CN111808807A - Mesenchymal stem cell serum-free medium without need of pre-coating and application thereof - Google Patents
Mesenchymal stem cell serum-free medium without need of pre-coating and application thereof Download PDFInfo
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Abstract
The invention relates to a mesenchymal stem cell serum-free culture medium without need of pre-coating and application thereof, wherein the mesenchymal stem cell serum-free culture medium without need of pre-coating comprises a basic culture medium and an additive, and the additive comprises recombinant human serum albumin, a recombinant human basic fibroblast growth factor, a transforming growth factor beta 1 and DGEA peptide. The culture medium is a culture system completely free from Xeno (Xeno-free), so that Xeno pollution is avoided; the DGEA peptide is creatively added and matched with additive components such as recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor beta 1 and the like, so that the mesenchymal stem cells are directly cultured without pre-coating a culture container during culturing the mesenchymal stem cells, the cells have higher anchorage rate, amplification times and cell activity rate, and the cell growth state is good. The method greatly simplifies the operation steps, saves the cost and is beneficial to large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of cell culture, relates to a mesenchymal stem cell serum-free culture medium and application thereof, and particularly relates to a mesenchymal stem cell serum-free culture medium without pre-coating and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent Cells that can differentiate into a variety of cell types, including: osteoblasts, chondrocytes and adipocytes, this ability to differentiate has been demonstrated in a variety of contexts, including specific cells and tissues in living animals and the corresponding cells and tissues grown in tissue culture. In recent years, MSCs have become a hotspot for research and clinical research due to their broad therapeutic use and their great potential to repair damaged or diseased tissues.
The MSC culture medium used at present contains essentially Fetal Bovine Serum (FBS) because it provides hormones and growth factors necessary for cell attachment and proliferation. Isolation and culture of MSCs therefore depends to a large extent on the use of animal products, in particular Fetal Bovine Serum (FBS). FBS is extracted from the blood of fetal cattle obtained in the meat processing industry and is added to the growth medium during MSC culture, usually at a concentration of 10% by volume. The major problem with the high volume use of FBS is the risk of transmission of infectious agents, which is not only present on the patient, but also to the product technicians and medical personnel (when handling the product). When using FBS-containing media, attention is paid to Bovine Spongiform Encephalopathy (BSE), which is commonly referred to as mad cow disease, and variant Creutzfeldt-Jakob disease (vCJD). In addition, FBS needs to be stored at-20 ℃ under refrigeration. And vary widely from batch to batch. In order to solve the problem of safe supply and use of FBS, research and development personnel research serum-free culture media and obtain certain results.
CN108300690A discloses a separation culture method of bone marrow mesenchymal stem cells and a serum-free culture medium, wherein the serum-free culture medium is a basal culture medium added with ascorbic acid, basic fibroblast growth factor, platelet-derived factor and human serum albumin, and the component concentration in the serum-free culture medium is 60-80mg/L of ascorbic acid, 10-30 mug/L of basic fibroblast growth factor, 10-30 mug/L of platelet-derived factor and 4-8g/L of human serum albumin. Can efficiently and quickly separate the adipose-derived mesenchymal stem cells, is beneficial to the growth of the adipose-derived mesenchymal stem cells, and greatly improves the proliferation rate of the adipose-derived mesenchymal stem cells.
CN108251359A discloses a serum-free culture medium for mesenchymal stem cells and a culture method, wherein the serum-free culture medium comprises a basic culture medium and additive components, the additive components comprise hypoxia inducible factor-1, and the concentration of the hypoxia inducible factor-1 is 10-20 ng/mL; also comprises a mechano-growth factor, wherein the concentration of the mechano-growth factor is 20-50 ng/mL. The serum-free culture medium provided by the invention has a synergistic promotion effect on the growth of the mesenchymal stem cells by matching with a hypoxic culture condition, the growth condition of the mesenchymal stem cells is greatly improved, and the 'dryness' of the mesenchymal stem cells and the capability of differentiating the mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes and other types of cells are still maintained after multiple generations.
