Serum-free polypeptide culture medium for promoting proliferation of mesenchymal stem cells and preparation method of serum-free polypeptide culture medium

Abstract

The invention relates to the technical field of cell culture, and particularly provides a serum-free polypeptide culture medium for promoting proliferation of mesenchymal stem cells and a preparation method thereof, and the serum-free polypeptide culture medium for promoting proliferation of mesenchymal stem cells is prepared from dendrobium officinale, semen cuscutae, pseudo-ginseng, astragalus membranaceus, polygonum multiflorum, ginseng, artificial musk, oyster polypeptide, radix cyathulae polypeptide and the like. The serum-free polypeptide culture medium for promoting proliferation of the mesenchymal stem cells and the preparation method of the serum-free polypeptide culture medium are researched by further aiming at the culture environment of the adipose-derived mesenchymal stem cells and combining polypeptide and traditional Chinese medicine extracts.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells are a type of adult stem cell that is widely present in various tissues throughout the body. They can be cultured and expanded in large quantities in vitro and differentiate into cells from the inner, middle and outer germ layers under specific conditions. Mesenchymal stem cells can be divided into many types according to their sources. Among them, adipose-derived mesenchymal stem cells (ADSCs) are a type of stem cells separated from adipose tissue with multidirectional differentiation potential. They are easy to separate and obtain and are currently more mature stem cells for industrialization and commercialization.

[0003] The proliferation and cultivation of adipose-derived mesenchymal stem cells is an important step in their application. The serum-based culture medium used in the prior art has the risk of viral contamination. Therefore, providing a serum-free polypeptide culture medium for promoting the proliferation of adipose-derived mesenchymal stem cells and a preparation method thereof have important practical significance. Summary of the invention

[0004] In order to solve the above problems, on the one hand, the present invention provides a method for preparing a serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells, wherein the method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells is: Take 15-19% by weight of Dendrobium officinale, 14-18% by weight of Cuscuta australis, 12-16% by weight of Panax notoginseng, 10-14% by weight of Astragalus, 6-10% by weight of Polygonum multiflorum, 3-6% by weight of Ginseng, 1-3% by weight of Artificial Musk and make up the balance of pure water, chop the above eight substances until they pass through a 50-mesh sieve, add a colloid mill with a power of 3kW and a discharge fineness of 300 mesh at a feeding rate of 100kg / h, and grind at 3°C ​​until the material is discharged to obtain an extract, add 95% ethanol with a mass of 5 times the mass of the extract at 10°C, mix and immerse for 10 hours, and then filter through 400 mesh to obtain a filtrate; distill the filtrate under reduced pressure at 77°C and a vacuum degree of 800mbar until no liquid flows out continuously, collect the remaining liquid and filter through a 0.22μm film to obtain the Chinese medicine extract; The serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells is obtained by mixing 23% by weight of the traditional Chinese medicine extract, 3% by weight of the oyster polypeptide, 4% by weight of the Cyathula capitata polypeptide and the rest of the serum-free culture medium.

[0005] Furthermore, the mass fraction of the Dendrobium officinale is 17%.

[0006] Furthermore, the mass fraction of the dodder seeds is 16%.

[0007] Furthermore, the mass fraction of Panax notoginseng is 14%.

[0008] Furthermore, the mass fraction of the astragalus is 12%.

[0009] Furthermore, the mass fraction of the Polygonum multiflorum is 8%.

[0010] Furthermore, the mass fraction of the ginseng is 4%.

[0011] Furthermore, the mass fraction of the artificial musk is 2%.

[0012] On the other hand, the present invention provides a serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells prepared by the method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells as described above.

[0013] The present invention can bring the following beneficial effects: Li Meiyuan mentioned in "Study on the Effect and Mechanism of Achyranthes bidentata Polypeptide on Promoting Schwann Cell Proliferation" that Achyranthes bidentata polypeptide can effectively promote cell proliferation. Zhang Aiguo et al. mentioned the promoting effect of various Chinese medicines on stem cell proliferation in "Effects of Four Chinese Medicine Decoctions and Astragalus Alcohol Extract on the Proliferation Activity of Bone Marrow Mesenchymal Stem Cells". The present invention further targets the environment for culturing adipose mesenchymal stem cells, combines the use of polypeptides and Chinese medicine extracts, and studies the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells and the preparation method thereof. Combined with the experimental data of the present invention, the specific proportions of Dendrobium officinale, Cuscuta australis, Panax notoginseng, Astragalus, Polygonum multiflorum, Ginseng, artificial musk, oyster polypeptide and Achyranthes bidentata polypeptide of the present invention are used together, and the effect is poor if one of them is missing. According to the unique extraction and preparation process of the present invention, after mixing with the serum-free culture medium according to the proportion of the present invention, the proliferation of mesenchymal stem cells can be promoted, specifically the proliferation of adipose mesenchymal stem cells. DETAILED DESCRIPTION

[0014] In order to more clearly explain the overall concept of the present invention, the overall scheme of the present invention is described in detail in the form of embodiments below; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention; however, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details; in other examples, in order to avoid confusion with the present invention, some technical features known in the art are not described.

