A method for culturing liver stem cells

Abstract

The application discloses a liver stem cell culture method, which comprises the following steps: obtaining liver tissue cells by separation, inoculating the cells in a culture medium, and obtaining proliferated liver stem cells after culture, wherein the culture medium comprises dihydromyricetin-7-glucoside and ursolic acid methyl ester. Through experiments, it is found that by scientifically compounding dihydromyricetin-7-glucoside, ursolic acid methyl ester, ergotyl, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C and a liver-derived proliferation-promoting polypeptide in an RPMI-1640 basic culture medium, efficient in-vitro expansion and high-purity culture of liver stem cells are realized, and the cell activity of the target cells is effectively maintained.

Description

[Technical Field]

[0001] This invention relates to the field of cell culture technology, specifically to a method for culturing liver stem cells. [Background Technology]

[0002] The liver is a vital metabolic and detoxification organ in the human body, responsible for regulating glucose and lipid metabolism, protein synthesis, and the removal of various toxic substances. Liver dysfunction not only jeopardizes bodily homeostasis but is also closely related to many common diseases such as viral hepatitis, fatty liver, and drug-induced liver injury. The incidence of liver-related diseases continues to rise globally, severely impacting patients' health and quality of life. For patients with advanced liver damage, liver transplantation remains the primary treatment, but it is limited by donor scarcity, immune rejection, and high medical costs.

[0003] With advancements in regenerative medicine and tissue engineering, liver stem cells, due to their multi-directional differentiation potential, are considered ideal seed cells for liver tissue repair and disease treatment. The in vitro expansion and functional maintenance of liver stem cells has become a research hotspot for alternatives to liver transplantation, promoting liver regeneration, exploring the mechanisms of liver diseases, and developing new cell therapy approaches.

[0004] However, the in vitro culture of liver stem cells under current technology faces multiple challenges, and the results are not satisfactory. [Summary of the Invention]

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for culturing liver stem cells. To achieve the above objective, this invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for culturing liver stem cells, comprising isolating and obtaining liver tissue cells, seeding the cells in a culture medium, and obtaining proliferated liver stem cells after culture, wherein the culture medium comprises dihydromyricetin-7-glucoside and methyl ursolic acid.

[0007] Preferably, the culture medium includes RPMI-1640 basal culture medium, and further includes the following components: dihydromyricetin-7-glucoside: 15-30 μM, methyl ursolic acid: 60-80 μM, ergosterol benzyl ester: 50-200 mg / L, trehalose: 20-50 mg / L, EGF: 5-15 ng / mL, bFGF: 3-10 ng / mL, glutamine: 20-50 mg / L, albumin: 20-40 g / L, taurine: 50-200 mg / L, and vitamin C: 50-150 mg / L.

[0008] Preferably, the culture medium further includes 5-10 μg / mL of a liver-derived proliferative polypeptide, the amino acid sequence of which is Ac-CLGSPYRKLCETNKC-NH2, and is prepared by solid-phase synthesis.

[0009] Preferably, the culture conditions are: temperature of 37°C, CO2 concentration of 5%, medium change every 2-3 days, and culture for a total of 8-10 days.

[0010] Preferably, the cell seeding density is 1×10⁻⁶. 4 ~5×10 4 pcs / cm 2 .

[0011] The present invention differs from the prior art in that it achieves the following technical effects:

[0012] This invention, through experiments, demonstrates that by scientifically combining dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and liver-derived proliferative peptides in RPMI-1640 basal medium, efficient in vitro expansion and high-purity culture of liver stem cells were achieved, while effectively maintaining the cell viability of the target cells. [Attached Image Description]

[0013] Figure 1 This is a photograph of the morphological observation of liver stem cells cultured in the early stage of proliferation according to Example 1 of the present invention.

[0014] Figure 2 This is a photograph of the morphological observation of liver stem cells cultured in the mid-stage of proliferation according to Example 1 of the present invention.

[0015] Figure 3 This is a photograph of the morphological observation of liver stem cells cultured in the later stage of proliferation according to Example 1 of the present invention.

Detailed Implementation Methods

[0016] The technical solutions and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in textbooks and experimental guides, or according to the manufacturer's recommendations, which are well-known or readily available to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The following embodiments are merely preferred embodiments of the present invention and do not limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0017] Source of raw materials: intact fetuses 3-4 months after miscarriage, donated voluntarily by the provider with informed consent; the fetuses tested negative for hepatitis virus markers, syphilis, and HIV.