For serum-free culture media, the existing products (including the patents CN108300690A and CN108251359A cited above) require pre-coating of the culture vessel, because if the pre-coating is not performed, the adherence of the cells is significantly lower than that of the serum-containing culture medium, but the pre-coating operation is not suitable for large-scale industrial production. Therefore, it is important to develop a culture medium that can promote cell adhesion without pre-coating the culture vessel.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a mesenchymal stem cell serum-free culture medium and application thereof, and particularly provides a mesenchymal stem cell serum-free culture medium without pre-coating and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a mesenchymal stem cell serum-free medium without need of pre-coating, which comprises a basic medium and additives, wherein the additives comprise recombinant human serum albumin, recombinant human basic fibroblast growth factor (bFGF2), transforming growth factor beta 1 (TGF-beta 1) and DGEA peptide.
The serum-free culture medium is a culture system completely free from foreign sources, so that foreign pollution is avoided; the DGEA peptide is creatively added and matched with additive components such as recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor beta 1 and the like, so that the mesenchymal stem cells are directly cultured without pre-coating a culture container, the cells still have higher adherence coefficient, amplification coefficient and cell survival rate, and the cell growth state is good. The method greatly simplifies the operation steps, saves the cost and is beneficial to large-scale industrial production. The DEGA peptide is a short peptide, and the CAS number is 134580-64-6.
Preferably, the concentration of the recombinant human serum albumin in the mesenchymal stem cell serum-free culture medium without pre-coating is 0.5-50 μ g/mL, such as 0.5 μ g/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 15 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL or 50 μ g/mL, and the like, and other specific points within the range can be selected, which are not described in detail herein, and are preferably 1-10 μ g/mL.
The serum-free medium has specific concentration requirements on the added recombinant human serum albumin, and when the concentration of the serum albumin exceeds the specific numerical range, the serum albumin can cause the phenomena of cell agglomeration, irregular shape and the like, and when the concentration of the serum albumin is less than the specific numerical range, the adherence rate of the cells can be obviously reduced, and the final product yield is reduced as a result. Wherein 1-10. mu.g/mL is a more preferable range.
Preferably, the concentration of the recombinant human basic fibroblast growth factor in the mesenchymal stem cell serum-free medium without pre-coating is 3-10ng/mL, such as 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 7ng/mL, 8ng/mL, 9ng/mL or 10ng/mL, and other specific points within the range can be selected, and detailed description is omitted here, and 3-5ng/mL is preferred.
The serum-free culture medium has specific concentration requirements on the added recombinant human basic fibroblast growth factor, and when the concentration of the added recombinant human basic fibroblast growth factor exceeds the specific numerical range, the cell division speed is too high, so that the differentiation phenomenon occurs, the aging speed is finally accelerated, and when the concentration of the added recombinant human basic fibroblast growth factor is less than the specific numerical range, the cell proliferation speed is slowed down, so that the culture efficiency is reduced. Wherein 3-5ng/mL is a more effective range.
Preferably, the concentration of the transforming growth factor beta 1 in the mesenchymal stem cell serum-free medium without pre-coating is 2-20ng/mL, such as 2ng/mL, 3ng/mL, 8ng/mL, 10ng/mL, 15ng/mL or 20ng/mL, and the like, and other specific points within the range can be selected, and detailed description is omitted here, and 3-8ng/mL is preferred.
The serum-free medium according to the present invention requires a specific concentration of the added transforming growth factor β 1, and when the concentration exceeds the above-mentioned specific range of values, the medium causes the cells to lose their original shape (the cells lose water due to an excessively high osmotic pressure), and when the concentration is less than the above-mentioned specific range of values, the medium causes the anchorage rate of the cells to decrease. Wherein 3-8ng/mL is the range with better effect.
Preferably, the concentration of the DGEA peptide in the serum-free culture medium of the mesenchymal stem cells without pre-coating is 10-50 μ g/mL, such as 10 μ g/mL, 15 μ g/mL, 20 μ g/mL, 25 μ g/mL, 30 μ g/mL, 35 μ g/mL, 40 μ g/mL, 45 μ g/mL or 50 μ g/mL, and the like, and other specific points within the range can be selected, and detailed description is omitted.
The serum-free medium according to the present invention requires a specific concentration of the added DGEA peptide, and when the concentration exceeds the above-mentioned specific numerical range, the serum-free medium adsorbs lipids and growth factors in the medium, so that the cells lack components necessary for proliferation, and when the concentration is less than the above-mentioned specific numerical range, the serum-free medium reduces the anchorage rate of the cells.
