular endothelial growth factor 0 - 20 μg / L, connective tissue growth factor 0 - 10 μg / L, stem cell growth factor 2 - 20 μg / L, recombinant human epidermal growth factor 5 - 20 μg / L, basic fibroblast growth factor 5 - 20 μg / L, transforming growth factor β3 0 - 10 μg / L, transferrin 5 - 25 mg / L, human serum albumin 1 - 10 g / L, fibronectin 1 - 10 mg / L, insulin 9 - 20 mg / L, dexamethasone 2 - 80 μg / L, hydrocortisone 0.05 - 0.5 mg / L, progesterone 0 - 6 μg / L, sodium selenite 5 - 8 μg / L, putrescine 0 - 10 mg / L, serotonin 0 - 10 mg / L, ethanolamine 1 - 4 mg / L, interleukin 0 - 20 μg / L, taurine 0.5 - 5 mg / L, non-essential amino acids 0 - 1% (V / V), deoxyadenosine 0 - 20 g / L, deoxycytidine 0 - 20 g / L, deoxyguanosine 0 - 20 g / L.

7. The culture medium according to claim 4, characterized in that, The added components and their contents in the medium are as follows: recombinant human epidermal growth factor 10 μg / L, basic fibroblast growth factor 10 μg / L, transferrin 5.5 mg / L, insulin 10 mg / L, sodium selenite 6.7 μg / L, ethanolamine 2 mg / L.

8. The culture medium according to claim 5, characterized in that, The added components and their contents in the medium are as follows: human serum albumin 1.25 g / L, fibronectin 5 mg / L, recombinant human epidermal growth factor 10 μg / L, basic fibroblast growth factor 10 μg / L, platelet-derived growth factor 2 μg / L, insulin-like growth factor 4 μg / L, transforming growth factor 2 μg / L, hepatocyte growth factor 4 μg / L, stem cell growth factor 4 μg / L, transferrin 5.5 mg / L, insulin 10 mg / L, dexamethasone 4 μg / L, hydrocortisone 150 μg / L, sodium selenite 6.7 μg / L, ethanolamine 2 mg / L, taurine 1.25 mg / L.

9. The culture medium according to claim 6, characterized in that, The added components and their contents in the culture medium are as follows: human serum albumin 1.25 g / L, fibronectin 5 mg / L, recombinant human epidermal growth factor 10 μg / L, basic fibroblast growth factor 10 μg / L, platelet-derived growth factor 2 μg / L, insulin-like growth factor 4 μg / L, vascular endothelial growth factor 4 μg / L, transforming growth factor-β1 2 μg / L, hepatocyte growth factor 4 μg / L, stem cell growth factor 4 μg / L, transforming growth factor-β3 2 μg / L, connective tissue growth factor 2 μg / L, transferrin 5.5 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, dexamethasone 4 μg / L, hydrocortisone 150 μg / L, putrescine 5.3 mg / L, taurine 1.25 mg / L, deoxyadenosine 10 g / L, deoxycytidine 11 g / L, deoxyguanosine 10 g / L, serotonin 5.5 mg / L, non-essential amino acids 1% (V / V), sodium selenite 6.7 μg / L, ethanolamine 2 mg / L, interleukin 1 μg / L.

10. Use of the culture medium according to any one of claims 1 to 9 in MSC culture and in exosome harvesting.

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