[0018] Example 1

[0019] Example 1 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0020] A method for culturing liver stem cells, the specific culturing process is as follows:

[0021] (1) Cell isolation:

[0022] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0023] (2) Cell inoculation and culture medium preparation:

[0024] Prepare the culture medium with the above components and proportions.

[0025] (3) Cultivation conditions:

[0026] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 9 days.

[0027] See cell morphology diagram. Figure 1-3 .

[0028] Example 2

[0029] Example 2 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 15 μM, methyl ursolic acid: 60 μM, ergosterol benzyl ester: 50 mg / L, trehalose: 20 mg / L, EGF: 5 ng / mL, bFGF: 3 ng / mL, glutamine: 20 mg / L, albumin: 20 g / L, taurine: 50 mg / L, and vitamin C: 50 mg / L.

[0030] A method for culturing liver stem cells, the specific culturing process is as follows:

[0031] (1) Cell isolation:

[0032] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0033] (2) Cell inoculation and culture medium preparation:

[0034] Prepare the culture medium with the above components and proportions.

[0035] (3) Cultivation conditions:

[0036] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 8 days.

[0037] Example 3

[0038] Example 3 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 30 μM, methyl ursolic acid: 80 μM, ergosterol benzyl ester: 200 mg / L, trehalose: 50 mg / L, EGF: 15 ng / mL, bFGF: 10 ng / mL, glutamine: 50 mg / L, albumin: 40 g / L, taurine: 200 mg / L, and vitamin C: 150 mg / L.

[0039] A method for culturing liver stem cells, the specific culturing process is as follows:

[0040] (1) Cell isolation:

[0041] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0042] (2) Cell inoculation and culture medium preparation:

[0043] Prepare the culture medium with the above components and proportions.

[0044] (3) Cultivation conditions:

[0045] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 10 days.

[0046] Example 4

[0047] Example 4 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and liver-derived proliferative peptide. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 8 μg / mL of liver-derived proliferative peptide.

[0048] The liver-derived proliferative polypeptide has the amino acid sequence Ac-CLGSPYRKLCETNKC-NH2, with an N-terminus acetylated and a C-terminus amidated, and contains cysteine ​​residues that can form disulfide bonds. It is prepared by solid-phase synthesis.

[0049] A method for culturing liver stem cells, the specific culturing process is as follows:

[0050] (1) Cell isolation:

[0051] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0052] (2) Cell inoculation and culture medium preparation:

[0053] Prepare the culture medium with the above components and proportions.

[0054] (3) Cultivation conditions:

[0055] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 9 days.

[0056] Example 5

[0057] Example 5 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 15 μM, methyl ursolic acid: 60 μM, ergosterol benzyl ester: 50 mg / L, trehalose: 20 mg / L, EGF: 5 ng / mL, bFGF: 3 ng / mL, glutamine: 20 mg / L, albumin: 20 g / L, taurine: 50 mg / L, vitamin C: 50 mg / L, and 5 μg / mL of liver-derived proliferative polypeptide.

[0058] Among them, the amino acid sequence of the liver-derived proliferative polypeptide is Ac-CLGSPYRKLCETNKC-NH2, which is prepared by solid-phase synthesis.

[0059] A method for culturing liver stem cells, the specific culturing process is as follows:

[0060] (1) Cell isolation:

[0061] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0062] (2) Cell inoculation and culture medium preparation:

[0063] Prepare the culture medium with the above components and proportions.

[0064] (3) Cultivation conditions:

[0065] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 9 days.

[0066] Example 6

[0067] Example 6 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 30 μM, methyl ursolic acid: 80 μM, ergosterol benzyl ester: 200 mg / L, trehalose: 50 mg / L, EGF: 15 ng / mL, bFGF: 10 ng / mL, glutamine: 50 mg / L, albumin: 40 g / L, taurine: 200 mg / L, vitamin C: 150 mg / L, and 10 μg / mL of liver-derived proliferative peptide.

[0068] Among them, the amino acid sequence of the liver-derived proliferative polypeptide is Ac-CLGSPYRKLCETNKC-NH2, which is prepared by solid-phase synthesis.

