Mesenchymal stem cell culture medium containing traditional Chinese medicine components as well as preparation method and application of mesenchymal stem cell culture medium

Abstract

The invention discloses a mesenchymal stem cell culture medium containing traditional Chinese medicine components and a preparation method and application thereof. The mesenchymal stem cell culture medium is a culture system composed of a basic culture medium and additives. DMEM / F12 is used as a basic culture medium, an additive solution comprises fetal calf serum, an American ginseng extract and a ganoderma spore extract, and the cell anti-inflammatory capacity, the repair promoting capacity and the angiogenesis promoting capacity are improved. The mesenchymal stem cell culture medium containing the traditional Chinese medicine components can be used for culturing mesenchymal stem cells, and after cells are inoculated for 24 hours, the original culture medium is abandoned, and the culture medium is replaced. By using the culture medium and the use method, the positive rate of the obtained mesenchymal stem cells CD73, CD90 and CD105 is greater than 95%, the positive rate of the obtained mesenchymal stem cells CD44, CD45, CD19, CD34, CD14 and HLA-DR is less than 2%, and the amplification efficiency is improved. Higher anti-inflammatory, repair-promoting and angiogenesis-promoting capacities are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to mesenchymal stem cell culture medium, in particular to a mesenchymal stem cell culture medium containing traditional Chinese medicine components and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of stem cells with multi-directional differentiation potential and self-renewal ability, which have the characteristics of wide source, easy to obtain and low immunogenicity. Under the regulation of specific signal pathways (such as Wnt and Notch pathways), MSCs can differentiate into osteoblasts, neural cells, epithelial cells, adipocytes and other types of cells. At the same time, MSCs have attracted widespread attention due to their functions of regulating immunity, inhibiting inflammatory response and anti-fibrosis. Therefore, stem cells have become a hot spot in the field of life science.

[0003] MSCs have great potential in the field of life science, mainly because they have the abilities of anti-inflammation and immune regulation, collagen synthesis and remodeling, and stimulating angiogenesis. MSCs can effectively promote collagen production, regulate inflammatory response and accelerate new blood vessel formation by activating PI3K / Akt, Notch and other signal pathways.

[0004] Panax quinquefolius L is a widely used ginseng herb of the Araliaceae family, and its extract is often used as a health product for stress, fatigue and anti-aging. Ginsenosides are the key active ingredients of Panax quinquefolius L extract, and the core structure is a tetracyclic triterpene saponin. According to its structure, it can be divided into three groups: protopanaxadiol (PPD), such as Rb1, Rb2, Rc and Rd; protopanaxatriol (PPT), such as Rg1, Re and Rf; oleanolic acid type ginsenoside; and other types (mostly derivatives of PPD / PPT after metabolism or processing). Among them, many types of ginsenosides have the functions of anti-aging and improving metabolism, such as Rg1 and Re can promote neuronal growth and reduce oxidative damage; Rb1 can inhibit inflammatory factors. By activating cell metabolic pathways, the proliferation rate and activity of MSCs are improved, and the role in tissue repair is improved. At the same time, its free radical scavenging ability can reduce oxidative damage in long-term culture and maintain genomic stability.

[0005] Ganoderma lucidum spore extract is an active ingredient extracted from Ganoderma lucidum spores, mainly containing polysaccharides, triterpenes, sterols and other substances, and has immune regulation, antioxidant and metabolic balance improvement. Triterpenes can activate the Nrf2 signaling pathway to enhance the activity of intracellular antioxidant enzymes, scavenge free radicals and reduce oxidative damage in MSC culture; Ganoderma lucidum polysaccharide promotes MSCs to secrete immune regulatory factors and enhances immune regulation ability, and at the same time, through the activation of P13K / Akt signaling pathway, it accelerates the cell cycle process and increases the proliferation rate. It can increase the transcription of Cdk4 mRNA to synthesize more cell cycle proteins, regulate the cell cycle and promote cell division and proliferation. At the same time, Ganoderma lucidum spore extract can also reduce tissue ischemia, hypoxia and edema, and improve the local microenvironment.