[0015] In the present invention, adipose-derived mesenchymal stem cells were purchased from Wuhan Yipu Biotechnology Co., Ltd. with the product number CM-H202 and the living cell specification of 5×10 5Cells / T25 culture flask; serum-free culture medium was purchased from Shanghai Qiming Biotechnology Co., Ltd., item number QMPM2011, specifically human adipose-derived mesenchymal stem cell serum-free culture medium; artificial musk was purchased from Taihe County Yushui Chinese Medicine Factory, which is a 50-mesh powder; oyster polypeptide was purchased from Xi'an Haoyu Kangze Biotechnology Co., Ltd., item number KZ-1214K, 80-mesh sterile powder; Sichuan Cyathula polypeptide was purchased from Snott Biological Co., Ltd., which is a 100-mesh sterile powder with 60% oligopeptide content and a molecular weight of about 1000 Daltons; Dendrobium officinale, Cuscuta, Panax notoginseng, Astragalus, Polygonum multiflorum and ginseng were purchased from Chengdu Deyinrunzhu Biotechnology Co., Ltd. with a water content of 5%. Ginseng was a 10-year-old forest ginseng. The above Chinese medicines met the standards of the State Food and Drug Administration's "Good Manufacturing Practice for Chinese Medicinal Materials" and the "Chinese Pharmacopoeia". The vacuum distiller was purchased from Shanghai Yuhua Instrument Equipment Co., Ltd., model RE-501, with a speed of 120 rpm and a maximum feed of 1 L each time. The colloid mill was purchased from Guangzhou Xingate Machinery Equipment Co., Ltd., model: XJT-L80A. The membrane filtration used a TST-BLGL membrane filter.

[0016] Unless otherwise specified, all raw material components in the following examples can be purchased through commercial channels, the experimental instruments used are conventional laboratory experimental instruments, and the performance testing methods are the testing methods known in the art.

[0017] The preferred implementation is as follows: Embodiment 1: The Chinese medicine extract is prepared by the following method: Take 17% by weight of Dendrobium officinale, 16% by weight of Cuscuta chinensis, 14% by weight of Panax notoginseng, 12% by weight of Astragalus, 8% by weight of Polygonum multiflorum, 4% by weight of Ginseng, 2% by weight of artificial musk and make up the balance of pure water, chop the above eight substances until they pass through a 50-mesh sieve, add a colloid mill with a power of 3kW and a discharge fineness of 300 mesh at a feeding rate of 100kg / h, and grind at 3°C ​​at low temperature until the material is discharged to obtain an extract, add 95% ethanol with a mass of 5 times the mass of the extract at 10°C, mix and immerse for 10 hours, and then filter through 400 mesh to obtain a filtrate; distill the filtrate under reduced pressure at 77°C and a vacuum degree of 800mbar until no liquid flows out continuously, collect the remaining liquid and filter through a 0.22μm film to obtain a traditional Chinese medicine extract.

[0018] Prepare serum-free peptide culture medium to promote mesenchymal stem cell proliferation as follows: The serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells is obtained by mixing 23% by weight of the traditional Chinese medicine extract, 3% by weight of the oyster polypeptide, 4% by weight of the Cyathula capitata polypeptide and the rest of the serum-free culture medium.

[0019] Embodiment 2-15: The only difference between Example 2 and Example 1 is that the mass fraction of Dendrobium officinale is 15%; The only difference between Example 3 and Example 1 is that the mass fraction of Dendrobium officinale is 19%; The only difference between Example 4 and Example 1 is that the mass fraction of Cuscuta australis is 14%; The only difference between Example 5 and Example 1 is that the mass fraction of Cuscuta australis is 18%; The only difference between Example 6 and Example 1 is that the mass fraction of Panax notoginseng is 12%; The only difference between Example 7 and Example 1 is that the mass fraction of Panax notoginseng is 16%; The only difference between Example 8 and Example 1 is that the mass fraction of Astragalus is 10%; The only difference between Example 9 and Example 1 is that the mass fraction of Astragalus is 14%; The only difference between Example 10 and Example 1 is that the mass fraction of Polygonum multiflorum is 6%; The only difference between Example 11 and Example 1 is that the mass fraction of Polygonum multiflorum is 10%; The only difference between Example 12 and Example 1 is that the mass fraction of ginseng is 3%; The only difference between Example 13 and Example 1 is that the mass fraction of ginseng is 6%; The only difference between Example 14 and Example 1 is that the mass fraction of artificial musk is 1%; The only difference between Example 15 and Example 1 is that the mass fraction of artificial musk is 3%.

