A serum-free culture method for umbilical cord mesenchymal stem cells and its application

Abstract

The present invention relates to the field of cell technology, and more particularly to a serum-free culture method for umbilical cord mesenchymal stem cells and its application. The mesenchymal stem cells are cultured in a composite MSCs culture medium, wherein the composite MSCs culture medium uses DMEM / F12 culture medium as a base component and is supplemented with a traditional Chinese medicine fermentation preparation, wherein the raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 2-3 parts of epimedium, 1-2 parts of zedoary zedoary, 1-1.5 parts of millettia reticulata, 1.5-2.5 parts of cornus officinalis, 0.8-1.2 parts of red clover, and 2-3 parts of prunella vulgaris.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to a serum-free culture method for umbilical cord mesenchymal stem cells and an application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with multidirectional differentiation potential. These stem cells can self-replicate, self-renew, stimulate tissue growth and repair, and have low pollution sources, are easy to obtain, have stable biological properties, and are more primitive in immunogenicity. Under appropriate in vitro experimental conditions, they are easy to expand and preserve. Mesenchymal stem cells cause the proliferation of immune cells in the body by changing the cytokines secreted by T lymphocytes, B lymphocytes, natural killer cells, and dendritic cells. Therefore, mesenchymal stem cells have extremely broad application prospects in the field of cell therapy. Umbilical cord mesenchymal stem cells have a strong proliferation capacity and can be rapidly expanded. They are also not easy to age and have multidirectional differentiation potential. They have broad clinical application prospects in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium. Umbilical cord mesenchymal stem cells (UCMSCs) can be isolated from human umbilical cords and offer advantages such as easy access. They possess strong immunomodulatory properties, promoting hematopoietic recovery and repairing diseased tissues and organs. They hold great clinical value in organ transplantation, autoimmune diseases, leukemia, and bone and muscle degeneration. However, existing UCMSC culture media require the addition of animal serum, such as fetal bovine serum. Stem cells grown in this medium may experience unknown changes or mutations in their internal cell structure. Animal serum also carries the risk of triggering immune rejection and contamination with pathogens such as bacteria and viruses, hindering both clinical application and basic research. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the present invention provides a serum-free culture method of umbilical cord mesenchymal stem cells and its application.

[0004] The present invention is achieved through the following technical solutions:

[0005] A serum-free culture method for umbilical cord mesenchymal stem cells comprises the following steps:

[0006] S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and resuspended in composite MSCs culture medium. The seeding density was adjusted to 1×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0007] S2. When cells reach 80% confluency, use 0.25% trypsin-EDTA solution at a ratio of 1:3 for digestion and passage. Continue culturing in MSCs-complex medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0008] Furthermore, the composite MSCs culture medium uses DMEM / F12 culture medium as a basic component, and the DMEM / F12 culture medium also includes the following ingredients: PALL serum substitute 5v / v%, traditional Chinese medicine fermentation preparation 4 g / L, L-glutamine 10 mM, vitamin C 200 μM, transferrin 3 μg / mL, bFGF 10 ng / mL, insulin 12 μg / mL, β-mercaptoethanol 50 μM, and EGF 20 ng / mL.

[0009] Furthermore, the raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 2-3 parts of epimedium, 1-2 parts of zedoary zedoary, 1-1.5 parts of millettia reticulata, 1.5-2.5 parts of cornus officinalis, 0.8-1.2 parts of red clover, and 2-3 parts of selfheal.

[0010] Furthermore, the preparation method of the traditional Chinese medicine fermentation preparation comprises the following steps:

[0011] L1. Wash and dry Epimedium, Curcuma, Millettia reticulata, Cornus officinalis, red clover and Prunella vulgaris, grind through an 80-100 mesh sieve, mix with 10-15 times the weight volume of deionized water, soak for 1 h, ultrasonically treat at 80-90 ° C, 500-600W for 1 h, cool to room temperature, filter to obtain a residue and filtrate;

[0012] L2. Dry the filter residue obtained in step L1, pass it through an 80-100 mesh sieve, mix it with 10-15 times the weight volume of deionized water, and sterilize it by high pressure. 6 CFU / mL and 1×10 6 CFU / mL inoculated with activated Lactobacillus reuteri and Bifidobacterium adolescentis, fermented at 37°C for 48 h, and centrifuged at 5000-6000 rpm for 20 min to obtain supernatant I;

[0013] L3. The filtrate obtained in step L1 was sterilized by high pressure at 5×10 6 The activated Lactobacillus rhamnosus was inoculated at an inoculum size of 100 CFU / mL, fermented at 37°C for 48 h, centrifuged at 10,000 rpm for 10-15 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was added at 5 mg / mL, sterilized at 115°C for 30 min, cooled to 30°C, filtered through a titanium rod filter, and freeze-dried to obtain a traditional Chinese medicine fermentation preparation.