[0006] The existing stem cell therapy has limitations, such as high level of oxidative stress, chronic inflammation and various metabolic toxins in the cell microenvironment, which significantly reduces the survival of transplanted cells and becomes a major obstacle to the function of MSCs. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients and its preparation method and application. By adding American ginseng extract and Ganoderma lucidum spore extract to the culture medium, the proliferation and differentiation of MSCs are promoted, the tissue repair ability is improved, the damage of oxidative stress to MSCs is reduced, the influence of inflammation is reduced, and the cell metabolic state is improved to provide energy support for cells.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is: a mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients, comprising a culture system composed of a basic culture medium and an additive, the basic culture medium is DMEM / F12, and the additive comprises fetal bovine serum, American ginseng extract and Ganoderma lucidum spore extract; The American ginseng extract is prepared: after irradiation sterilization, 1g of powder is added to 5mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant is taken. The Ganoderma lucidum spore extract is prepared: after irradiation sterilization, 1g of powder is added to 15mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant is taken.

[0009] Further, the culture system is DMEM / F12 containing 5%-15% volume fraction of fetal bovine serum, 0.5%-1% volume fraction of American ginseng extract and 0.1%-0.5% volume fraction of Ganoderma lucidum spore extract.

[0010] Further, in the preparation of American ginseng extract and Ganoderma lucidum spore extract: incubate at 37℃ for 1 hour, centrifuge at 3000g.

[0011] A preparation method of a mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients, the preparation steps comprising: (1) American ginseng extract preparation: after irradiation sterilization of American ginseng powder, 1g of powder is added to 5mL of preheated DMSO to 37℃, incubated at 37℃, centrifuged, and the supernatant is taken; (2) Ganoderma spore extract preparation: after irradiation sterilization of Ganoderma spore powder, 1g of powder is added to 15mL of preheated DMSO to 37℃, incubated at 37℃, centrifuged, and the supernatant is taken; (3) Take the base medium DMEM / F12, add fetal bovine serum at a volume fraction of 5%-15%, add American ginseng extract at a volume fraction of 0.5%-1%, and add Ganoderma spore extract at a volume fraction of 0.1%-0.5%; mix thoroughly and store in the refrigerator.

[0012] Further, in the preparation of American ginseng extract and Ganoderma spore extract: incubate at 37℃ for 1 hour, and centrifuge at 3000g.

[0013] Further, the temperature of the refrigerated storage is 0-8℃.

[0014] A mesenchymal stem cell culture method, comprising the following steps: (1) Take umbilical cord mesenchymal stem cells, resuspend the cell pellet with umbilical cord mesenchymal stem cell culture medium, filter, count, and according to the counting result, inoculate the cells into culture bottles or dishes according to (2-3)×10 4 / cm 2 Inoculate the cells into culture bottles or dishes, add umbilical cord mesenchymal stem cell culture medium, and place in an incubator for culture, with the incubator parameters being 37℃ and 5% CO2; (2) After 24 hours of cell culture, discard the original culture medium and replace it with the above-mentioned mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients or the above-mentioned preparation method to obtain the mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients, and place in an incubator for culture, with the incubator parameters being 37℃ and 5% CO2; (3) When the cell confluence reaches more than 90%, recover the cells.

[0015] Further, the mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients needs to be warmed to 37℃ before use.

[0016] Further, the use amount of the mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients is 0.2mL / cm 2 .

[0017] The beneficial effects of this invention are that stem cells possess anti-inflammatory and immunomodulatory abilities, promote collagen synthesis and remodeling, and stimulate angiogenesis. Adding American ginseng extract and Ganoderma lucidum spore extract to the basal culture medium, with active ingredients such as ginsenosides, triterpenes, polysaccharides, and sterols, demonstrates significant advantages in reducing oxidative damage, optimizing the microenvironment, activating endogenous repair, and supporting energy metabolism. After induced culture, these substances significantly enhance the cells' anti-inflammatory and immunomodulatory abilities, promote collagen synthesis and remodeling, and stimulate angiogenesis, providing a foundation for stem cell research. Attached Figure Description

[0018] Figure 1 These are morphological images of routinely cultured A-group P3~P6 generation umbilical cord mesenchymal stem cells.

[0019] Figure 2 These are morphological images of P3-P6 generation umbilical cord mesenchymal stem cells from group B obtained using the method of this invention.

[0020] Figure 3 This is a graph showing the expansion curves of P3-P6 generation umbilical cord mesenchymal stem cells from Group B using the method of this invention and Group A using the conventional culture method.

[0021] Figure 4 This is a graph showing the viability of P3-P6 generation umbilical cord mesenchymal stem cells from group B, which uses the method of this invention, and group A, which uses the conventional culture method.

[0022] Figure 5 This is a cell surface marker detection image of routinely cultured A-group P3~P6 generation umbilical cord mesenchymal stem cells.

