CN109777771B - Serum-free medium for primary umbilical cord mesenchymal stem cells and method of using the same - Google Patents

Abstract

The invention relates to a serum-free culture medium of primary umbilical cord mesenchymal stem cells and a use method thereof, wherein the serum-free culture medium of the primary umbilical cord mesenchymal stem cells comprises the following components: alpha-MEM culture, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF. Fetal calf serum is not added into the culture medium, so that immunological rejection possibly generated in clinical application is avoided, and the risk of pathogenic microorganism pollution is reduced; by adding cytokines such as IL-3, IL-6, G-CSF, SCF and the like, the strong proliferation effect can be generated by the cooperation with FL, and the differentiation of umbilical cord mesenchymal stem cells can be inhibited; the EGF is added to cooperate with the FGF-2 to promote the proliferation and differentiation of the umbilical cord mesenchymal stem cells; by adding BMP-4 as a nutritional factor for the proliferation and differentiation of stem cells, the normal proliferation state of umbilical cord mesenchymal stem cells can be maintained and regulated.

Description

Serum-free medium for primary umbilical cord mesenchymal stem cells and method of using the same

technical field

The invention relates to the field of biotechnology, in particular to a serum-free medium for primary umbilical cord mesenchymal stem cells and a method for using the same.

Background technique

Umbilical cord mesenchymal stem cells (MSCs) have high differentiation potential and can differentiate in multiple directions. It has broad clinical application prospects in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle. Umbilical cord mesenchymal stem cells can be isolated from human umbilical cord, which has the advantages of convenient acquisition and no ethical controversy. Umbilical cord mesenchymal stem cells have strong immune regulation, can promote hematopoietic recovery and repair diseased tissues and organs, and have high clinical applications in organ transplantation, autoimmune diseases, leukemia, bone and muscle degeneration diseases, etc. value.

How to achieve rapid expansion of primary umbilical cord mesenchymal stem cell culture without affecting its cell viability is the key to umbilical cord mesenchymal stem cell culture. The primary cells have slow proliferation and low cell viability.

The existing umbilical cord mesenchymal stem cell culture medium needs to be supplemented with animal serum such as fetal bovine serum. The internal structure of the stem cells grown in this medium may undergo some unknown transformation or mutation, and animal serum may also cause immune rejection. There is a risk of being contaminated by pathogenic microorganisms such as bacteria and viruses, which is not conducive to the clinical application and basic research of stem cells.

The process of proliferation, differentiation, development and maturation of umbilical cord mesenchymal stem cells is quite complex, and it depends on the regulation of various hematopoietic factors, among which the role of cell-stimulating factors is extremely critical.

SUMMARY OF THE INVENTION

Based on this, the present invention provides a serum-free medium for primary umbilical cord mesenchymal stem cells and a method for using the same, which achieves rapid expansion without affecting the cell viability and avoids the risk of rejection and pathogenic microorganism contamination caused by animal serum .

A serum-free medium for primary umbilical cord mesenchymal stem cells, comprising the following components: α-MEM medium, AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.

In one embodiment, the serum-free medium for the primary umbilical cord mesenchymal stem cells further comprises one or more of dexamethasone, transferrin, total Panax notoginseng saponins, and a combination of cytokines.

In one embodiment, the concentration of dexamethasone is 0.1-0.5 μg/ml; the concentration of transferrin is 0.01-0.1 μg/ml; and the concentration of Panax notoginseng saponins is 5-15 μg/ml. Dexamethasone combined with AMD3100 has a synergistic effect to promote the mobilization, crawling and proliferation of umbilical cord mesenchymal stem cells from the tissue. The traditional Chinese medicine ingredient Panax notoginseng saponins can accelerate the proliferation of umbilical cord mesenchymal stem cells.

In one embodiment, the cytokine combination comprises IL-3, SCF, G-CSF, FL and EGF; the concentrations of IL-3, SCF, G-CSF, FL and EGF are: IL-3 10- 20ng/ml; SCF 50-150ng/ml; G-CSF 1-100ng/ml; FL 1-50ng/ml; EGF 5-15ng/ml. FL is a key cytokine that can regulate early stem cells. Combined with cytokines such as IL-3, IL-6, G-CSF and SCF, it has a strong proliferation effect on umbilical cord mesenchymal stem cells.

In one embodiment, the α-MEM medium is a serum-free medium. Provides essential nutrients for stem cell growth and proliferation.

In one of the embodiments, the concentration of AMD3100 is 0.5-5 μg/ml. Promote the growth and proliferation of umbilical cord mesenchymal stem cells.