Preferably, the basal medium comprises α - Μ Ε medium.
The basic culture medium related to the invention includes, but is not limited to, culture medium of alpha- Μ Ε Μ.
Preferably, the additive further comprises an antioxidant, a diabody, dexamethasone, a lipid substance, and transferrin.
Preferably, the antioxidant comprises ascorbic acid 2-phosphate.
Preferably, the diabodies include penicillins and streptomycins.
Preferably, the additive further comprises ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances, and transferrin.
Preferably, the additive further comprises 100-200 μ M ascorbic acid 2-phosphate, 50-150U/mL penicillin, 50-150U/mL streptomycin, 50-100nM dexamethasone, 30-50pg/mL lipid material and 0.8-1.2% transferrin.
The concentration of the ascorbic acid 2-phosphate can be 100 μ M, 120 μ M, 150 μ M, 180 μ M or 200 μ M, and other specific values in the range can be selected, and detailed description is omitted here.
The concentration of the penicillin can be 50U/mL, 80U/mL, 100U/mL, 120U/mL or 150U/mL, and other specific values in the range can be selected, and detailed description is omitted here.
The concentration of the streptomycin can be 50U/mL, 80U/mL, 100U/mL, 120U/mL or 150U/mL, and other specific values in the range can be selected, and detailed description is omitted here.
The concentration of dexamethasone can be 50nM, 60nM, 70nM, 80nM, 90nM or 100nM, and other specific values in the range can be selected, which is not described in detail herein.
The concentration of the lipid substance can be 30pg/mL, 35pg/mL, 40pg/mL, 45pg/mL or 50pg/mL, and other specific values within the range can be selected, and detailed description is omitted here.
The mass percentage content of the transferrin can be 0.8%, 0.9%, 1.0%, 1.1%, or 1.2%, and other specific values in the range can be selected, and detailed description is omitted here.
The serum-free culture medium has specific concentration requirements on added ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and transferrin, and has significant influence on the culture medium in the aspects of maintaining the anchorage rate, the expansion multiple and the cell viability of the mesenchymal stem cells.
As a preferred technical scheme of the invention, the mesenchymal stem cell serum-free culture medium without being coated in advance comprises an alpha- Μ Ε Μ culture medium, 0.5-50 μ g/mL recombinant human serum albumin, 3-10ng/mL recombinant human basic fibroblast growth factor, 2-20ng/mL transforming growth factor beta 1, 10-50 μ g/mL DGEA peptide, 100-200 μ M ascorbic acid 2-phosphate, 50-150U/mL penicillin, 50-150U/mL streptomycin, 50-100nM dexamethasone, 30-50pg/mL lipid substances and 0.8-1.2% transferrin.
The preparation method of the serum-free feeder layer-free embryonic stem cell or pluripotent stem cell culture medium comprises the following steps:
dissolving the additive components in the culture medium according to respective dissolution characteristics, filtering with a filter membrane for sterilization, adding into the basic culture medium at normal temperature (20-30 deg.C), and mixing.
In another aspect, the invention provides an application of the mesenchymal stem cell serum-free medium without pre-coating in mesenchymal stem cell culture.
Compared with the prior art, the invention has the following beneficial effects:
the serum-free culture medium is a culture system completely free from foreign sources, so that foreign pollution is avoided; the DGEA peptide is creatively added and is matched with additive components with low content such as recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor beta 1 and the like, so that a culture container is not required to be pre-coated and the mesenchymal stem cells are directly cultured, the cells still have high adherence coefficient, amplification coefficient and cell viability, the adherence coefficient can be up to more than 1.80, the amplification coefficient can be up to more than 2.30, the cell viability is 93.48-97.71%, and the cell growth state is good. The method greatly simplifies the operation steps, saves the cost and is beneficial to large-scale industrial production.