[0023] Figure 6 This is a cell surface marker detection image of P3-P6 generation umbilical cord mesenchymal stem cells in group B using the method of this invention.

[0024] Figure 7 These are osteogenic induction staining images of group B (using the method of this invention) and group A (using the conventional culture method) at generation P6.

[0025] Figure 8 These are staining images of P6 generation chondrogenic induction in group B using the method of this invention and group A using the conventional culture method.

[0026] Figure 9 These are adipogenic induction staining images of group B, which was cultured using the method of this invention, and group A, which was cultured using the conventional method, at generation P6.

[0027] Figure 10 The diagram shows the factor secretion of groups B, D, and F using the method of this invention, and groups A, C, and E using the conventional culture method.

[0028] Figure 11The graph shows the oxidative stress (ATP and MDA concentrations) of groups B, D, and F using the method of this invention and groups A, C, and E using the conventional culture method.

[0029] Figure 12 The graphs show the oxidative stress (SOD enzyme activity) detection of groups B, D, and F using the method of this invention and groups A, C, and E using the conventional culture method. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] This invention provides a mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients, comprising a culture system consisting of a basal culture medium and additives. The basal culture medium is DMEM / F12, and the additives include fetal bovine serum, American ginseng extract, and Ganoderma lucidum spore extract. Preparation of the American ginseng extract: American ginseng powder, after being sterilized by irradiation, 1g of powder was added to 5mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant was collected. Preparation of Ganoderma lucidum spore extract: After sterilization by irradiation, 1g of Ganoderma lucidum spore powder is added to 15mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant is collected.

[0032] Furthermore, the culture system is DMEM / F12 containing 5%-15% fetal bovine serum, 0.5%-1% American ginseng extract, and 0.1%-0.5% Ganoderma lucidum spore extract.

[0033] Furthermore, in the preparation of the American ginseng extract and the Ganoderma lucidum spore extract: incubation at 37°C for 1 hour, followed by centrifugation at 3000g.

[0034] A method for preparing a mesenchymal stem cell culture medium containing traditional Chinese medicine components, comprising the following preparation steps: (1) Preparation of American ginseng extract: American ginseng powder, after being sterilized by irradiation, 1g of powder was added to 5mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant was collected; (2) Preparation of Ganoderma lucidum spore extract: After sterilization by irradiation, 1g of Ganoderma lucidum spore powder was added to 15mL of DMSO preheated to 37℃, incubated at 37℃, centrifuged, and the supernatant was collected. (3) Take the basic culture medium DMEM / F12, add fetal bovine serum at a volume fraction of 5%-15%, American ginseng extract at a volume fraction of 0.5%-1%, and Ganoderma lucidum spore extract at a volume fraction of 0.1%-0.5%; mix thoroughly and store under cold.

[0035] Furthermore, in the preparation of the American ginseng extract and the Ganoderma lucidum spore extract: incubation at 37°C for 1 hour, followed by centrifugation at 3000g.

[0036] Furthermore, the refrigerated storage temperature is 0-8℃.

[0037] A method for culturing mesenchymal stem cells includes the following steps: (1) Take umbilical cord mesenchymal stem cells, resuspend the cell pellet in umbilical cord mesenchymal stem cell culture medium, filter, count, and according to the counting results, divide according to (2-3)×10 4 / cm 2 Inoculation amount: Inoculate cells into culture flasks or culture dishes, add umbilical cord mesenchymal stem cell culture medium, and place them in an incubator with incubator parameters of 37℃ and 5% CO2; (2) After culturing the cells for 24 hours, discard the original culture medium and replace it with the mesenchymal stem cell culture medium containing traditional Chinese medicine components or the mesenchymal stem cell culture medium containing traditional Chinese medicine components prepared by the above preparation method. Place the cells in an incubator with the parameters of 37℃ and 5% CO2. (3) When the cell fusion rate reaches 90% or more, the cells are recovered.

[0038] Furthermore, the mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients needs to be warmed to 37°C before use.

[0039] Furthermore, the amount of mesenchymal stem cell culture medium containing traditional Chinese medicine components used is 0.2 mL / cm³. 2 .

