CN116836920A - Serum-free culture medium and method for preparing mesenchymal stem cells by using same - Google Patents

Serum-free culture medium and method for preparing mesenchymal stem cells by using same Download PDF

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CN116836920A
CN116836920A CN202311050366.5A CN202311050366A CN116836920A CN 116836920 A CN116836920 A CN 116836920A CN 202311050366 A CN202311050366 A CN 202311050366A CN 116836920 A CN116836920 A CN 116836920A
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serum
mesenchymal stem
stem cells
culture medium
free
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肖碧波
杨安平
魏晓滨
陈鑫
肖景棠
杨海健
朱锐生
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Foshan Biomedical Engineering Society
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    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2501/2306—Interleukin-6 (IL-6)

Abstract

The invention provides a serum-free culture medium and a method for preparing mesenchymal stem cells by using the same. The serum-free culture medium provided by the invention has definite components, can avoid the interference of serum on cell culture, and reduces the allergen; the serum-free culture medium provided by the invention can effectively promote the in-vitro amplification efficiency of mesenchymal stem cells, improve the amplification times of the mesenchymal stem cells, and simultaneously has lower apoptosis rate in the culture stage of the mesenchymal stem cells, effectively improves the in-vitro amplification survival rate of the mesenchymal stem cells, and fully proves that the serum-free culture medium provided by the invention has the advantage of outstanding culture.