Drawings
FIG. 1 is a diagram showing the observation of cell morphology in the groups of cells of examples 1 to 5, which were cultured for 1, 2 and 3 days;
FIG. 2 is a diagram showing the observation of cell morphology in the groups of cells of examples 1 to 9 and comparative example 1, after culturing for 1, 2 and 3 days.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The components and reagents involved in the following examples are all conventional commercial products, wherein α - Μ Ε Μ medium is purchased from Gibco; recombinant human serum albumin was purchased from Peprotech; the recombinant human basic fibroblast growth factor is purchased from Peprotech company; transforming growth factor beta 1 was purchased from Peprotech; DGEA peptides were purchased from Kinsley Biotechnology, Inc.; ascorbic acid 2-phosphate was purchased from Sigma; penicillin/streptomycin was purchased from BI corporation; dexamethasone was purchased from Sigma; lipids were purchased from Gibco (11905-; ITS (transferrin) was purchased from Gibco; the mesenchymal stem cells are umbilical cord mesenchymal stem cells, the human newborn umbilical cord is taken out within 24 hours, washed by PBS under aseptic condition, and bloodstain is removed; shearing the cleaned umbilical cord into tissue blocks with sterile scissors in an animal cell culture medium, removing blood vessels, crushing, mixing the precipitate obtained by centrifugation with a DMEM/F12 complete culture medium, shaking uniformly, planting in a cell culture dish, and culturing in a 5% CO2 incubator at 37 ℃; and on the 14 th day of culture, completely sucking out the tissue blocks, discarding the tissue blocks, supplementing a DMEM/F12 complete culture medium, and then replacing the culture medium every 3 days to continue culturing to obtain the umbilical cord mesenchymal stem cells.
Example 1
The present embodiment provides a mesenchymal stem cell serum-free medium without pre-coating, and the formula thereof is shown in the following table:
the preparation method comprises the following steps: the recombinant human serum albumin, the recombinant human basic fibroblast growth factor, the transforming growth factor beta 1, the DGEA peptide, the ascorbic acid 2-phosphate, the penicillin/streptomycin solution, the dexamethasone and the lipid with the formula amount are sequentially added into 500mL of alpha-MEM culture medium.
Example 2
The present embodiment provides a mesenchymal stem cell serum-free medium without pre-coating, and the formula thereof is shown in the following table:
the preparation process is as in example 1.
Example 3
The present embodiment provides a mesenchymal stem cell serum-free medium without pre-coating, and the formula thereof is shown in the following table:
the preparation process is as in example 1.
Example 4
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation of the serum-free medium is different from that of example 1 only in that the recombinant human serum albumin has a mass percentage of 60 μ g/mL, and the rest are consistent.
Example 5
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation of the serum-free medium is different from that of example 1 only in that the recombinant human serum albumin has a mass percentage of 20 μ g/mL, and the rest are consistent.
Example 6
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation differs from example 1 only in that the concentration of recombinant human basic fibroblast growth factor is 12ng/mL, and the others are consistent.
Example 7
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation differs from example 1 only in that the concentration of recombinant human basic fibroblast growth factor is 8ng/mL, and the rest are consistent.
Example 8
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation of the serum-free medium is different from that of example 1 only in that the mass percentage content of the DGEA peptide is 60 μ g/mL, and the others are consistent.
Example 9
The present example provides a serum-free medium for mesenchymal stem cells without pre-coating, and the formulation of the serum-free medium is different from that of example 1 only in that the mass percentage content of the DGEA peptide is 5 μ g/mL, and the others are consistent.
Comparative example 1
The comparison example provides a serum-free culture medium for mesenchymal stem cells, and the formula of the serum-free culture medium is different from that of the serum-free culture medium in example 1 only in that the serum-free culture medium does not contain the DGEA peptide, and other components and concentrations are kept unchanged.
The preparation process is as in example 1.
Evaluation test:
(1) taking out the frozen mesenchymal stem cells of the generation 3, and unfreezing in water bath at 37 ℃;
(2) transferring the cell suspension to a super clean bench, and slowly adding the cell suspension into a prepared centrifuge tube (10 mLDPBS is added in advance); sampling and counting after uniform resuspension;
(3) centrifuging at 1200rpm for 5min, and removing the supernatant;
(4) adding a proper amount of DPBS (platelet-derived growth medium) for resuspension of cells, centrifuging at 1200rpm for 5min, discarding the supernatant, and removing residual FBS;
(5) the culture media of examples 1 to 9 and comparative example 1 were added to the cell suspension to a concentration of 1.0X 10, respectively, according to the counting results6Individual cells/mL;
(6) then, according to a 1.0 × 104Individual cell/cm2Uniformly inoculating the seeds into a 6-hole plate, and arranging 3 parallel holes;
(7) standing at 37 deg.C and 5% CO2Culturing in a cell culture box, and changing the culture solution every day on the 2 nd day. The following evaluation tests were then carried out:
(I) cell morphology observation
The operation method comprises the following steps: the cells cultured to day 1, day 2 and day 3 in each group were inverted respectively to observe the cell morphology of each group under a microscope and to collect images.