[0014] Furthermore, in step L2, the Lactobacillus reuteri and Bifidobacterium adolescentis were purchased from Guangdong Provincial Microbiological Culture Collection Center, with numbers GDMCC NO.1.614 and GDMCC NO.1.1262, respectively.

[0015] Furthermore, in step L3, the Lactobacillus rhamnosus was purchased from Guangdong Provincial Microbiological Culture Collection Center with a number of GDMCC NO.1.1732.

[0016] Furthermore, the present invention also provides the use of the umbilical cord mesenchymal stem cells in the preparation of a drug for treating thrombocytopenia.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a kind of umbilical cord mesenchymal stem cell serum-free culture method and application thereof, by the synergistic application of specific serum-free culture medium formula and Chinese medicine fermentation preparation, significantly improve the proliferation activity, immunomodulatory function and thrombocytopenia treatment effect of umbilical cord mesenchymal stem cells.Traditional culture system relies on fetal bovine serum, and there are risks such as pathogen contamination and immune rejection.The present invention is based on DMEM / F12, adds PALL serum substitute, and combines with specific components (including bFGF, EGF, transferrin etc.), constructs serum-free culture system.The present invention adds Chinese medicine fermentation preparation, and Chinese medicine epimedium, zedoary turmeric, millettia reticulata, cornus officinalis, red clover and selfheal are extracted with water, and the filter residue is fermented by Lactobacillus reuteri+Bifidobacterium adolescentis, and the filtrate is fermented by Lactobacillus rhamnosus, and the active substance in fermentation product synergizes with the serum substitute, growth factor and other components in culture medium, significantly improves cell viability, improves the culture effect of umbilical cord mesenchymal stem cells. After fermentation with probiotics, the effective ingredients in traditional Chinese medicine can release small molecule active substances that are more easily absorbed by cells. They combine with bacterial metabolites to improve the cellular microenvironment. The traditional Chinese medicine fermentation preparation works synergistically with each component to effectively enhance the proliferation capacity and biological activity of umbilical cord mesenchymal stem cells. The components of the traditional Chinese medicine fermentation preparation of the present invention synergize and enhance the effect. After the traditional Chinese medicine is fermented with Lactobacillus reuteri, Bifidobacterium adolescentis and Lactobacillus rhamnosus, a variety of bioactive substances are released to regulate the activity of umbilical cord mesenchymal stem cells. Moreover, after fermentation, the bioavailability of the traditional Chinese medicine ingredients is improved, which can promote the regulation of immune balance of umbilical cord mesenchymal stem cells, upregulate the anti-inflammatory factor IL-10, inhibit the pro-inflammatory factor IFN-γ, improve the immune imbalance of thrombocytopenia, and promote the recovery of platelet and spleen indexes. The traditional Chinese medicine fermentation preparation of the present invention adopts the "water extraction-two-stage fermentation" process. First, the filter residue is fermented, and Lactobacillus reuteri and Bifidobacterium adolescentis degrade plant fiber and release active substances. Then the filtrate is fermented, and Lactobacillus rhamnosus further metabolizes water-soluble components, stabilizes the structure of active substances, and at the same time enhances antioxidant capacity, thereby increasing the yield and bioavailability of active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The growth conditions of the mesenchymal stem cells described in Example 1 and Comparative Examples 1-3 of the present invention;

[0021] Figure 2 The effects of the mesenchymal stem cells described in Example 2 and Comparative Examples 1-3 of the present invention on the platelet levels in mice;

[0022] Figure 3 The effects of the mesenchymal stem cells described in Example 2 and Comparative Examples 1-3 of the present invention on the spleen index of mice;