[0040] This invention involves adding American ginseng extract and Ganoderma lucidum spore extract to a basal culture medium. After reaching a certain degree of confluence with conventional culture medium, the medium is replaced with this new medium, resulting in cultured MSCs with enhanced anti-inflammatory, repair-promoting, and angiogenesis-promoting capabilities. Studies have found that MSCs possess anti-inflammatory and immunomodulatory properties, promote collagen synthesis and remodeling, and stimulate angiogenesis. Furthermore, the main active components of American ginseng extract and Ganoderma lucidum spore extract, such as ginsenosides, triterpenes, polysaccharides, and sterols, exhibit significant advantages in reducing oxidative damage, optimizing the microenvironment, activating endogenous repair, and supporting energy metabolism. This invention adds American ginseng extract and Ganoderma lucidum spore extract to the MSC basal culture system, altering the MSC culture conditions and promoting their anti-inflammatory, repair-promoting, and angiogenesis-promoting capabilities.

[0041] This invention is used to cultivate functionally enhanced MSCs that have anti-inflammatory and immunomodulatory effects, promote collagen synthesis and remodeling, and stimulate angiogenesis, thereby improving MSC function.

[0042] Example 1 (1) Take P2 generation umbilical cord mesenchymal stem cells, resuspend the cell pellet in conventional umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 10% fetal bovine serum (Excell / FSP500)), filter, count, and according to the counting results, divide according to 2×10 4 / cm 2 The cells were seeded into two T-25 culture flasks, A and B (P3 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0043] (2) Preparation of mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients Preparation of American ginseng extract: American ginseng powder, after being sterilized by irradiation, 1g of powder was added to 5mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Preparation of Ganoderma lucidum spore extract: After sterilization by irradiation, 1g of Ganoderma lucidum spore powder was added to 15mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Take the basic culture medium DMEM / F12 (Gibco / C11330500BT), add fetal bovine serum at a volume fraction of 10%; add American ginseng extract at a volume fraction of 0.8%; add Ganoderma lucidum spore extract at a volume fraction of 0.3%; mix thoroughly and warm to 37℃ before use.

[0044] (3) After 24 hours of cell culture, take group B cells, discard the original culture medium, add 5 mL of the culture medium prepared in step (2) that has been warmed to 37°C (DMEM / F12 culture medium (Gibco / C11330500BT), containing 10% fetal bovine serum (Excell / FSP500), 0.8% American ginseng extract, and 0.3% Ganoderma lucidum spore extract), and put it into an incubator for culture; the incubator parameters are 37°C and 5% CO2.

[0045] (4) After culturing A and B cells for 3 days, when the cell confluence reached 90%, the supernatant was discarded. 1 mL of Lyple digestion solution (Gibco / 12563029) was added to each flask to digest the cells, causing them to detach from the bottom of the culture flask. The cells were then collected into separate 50 mL centrifuge tubes. After centrifugation at 300 g for 8 minutes, the supernatant was discarded. The cells were resuspended in standard umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 10% fetal bovine serum (Excell / FSP500)) and filtered into new centrifuge tubes. Cells were counted (see Table 1) and cultured at 2 × 10⁻⁶. 4 / cm 2 The cells were seeded into four T-25 culture flasks, labeled A1, A2, B1, and B2 (P4 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0046] (5) Repeat steps (3)-(4) until P6 generation cells are recovered; recover the supernatant and cells, and perform relevant tests. The main test indicators are: Cell morphology ( Figures 1-2 ); Cell expansion curve, cell viability ( Figures 3-4 ); Flow cytometry was used to determine the expression levels of CD73, CD90, CD105, CD34, and CD45. Figures 5-6 ); Identification of cell differentiation into adipocytes, osteoblasts, and chondrocytes ( Figures 7-9 ;) Detection of EGF and VEGF secretion ( Figure 10 ); Detection of IL-10 and PGE2 secretion ( Figure 10 ); Oxidative stress detection (including ATP detection, SOD detection, and MDA detection) Figures 11-12 ).

[0047] Example 2 (1) Take P2 generation umbilical cord mesenchymal stem cells, resuspend the cell pellet in conventional umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 5% fetal bovine serum (Excell / FSP500)), filter, count, and according to the counting results, divide according to 2×10 4 / cm 2 The cells were seeded into two T-25 culture flasks, designated as C and D (P3 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0048] (2) Preparation of mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients Preparation of American ginseng extract: American ginseng powder, after being sterilized by irradiation, 1g of powder was added to 5mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Preparation of Ganoderma lucidum spore extract: After sterilization by irradiation, 1g of Ganoderma lucidum spore powder was added to 15mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Take the basal culture medium DMEM / F12 (Gibco / C11330500BT), add fetal bovine serum at a volume fraction of 5%; add American ginseng extract at a volume fraction of 0.5%; add Ganoderma lucidum spore extract at a volume fraction of 0.1%; mix thoroughly and warm to 37℃ before use.