As a result, as shown in fig. 1 and 2, it can be seen that: the serum-free culture medium can ensure that cells grow in an adherent manner on the premise of not being coated, and the recombinant human serum albumin and the DGEA peptide are necessary components for the cell to grow in an adherent manner and have a synergistic effect when being used together; when the concentration of the recombinant human serum albumin is too high, the cells have the phenomena of agglomeration, irregular shape and the like, and the subsequent proliferation is influenced.
(II) measurement of cell adherence coefficient
The operation method comprises the following steps: after each group is cultured for 18h, a small amount of culture medium is extracted, and the number of cells remaining in the culture medium is detected by cell counting, so that the cell amount of successful adherence is obtained.
The results are shown in table 1 (data are presented as relative adherence coefficients, 3 replicates per set of data, expressed as mean ± standard deviation), and it can be seen that: in contrast to the comparative examples, recombinant human serum albumin is an essential component for cell adhesion and has a synergistic effect when used together with DGEA peptides. When the concentration of the recombinant human serum albumin is deficient, the adherence efficiency is reduced. Likewise, DGEA peptides have an important role in cell adhesion. From the adherence coefficient corresponding to each group, the action of the recombinant albumin is dependent on DGEA, and when DGEA is deficient, the adherence action of the recombinant human albumin to cells is weakened. When the concentration of the DGEA and the recombinant albumin is increased to a certain concentration, the adherence effect of the cells is not obviously increased.
TABLE 1
| Experimental group | Relative adherence coefficient (compared to comparative example) |
| Comparative example 1 | 1.00±0.02 |
| Example 1 | 1.80±0.04** |
| Example 2 | 1.76±0.05** |
| Example 3 | 1.79±0.05** |
| Example 4 | 1.81±0.09** |
| Example 5 | 1.77±0.02** |
| Example 6 | 1.78±0.01** |
| Example 7 | 1.70±0.05** |
| Example 8 | 1.81±0.06** |
| Example 9 | 1.25±0.01** |
Indicates that there is a significant difference in the experimental data compared to the comparative example (P <0.01)
(III) cell expansion fold assay
The operation method comprises the following steps: and collecting cells after culturing for 3 days in each group, counting by using a blood counting chamber, and calculating the collection amount, wherein the ratio of the collection amount to the inoculation amount is the amplification multiple of the cells.
The results are shown in table 2 (data are presented as relative amplification factor, 3 replicates for each set of data, and the data are the average of 3), showing that: at a proper concentration, bFGF has a promoting effect on cell proliferation, while recombinant human serum albumin and DGEA peptide have an insignificant effect on cell proliferation, but high concentrations of proteins have an adverse effect on cell proliferation, presumably because the high concentrations of proteins have an adsorption effect on nutrients in a culture medium and form a competitive inhibition effect with the uptake of nutrients by cells. bFGF is positively correlated with the proliferation of cells under a certain concentration, and when adherence-related proteins (recombinant human serum albumin and DGEA peptide) are lacked, the expansion fold is reduced because cells cannot adhere normally.
TABLE 2
| Experimental group | Relative amplification factor (compared to comparative example) |
| Comparative example | 1.00±0.04 |
| Example 1 | 2.30±0.07** |
| Example 2 | 2.21±0.01** |
| Example 3 | 2.13±0.05** |
| Example 4 | 0.87±0.01** |
| Example 5 | 1.05±0.09 |
| Example 6 | 2.50±0.06** |
| Example 7 | 2.48±0.07** |
| Example 8 | 1.03±0.11** |
| Example 9 | 0.98±0.13 |
Indicates that there is a significant difference in the experimental data compared to the comparative example (P <0.01)
(IV) cell viability assay
The operation method comprises the following steps: cells were collected after 3 days of culture for each group, and the amount collected was counted using a hemocytometer and the rate of viability was read.