Description

Serum-free culture medium and method for preparing mesenchymal stem cells by using same
Technical Field
The invention relates to the technical field of culture media, in particular to a serum-free culture medium and a method for preparing mesenchymal stem cells by using the serum-free culture medium.
Background
The mesenchymal stem cells are multipotent stem cells derived from mesoderm, are separated from bone marrow at the earliest, and have strong clinical application prospect in aspects of immunoregulation and tissue regeneration because of easy in-vitro amplification and no tumorigenicity. MSCs have two main features: self-renewal and differentiation potential; meanwhile, it can secrete some important cytokines, express specific receptors and can be genetically modified so as to change the molecular susceptibility of the natural behavior of the receptor. Therefore, MSCs are increasingly receiving attention. Many scholars sequentially isolate similar cells from human umbilical cord Wharton's jelly and umbilical cord perivascular tissues by different methods.
The existing formula of the mesenchymal stem cell culture medium contains human serum or animal serum, has a large number of unknown components in the serum, and even possibly contains some unknown allergic sources and pathogens, so that on one hand, experimental results can be interfered, and on the other hand, the safety of the cultured cells cannot be ensured, and the mesenchymal stem cell culture medium cannot be applied to clinical researches. The existing serum-free culture medium has excessively complex components, the cell amplification effect is not ideal, and large-scale in-vitro amplification is difficult to realize.
Therefore, serum-free media with defined composition, controllable quality and suitable for primary culture of mesenchymal stem cells are a necessary choice for MSC culture for clinical application. The invention aims to provide a serum-free culture medium for mesenchymal stem cells, which has remarkable amplification effect and high safety and is suitable for culturing clinical application.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a serum-free culture medium and a method for preparing mesenchymal stem cells by using the same. The serum-free culture medium has definite components, can avoid the interference of serum on cell culture, and can effectively promote the in-vitro amplification of mesenchymal stem cells, so that the serum-free culture medium has prominent culture advantages.
Specifically, the invention firstly provides a serum-free culture medium, which is characterized by comprising the following components: SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6, L-glutamine and basal medium.
Preferably, the concentration of SCF is 20-50ng/ml.
Preferably, the EPO is present in a concentration of 10-20ng/ml.
Preferably, the concentration of GM-CSF is in the range of 20-30ng/ml.
Preferably, the concentration of MCP-1 is 20-40ng/ml.
Preferably, the IL-3 concentration is 1-20ng/ml.
Preferably, the IL-4 concentration is 1-20ng/ml.
Preferably, the IL-6 concentration is 1-20ng/ml
Preferably, the L-glutamine is 5-10mM.
Preferably, the basal medium is low sugar DMEM-F12.
In another aspect, the invention provides a method for preparing a serum-free medium, which is characterized by comprising the following steps:
1) DMEM-F12 is used as a basic culture medium;
2) Sequentially weighing SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6 and L-glutamine according to a formula, adding into the DMEM-F12 culture medium in the step 1), and uniformly mixing until no obvious precipitate exists;
3) Filtering and sterilizing the mixed solution obtained in the step 2) through a microporous filter membrane to obtain a serum-free culture medium, and storing the serum-free culture medium at 4 ℃.
In another aspect, the present invention provides a method for culturing mesenchymal stem cells, wherein the mesenchymal stem cells are inoculated into the serum-free medium or cultured.
Preferably, the conditions of the culture are 37℃and 5% CO 2 。
Preferably, the seeded cell density is 1X 10 4 -1×10 5 And each mL.
Preferably, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
In another aspect, the invention provides the use of the serum-free medium in the preparation of mesenchymal stem cell products.
The invention has the following advantages: the serum-free culture medium provided by the invention has definite components, can avoid the interference of serum on cell culture, and reduces the allergen; the serum-free culture medium provided by the invention can effectively promote the in-vitro amplification efficiency of mesenchymal stem cells, improve the amplification times of the mesenchymal stem cells, and simultaneously has lower apoptosis rate in the culture stage of the mesenchymal stem cells, effectively improves the in-vitro amplification survival rate of the mesenchymal stem cells, and fully proves that the serum-free culture medium provided by the invention has the advantage of outstanding culture.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
A serum-free culture medium comprises 20-50ng/ml SCF, 10-20ng/ml EPO, 20-30ng/ml GM-CSF, 20-40ng/ml MCP-1, 1-20ng/ml IL-3, 1-20ng/ml IL-4, 1-20ng/ml IL-6, 5-10mM L-glutamine, and the balance DMEM-F12 basal medium, and is configured to be 1L for later use.
The preparation method of the serum-free culture medium comprises the following steps:
1) DMEM-F12 is used as a basic culture medium;
2) Sequentially weighing SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6 and L-glutamine according to a formula, adding into the DMEM-F12 culture medium in the step 1), and uniformly mixing until no obvious precipitate exists;
3) Filtering and sterilizing the mixed solution obtained in the step 2) through a microporous filter membrane to obtain a serum-free culture medium, and storing the serum-free culture medium at 4 ℃.
Example 2
A serum-free medium comprises 20ng/ml SCF, 10ng/ml EPO, 20ng/ml GM-CSF, 20ng/ml MCP-1, 1ng/ml IL-3, 1ng/ml IL-4, 1ng/ml IL-6, 5mM L-glutamine, the balance being DMEM-F12 basal medium and is configured for 1L use.
The preparation method of the serum-free culture medium comprises the following steps:
1) DMEM-F12 is used as a basic culture medium;
2) Sequentially weighing SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6 and L-glutamine according to a formula, adding into the DMEM-F12 culture medium in the step 1), and uniformly mixing until no obvious precipitate exists;
3) Filtering and sterilizing the mixed solution obtained in the step 2) through a microporous filter membrane to obtain a serum-free culture medium, and storing the serum-free culture medium at 4 ℃.
Example 3