[0020] Comparative Examples 1-37: The difference between Comparative Example 1 and Example 1 is that the mass fraction of Dendrobium officinale is 7%; The difference between Comparative Example 2 and Example 1 is that the mass fraction of Dendrobium officinale is 33%; The difference between Comparative Example 3 and Example 1 is that the mass fraction of Cuscuta australis is 7%; The difference between Comparative Example 4 and Example 1 is that the mass fraction of Cuscuta australis is 32%; The difference between Comparative Example 5 and Example 1 is that the mass fraction of Panax notoginseng is 6%; The only difference between Comparative Example 6 and Example 1 is that the mass fraction of Panax notoginseng is 30%; The difference between Comparative Example 7 and Example 1 is that the mass fraction of Astragalus is 5%; The only difference between Comparative Example 8 and Example 1 is that the mass fraction of Astragalus is 29%; The difference between Comparative Example 9 and Example 1 is that the mass fraction of Polygonum multiflorum is 3%; The only difference between Comparative Example 10 and Example 1 is that the mass fraction of Polygonum multiflorum is 20%; The only difference between Comparative Example 11 and Example 1 is that the mass fraction of ginseng is 1%; The only difference between Comparative Example 12 and Example 1 is that the mass fraction of ginseng is 12%; The only difference between Comparative Example 13 and Example 1 is that the mass fraction of artificial musk is 0.5%; The only difference between Comparative Example 14 and Example 1 is that the mass fraction of artificial musk is 6%; The only difference between Comparative Example 15 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 10%; The only difference between Comparative Example 16 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 40%; The only difference between Comparative Example 17 and Example 1 is that the mass fraction of oyster polypeptide is 1%; The only difference between Comparative Example 18 and Example 1 is that the mass fraction of oyster polypeptide is 6%; The difference between Comparative Example 19 and Example 1 is that the mass fraction of Cyathula capitata polypeptide is 1%; The only difference between Comparative Example 20 and Example 1 is that the mass fraction of Cyathula officinalis polypeptide is 10%; The only difference between Comparative Example 21 and Example 1 is that the cryogenic grinding temperature is 20°C; The difference between Comparative Example 22 and Example 1 is that the vacuum distillation temperature is 55°C; The difference between Comparative Example 23 and Example 1 is that the vacuum distillation temperature is 88°C; The difference between Comparative Example 24 and Example 1 is that Dendrobium candidum is replaced with Cuscuta australis, Panax notoginseng, Astragalus, Polygonum multiflorum, Ginseng and artificial musk in the same mass ratio as that of Example 1; The difference between Comparative Example 25 and Example 1 is that the dodder seed is replaced with Dendrobium officinale, Panax notoginseng, Astragalus, Polygonum multiflorum, ginseng and artificial musk of the same mass ratio as that of Example 1; The difference between Comparative Example 26 and Example 1 is that Panax notoginseng is replaced by Dendrobium officinale, Cuscuta australis, Astragalus, Polygonum multiflorum, Ginseng and artificial musk of the same mass ratio as that of Example 1; The difference between Comparative Example 27 and Example 1 is that Astragalus is replaced with Dendrobium officinale, Cuscuta australis, Panax notoginseng, Polygonum multiflorum, ginseng and artificial musk of the same mass ratio as that of Example 1; The difference between Comparative Example 28 and Example 1 is that Polygonum multiflorum is replaced with Dendrobium officinale, Cuscuta australis, Panax notoginseng, Astragalus, Ginseng and artificial musk of the same mass ratio as that of Example 1; The difference between Comparative Example 29 and Example 1 is that ginseng is replaced by Dendrobium officinale, Cuscuta australis, Panax notoginseng, Astragalus, Polygonum multiflorum and artificial musk of the same mass ratio as that of Example 1; The difference between Comparative Example 30 and Example 1 is that the artificial musk is replaced by Dendrobium officinale, Cuscuta australis, Panax notoginseng, Astragalus, Polygonum multiflorum and Ginseng in the same mass ratio as that in Example 1; The difference between Comparative Example 31 and Example 1 is that the Chinese medicine extract is replaced with oyster polypeptide and Sichuan Cyathula polypeptide of the same mass ratio as that of Example 1; The difference between Comparative Example 32 and Example 1 is that the oyster polypeptide is replaced with a Chinese herbal medicine extract and a Sichuan Cyathula polypeptide of the same mass ratio as that of Example 1; The difference between Comparative Example 33 and Example 1 is that the Cyathula officinalis polypeptide is replaced with a Chinese herbal medicine extract and an oyster polypeptide of the same mass ratio as that of Example 1; The difference between Comparative Example 34 and Example 1 is that the Chinese medicine extract, oyster polypeptide and Cyathula officinalis polypeptide are replaced with pure water of equal mass; The difference between Comparative Example 35 and Example 1 is that the Chinese herbal medicine extract, oyster polypeptide and Cyathula officinalis polypeptide are replaced with serum-free culture medium of equal mass; The only difference between Comparative Example 36 and Example 1 is that the later experiments were conducted on CP-H166 human bone marrow mesenchymal stem cells from Wuhan Yipu Biotechnology Co., Ltd.; The only difference between Comparative Example 37 and Example 1 is that the later experiments were conducted on CP-H166 human bone marrow mesenchymal stem cells from Wuhan Yipu Biotechnology Co., Ltd., and the culture medium used was the human bone marrow mesenchymal stem cell serum-free culture medium from Shanghai Yubo Biotechnology Co., Ltd.

