Adipose-derived stem cell culture medium containing wormwood extract and preparation method of adipose-derived stem cell culture medium

Abstract

The invention relates to an adipose-derived stem cell culture medium containing wormwood extract and a preparation method of the adipose-derived stem cell culture medium, and belongs to the technical field of biology. The adipose-derived stem cell culture medium contains a DMEM (Dulbecco Modified Eagle Medium) basal culture medium, and also contains the following components: 5 to 7 [mu] g / mL of wormwood extract, 1 to 1.5 [mu] g / mL of phosphatidylserine, 0.02 to 0.03 [mu] g / mL of D-calcium pantothenate, 1.2 to 1.5 [mu] g / mL of sialic acid, 0.06 to 0.08 [mu] g / mL of eucommia male flower peptide, 0.3 to 0.35 [mu] g / mL of transferrin and 0.3 to 0.4 mg / mL of bovine serum albumin. The prepared adipose-derived stem cell culture medium has a good promoting effect on proliferation of adipose-derived stem cells from rabbits and mice, the added wormwood polysaccharide extract has a good bacteriostatic effect, and the safety and stability of the culture medium can be effectively maintained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a culture medium for adipose stem cells containing Artemisia argyi extract and its preparation method. Background Technology

[0002] Stem cells are undifferentiated cells with high proliferative and differentiation potential. Under different induction conditions, they can differentiate into highly specialized somatic cells with different specific functions, that is, a type of cell with multi-lineage differentiation and self-replication capabilities. According to developmental stage, stem cells can be classified into embryonic stem cells and adult stem cells.

[0003] Recent studies have found that adipose-derived stem cells (ADSCs) can be isolated and cultured from adipose tissue. These cells can differentiate into cells from different germ layers, such as chondrocytes, osteoblasts, myocytes, nerve cells, and adipocytes. They possess self-renewal capacity and multi-lineage differentiation potential, similar to bone marrow mesenchymal stem cells. They can be obtained in large quantities from subcutaneous adipose tissue under local anesthesia and cultured in vitro. Due to their advantages of easy large-scale acquisition, readily available autologous material, and wide availability, they are an ideal seed cell for tissue engineering. Karyotype analysis shows that after multiple passages, human adipose-derived stem cells (ADSCs) still possess a normal diploid karyotype, which does not change with increasing passage number, indicating that ADSCs have genetic stability. Furthermore, ADSCs do not express major histocompatibility complex II or human leukocyte DR antigen, and when co-cultured with allogeneic peripheral blood cells, they do not stimulate a mixed lymphocyte response, making them suitable for allogeneic transplantation. Therefore, they have become a research hotspot in the medical field.

[0004] Existing methods for culturing adipose-derived stem cells are complex, yielding a small number of stem cells with low purity, resulting in slow proliferation and susceptibility to contamination. Therefore, it is necessary to develop a novel, suitable, and effective culture medium. Summary of the Invention

[0005] The main objective of this invention is to provide a novel adipose-derived stem cell culture medium, namely an adipose-derived stem cell culture medium containing Artemisia argyi extract and its preparation method. The culture medium provided by this invention can promote the proliferation of adipose-derived stem cells and can maintain the safety and stability of the culture medium for a long time.

[0006] The present invention employs the following technical solutions to achieve the above objectives: A culture medium for adipose-derived stem cells, containing DMEM basal medium and the following components: Artemisia argyi extract 5-7 μg / mL, phosphatidylserine 1-1.5 μg / mL, D-calcium pantothenate 0.02-0.03 μg / mL, sialic acid 1.2-1.5 μg / mL, Eucommia ulmoides male flower peptide 0.06-0.08 μg / mL, transferrin 0.3-0.35 μg / mL, and bovine serum albumin 0.3-0.4 mg / mL.

[0007] Preferably, the DMEM basal medium is a low-sugar DMEM basal medium.

[0008] Preferably, the mugwort extract is a mugwort polysaccharide extract.

[0009] Preferably, the preparation method of the Artemisia argyi polysaccharide extract includes the following steps: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Mix the Artemisia argyi powder with water, adjust the pH to 6.5-7.0, add Lactococcus lactis solution and mix well. Perform anaerobic fermentation at 30℃ for 10-12 hours. Then add 1L of 10% ethanol solution, heat and reflux for 2 hours, cool, and obtain the mixture for later use. Step 3: Adjust the pH of the mixture to 5.0, add cellulase, and enzymatically hydrolyze at 50℃ for 5-6 hours. Inactivate the enzyme at 90℃ for 10 minutes, cool, then adjust the pH to 7.5, add streptomycin, and enzymatically hydrolyze at 40℃ for 2-3 hours. Inactivate the enzyme at 90℃ for 10 minutes, cool, filter, and use the extract for later use. Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, adjust the pH to 5.0, add xylanase and hydrolyze at 50℃ for 1.5-2 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain the crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0010] More preferably, in step 2, the ratio of Artemisia argyi powder, water, Lactococcus lactis solution, and ethanol solution is 1:1:(0.01-0.012):1.

