Culture medium for amniotic mesenchymal stem cells as well as preparation method and application of culture medium

Abstract

The invention discloses a culture medium for amniotic mesenchymal stem cells as well as a preparation method and application of the culture medium for the amniotic mesenchymal stem cells, and the culture medium for the amniotic mesenchymal stem cells consists of a basic culture medium and an additive, the additive is prepared from the following components: 5 to 10 ng / mL of basic fibroblast growth factors, 4 to 8 vol% of fetal calf serum, 15 to 25 ng / mL of vascular endothelial growth factors, 0.8 to 1.2 vol% of double antibodies, 0.01 to 0.02 vol% of CHIR-99021, 2 to 5 mg / mL of notoginsenoside and 1 to 3 mg / mL of vitamin C. The invention further discloses a preparation method of the additive. The culture medium for the amniotic mesenchymal stem cells is based on the basic culture medium and supplemented with various growth factors, fetal calf serum, CHIR-99021, notoginsenoside and vitamin C, proliferation of the amniotic mesenchymal stem cells can be remarkably promoted, under the combined action of all the components, the amplification efficiency of the amniotic mesenchymal stem cells is improved, and the proliferation rate of the amniotic mesenchymal stem cells is increased. A large number of amniotic mesenchymal stem cells can be rapidly obtained, and market requirements can be met.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium for cell culture, and in particular to a culture medium for amniotic mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] The amnion is a thin membrane composed of a single layer of epithelial cells connected to each other. The single layer of epithelial cells has the function of secreting amniotic fluid. As the embryo grows and develops, the amnion and the chorion are closely attached to form the fetal membrane. As the amniotic cavity gradually expands, the fetal membrane becomes a translucent film with no blood vessels and is tough (the fetal membrane is also the cyst wall of the amniotic cavity). The liquid filled in the amniotic cavity is amniotic fluid. The amnion only covers the fetal surface of the placenta and does not penetrate into the placental tissue. As the pregnancy progresses, the amniotic cavity gradually expands and occupies the entire uterine cavity. The amnion is the innermost layer of the fetal membrane. It is a translucent film that is connected to the amniotic layer covering the placenta and umbilical cord.

[0003] Amniotic mesenchymal stem cells are a multifunctional stem cell with the ability to self-renew and regenerate. They can differentiate into various types of cells under certain conditions and have a wide range of medical application potential. The main effects of amniotic mesenchymal stem cells include: Tissue repair and regeneration: Amniotic mesenchymal stem cells can differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes, thereby promoting tissue repair and regeneration. This plays an important role in fracture healing, cartilage repair, and skin regeneration. Immunomodulation: Amniotic mesenchymal stem cells have immunomodulatory functions, which can inhibit inflammatory responses and regulate the activity of immune cells. This helps to reduce the occurrence and development of autoimmune diseases and reduce rejection reactions in situations such as organ transplantation. Angiogenesis: These stem cells have the ability to promote the formation of new blood vessels, which can improve blood circulation and promote tissue repair. This is very important in the treatment of cardiovascular diseases, ischemic diseases, and vascular damage. Anti-tumor effects: Recent studies have shown that amniotic mesenchymal stem cells may have anti-tumor effects, which can inhibit tumor growth and spread and improve the efficacy of chemotherapy and radiotherapy.

[0004] At present, the culture effect of amniotic mesenchymal stem cells is not ideal, the proliferation rate is slow, and it cannot meet the demand for a large number of amniotic mesenchymal stem cells.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a culture medium for amniotic mesenchymal stem cells and a preparation method and application thereof. The present invention can significantly promote the proliferation of amniotic mesenchymal stem cells and quickly obtain sufficient amniotic mesenchymal stem cells by adding specific additive components to the basic culture medium.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The first aspect of the present invention provides a culture medium for amniotic mesenchymal stem cells, the culture medium for amniotic mesenchymal stem cells is composed of a basal culture medium and additives, and the additives include the following components in terms of final concentration:

[0009] Basic fibroblast growth factor 5-10 ng / mL, fetal bovine serum 4 vol%-8 vol%, vascular endothelial growth factor 15-25 ng / mL, double antibody 0.8 vol%-1.2 vol%, CHIR-99021 0.01 vol%-0.02 vol%, notoginseng saponin 2-5 mg / mL and vitamin C 1-3 mg / mL.

[0010] Preferably, the additive comprises the following components:

[0011] Basic fibroblast growth factor 6-8 ng / mL, fetal bovine serum 5 vol%-7 vol%, vascular endothelial growth factor 18-22 ng / mL, double antibody 0.9 vol%-1.1 vol%, CHIR-99021 0.015 vol%-0.02 vol%, notoginseng saponin 3-4 mg / mL and vitamin C 2-3 mg / mL.