[0023] Figure 4 This is the effect of the mesenchymal stem cells described in Example 3 of the present invention and Comparative Examples 1-3 on the cytokine levels in mice. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0025] Example 1: A serum-free culture method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0026] S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and resuspended in composite MSCs culture medium. The seeding density was adjusted to 1×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0027] S2. When cells reach 80% confluency, use 0.25% trypsin-EDTA solution at a ratio of 1:3 for digestion and passage. Continue culturing in MSCs-complex medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0028] The composite MSCs culture medium is based on DMEM / F12 medium, which also includes the following components: PALL serum replacement 5v / v%, traditional Chinese medicine fermentation preparation 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0029] The raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 3 parts of epimedium, 2 parts of zedoary turmeric, 1.5 parts of millettia reticulata, 2.5 parts of cornus officinalis, 1.2 parts of red clover, and 3 parts of selfheal.

[0030] The preparation method of the traditional Chinese medicine fermentation preparation comprises the following steps:

[0031] L1. Wash and dry 3 g of Epimedium, 2 g of Curcuma, 1.5 g of Millettia reticulata, 2.5 g of Cornus officinalis, 1.2 g of Red Medicago serrata, and 3 g of Prunella vulgaris. Grind through a 100-mesh sieve and mix thoroughly with 15 times the weight by volume of deionized water. Soak for 1 hour, sonicate at 90°C at 600 W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0032] L2. The filter residue obtained in step L1 was dried, passed through a 100-mesh sieve, mixed with 15 times the weight volume of deionized water, and sterilized by autoclave. 6 CFU / mL and 1×10 6 The activated Lactobacillus reuteri and Bifidobacterium adolescentis were inoculated with an inoculum size of 100 CFU / mL, fermented at 37°C for 48 h, and centrifuged at 6000 rpm for 20 min to obtain supernatant I. Lactobacillus reuteri and Bifidobacterium adolescentis were purchased from Guangdong Provincial Microbiological Culture Collection, with numbers GDMCC NO.1.614 and GDMCC NO.1.1262, respectively.

[0033] L3. The filtrate obtained in step L1 was sterilized by high pressure at 5×10 6 CFU / mL was inoculated with activated Lactobacillus rhamnosus, fermented at 37°C for 48 h, centrifuged at 10,000 rpm for 15 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was added at 5 mg / mL, sterilized at 115°C for 30 min, cooled to 30°C, filtered through a titanium rod filter, and freeze-dried to obtain a traditional Chinese medicine fermentation preparation; Lactobacillus rhamnosus was purchased from Guangdong Provincial Microbiological Culture Collection Center with the number GDMCC NO.1.1732.

[0034] Example 2: A serum-free culture method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0035] S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and resuspended in composite MSCs culture medium. The seeding density was adjusted to 1×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0036] S2. When cells reach 80% confluency, use 0.25% trypsin-EDTA solution at a ratio of 1:3 for digestion and passage. Continue culturing in MSCs-complex medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0037] The composite MSCs culture medium is based on DMEM / F12 medium, which also includes the following components: PALL serum replacement 5v / v%, traditional Chinese medicine fermentation preparation 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0038] The raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 2 parts of epimedium, 1 part of zedoaria, 1 part of millettia reticulata, 1.5 parts of cornus officinalis, 0.8 part of red clover, and 2 parts of selfheal.

[0039] Furthermore, the preparation method of the traditional Chinese medicine fermentation preparation comprises the following steps:

[0040] L1. Wash and dry 2 g of Epimedium, 1 g of Curcuma, 1 g of Millettia reticulata, 1.5 g of Cornus officinalis, 0.8 g of Red Medicago serrata, and 2 g of Prunella vulgaris. Grind through an 80-mesh sieve and mix thoroughly with 10 times the weight by volume of deionized water. Soak for 1 hour, sonicate at 80°C and 500W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0041] L2. Dry the filter residue obtained in step L1, pass it through an 80-mesh sieve, mix it with 10 times the weight volume of deionized water, and sterilize it by high pressure. 6 CFU / mL and 1×10 6 The activated Lactobacillus reuteri and Bifidobacterium adolescentis were inoculated with an inoculum size of 100 CFU / mL, fermented at 37°C for 48 h, and centrifuged at 5000 rpm for 20 min to obtain supernatant I. Lactobacillus reuteri and Bifidobacterium adolescentis were purchased from Guangdong Provincial Microbiological Culture Collection Center, with numbers GDMCC NO.1.614 and GDMCC NO.1.1262, respectively.