[0011] More preferably, the amount of cellulase added in step 3 is 2-3% of the weight of the Artemisia argyi powder.

[0012] More preferably, the amount of streptomycin added in step 3 is 1-1.5% of the weight of the Artemisia argyi powder.

[0013] More preferably, the amount of xylanase added in step 5 is 5-6% of the precipitate weight.

[0014] The present invention has the following beneficial effects: This invention provides a novel adipose-derived stem cell culture medium. Compared with traditional cell culture media, the medium provided by this invention can promote the proliferation of adipose-derived stem cells, and the cultured adipose-derived stem cells can maintain good stem cell morphology and have a better cell proliferation rate. In the medium of this invention, Artemisia argyi polysaccharide extract, sialic acid, Eucommia ulmoides male flower peptide, and other components are added to the traditional DMEM medium to promote cell regeneration, and the cultured adipose-derived stem cells have good cell performance. At the same time, the Artemisia argyi polysaccharide extract added to the medium of this invention has a good antibacterial effect and can be used as an antibacterial component when used in the adipose-derived stem cell culture medium to ensure the safety and stability of the medium. Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0016] Example 1 A fat stem cell culture medium containing low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 7 μg / mL, phosphatidylserine 1 μg / mL, D-calcium pantothenate 0.03 μg / mL, sialic acid 12 μg / mL, Eucommia ulmoides male flower peptide 0.08 μg / mL, transferrin 0.3 μg / mL, and bovine serum albumin 0.4 mg / mL.

[0017] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 7.0, then add 30 mL of Lactococcus lactis solution (the commercial strain CGMCC 1.15072 purchased from the China General Microbiological Culture Collection Center was activated according to the instructions and cultured until the effective viable count was approximately 1 × 10⁻⁶). 10 (CFU / mL), anaerobic fermentation at 30℃ for 12h, then 1L of 10% ethanol solution was added, heated and refluxed for 2h, cooled, and the mixture was used for later use; Step 3: Adjust the pH of the mixture to 5.0, add 30 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), hydrolyze at 50℃ for 5 h, inactivate the enzyme at 90℃ for 10 min, cool, then adjust the pH to 7.5, add 15 mg of streptomycin (CAS No. 9036-06-0, 7000 U / g), hydrolyze at 40℃ for 2 h, inactivate the enzyme at 90℃ for 10 min, cool, filter, and use the extract for later use; Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, add xylanase (CAS No. 9025-57-4, 300,000 U / g) at 6% of the precipitate weight, enzymatically hydrolyze at 50℃ for 1.5 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0018] Example 2 A fat stem cell culture medium containing low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 5 μg / mL, phosphatidylserine 1.5 μg / mL, D-calcium pantothenate 0.02 μg / mL, sialic acid 15 μg / mL, Eucommia ulmoides male flower peptide 0.06 μg / mL, transferrin 0.35 μg / mL, and bovine serum albumin 0.3 mg / mL.

[0019] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 6.5, then add 35 mL of Lactococcus lactis solution (the commercial strain CGMCC 1.15072 purchased from the China General Microbiological Culture Collection Center was activated according to the instructions and cultured until the effective viable count was approximately 1 × 10⁻⁶). 10 (CFU / mL), anaerobic fermentation at 30℃ for 10h, then 1L of 10% ethanol solution was added, heated and refluxed for 2h, cooled, and the mixture was used for later use; Step 3: Adjust the pH of the mixture to 5.0, add 20 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), hydrolyze at 50℃ for 6 h, inactivate the enzyme at 90℃ for 10 min, cool, then adjust the pH to 7.5, add 10 mg of streptomycin (CAS No. 9036-06-0, 7000 U / g), hydrolyze at 40℃ for 3 h, inactivate the enzyme at 90℃ for 10 min, cool, filter, and use the extract for later use; Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, add xylanase (CAS No. 9025-57-4, 300,000 U / g) at 5% of the precipitate weight, enzymatically hydrolyze at 50℃ for 2 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0020] Example 3 A fat stem cell culture medium containing low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 6 μg / mL, phosphatidylserine 1.3 μg / mL, D-calcium pantothenate 0.02 μg / mL, sialic acid 14 μg / mL, Eucommia ulmoides male flower peptide 0.07 μg / mL, transferrin 0.33 μg / mL, and bovine serum albumin 0.35 mg / mL.