[0049] (3) After 24 hours of cell culture, take group B cells, discard the original culture medium, add 5 mL of the culture medium prepared in step (2) that has been warmed to 37°C (DMEM / F12 medium (Gibco / C11330500BT), containing 5% fetal bovine serum (Excell / FSP500), 0.5% American ginseng extract, and 0.1% Ganoderma lucidum spore extract), and put it into an incubator for culture; the incubator parameters are 37°C and 5% CO2.

[0050] (4) After culturing A and B cells for 3 days, when the cell confluence reached 90%, the supernatant was discarded. 1 mL of Lyple digestion solution (Gibco / 12563029) was added to each flask to digest the cells, causing them to detach from the bottom of the culture flask. The cells were then collected into separate 50 mL centrifuge tubes. After centrifugation at 300 g for 8 minutes, the supernatant was discarded. The cells were resuspended in standard umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 5% fetal bovine serum (Excell / FSP500)) and filtered into new centrifuge tubes. Cells were counted (see Table 1) and cultured at 2 × 10⁻⁶ cells / mL. 4 / cm 2 The cells were seeded into four T-25 culture flasks, labeled C1, C2, D1, and D2 (P4 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0051] (5) Repeat steps (3)-(4) until P6 generation cells are recovered; recover the supernatant and perform relevant tests. The main test indicators are: Detection of EGF and VEGF secretion (Figure 10 ); Detection of IL-10 and PGE2 secretion ( Figure 10 ); Oxidative stress detection (including ATP detection, SOD detection, and MDA detection) Figures 11-12 ).

[0052] Example 3 (1) Take P2 generation umbilical cord mesenchymal stem cells, resuspend the cell pellet in conventional umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 15% fetal bovine serum (Excell / FSP500)), filter, count, and according to the counting results, divide according to 3×10 4 / cm 2 The cells were seeded into two T-25 culture flasks, designated as E and F (P3 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0053] (2) Preparation of mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients Preparation of American ginseng extract: American ginseng powder, after being sterilized by irradiation, 1g of powder was added to 5mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Preparation of Ganoderma lucidum spore extract: After sterilization by irradiation, 1g of Ganoderma lucidum spore powder was added to 15mL of DMSO (WAK / WAK-DMSO-70) preheated to 37℃, incubated at 37℃ for 1 hour, centrifuged at 3000g, and the supernatant was collected. Take the basic culture medium DMEM / F12 (Gibco / C11330500BT), add fetal bovine serum at a volume fraction of 15%; add American ginseng extract at a volume fraction of 1%; add Ganoderma lucidum spore extract at a volume fraction of 0.5%; ⑥ mix thoroughly and warm to 37℃ before use.

[0054] (3) After 24 hours of cell culture, take group B cells, discard the original culture medium, add 5 mL of the culture medium prepared in (2) that has been warmed to 37℃ (DMEM / F12 culture medium (Gibco / C11330500BT), containing 15% volume fraction fetal bovine serum (Excell / FSP500), 1% volume fraction American ginseng extract, and 0.5% volume fraction Ganoderma lucidum spore extract), and put it into an incubator for culture; the incubator parameters are 37℃ and 5% CO2.

[0055] (4) After culturing A and B cells for 3 days, when the cell confluence reached 90%, the supernatant was discarded. 1 mL of Lyple digestion solution (Gibco / 12563029) was added to each flask to digest the cells, causing them to detach from the bottom of the culture flask. The cells were then collected into separate 50 mL centrifuge tubes. After centrifugation at 300 g for 8 minutes, the supernatant was discarded. The cells were resuspended in standard umbilical cord mesenchymal stem cell culture medium (DMEM / F12 medium (Gibco / C11330500BT), containing 15% fetal bovine serum (Excell / FSP500)) and filtered into new centrifuge tubes. Cells were counted (Table 1) and cultured at 3 × 10⁻⁶ cells / mL. 4 / cm 2 The cells were seeded into four T-25 culture flasks, labeled E1, E2, F1, and F2 (P4 generation). Each flask was supplemented with 5 mL of standard umbilical cord mesenchymal stem cell culture medium and placed in an incubator with the following parameters: 37°C and 5% CO2.