[0042] L3. The filtrate obtained in step L1 was sterilized by high pressure at 5×10 6 CFU / mL was inoculated with activated Lactobacillus rhamnosus, fermented at 37°C for 48 h, centrifuged at 10,000 rpm for 10 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was added at 5 mg / mL, sterilized at 115°C for 30 min, cooled to 30°C, filtered through a titanium rod filter, and freeze-dried to obtain a traditional Chinese medicine fermentation preparation; Lactobacillus rhamnosus was purchased from Guangdong Provincial Microbiological Culture Collection Center with the number GDMCC NO.1.1732.

[0043] Example 3: A serum-free culture method for umbilical cord mesenchymal stem cells, comprising the following steps:

[0044] S1. Umbilical cord mesenchymal stem cells were prepared by isolating umbilical cord tissue and resuspended in composite MSCs culture medium. The seeding density was adjusted to 1×10 6 cells / mL, cultured in a 5% CO2, 37°C incubator, with the medium changed every 2 days;

[0045] S2. When cells reach 80% confluency, use 0.25% trypsin-EDTA solution at a ratio of 1:3 for digestion and passage. Continue culturing in MSCs-complex medium at 5% CO2 and 37°C, changing the medium every 2 days.

[0046] The composite MSCs culture medium is based on DMEM / F12 medium, which also includes the following components: PALL serum replacement 5v / v%, traditional Chinese medicine fermentation preparation 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, and EGF 20ng / mL.

[0047] The raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 2.5 parts of epimedium, 1.5 parts of zedoaria, 1.2 parts of millettia reticulata, 2 parts of cornus officinalis, 1 part of red clover, and 2.5 parts of selfheal.

[0048] The preparation method of the traditional Chinese medicine fermentation preparation comprises the following steps:

[0049] L1. Wash and dry 2.5 g of Epimedium, 1.5 g of Curcuma, 1.2 g of Millettia reticulata, 2 g of Cornus officinalis, 1 g of Red Medicago, and 2.5 g of Prunella vulgaris. Grind through a 90-mesh sieve and mix thoroughly with 12 times the weight by volume of deionized water. Soak for 1 hour, sonicate at 85°C at 550W for 1 hour, cool to room temperature, and filter to obtain the residue and filtrate.

[0050] L2. The filter residue obtained in step L1 was dried, passed through a 90-mesh sieve, mixed with 12 times the weight volume of deionized water, and sterilized by autoclave. 6 CFU / mL and 1×10 6 The activated Lactobacillus reuteri and Bifidobacterium adolescentis were inoculated with an inoculum size of 100 CFU / mL, fermented at 37°C for 48 h, and centrifuged at 5500 rpm for 20 min to obtain supernatant I. Lactobacillus reuteri and Bifidobacterium adolescentis were purchased from Guangdong Provincial Microbiological Culture Collection, with numbers GDMCC NO.1.614 and GDMCC NO.1.1262, respectively.

[0051] L3. The filtrate obtained in step L1 was sterilized by high pressure at 5×106 CFU / mL was inoculated with activated Lactobacillus rhamnosus, fermented at 37°C for 48 h, centrifuged at 10,000 rpm for 12 min, the supernatant was mixed with the supernatant I obtained in step L2, and distilled under reduced pressure at 72°C and a vacuum degree of 800 mbar until no liquid continuously dripped out. Activated carbon for injection was added at 5 mg / mL, sterilized at 115°C for 30 min, cooled to 30°C, filtered through a titanium rod filter, and freeze-dried to obtain a traditional Chinese medicine fermentation preparation; Lactobacillus rhamnosus was purchased from Guangdong Provincial Microbiological Culture Collection Center with the number GDMCC NO.1.1732.

[0052] The only difference between Comparative Example 1 and Example 1 is that no Chinese medicinal fermentation preparation is added.