[0069] A method for culturing liver stem cells, the specific culturing process is as follows:

[0070] (1) Cell isolation:

[0071] Aseptically harvest liver tissue, rinse 3-5 times with PBS, and mince. Add the tissue pieces to a PBS mixture containing 0.02% (w / v) type I collagenase and 0.02% type II collagenase, and gently digest at 37°C for 30 min. After digestion, filter through a 200-mesh sieve and collect the filtrate. Centrifuge at 200g for 5 min, discard the supernatant, and wash the cell pellet twice with PBS. Count the cells using trypan blue staining and adjust the density to 3 × 10⁻⁶. 4 pcs / cm 2 .

[0072] (2) Cell inoculation and culture medium preparation:

[0073] Prepare the culture medium with the above components and proportions.

[0074] (3) Cultivation conditions:

[0075] Cultured at 37℃, 5% CO2, in a moist environment, with the medium changed every 2 days, for a total of 9 days.

[0076] Comparative Example 1

[0077] Comparative Example 1 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0078] The culture medium for Comparative Example 1 used dihydromyricetin instead of dihydromyricetin-7-glucoside, and the rest was the same as in Example 1.

[0079] Comparative Example 2

[0080] Comparative Example 2 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0081] The culture medium for Comparative Example 2 did not use methyl ursolic acid, but was otherwise the same as in Example 1.

[0082] Comparative Example 3

[0083] Comparative Example 3 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0084] The culture medium for Comparative Example 3 did not use ergobenzyl ester and trehalose, but was otherwise the same as in Example 1.

[0085] Comparative Example 4

[0086] Comparative Example 4 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 7 μM, methyl ursolic acid: 30 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0087] The culture medium for Comparative Example 4 had a reduced amount of dihydromyricetin-7-glucoside and methyl ursolic acid, but the rest was the same as in Example 1.

[0088] Comparative Example 5

[0089] Comparative Example 5 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, and vitamin C. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 45 μM, methyl ursolic acid: 130 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, and vitamin C: 94 mg / L.

[0090] The culture medium for Comparative Example 5 was modified by increasing the amounts of dihydromyricetin-7-glucoside and methyl ursolic acid, while the rest was the same as in Example 1.

[0091] Comparative Example 6

[0092] Comparative Example 6 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and liver-derived proliferative peptide. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 2 μg / mL of liver-derived proliferative peptide.

[0093] The culture medium in Comparative Example 6 had a reduced amount of liver-derived proliferative peptides, but the rest was the same as in Example 4.

[0094] Comparative Example 7

[0095] Comparative Example 7 provides a culture medium for liver stem cells, which consists of basal medium RPMI-1640 and the following components added to RPMI-1640: dihydromyricetin-7-glucoside, methyl ursolic acid, ergosterol benzyl ester, trehalose, EGF, bFGF, glutamine, albumin, taurine, vitamin C, and liver-derived proliferative peptide. The contents of the above components in RPMI-1640 are as follows: dihydromyricetin-7-glucoside: 21 μM, methyl ursolic acid: 73 μM, ergosterol benzyl ester: 108 mg / L, trehalose: 38 mg / L, EGF: 9 ng / mL, bFGF: 6 ng / mL, glutamine: 35 mg / L, albumin: 33 g / L, taurine: 129 mg / L, vitamin C: 94 mg / L, and 24 μg / mL of liver-derived proliferative peptide.

[0096] The culture medium in Comparative Example 7 had an increased amount of liver-derived proliferative peptide, while the rest was the same as in Example 4.

[0097] Experimental Test

[0098] (1) Cell proliferation rate

[0099] The number of liver stem cells obtained in Examples 1-6 and Comparative Examples 1-7 after the culture was completed was counted using a Countster cell counter, and the cell proliferation rate was calculated. All data were taken from three independent experiments, with each experiment being a three-well replicate. Data are expressed as mean ± standard deviation, and the results are shown in Table 1 below.

[0100]

[0101]

[0102] Analysis of Table 1 shows that the cell proliferation folds of Examples 4, 6, and 5 were significantly higher than those of all control groups; indicating that the combination of liver-derived proliferative peptides with dihydromyricetin-7-glucoside and methyl ursolic acid has a synergistic effect and can significantly enhance the in vitro expansion capacity of liver stem cells.

[0103] Although Examples 1, 3, and 2 did not contain liver-derived proliferative peptides, their proliferation rates were significantly better than those of the traditional comparative examples. This demonstrates that the combination of dihydromyricetin-7-glucoside and methyl ursolic acid, along with dosage optimization, plays a crucial role in promoting the proliferation of liver stem cells.