[0021] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 7.0, then add 33 mL of Lactococcus lactis solution (the commercial strain CGMCC 1.15072 purchased from the China General Microbiological Culture Collection Center was activated according to the instructions and cultured until the effective viable count was approximately 1 × 10⁻⁶). 10 (CFU / mL), anaerobic fermentation at 30℃ for 11 h, then 1 L of 10% ethanol solution was added, heated and refluxed for 2 h, cooled, and the mixture was used for later use; Step 3: Adjust the pH of the mixture to 5.0, add 25 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), and hydrolyze at 50℃ for 5.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, then adjust the pH to 7.5, add 13 mg of streptomycin (CAS No. 9036-06-0, 7000 U / g), and hydrolyze at 40℃ for 2.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, filter, and reserve the extract. Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, add xylanase (CAS No. 9025-57-4, 300,000 U / g) at 6% of the precipitate weight, enzymatically hydrolyze at 50℃ for 1.5 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0022] Comparative Example 1 An adipose-derived stem cell culture medium containing a low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 6 μg / mL, phosphatidylserine 1.3 μg / mL, D-calcium pantothenate 0.02 μg / mL, transferrin 0.33 μg / mL, and bovine serum albumin 0.35 mg / mL.

[0023] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 7.0, then add 33 mL of Lactococcus lactis solution (the commercial strain CGMCC 1.15072 purchased from the China General Microbiological Culture Collection Center was activated according to the instructions and cultured until the effective viable count was approximately 1 × 10⁻⁶). 10 (CFU / mL), anaerobic fermentation at 30℃ for 11 h, then 1 L of 10% ethanol solution was added, heated and refluxed for 2 h, cooled, and the mixture was used for later use; Step 3: Adjust the pH of the mixture to 5.0, add 25 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), and hydrolyze at 50℃ for 5.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, then adjust the pH to 7.5, add 13 mg of streptomycin (CAS No. 9036-06-0, 7000 U / g), and hydrolyze at 40℃ for 2.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, filter, and reserve the extract. Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, add xylanase (CAS No. 9025-57-4, 300,000 U / g) at 6% of the precipitate weight, enzymatically hydrolyze at 50℃ for 1.5 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0024] Comparative Example 2 A fat stem cell culture medium containing low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 6 μg / mL, phosphatidylserine 1.3 μg / mL, D-calcium pantothenate 0.02 μg / mL, sialic acid 14 μg / mL, Eucommia ulmoides male flower peptide 0.07 μg / mL, transferrin 0.33 μg / mL, and bovine serum albumin 0.35 mg / mL.

[0025] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 5.0, add 25 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), and enzymatically hydrolyze at 50℃ for 5.5 h. Inactivate the enzyme by incubation at 90℃ for 10 min, cool, and then adjust the pH to 7.0. Add 1 L of 10% ethanol solution, heat and reflux for 2 h, cool, filter, and obtain the extract for later use. Step 3: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and obtain the Artemisia argyi polysaccharide extract.

[0026] Comparative Example 3 A fat stem cell culture medium containing low-glucose DMEM basal medium and the following components: Artemisia argyi polysaccharide extract 6 μg / mL, phosphatidylserine 1.3 μg / mL, D-calcium pantothenate 0.02 μg / mL, sialic acid 14 μg / mL, Eucommia ulmoides male flower peptide 0.07 μg / mL, transferrin 0.33 μg / mL, and bovine serum albumin 0.35 mg / mL.

[0027] The preparation method of the Artemisia argyi polysaccharide extract is as follows: Step 1: Crush commercially available mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Add 1 kg of Artemisia argyi powder to 1 L of water and mix well. Adjust the pH to 7.0, then add 33 mL of Lactococcus lactis solution (the commercial strain CGMCC 1.15072 purchased from the China General Microbiological Culture Collection Center was activated according to the instructions and cultured until the effective viable count was approximately 1 × 10⁻⁶). 10(CFU / mL), anaerobic fermentation at 30℃ for 11 h, then 1 L of 10% ethanol solution was added, heated and refluxed for 2 h, cooled, and the mixture was used for later use; Step 3: Adjust the pH of the mixture to 5.0, add 25 mg of cellulase (CAS No. 9012-54-8, 50 U / mg), and hydrolyze at 50℃ for 5.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, then adjust the pH to 7.5, add 13 mg of streptomycin (CAS No. 9036-06-0, 7000 U / g), and hydrolyze at 40℃ for 2.5 h. Inactivate the enzyme at 90℃ for 10 min, cool, filter, and reserve the extract. Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, add xylanase (CAS No. 9025-57-4, 300,000 U / g) at 6% of the precipitate weight, enzymatically hydrolyze at 50℃ for 1.5 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with molecular weights <8000 and >12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

[0028] Performance testing All the following experiments were conducted based on existing literature. All materials and reagents used in the following experiments are commercially available products.