[0053] The difference between Comparative Example 2 and Example 1 is that the supernatant I obtained in step L2 is distilled under reduced pressure at 72 ° C and a vacuum degree of 800 mbar until no liquid continuously drips out, activated carbon for injection is added at 5 mg / mL, sterilized at 115 ° C for 30 min, cooled to 30 ° C, filtered through a titanium rod filter, and freeze-dried. The resulting product replaces the traditional Chinese medicine fermentation preparation.

[0054] The only difference between Comparative Example 3 and Example 1 is that the filtrate obtained in step L1 is sterilized by high pressure and heated to 5×10 6 Activated Lactobacillus rhamnosus was inoculated with an inoculum size of 100 CFU / mL, fermented at 37°C for 48 h, centrifuged at 10,000 rpm for 15 min, and the supernatant was distilled under reduced pressure at 72°C and 800 mbar vacuum until no liquid continuously dripped out. Injectable activated carbon was added at 5 mg / mL, sterilized at 115°C for 30 min, cooled to 30°C, filtered through a titanium rod filter, and freeze-dried. The resulting product was used to replace the traditional Chinese medicine fermentation preparation.

[0055] Experimental Example 1: The P3 generation umbilical cord mesenchymal stem cells obtained by culture in Example 1 and Comparative Examples 1-3 were collected and prepared into 1×10 4 A cell suspension of 100 μg / mL was inoculated into a 96-well plate, 200 μL per well, and placed in a 37°C, 5% CO2 incubator. Six wells were randomly selected 4 hours before the end of culture on days 1, 2, 3, 4, 5, 6, and 7, and 20 μL of 5 g / L MTT was added to each well. After the incubation period, 150 μL of DMSO solution was added to each well and shaken for 10 minutes. The absorbance (OD) of each well at 450 nm was measured using a microplate reader, and a growth curve was plotted. The results are shown in the figure. Figure 1 shown.

[0056] Figure 1The results showed that compared with Example 1, Comparative Examples 1-3 changed the components in the culture medium, and the cell proliferation rate showed a downward trend, lower than that of Example 1. The above results indicate that the Chinese medicinal fermentation preparation in the culture medium of Example 1 group works together with the various components in the culture medium to effectively enhance the proliferation capacity of umbilical cord mesenchymal stem cells.

[0057] Experimental Example 2: Female BALB / c mice (weight 20±2 g, 8 weeks old) were randomly divided into 6 groups: normal group (Nor), model group (Mod), Example 2 treatment group (Example 2), Comparative Example 1 treatment group (Comparative Example 1), Comparative Example 2 treatment group (Comparative Example 2), Comparative Example 3 treatment group (Comparative Example 3), with 10 mice in each group. Blood was collected from the eyeballs of BALB / c mice to collect platelets, which were suspended in physiological saline and adjusted to 1×10 9 FCA and FICA antigens were prepared by mixing the FCA (complete Freund's adjuvant) and FICA (incomplete Freund's adjuvant) with equal volumes of the FCA and FICA antigens, respectively. Guinea pigs (male, weighing 250 ± 20 g, 8 weeks old) were selected and acclimated for one week. At week 0, 1 mL of FCA antigen was injected subcutaneously into at least four sites on the paws, back, and abdomen of the guinea pigs. Separately, 1 mL of FICA antigen was injected subcutaneously into at least four sites on the paws, back, and abdomen of the guinea pigs at weeks 1, 2, 3, and 4. After each injection, the guinea pigs were disinfected with chloramphenicol to prevent infection. At week 5, the guinea pigs were anesthetized with 3% sodium barbital (30-45 mg / kg), and blood was collected from the heart. The supernatant was centrifuged at 3500 rpm for 10 minutes, and the supernatant was used as guinea pig anti-mouse platelet serum (GP-APS). The GP-APS was placed in a 56°C water bath for 30 min, an equal volume of 5% BALB / c mouse red blood cell suspension in normal saline was added, the mixture was incubated at 37°C for 1 h, centrifuged at 3500 r / min for 10 min, the supernatant was taken, an equal volume of 5% BALB / c mouse red blood cell suspension in normal saline was added, the mixture was incubated at 37°C for 1 h, centrifuged at 3500 r / min for 10 min, the supernatant was taken, and the GP-APS solution was obtained. The BALB / c mice in groups Mod, Example 2, and Comparative Examples 1-3 were intraperitoneally injected with GP-APS solution (10 μL / g body weight) on days 1, 3, 5, 7, 9, 11, and 13 to establish a mouse immune thrombocytopenia (ITP) model. On day 15, 2×10 6 The P4 umbilical cord mesenchymal stem cells were injected in a volume of 200 μL. On day 21, all mice were sacrificed and the spleen index and platelet concentration were measured. Figure 2 and Figure 3 shown.