[0012] Preferably, the additive comprises the following components:

[0013] Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, vascular endothelial growth factor 20ng / mL, double antibody 1vol%, CHIR-99021 0.015vol%, Panax notoginseng saponin 4mg / mL and vitamin C 2mg / mL.

[0014] Preferably, the basal culture medium is DMEM / F12 culture medium.

[0015] The second aspect of the present invention provides a method for preparing the culture medium for amniotic mesenchymal stem cells, the method comprising the following steps:

[0016] (a) Weigh each component according to the ratio;

[0017] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0018] The third aspect of the present invention provides a use of the above-mentioned culture medium for amniotic mesenchymal stem cells in the expansion culture of amniotic mesenchymal stem cells.

[0019] A fourth aspect of the present invention provides a method for expanding and culturing amniotic mesenchymal stem cells, the method comprising the following steps:

[0020] The amniotic membrane mesenchymal stem cells are inoculated into the amniotic membrane mesenchymal stem cell culture medium for culture.

[0021] Preferably, the inoculation concentration of the amniotic mesenchymal stem cells is (2-6)×10 4 cfu / mL.

[0022] Preferably, the culture temperature is 37°C and the CO2 concentration is 5%.

[0023] Compared with the prior art, the beneficial effects of the present invention include at least:

[0024] The culture medium for amniotic mesenchymal stem cells of the present invention is based on a basal culture medium and supplemented with various growth factors, fetal bovine serum, CHIR-99021, notoginseng saponin and vitamin C, which can significantly promote the proliferation of amniotic mesenchymal stem cells. Under the joint action of various components, the expansion efficiency of amniotic mesenchymal stem cells is improved, a large number of amniotic mesenchymal stem cells are quickly obtained, and market demand can be met. DETAILED DESCRIPTION

[0025] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0026] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the invention belongs.

[0027] The embodiment of the present invention provides a culture medium for amniotic mesenchymal stem cells, wherein the culture medium for amniotic mesenchymal stem cells is composed of a basal culture medium and additives, wherein the additives include the following components in terms of final concentration:

[0028] Basic fibroblast growth factor 5-10 ng / mL, fetal bovine serum 4 vol%-8 vol%, vascular endothelial growth factor 15-25 ng / mL, double antibody 0.8 vol%-1.2 vol%, CHIR-99021 0.01 vol%-0.02 vol%, notoginseng saponin 2-5 mg / mL and vitamin C 1-3 mg / mL.

[0029] The culture medium for amniotic mesenchymal stem cells of the present invention is based on a basal culture medium and supplemented with various growth factors, fetal bovine serum, CHIR-99021, notoginseng saponin and vitamin C, which can significantly promote the proliferation of amniotic mesenchymal stem cells. Under the joint action of various components, the expansion efficiency of amniotic mesenchymal stem cells is improved, a large number of amniotic mesenchymal stem cells are quickly obtained, and market demand can be met.

[0030] In some embodiments, the additive includes the following components:

[0031] Basic fibroblast growth factor 6-8 ng / mL, fetal bovine serum 5 vol%-7 vol%, vascular endothelial growth factor 18-22 ng / mL, double antibody 0.9 vol%-1.1 vol%, CHIR-99021 0.015 vol%-0.02 vol%, notoginseng saponin 3-4 mg / mL and vitamin C 2-3 mg / mL.

[0032] In one embodiment, the additive comprises the following components:

[0033] Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, vascular endothelial growth factor 20ng / mL, double antibody 1vol%, CHIR-99021 0.015vol%, Panax notoginseng saponin 4mg / mL and vitamin C 2mg / mL.

[0034] In one embodiment, the basal culture medium is DMEM / F12 medium.

[0035] Another embodiment of the present invention provides a method for preparing the culture medium for amniotic mesenchymal stem cells, the method comprising the following steps:

[0036] (a) Weigh each component according to the ratio;

[0037] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0038] Another embodiment of the present invention provides a use of the above-mentioned culture medium for amniotic membrane mesenchymal stem cells in the expansion culture of amniotic membrane mesenchymal stem cells.

[0039] Another embodiment of the present invention provides a method for expanding and culturing amniotic mesenchymal stem cells, the method comprising the following steps:

[0040] The amniotic membrane mesenchymal stem cells are inoculated into the amniotic membrane mesenchymal stem cell culture medium for culture.

[0041] In some embodiments, the inoculation concentration of the amniotic mesenchymal stem cells is (2-6)×10 4 cfu / mL.