The results are shown in table 3, which shows that: the cell survival rate is related to the adherent state of the cells, and the proliferation of the mesenchymal stem cells depends on effective cell adherence and proper nutrient components. In the absence of recombinant human serum albumin and DGEA, the adherence efficiency of cells is reduced, and the cells cannot normally proliferate and die. When the anchorage-associated protein is too high, nutrient components in the culture medium can be competitively adsorbed, so that the cells are lack of nutrition and apoptosis is promoted. bFGF can effectively promote the proliferation of cells, and the cells are in a rapid proliferation state at high concentration, which is shown by the fact that the survival rate is increased.
TABLE 3
| Experimental group | Rate of cell viability |
| Comparative example | 92.83％ |
| Example 1 | 96.45％ |
| Example 2 | 96.84％ |
| Example 3 | 96.49％ |
| Example 4 | 93.48％ |
| Example 5 | 94.14％ |
| Example 6 | 97.71％ |
| Example 7 | 96.84％ |
| Example 8 | 95.01％ |
| Example 9 | 93.51％ |
The applicant states that the invention is illustrated by the above examples, but the invention is not limited to the above examples, i.e. the invention is not necessarily dependent on the above examples to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. The mesenchymal stem cell serum-free medium without being coated in advance is characterized by comprising a basic medium and additives, wherein the additives comprise recombinant human serum albumin, a recombinant human basic fibroblast growth factor, a transforming growth factor beta 1 and DGEA peptide.
2. The mesenchymal stem cell serum-free medium without pre-coating of claim 1, wherein the concentration of the recombinant human serum albumin in the mesenchymal stem cell serum-free medium without pre-coating is 0.5-50 μ g/mL, preferably 1-10 μ g/mL.
3. The mesenchymal stem cell serum-free medium without pre-coating of claim 1 or 2, wherein the concentration of the recombinant human basic fibroblast growth factor in the mesenchymal stem cell serum-free medium without pre-coating is 3-10ng/mL, preferably 3-5 ng/mL.
4. The mesenchymal stem cell serum-free medium without pre-coating of any one of claims 1-3, wherein the concentration of the transforming growth factor beta 1 in the mesenchymal stem cell serum-free medium without pre-coating is 2-20ng/mL, preferably 3-8 ng/mL.
5. The mesenchymal stem cell serum-free medium without pre-coating of any one of claims 1-4, wherein the concentration of the DGEA peptide in the mesenchymal stem cell serum-free medium without pre-coating is 10-50 μ g/mL.
6. The mesenchymal stem cell serum-free culture medium without prior coating of any one of claims 1-5, wherein the basal medium comprises a culture medium of a- Μ Ε Μ.
7. The mesenchymal stem cell serum-free medium without prior coating of any one of claims 1-6, wherein the additive further comprises an antioxidant, a diabody, dexamethasone, a lipid substance and transferrin;
preferably, the antioxidant comprises ascorbic acid 2-phosphate.
8. The serum-free medium for mesenchymal stem cells without prior coating of claim 7, wherein the additives further comprise ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and transferrin;
preferably, the additive further comprises 100-200 μ M ascorbic acid 2-phosphate, 50-150U/mL penicillin, 50-150U/mL streptomycin, 50-100nM dexamethasone, 30-50pg/mL lipid material and 0.8-1.2% transferrin.
9. The mesenchymal stem cell serum-free medium without prior coating of any one of claims 1-8, wherein the mesenchymal stem cell serum-free medium without prior coating comprises a- Μ medium, 0.5-50 μ g/mL recombinant human serum albumin, 3-10ng/mL recombinant human basic fibroblast growth factor, 2-20ng/mL transforming growth factor β 1, 10-50 μ g/mL DGEA peptide, 100-200 μ M ascorbic acid 2-phosphate, 50-150U/mL penicillin, 50-150U/mL streptomycin, 50-100nM dexamethasone, 30-50pg/mL lipid substance and 0.8-1.2% transferrin.
10. Use of the mesenchymal stem cell serum-free medium without prior coating of any one of claims 1-9 in culturing mesenchymal stem cells.
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