[0029] I. Antibacterial effects of different Artemisia argyi polysaccharides The Artemisia argyi polysaccharides obtained in Examples 1, 2, and 3 were prepared into 1 mg / mL polysaccharide solutions. Filter paper was soaked in different polysaccharide solutions, streptomycin solution, and sterile water for 1 h. The culture broths of Staphylococcus aureus and Escherichia coli were evenly spread on the surface of the culture medium. The soaked filter paper was then placed in the culture medium and cultured at 37°C for 24 h. The diameter of the inhibition zone was measured.

[0030] Results and analysis: As can be seen from the results in Table 1, the polysaccharides obtained in Example 1 have good inhibitory effects on Staphylococcus aureus and Escherichia coli. The polysaccharides in Comparative Example 1 are obtained by ordinary extraction methods, and the polysaccharides in Comparative Example 2 are different from those in this invention, and their antibacterial effects are poor.

[0031] Table 1. Antibacterial properties of different Artemisia argyi polysaccharides II. Effects of different culture media on the proliferation of rabbit-derived adipose-derived stem cells 1. Sources and Cultivation After anesthetizing the rabbits, adipose tissue was removed from both sides of the abdominal groin, cut into granules, homogenized, and digested with 0.075% collagenase. After filtration, the tissue was centrifuged at 4°C and 1200 rpm for 5 min, and the upper fat and supernatant were discarded. The precipitated tissue was washed three times with phosphate-buffered saline (PBS), diluted with low-glucose DMEM medium containing 10% fetal bovine serum, centrifuged, and the precipitated tissue was removed.

[0032] The precipitated tissue was seeded into low-glucose DMEM medium containing 10% fetal bovine serum and incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The original culture medium was then discarded, and the cells were washed twice with PBS to remove adherent debris. DMEM medium containing 15% fetal bovine serum was then added and cultured again, with the medium changed twice weekly. When the flask walls were essentially confluent with cells, the cells were digested with 0.25% trypsin and passaged.

[0033] Select well-grown third-generation cells, digest them with 0.25% trypsin to prepare a cell suspension, and adjust the concentration to 2×10⁻⁶. 9 Cells in culture flasks were seeded into 96-well culture plates with different culture media and cultured for 24h, 48h, and 72h to investigate the effects of different culture media on the proliferation of rabbit adipose stem cells.

[0034] MTT assay for cell proliferation: After culturing for 24 h, 48 h, and 72 h, the concentration was adjusted to 5 × 10⁻⁶. 7 / L; Transfer each well of culture medium to a 96-well cell culture plate, with each well containing 100 μL of cell suspension. Add 20 μL of 5 mg / mL MTT solution to each well. Continue culturing at 37°C and 5% CO2 for 4 h. After terminating the culture, discard the supernatant. Add 150 μL of dimethyl sulfoxide to each well, vortex to mix thoroughly, and measure the absorbance (A) at 570 nm. Repeat the measurement and take the average value as the final result.

[0035] 2. Results and Analysis As shown in Table 2, compared with the traditional low-glucose DMEM medium, the medium prepared in Examples 1-3 can significantly promote the proliferation of rabbit adipose stem cells. Comparative Example 1 lacks sialic acid and Eucommia ulmoides male flower peptide. Although it can also promote cell proliferation, the effect is worse than that of Examples 1-3. The Artemisia argyi polysaccharide extract in Comparative Example 2 is obtained by traditional enzymatic hydrolysis, water extraction and alcohol precipitation. Its polysaccharide composition is complex and has little effect on the proliferation of adipose stem cells. It may even have an inhibitory effect. The Artemisia argyi polysaccharide extract in Comparative Example 3 has a different molecular weight than that in this invention. Its effect on the proliferation of adipose stem cells is not obvious.

[0036] Table 2. Effects of different culture media on the proliferation of rabbit adipose-derived stem cells Note: * indicates P < 0.05 compared to low-glucose DMEM medium.