[0042] In one embodiment, the culture temperature is 37°C and the CO2 concentration is 5%.

[0043] The technical solution of the present invention is further described in detail below through specific embodiments.

[0044] Example 1

[0045] This embodiment is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0046] Basic fibroblast growth factor 5ng / mL, fetal bovine serum 8vol%, vascular endothelial growth factor 25ng / mL, double antibody 0.8vol%, CHIR-99021 0.02vol%, notoginseng saponin 5mg / mL and vitamin C 1mg / mL;

[0047] The basal culture medium was DMEM / F12 medium.

[0048] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0049] (a) Weigh each component according to the ratio;

[0050] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0051] Example 2

[0052] This embodiment is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0053] Basic fibroblast growth factor 10ng / mL, fetal bovine serum 4vol%, vascular endothelial growth factor 15ng / mL, double antibody 1.2vol%, CHIR-99021 0.01vol%, notoginseng saponin 2mg / mL and vitamin C 3mg / mL;

[0054] The basal culture medium was DMEM / F12 medium.

[0055] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0056] (a) Weigh each component according to the ratio;

[0057] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0058] Example 3

[0059] This embodiment is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0060] Basic fibroblast growth factor 6ng / mL, fetal bovine serum 7vol%, vascular endothelial growth factor 18ng / mL, double antibody 1.1vol%, CHIR-99021 0.015vol%, notoginseng saponin 4mg / mL and vitamin C 2mg / mL;

[0061] The basal culture medium was DMEM / F12 medium.

[0062] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0063] (a) Weigh each component according to the ratio;

[0064] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0065] Example 4

[0066] This embodiment is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0067] Basic fibroblast growth factor 8ng / mL, fetal bovine serum 5vol%, vascular endothelial growth factor 22ng / mL, double antibody 0.9vol%, CHIR-99021 0.02vol%, notoginseng saponin 3mg / mL and vitamin C 3mg / mL;

[0068] The basal culture medium was DMEM / F12 medium.

[0069] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0070] (a) Weigh each component according to the ratio;

[0071] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0072] Example 5

[0073] This embodiment is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0074] Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, vascular endothelial growth factor 20ng / mL, double antibody 1vol%, CHIR-99021 0.015vol%, notoginseng saponin 4mg / mL and vitamin C 2mg / mL;

[0075] The basal culture medium was DMEM / F12 medium.

[0076] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0077] (a) Weigh each component according to the ratio;

[0078] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0079] Example 6

[0080] This embodiment is a method for expanding and culturing amniotic mesenchymal stem cells, and the method for expanding and culturing amniotic mesenchymal stem cells comprises the following steps:

[0081] The amniotic membrane mesenchymal stem cells were inoculated in the medium for amniotic membrane mesenchymal stem cells of Example 3 for culture, wherein the inoculation concentration of the amniotic membrane mesenchymal stem cells was 2×10 4 cfu / mL; the culture temperature was 37°C and the CO2 concentration was 5%.

[0082] Example 7

[0083] This embodiment is a method for expanding and culturing amniotic mesenchymal stem cells, and the method for expanding and culturing amniotic mesenchymal stem cells comprises the following steps:

[0084] The amniotic membrane mesenchymal stem cells were inoculated in the amniotic membrane mesenchymal stem cell culture medium of Example 4 for culture, wherein the inoculation concentration of the amniotic membrane mesenchymal stem cells was 6×10 4 cfu / mL; the culture temperature was 37°C and the CO2 concentration was 5%.

[0085] Example 8

[0086] This embodiment is a method for expanding and culturing amniotic mesenchymal stem cells, and the method for expanding and culturing amniotic mesenchymal stem cells comprises the following steps:

[0087] The amniotic membrane mesenchymal stem cells were inoculated in the amniotic membrane mesenchymal stem cell culture medium of Example 5 for culture, wherein the inoculation concentration of the amniotic membrane mesenchymal stem cells was 5×10 4 cfu / mL; the culture temperature was 37°C and the CO2 concentration was 5%.

[0088] Comparative Example 1

[0089] This comparative example is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0090] Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, vascular endothelial growth factor 20ng / mL, double antibody 1vol%, notoginseng saponin 4mg / mL and vitamin C 2mg / mL;

[0091] The basal culture medium was DMEM / F12 medium.

[0092] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0093] (a) Weigh each component according to the ratio;

[0094] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0095] Comparative Example 2

[0096] This comparative example is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0097] basic fibroblast growth factor 7 ng / mL, fetal bovine serum 6 vol%, vascular endothelial growth factor 20 ng / mL, double antibody 1 vol%, CHIR-99021 0.015 vol% and vitamin C 2 mg / mL;

[0098] The basal culture medium was DMEM / F12 medium.