[0058] Figure 2The results showed that the platelet level in the model group was significantly lower than that in the normal group, and the platelet levels in Example 2 and Comparative Examples 1-3 were significantly higher than those in the model group, with Example 2 showing better results than Comparative Examples 1-3. Figure 3 The results showed that the spleen index of the model group was significantly higher than that of the normal group, and the spleen index of Example 2 and Comparative Examples 1-3 groups was significantly lower than that of the model group, among which the effect of Example 2 group was better than that of Comparative Examples 1-3. The above results indicate that the present invention can improve the therapeutic effect of thrombocytopenia by adding a traditional Chinese medicine fermentation preparation to the culture medium to culture umbilical cord mesenchymal stem cells.

[0059] Experimental Example 3: Mice were modeled according to the method of Experimental Example 2 and divided into groups. The treatment group was injected with P4 umbilical cord mesenchymal stem cells of Example 3 and Comparative Examples 1-3. On the 21st day, the levels of IFN-γ and IL-10 in the peripheral blood serum of the mice were quantified using an enzyme-linked immunosorbent assay (ELISA) kit. The results are as follows: Figure 4 shown.

[0060] Figure 4 The results showed that the IFN-γ level in the model group was significantly higher than that in the normal group, while the IFN-γ levels in Example 3 and Comparative Examples 1-3 were significantly lower than those in the model group; the IL-10 level in the model group was significantly lower than that in the normal group, while the IL-10 levels in Example 3 and Comparative Examples 1-3 were significantly higher than those in the model group. The above results indicate that the umbilical cord mesenchymal stem cells prepared by the culture method of the present invention can promote IL-10, inhibit IFN-γ, improve ITP immune imbalance, and restore platelet levels.

[0061] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A serum-free culture method for umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: S1. Umbilical cord mesenchymal stem cells were isolated from umbilical cord tissue and cultured in a composite MSCs medium. S2. Umbilical cord mesenchymal stem cells were digested and passaged, and then cultured in the composite MSCs medium. The composite MSCs culture medium uses DMEM / F12 culture medium as a basic component, and the DMEM / F12 culture medium further includes the following components: PALL serum replacement 5v / v%, traditional Chinese medicine fermentation preparation 4g / L, L-glutamine 10mM, vitamin C 200μM, transferrin 3μg / mL, bFGF 10ng / mL, insulin 12μg / mL, β-mercaptoethanol 50μM, EGF 20ng / mL; The raw materials for preparing the traditional Chinese medicine fermentation preparation include the following components in parts by weight: 2-3 parts of epimedium, 1-2 parts of zedoary turmeric, 1-1.5 parts of millettia repens, 1.5-2.5 parts of cornus officinalis, 0.8-1.2 parts of red clover, and 2-3 parts of prunella vulgaris; The preparation method of the traditional Chinese medicine fermentation preparation comprises the following steps: L1 parts by weight of Epimedium, Curcuma, Millettia, Cornus officinalis, red clover and Prunella vulgaris water extraction and filtration to obtain a residue and a filtrate; L2 The residue obtained in step L1 was inoculated with Lactobacillus reuteri and Bifidobacterium adolescentis, fermented, and centrifuged to obtain a supernatant I, the Lactobacillus reuteri and Bifidobacterium adolescentis numbers are GDMCC NO.1.614 and GDMCC NO.1.1262; L3. The filtrate obtained in step L1 was inoculated with Lactobacillus rhamnosus, fermented, centrifuged, and the supernatant was mixed with the supernatant I obtained in step L2, distilled under reduced pressure, added with activated carbon, sterilized, filtered, and freeze-dried to obtain a traditional Chinese medicine fermentation preparation. The Lactobacillus rhamnosus strain is numbered GDMCC NO.1.1732.

2. Use of the umbilical cord mesenchymal stem cells obtained by the culture method according to claim 1 in the preparation of a drug for treating thrombocytopenia.

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