In one embodiment, the concentration of BMP-4 is 0.01-0.1 ng/ml; the concentration of insulin is 10-20 μg/ml; the concentration of FGF-2 is 1-12 ng/ml. FGF-2 and BMP-4 promote the proliferation of umbilical cord mesenchymal stem cells. Insulin better facilitates the absorption and utilization of glucose by cells.

In one embodiment, the concentration of IL-6 is 0.05-0.15 μg/ml; the concentration of LIF is 0.01-0.1 μg/ml. Leukemia inhibitory factor LIF can inhibit stem cell differentiation and promote stem cell proliferation.

The use method of the serum-free medium for primary umbilical cord mesenchymal stem cells as described in any one of the above, the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, and the use method Include the following steps:

a. Add AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF to the α-MEM medium according to the concentration to make the medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days.

The method for using the serum-free medium for primary umbilical cord mesenchymal stem cells according to any one of the above, wherein the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the use of The method includes the following steps:

a. Add AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginseng saponins according to the concentration in the α-MEM medium to make the medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days.

The method for using the serum-free medium for primary umbilical cord mesenchymal stem cells according to any one of the above, wherein the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the use of The method includes the following steps:

a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days.

The method for using the serum-free medium for primary umbilical cord mesenchymal stem cells according to any one of the above, wherein the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the use of The method includes the following steps:

a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. IL-3, SCF, EGF, G-CSF and FL were added before culturing and stopped after 10 days.

The method for using the serum-free medium for primary umbilical cord mesenchymal stem cells according to any one of the above, wherein the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the use of The method includes the following steps:

a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. IL-3, SCF, EGF, G-CSF were added before culture until 10 days later, and FL was added on the 5th day of culture until 15 days later.

The method for using the serum-free medium for primary umbilical cord mesenchymal stem cells according to any one of the above, wherein the serum-free medium for primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the use of The method includes the following steps:

a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity under cultivation;

c. IL-3, EGF and G-CSF were added before culturing until 10 days later, and SCF and FL were added on the 5th day of culture until 15 days later.

Supplementary Instructions

IL-3 is Interleukin3 Interleukin 3;

SCF is stem cell factor;

G-CSF is granulocyte colony-stimulating factor;

FL is a (flt3-ligand) type III tyrosine kinase receptor ligand;

EGF is epidermal growth factor epidermal growth factor;

BMP-4 is Bone Morphogenetic Protein 4;

FGF-2 is Fibroblast Growth Factor 2;

IL-6 is Interleukin6 Interleukin 6;

LIF is leukemia inhibitory factor leukemia inhibitory factor;

α-MEM is a modified version of Dulbecco's minimum essential medium;

FBS is fetal bovine serum;

Wharton's jelly refers to the gel-like filler between the amniotic membrane and blood vessels of the umbilical cord, which contains more umbilical cord mesenchymal stem cells;

Cell viability (%) = viable cell density/(viable cell density + dead cell density) × 100%.

Beneficial effects of the present invention:

1. In the present invention, by developing a serum-free medium, that is, no fetal bovine serum is added to the medium, the immune rejection that may occur in clinical application is avoided, and the risk of pathogenic microorganism contamination is reduced;

2. In the present invention, by adding cytokines such as IL-3, IL-6, G-CSF and SCF, it can synergize with FL to produce a strong proliferation effect while inhibiting the differentiation of umbilical cord mesenchymal stem cells; by adding EGF, it can synergize with FGF-2 to promote the umbilical cord Proliferation and differentiation of mesenchymal stem cells; by adding BMP-4 as a nutritional factor for the proliferation and differentiation of stem cells, it can maintain and regulate the normal proliferation state of umbilical cord mesenchymal stem cells; by adding the traditional Chinese medicine ingredient Panax notoginseng saponins, the umbilical cord mesenchymal stem cells can proliferate Effect; adding dexamethasone can synergize with AMD3100 to have an auxiliary effect on the proliferation and differentiation of umbilical cord mesenchymal stem cells; by changing the order of adding cytokines, it can weaken the inhibitory effect of FL on the early proliferation of umbilical cord mesenchymal stem cells, and at the same time inhibit the middle and late umbilical cord interstitial cells. Differentiation of mesenchymal stem cells, thereby improving the activity of umbilical cord mesenchymal stem cells in vitro proliferation and differentiation, shortening the proliferation cycle, and improving cell viability.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

The present invention provides a serum-free medium for primary umbilical cord mesenchymal stem cells, comprising the following components: α-MEM medium, AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.