A serum-free medium is formulated to include 50ng/ml SCF, 20ng/ml EPO, 30ng/ml GM-CSF, 40ng/ml MCP-1, 20ng/ml IL-3, 20ng/ml IL-4, 20ng/ml IL-6, 10mM L-glutamine, the balance being DMEM-F12 basal medium and configured for 1L use.
The preparation method comprises the following steps:
1) DMEM-F12 is used as a basic culture medium;
2) Sequentially weighing SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6 and L-glutamine according to a formula, adding into the DMEM-F12 culture medium in the step 1), and uniformly mixing until no obvious precipitate exists;
3) Filtering and sterilizing the mixed solution obtained in the step 2) through a microporous filter membrane to obtain a serum-free culture medium, and storing the serum-free culture medium at 4 ℃.
Comparative example 1
A serum-free medium was formulated differently from example 2 in that its cytokine consisted of 50ng/ml SCF, 20ng/ml EPO, 30ng/ml GM-CSF, 40ng/ml MCP-1.
Comparative example 2
A serum-free medium was formulated differently from example 2 in that the cytokines consisted of 20ng/ml IL-3, 20ng/ml IL-4, 20ng/ml IL-6.
Comparative example 3
A serum-free medium was formulated differently from example 2 in that it had a basal medium consisting of only 10mM L-glutamine.
Comparative example 4
A serum-free medium was formulated differently from comparative example 1 in that it further contained a 10mM L-glutamine composition.
Comparative example 5
A serum-free medium was formulated differently from comparative example 2 in that it further contained a 10mM L-glutamine composition.
Test example 1
Each treatment was repeated 3 times in 2mL of serum-free medium of the above examples and comparative examples one by one in a 24-well plate. Then umbilical cord blood mesenchymal stem cells are taken and washed by PBS to adjust the cell density to 5 multiplied by 10 4 ce L/mL, gently beating, inoculating 100 μL of the mixture into corresponding well plate containing serum-free medium, and inoculating 5% CO at 37deg.C 2 Is cultured in an incubator of (a). Cell expansion was calculated at the beginning of culture, day 7, day 14, and day 21, respectively.
The statistical results are shown in table 1 below: in examples 2 and 3 of the present invention, the expansion factor of the umbilical cord blood mesenchymal stem cells after continuous 7 days of culture can reach up to 1562.8 times, and the serum-free medium of the present invention has superior cell expansion capacity compared with other comparative examples. Especially, when the cell culture time is increased to 21 days, the expansion times of the mesenchymal stem cells of the umbilical cord blood can reach 9956.2 times. The best amplification effect of the serum-free medium of the invention is fully proved.
TABLE 1 cell expansion fold of umbilical cord blood mesenchymal stem cells in serum-free medium
Group of For 7 days 14 days 21 days
Example 2 1562.8±100.2 7894.5±90.6 9956.2±151.4
Example 3 1326.5±44.6 5643.2±120.7 8475.3±203.8
Comparative example 1 152.3±50.5 654.2±40.2 842.1±56.7
Comparative example 2 30.2±9.5 57.9±10.2 100.5±7.6
Comparative example 3 359.5±21.5 947.6±56.6 1000.1±84.4
Comparative example 4 100.4±12.7 356.2±25.5 663.5±36.6
Comparative example 5 16.2±8.1 24.3±9.6 21.2±5.4
Apoptosis rate analysis of umbilical cord blood mesenchymal stem cells in the serum-free medium described above gave the results shown in table 2: in the embodiments 2 and 3 of the present invention, the apoptosis rate of the umbilical cord blood mesenchymal stem cells is obviously lower than that of other serum-free media, and the apoptosis rate is only about 8.2 after continuous 21 days of culture, which further proves that the serum-free media of the present invention has the advantage of protruding culture.
TABLE 2 apoptosis of umbilical cord blood mesenchymal stem cells in serum-free Medium (%)
Group of For 7 days 14 days 21 days
Example 2 5.2 6.5 8.2
Example 3 5.4 7.1 8.3
Comparative example 1 6.5 9.2 15.3
Comparative example 2 8.8 12.3 20.5
Comparative example 3 6.2 8.2 10.3
Comparative example 4 6.5 11.5 18.2
Comparative example 5 6.5 38.7 57.8
In addition to the umbilical cord blood mesenchymal stem cells, the inventors performed the same cell expansion factor and apoptosis rate detection analysis for bone marrow mesenchymal stem cells and adipose mesenchymal stem cells, and the results were the same as the experimental analysis results for umbilical cord mesenchymal stem cells. The serum-free culture medium has good amplification activity, can meet the mass production and preparation of mesenchymal stem cells, and improves the clinical utilization efficiency of the mesenchymal stem cells.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A serum-free medium comprising: SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6, L-glutamine and basal medium.
2. The serum-free medium of claim 1, wherein the concentration of SCF is 20-50ng/ml, the concentration of EPO is 10-20ng/ml, the concentration of GM-CSF is 20-30ng/ml, and the concentration of MCP-1 is 20-40ng/ml.
3. The serum-free medium of claim 1, wherein said IL-3 is at a concentration of 1-20ng/ml, said IL-4 is at a concentration of 1-20ng/ml, said IL-6 is at a concentration of 1-20ng/ml, and said L-glutamine is 5-10mM.
4. A serum-free medium according to any one of claims 1 to 3, wherein the basal medium is low sugar DMEM-F12.
5. The method for preparing a serum-free medium according to any one of claims 1 to 4, comprising the steps of:
1) DMEM-F12 is used as a basic culture medium;
2) Sequentially weighing SCF, EPO, GM-CSF, MCP-1, IL-3, IL-4, IL-6 and L-glutamine according to a formula, adding into the DMEM-F12 culture medium in the step 1), and uniformly mixing until no obvious precipitate exists;
3) Filtering and sterilizing the mixed solution obtained in the step 2) through a microporous filter membrane to obtain a serum-free culture medium, and storing the serum-free culture medium at 4 ℃.
6. A method of culturing mesenchymal stem cells, comprising inoculating the mesenchymal stem cells into the serum-free medium of any one of claims 1 to 4 for culturing.
7. The method according to claim 6, wherein the culturing is carried out at 37℃under 5% CO 2 。
8. The method of claim 6, wherein the seeded cell density is 1 x 10 4 -1×10 5 And each mL.
9. The method of claim 6, wherein the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose mesenchymal stem cells.
10. Use of the serum-free medium according to any one of claims 1-4 for the preparation of a mesenchymal stem cell product.
CN202311050366.5A 2023-08-21 2023-08-21 Serum-free culture medium and method for preparing mesenchymal stem cells by using same Pending CN116836920A (en)

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