[0056] (5) Repeat steps (3)-(4) until P6 generation cells are recovered; recover the supernatant and perform relevant tests. The main test indicators are: Detection of EGF and VEGF secretion ( Figure 10 ); Detection of IL-10 and PGE2 secretion ( Figure 10 ); Oxidative stress detection (including ATP detection, SOD detection, and MDA detection) Figures 11-12 ).

[0057] Table 1

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mesenchymal stem cell culture medium containing a traditional Chinese medicine ingredient, characterized in that, The culture system comprises a basic medium and additives, the basic medium is DMEM / F12, and the additives comprise fetal bovine serum, American ginseng extract and ganoderma spore extract; The American ginseng extract is prepared as follows: after irradiation sterilization, 1 g of American ginseng powder is added into 5 mL of DMSO preheated to 37 DEG C, and then incubated at 37 DEG C, centrifuged, and the supernatant is taken; The ganoderma spore extract is prepared as follows: after irradiation sterilization, 1 g of American ginseng powder is added into 5 mL of DMSO preheated to 37 DEG C, and then incubated at 37 DEG C, centrifuged, and the supernatant is taken.

2. The mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients according to claim 1, characterized in that, The culture system is DMEM / F12 containing 5%-15% fetal bovine serum by volume fraction, 0.5%-1% American ginseng extract by volume fraction and 0.1%-0.5% ganoderma spore extract by volume fraction.

3. The mesenchymal stem cell culture medium containing traditional Chinese medicine ingredients according to claim 1, characterized in that, In the preparation of the American ginseng extract and the ganoderma spore extract, the incubation is performed at 37 DEG C for 1 hour, and the centrifugation is performed at 3000g.

4. A method for preparing a mesenchymal stem cell culture medium containing a traditional Chinese medicine ingredient, characterized in that, The preparation steps comprise: (1) American ginseng extract preparation: after irradiation sterilization, 1 g of American ginseng powder is added into 5 mL of DMSO preheated to 37 DEG C, and then incubated at 37 DEG C, centrifuged, and the supernatant is taken; (2) ganoderma spore extract preparation: after irradiation sterilization, 1 g of American ginseng powder is added into 5 mL of DMSO preheated to 37 DEG C, and then incubated at 37 DEG C, centrifuged, and the supernatant is taken; (3) DMEM / F12 is taken as the basic medium, and fetal bovine serum is added at a volume fraction of 5%-15%, American ginseng extract is added at a volume fraction of 0.5%-1%, and ganoderma spore extract is added at a volume fraction of 0.1%-0.5%; and the mixture is uniformly mixed and stored in a refrigerator.

5. The method of claim 4, wherein the medium for culturing mesenchymal stem cells containing the Chinese medicine ingredient is prepared by adding the Chinese medicine ingredient to the medium for culturing mesenchymal stem cells. In the preparation of the American ginseng extract and the ganoderma spore extract, the incubation is performed at 37 DEG C for 1 hour, and the centrifugation is performed at 3000g.

6. The method for preparing the mesenchymal stem cell culture medium containing traditional Chinese medicine components according to claim 4, characterized in that, The temperature of the storage in the refrigerator is 0-8 DEG C.

7. A method for culturing mesenchymal stem cells, characterized by, The steps comprise: (1) Take umbilical cord mesenchymal stem cells, resuspend the cell precipitate with umbilical cord mesenchymal stem cell culture medium, filter, count, and according to the counting result, inoculate (2-3) x 10 4 / cm 2 Inoculate the cells into a culture flask or culture dish, supplement with umbilical cord mesenchymal stem cell culture medium, and place in an incubator for culture. The incubator parameters are 37℃, 5% CO2. (2) after the cells are cultured for 24 hours, the original culture medium is discarded, and the mesenchymal stem cell culture medium containing traditional Chinese medicine components prepared by the preparation method of any one of claims 1-3 or the mesenchymal stem cell culture medium containing traditional Chinese medicine components of any one of claims 4-6 is added, and then the cells are cultured in a culture box, and the parameters of the culture box are 37 DEG C and 5% CO2; (3) when the cell fusion degree is greater than 90%, the cells are recovered.

8. The culture method of mesenchymal stem cells according to claim 7, wherein, Before use, the mesenchymal stem cell culture medium containing traditional Chinese medicine components needs to be warmed to 37 DEG C.

9. The culture method of claim 7, wherein the mesenchymal stem cells are derived from the umbilical cord blood. The amount of the mesenchymal stem cell culture medium containing the traditional Chinese medicine ingredient is 0.2 mL / cm 2 .

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