[0021] Adipose-derived mesenchymal stem cells were resuspended in serum-free medium at 1×10 6 Inoculate each cell into a corresponding T175 culture flask, add 40 ml of the serum-free peptide culture medium for promoting the proliferation of mesenchymal stem cells in each of the above examples, place in a CO2 incubator at 37°C, 5% CO2, and 95% humidity, culture for 72 hours, resuspend the cells after trypsin digestion, and use the hemocytometer method to estimate the total number of living cells after trypan blue staining. The number of living cells is rounded to one significant figure after the decimal point, unit 10 7 indivual.

[0022] Table 1: Observation results of viable cell numbers for each example:

[0023] It can be seen from the data in Table 1 that, compared with other examples, the embodiments of the present invention, especially Example 1 of the present invention, culture adipose mesenchymal stem cells to obtain a larger number of living cells, that is, the embodiments of the present invention, especially Example 1 of the present invention, have a better effect on promoting the proliferation of adipose mesenchymal stem cells. The present invention provides a serum-free polypeptide culture medium that promotes the proliferation of adipose mesenchymal stem cells and a preparation method thereof. Comparative Examples 36 and 37 demonstrate the unique advantages of the embodiments of the present invention for adipose mesenchymal stem cells, and have a poor effect on promoting the proliferation of other similar stem cells.

[0024] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells, characterized in that: The preparation method of the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells is as follows: Take 15-19% by weight of Dendrobium officinale, 14-18% by weight of Cuscuta australis, 12-16% by weight of Panax notoginseng, 10-14% by weight of Astragalus, 6-10% by weight of Polygonum multiflorum, 3-6% by weight of Ginseng, 1-3% by weight of Artificial Musk and make up the balance of pure water, chop the above eight substances until they pass through a 50-mesh sieve, add a colloid mill with a power of 3kW and a discharge fineness of 300 mesh at a feeding rate of 100kg / h, and grind at 3°C ​​until the material is discharged to obtain an extract, add 95% ethanol with a mass of 5 times the mass of the extract at 10°C, mix and immerse for 10 hours, and then filter through 400 mesh to obtain a filtrate; distill the filtrate under reduced pressure at 77°C and a vacuum degree of 800mbar until no liquid flows out continuously, collect the remaining liquid and filter through a 0.22μm film to obtain a Chinese medicine extract; The serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells is obtained by mixing 23% by weight of the traditional Chinese medicine extract, 3% by weight of the oyster polypeptide, 4% by weight of the Cyathula capitata polypeptide and the rest of the serum-free culture medium.

2. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the Dendrobium officinale is 17%.

3. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the dodder seed is 16%.

4. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the Panax notoginseng is 14%.

5. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the astragalus root is 12%.

6. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the Polygonum multiflorum is 8%.

7. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the ginseng is 4%.

8. The method for preparing the serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the artificial musk is 2%.

9. A serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells prepared by the method for preparing a serum-free polypeptide culture medium for promoting the proliferation of mesenchymal stem cells as claimed in any one of claims 1 to 8.

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