[0104] Although Comparative Example 6 contained liver-derived proliferative polypeptides, the proliferation effect was significantly lower than that of the optimal embodiment due to the low polypeptide dosage. In Comparative Examples 3, 4, 5, and 7, the cell proliferation capacity was inhibited or limited to varying degrees due to the absence of key components of the present invention or unreasonable dosage settings, further illustrating that the key components are indispensable and that there is a window effect in dosage.

[0105] Comparative Examples 1 and 2, respectively, lacked the key components of the present invention and had the weakest proliferation ability.

[0106] Therefore, based on the comparative results, it is further verified that the key components and dosage ratios in the formula selected in this invention are the optimal choices. They should not be reduced arbitrarily, nor should they be increased blindly, otherwise the cell proliferation effect will decrease.

[0107] (2) Cell purity analysis

[0108] Liver stem cells from Examples 4, 1, 1, 3, and 6 were collected, digested with 0.25% trypsin containing 1 mmol / L EDTA, centrifuged, and washed twice with PBS. The cells were then resuspended in PBS buffer and the cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6 The concentration of antibodies was measured at 100 μL / mL and dispensed into 100 μL tubes. FITC- or PE-labeled monoclonal antibodies against CD34, CD44, CD90, CD105, CD13, HLA-ABC, and HLA-DR were added, and the mixture was gently mixed and incubated at room temperature in the dark for 15–20 minutes. A negative control tube (without antibody, only an equal volume of PBS buffer) was prepared. After washing once with PBS, the tubes were resuspended in 400 μL of PBS and analyzed using a BD flow cytometer. Flow cytometry data were analyzed using software. Each group was repeated three times, and the mean ± standard deviation of the positivity rate for each antigen was calculated. The results are shown in Table 2 below.

[0109]

[0110] In Example 4, the expression levels of typical surface markers CD44, CD90, CD105, and CD13 in liver stem cells were the highest, while the expression levels of negative markers CD34 and HLA-DR were the lowest, significantly higher than in other examples and comparative groups. This fully demonstrates that the culture method of the present invention not only expands the number of cells but also significantly improves the phenotypic specificity and purity of stem cells. Although the key polypeptide was not added in Example 1, the stem cell phenotype of the cells was still high, significantly higher than in all comparative groups, highlighting the importance of the core small molecule combination (dihydromyricetin-7-glucoside + methyl ursolic acid) of the present invention. The stem cell phenotype of Comparative Example 3 was... The expression of stem cell markers was significantly reduced, while the expression of CD34 and HLA-DR was significantly increased, indicating that ergosterol and trehalose play an irreplaceable auxiliary role in maintaining cell stemness, and their absence significantly reduced cell purity. In Comparative Example 2, positive stem cell markers were further reduced, while CD34 and HLA-DR were significantly increased, indicating that methyl ursolic acid is crucial for maintaining stem cell phenotype. Comparative Example 1 showed the worst performance, with the lowest stem cell-specific markers and the highest CD34 and HLA-DR markers for differentiated or impure cells, showing that dihydromyricetin-7-glucoside is an essential key component for maintaining stem cell characteristics and cannot be arbitrarily replaced.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for culturing liver stem cells, comprising isolating liver tissue cells, inoculating the cells in a culture medium, and obtaining proliferated liver stem cells after culturing, characterized in that: The medium component comprises 15-30 μM of dihydromyricetin-7-glucoside and 60-80 μM of methyl ursolate.

2. The liver stem cell culture method according to claim 1, wherein: The medium comprises a basic medium RPMI-1640, and further comprises the following components: ergocryptine: 50-200 mg / L, trehalose: 20-50 mg / L, EGF: 5-15 ng / mL, bFGF: 3-10 ng / mL, glutamine: 20-50 mg / L, albumin: 20-40 g / L, taurine: 50-200 mg / L, vitamin C: 50-150 mg / L.

3. The liver stem cell culture method according to claim 2, wherein: The medium further comprises 5-10 μg / mL of a liver-derived proliferation-promoting polypeptide with an amino acid sequence of Ac-CLGSPYRKLCETNKC-NH2, which is prepared by a solid-phase synthesis method.

4. The liver stem cell culture method according to claim 1, wherein, The culture condition is that the temperature is 37℃, the CO2 concentration is 5%, the medium is replaced every 2-3 days, and the culture is performed for 8-10 days.

5. The liver stem cell culture method according to claim 1, wherein, The cell seeding density is 1 x 10 4 5 x 10 4 cm-2 2 .

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