In a preferred embodiment, the serum-free medium for primary umbilical cord mesenchymal stem cells further comprises one or more of dexamethasone, transferrin, total saponins of Panax notoginseng and a combination of cytokines. By adding different combinations of AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins to the α-MEM medium, while adjusting IL-3, SCF, EGF, G-CSF, FL and other cytokines are added and stopped to achieve a serum-free medium for primary umbilical cord mesenchymal stem cells. Fetal bovine serum is not added to the medium, thereby avoiding the need for clinical applications. Possible immune rejection, reducing the risk of contamination by pathogenic microorganisms.

The preferred 6 groups of examples are as follows:

Example 1:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: α-MEM medium, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF, and the concentration of each component As shown in Table 1.

Table 1

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

BMP-4 0.05ng/ml SCF 100ng/ml

Insulin 10μg/ml EGF 7ng/ml

LIF 0.05μg/ml G-CSF 25ng/ml

FL 30ng/ml

Remarks: The α-MEM is used as the base culture medium, and the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the final concentration is implemented in accordance with this table.

The serum-free medium of the above-mentioned primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, and the culturing method includes the following steps:

a. Preparation of umbilical cord mesenchymal stem cell culture medium: AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, and LIF were added to α-MEM medium to make umbilical cord mesenchymal stem cell culture medium;

b. Cultivate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's gel suspension of α-MEM culture solution, the density is 0.25g/ml, at 37°C, 5% CO2 full saturation humidity cultured at the bottom; the medium was changed on the 5th, 10th, 13th, and 15th days after the start of the culture;

c. Add cytokines: add IL-3, SCF, EGF, G-CSF, FL to the fresh medium with the concentration in Table 1 before culturing until the end of 15 days.

Example 2:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng saponins, each group The concentrations of the fractions are shown in Table 2.

Table 2

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

Transferrin 0.02μg/ml SCF 100ng/ml

BMP-4 0.05ng/ml EGF 7ng/ml

Insulin 10μg/ml G-CSF 25ng/ml

IL-6 0.1μg/ml FL 30ng/ml

FGF-2 5ng/ml

LIF 0.05μg/ml

Panax notoginseng saponins 8μg/ml

Remarks: The α-MEM is used as the base culture medium, and the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the final concentration is implemented in accordance with this table.

a. Preparation of umbilical cord mesenchymal stem cell culture medium: AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins were added to the α-MEM medium according to the concentration to make umbilical cord Mesenchymal stem cell culture medium;

b. Add IL-3, SCF, EGF, G-CSF, and FL to the fresh medium of the exchanged medium at the concentration in Table 2 before culturing until the end of 15 days.

Example 3:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng Saponins, the concentrations of each component are shown in Table 3.

table 3

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

Dexamethasone 0.2μg/ml SCF 100ng/ml

Transferrin 0.02μg/ml EGF 7ng/ml

BMP-4 0.05ng/ml G-CSF 25ng/ml

Insulin 10μg/ml FL 30ng/ml

IL-6 0.1μg/ml

FGF-2 5ng/ml

LIF 0.05μg/ml

Panax notoginseng saponins 15μg/ml

Remarks: The α-MEM is used as the basic culture medium, and the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the final concentration is implemented in accordance with this table.

a. Preparation of umbilical cord mesenchymal stem cell culture medium: add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng to the α-MEM medium according to the concentration Saponins are made into umbilical cord mesenchymal stem cell culture medium;

b. Add IL-3, SCF, EGF, G-CSF, and FL to the fresh medium of the exchanged medium according to the concentration in Table 3 before culturing until the end of 15 days.

Example 4:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: α-MEM medium, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6 , LIF, Panax notoginseng saponins, the concentration of each component is shown in Table 4.

Table 4

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

Dexamethasone 0.2μg/ml SCF 100ng/ml

Transferrin 0.02μg/ml EGF 7ng/ml

BMP-4 0.05ng/ml G-CSF 25ng/ml

Insulin 10μg/ml FL 30ng/ml

IL-6 0.1μg/ml

FGF-2 5ng/ml

LIF 0.05μg/ml

Panax notoginseng saponins 5μg/ml

Remarks: With α-MEM as the basic culture medium, the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the concentration is implemented in accordance with this table.

a. Preparation of umbilical cord mesenchymal stem cell culture medium: add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng to the α-MEM medium according to the concentration Saponins are made into umbilical cord mesenchymal stem cell culture medium;

b. Add IL-3, SCF, EGF, G-CSF, and FL to the fresh medium of the exchanged medium according to the concentration in Table 4 before culturing until it is stopped after 10 days.