[0099] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0100] (a) Weigh each component according to the ratio;

[0101] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0102] Comparative Example 3

[0103] This comparative example is a culture medium for amniotic mesenchymal stem cells, which is composed of a basal culture medium and additives. The additives include the following components in terms of final concentration:

[0104] Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, double antibody 1vol%, CHIR-990210.015vol%, notoginseng saponin 4mg / mL and vitamin C 2mg / mL;

[0105] The basal culture medium was DMEM / F12 medium.

[0106] The preparation method of the above-mentioned culture medium for amniotic mesenchymal stem cells comprises the following steps:

[0107] (a) Weigh each component according to the ratio;

[0108] (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

[0109] Experimental example

[0110] Obtaining the culture medium for amniotic mesenchymal stem cells of Example 5 and Comparative Examples 1 to 3;

[0111] The amniotic membrane mesenchymal stem cells were inoculated into 10 ml of culture medium for culture. The inoculation concentration of the amniotic membrane mesenchymal stem cells was 5×10 4 cfu / mL; the culture temperature was 37°C and the CO2 concentration was 5%; the culture medium was replaced every 3 days, and the cells were digested after 96 hours of culture, and then the cells were counted. The counting results are shown in Table 1;

[0112] Table 1

[0113] Group Number of cells Example 5 <![CDATA[1.06×10 8 ]]> Comparative Example 1 <![CDATA[4.58×10 7 ]]> Comparative Example 2 <![CDATA[2.12×10 7 ]]> Comparative Example 3 <![CDATA[5.81×10 7 ]]>

[0114] From Table 1, we can see that:

[0115] Compared with the comparative example, the embodiment of the present invention can better promote the proliferation of amniotic mesenchymal stem cells and improve the proliferation efficiency thereof by limiting the raw material components in the culture medium.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A culture medium for amniotic mesenchymal stem cells, characterized in that The culture medium for amniotic mesenchymal stem cells consists of a basal culture medium and additives. The additives include the following components in terms of final concentration: Basic fibroblast growth factor 5-10 ng / mL, fetal bovine serum 4 vol%-8 vol%, vascular endothelial growth factor 15-25 ng / mL, double antibody 0.8 vol%-1.2 vol%, CHIR-99021 0.01 vol%-0.02 vol%, notoginseng saponin 2-5 mg / mL and vitamin C 1-3 mg / mL.

2. The culture medium for amniotic mesenchymal stem cells according to claim 1, characterized in that Said additives include the following components: Basic fibroblast growth factor 6-8 ng / mL, fetal bovine serum 5 vol%-7 vol%, vascular endothelial growth factor 18-22 ng / mL, double antibody 0.9 vol%-1.1 vol%, CHIR-99021 0.015 vol%-0.02 vol%, notoginseng saponin 3-4 mg / mL and vitamin C 2-3 mg / mL.

3. The culture medium for amniotic mesenchymal stem cells according to claim 1, characterized in that Said additives include the following components: Basic fibroblast growth factor 7ng / mL, fetal bovine serum 6vol%, vascular endothelial growth factor 20ng / mL, double antibody 1vol%, CHIR-99021 0.015vol%, Panax notoginseng saponin 4mg / mL and vitamin C 2mg / mL.

4. The culture medium for amniotic mesenchymal stem cells according to claim 1, characterized in that The basic culture medium is DMEM / F12 culture medium.

5. The method for preparing a culture medium for amniotic mesenchymal stem cells according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (a) Weigh each component according to the ratio; (b) adding each component in the additive to the basal culture medium, mixing, filtering and sterilizing, thereby obtaining the culture medium for amnion mesenchymal stem cells.

6. Use of the culture medium for amniotic mesenchymal stem cells according to any one of claims 1 to 4 in the expansion culture of amniotic mesenchymal stem cells.

7. A method for expanding and culturing amniotic mesenchymal stem cells, characterized in that: The amniotic membrane mesenchymal stem cell expansion and culture method comprises the following steps: The amniotic membrane mesenchymal stem cells are inoculated in the medium for amniotic membrane mesenchymal stem cells according to any one of claims 1 to 4 and cultured.

8. The method for expanding and culturing amniotic mesenchymal stem cells according to claim 7, characterized in that: The inoculation concentration of the amniotic membrane mesenchymal stem cells is (2-6)×10 4 cfu / mL.

9. The method for expanding and culturing amniotic mesenchymal stem cells according to claim 7, characterized in that: The culture temperature was 37°C and the CO2 concentration was 5%.