Example 5:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: α-MEM medium, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6 , LIF, Panax notoginseng saponins, the concentration of each component is shown in Table 5.

table 5

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

Dexamethasone 0.2μg/ml SCF 100ng/ml

Transferrin 0.02μg/ml EGF 7ng/ml

BMP-4 0.05ng/ml G-CSF 25ng/ml

Insulin 10μg/ml FL 30ng/ml

IL-6 0.1μg/ml

FGF-2 5ng/ml

LIF 0.05μg/ml

Panax notoginseng saponins 7μg/ml

Remarks: The α-MEM is used as the base culture medium, and the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the final concentration is implemented in accordance with this table.

a. Preparation of umbilical cord mesenchymal stem cell culture medium: add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng to the α-MEM medium according to the concentration Saponins are made into umbilical cord mesenchymal stem cell culture medium;

b. Add IL-3, SCF, EGF, G-CSF to the fresh medium with the concentration in Table 5 before culturing until 10 days later, and add FL on the 5th day of culturing until 15 days later.

Example 6:

The serum-free medium for primary umbilical cord mesenchymal stem cells in this example includes the following components: α-MEM medium, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6 , LIF, Panax notoginseng saponins, the concentration of each component is shown in Table 6.

Table 6

Medium Component Concentration Cytokine Concentration

AMD3100 1μg/ml IL-3 10ng/ml

Dexamethasone 0.2μg/ml SCF 100ng/ml

Transferrin 0.02μg/ml EGF 7ng/ml

BMP-4 0.05ng/ml G-CSF 25ng/ml

Insulin 10μg/ml FL 30ng/ml

IL-6 0.1μg/ml

FGF-2 5ng/ml

LIF 0.05μg/ml

Panax notoginseng saponins 12μg/ml

Remarks: The α-MEM is used as the base culture medium, and the culture medium is prepared according to the requirements of the addition of different components and cytokines in each stage of the example, and the final concentration is implemented in accordance with this table.

a. Preparation of umbilical cord mesenchymal stem cell culture medium: add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginseng to the α-MEM medium according to the concentration Saponins are made into umbilical cord mesenchymal stem cell culture medium;

b. Add IL-3, EGF, G-CSF to the fresh medium with the concentration in Table 6 before culturing until 10 days later, and add SCF and FL on the 5th day of culturing until 15 days later.

Control group: Umbilical cord mesenchymal stem cell culture medium was prepared: 15% FBS was added to the α-MEM medium.

The umbilical cord mesenchymal stem cell culture medium of Examples 1 to 6 and the control group was tested, and the cell count and cell survival rate were detected.

Cell counting and cell viability detection methods:

1. After culturing for 10 days, check the confluence of cells every day. When the confluence is 85%-90%, take out the culture flask and shake it in a clean workbench to make the tissue pieces fall off;

2. Pour the tissue block and culture medium into a 50ml centrifuge tube, centrifuge at 800g for 5min, and use the first supernatant as a stop solution for backup;

3. Gently rinse the cells twice with 10ml of normal saline in each culture flask, and pour out the washed saline;

4. Add 5ml of trypsin digestion solution to each culture flask, shake and digest until most of the adherent spindle cells begin to fall off and float in a circular shape (observed under an inverted microscope), digest for about 4-6 minutes, and then go to the Add 6ml of stop solution to each culture flask to stop digestion;

5. Filter the cells and suspension together into a 50ml centrifuge tube, wash the culture flask with 10ml of physiological saline, then filter and pour into the same centrifuge tube, centrifuge at 300g for 10min, then resuspend with physiological saline to 50ml, and mix well , for cell count and cell viability assay.

6. Calculation of cell viability:

Cell viability is the proportion of viable cells, and the present invention uses trypan blue exclusion method to count viable cells:

1) Take 20ul of cell suspension and add it to 80ul of trypan blue solution, and dilute it 5 times. Vortex to mix the diluted cell samples;

2) Aspirate 10ul of the diluent and count with a hemocytometer;

3) Dead cells are stained blue dead cells, live cells are transparent with refraction (unstained).

4), cell density calculation method:

The mean of the total number of viable cells in each square × dilution factor × 10 = the number of viable cells/mL.

5) The calculation method of the number of cells is as follows:

(density of live cells + density of dead cells) × 5 (diluted 5 times) × 50 (constituted to 50ml) = number of cells

6) The calculation method of cell viability is as follows:

Viable cell density/(live cell density+dead cell density)×100%=cell viability.

7. Cytometry

All cells cultured in the present invention are subjected to flow detection, which can ensure that the purity of the umbilical cord mesenchymal stem cells does not change greatly.

8. The experimental methods used in the following examples are conventional methods unless otherwise specified.

9. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Result statistics and analysis:

Group Confluence Time to Standard (d) Cell Number Cell Viability

Example 1 14 0.653×107 83.2%

Example 2 13 0.678×107 82.9%

Example 3 12 0.659×107 87.1%

Example 4 12 0.67×107 91.6%

Example 5 12 0.67×107 95.8%

Example 6 11 0.683×107 98.3%

Control group 15 0.627×107 93.6%

Analysis of the results: In terms of cell expansion, the time for the number of cells to reach the standard in Examples 1-6 was lower than that in the control group. In terms of cell viability, the cell viability of Examples 5-6 was better than that of the control group. The comparison shows that the amplification time of Example 5 and Example 6 is short (the number of reaching the target in the shortest time) and the cell activity is high, which are both better than those of the control group.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. a serum-free substratum of primary umbilical cord mesenchymal stem cells, is characterized in that, comprises the following components: α-MEM culture fluid, AMD3100, BMP-4, insulin, FGF-2, IL-6, LIF, In combination with Panax notoginseng saponins and cytokines, the concentration of AMD3100 is 0.5-5 μg/ml, the concentration of BMP-4 is 0.01-0.1 ng/ml; the concentration of insulin is 10-20 μg/ml; The concentration of FGF-2 is 1-12ng/ml, the concentration of IL-6 is 0.05-0.15μg/ml; the concentration of LIF is 0.01-0.1μg/ml, and the concentration of total saponins of Panax notoginseng is 5- 15 μg/ml, the cytokine combination includes IL-3, SCF, G-CSF, FL and EGF; the concentrations of the IL-3, SCF, G-CSF, FL and EGF are respectively: IL-3 10-20ng /ml; SCF 50-150ng/ml; G-CSF 1-100ng/ml; FL 1-50ng/ml; EGF 5-15ng/ml. 2. the serum-free medium of primary umbilical cord mesenchymal stem cells according to claim 1, is characterized in that, the serum-free medium of described primary umbilical cord mesenchymal stem cells also comprises in dexamethasone, transferrin. one or more of them. 3. The serum-free medium for primary umbilical cord mesenchymal stem cells according to claim 2, wherein the concentration of the dexamethasone is 0.1-0.5 μg/ml; the concentration of the transferrin is 0.01 -0.1 μg/ml. 4 . The serum-free medium for primary umbilical cord mesenchymal stem cells according to claim 1 , wherein the α-MEM medium is a serum-free medium. 5 . 5. the using method of the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, is characterized in that, the serum-free medium of described primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF to the α-MEM medium according to the concentration to make the medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's jelly with a suspension of α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days. 6. The method for using the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, wherein the serum-free medium of the primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginseng saponins according to the concentration in the α-MEM medium to make the medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's jelly with a suspension of α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days. 7. The using method of the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, wherein the serum-free medium of the primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's jelly with a suspension of α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. Add IL-3, SCF, EGF, G-CSF, FL before culturing until the end of 15 days. 8. The method for using the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, wherein the serum-free medium of the primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution in each flask, inoculate a suspension of 2ml of Wharton's gel with α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. IL-3, SCF, EGF, G-CSF and FL were added before culturing and stopped after 10 days. 9. The method for using the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, wherein the serum-free medium of the primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in α-MEM medium to make culture medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's jelly with a suspension of α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. IL-3, SCF, EGF, G-CSF were added before culture until 10 days later, and FL was added on the 5th day of culture until 15 days later. 10. The method for using the serum-free medium of primary umbilical cord mesenchymal stem cells according to any one of claims 1 to 4, wherein the serum-free medium of the primary umbilical cord mesenchymal stem cells is used for Culturing umbilical cord mesenchymal stem cells, the use method includes the following steps: a. Add AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginseng saponins according to the concentration in the α-MEM medium to make the medium; b. Incubate in T75 culture flasks, inoculate 8ml of culture solution per flask, inoculate 2ml of Wharton's jelly with a suspension of α-MEM culture solution at a density of 0.25g/ml, at 37°C, fully saturated with 5% CO ₂ Cultivated under humidity; c. IL-3, EGF and G-CSF were added before culturing until 10 days later, and SCF and FL were added on the 5th day of culture until 15 days later.