[0037] III. Effects of different culture media on the proliferation of adipose-derived stem cells from SD rats 1. Sources and Cultivation After euthanizing SD rats, inguinal fat was separated, cut into granules, homogenized, and then digested with type I collagenase at 37°C for 60 min. After filtration, the cells were centrifuged at 1000 r / min for 10 min, resuspended in DMEM, and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cells reached 80% confluence, they were digested with trypsin and passaged.

[0038] Adipose-derived stem cells in the logarithmic growth phase were digested with trypsin, centrifuged, and the supernatant was discarded. 100 μL of a 2×10⁻⁶ concentration was collected. 10 Cell suspensions of 1 / L were transferred into 96-well plates, and 100 μL of different culture media were added to each well.

[0039] Cell proliferation capacity was observed using the CCK-8 assay. Cells from each group were cultured for 24, 48, and 72 hours. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1.5 hours. The OD value at 450 nm was measured using a microplate reader, and the OD value was used to represent cell proliferation capacity.

[0040] 2. Results and Analysis As can be seen from the results in Table 3, the culture media in Examples 1-3 have a better promoting effect on the proliferation of mouse adipose stem cells, which is better than the traditional low-glucose DMEM culture medium and the culture media in Comparative Examples 1-3. The overall data trend is the same as the above results of rabbit adipose stem cell culture.

[0041] Table 3 Effects of different culture media on the proliferation of mouse adipose-derived stem cells Note: * indicates P < 0.05 compared to low-glucose DMEM medium.

Claims

1. A culture medium for adipose-derived stem cells, characterized in that, It contains DMEM basal medium and the following ingredients: Artemisia argyi extract 5-7 μg / mL, phosphatidylserine 1-1.5 μg / mL, D-calcium pantothenate 0.02-0.03 μg / mL, sialic acid 1.2-1.5 μg / mL, Eucommia ulmoides male flower peptide 0.06-0.08 μg / mL, transferrin 0.3-0.35 μg / mL, and bovine serum albumin 0.3-0.4 mg / mL.

2. The adipose-derived stem cell culture medium as described in claim 1, characterized in that, The DMEM basal medium is a low-sugar DMEM basal medium.

3. The adipose-derived stem cell culture medium as described in claim 1, characterized in that, The mugwort extract is a mugwort polysaccharide extract.

4. The adipose-derived stem cell culture medium as described in claim 3, characterized in that, The preparation method of the Artemisia argyi polysaccharide extract includes the following steps: Step 1: Crush the mugwort into powder and pass it through an 80-mesh sieve to obtain mugwort powder; Step 2: Mix the Artemisia argyi powder with water, adjust the pH to 6.5-7.0, add Lactococcus lactis solution and mix well. Perform anaerobic fermentation at 30℃ for 10-12 hours. Then add 1L of 10% ethanol solution, heat and reflux for 2 hours, cool, and obtain the mixture for later use. Step 3: Adjust the pH of the mixture to 5.0, add cellulase, and enzymatically hydrolyze at 50℃ for 5-6 hours. Inactivate the enzyme at 90℃ for 10 minutes, cool, then adjust the pH to 7.5, add streptomycin, and enzymatically hydrolyze at 40℃ for 2-3 hours. Inactivate the enzyme at 90℃ for 10 minutes, cool, filter, and use the extract for later use. Step 4: Adjust the ethanol content of the extract to 30%, 50%, and 80% respectively. After each adjustment, let it stand for 2-3 hours, centrifuge, collect the precipitate, and set it aside for later use. Step 5: Redissolve the precipitate in water at a ratio of 1:10 g / mL, adjust the pH to 5.0, add xylanase and hydrolyze at 50℃ for 1.5-2 h, inactivate the enzyme at 90℃ for 10 min, cool, concentrate, and dry to obtain the crude extract. Step 6: After redissolving the crude extract in a small amount of water, load the sample onto a Sephadex G-100 dextran gel column and elute with 2% acetic acid solution. Analyze the eluent and retain the eluent containing polysaccharides with a molecular weight of 8000-12000. Combine the eluents and dry them to obtain Artemisia argyi polysaccharide extract.

5. The adipose-derived stem cell culture medium as described in claim 4, characterized in that, In step 2, the ratio of Artemisia argyi powder, water, Lactococcus lactis solution, and ethanol solution is 1:1:(0.01-0.012):1, expressed in kg / L / L / L.

6. The adipose-derived stem cell culture medium as described in claim 4, characterized in that, In step 3, the amount of cellulase added is 2-3% of the weight of the Artemisia argyi powder.

7. The adipose-derived stem cell culture medium as described in claim 4, characterized in that, In step 3, the amount of streptomycin added is 1-1.5% of the weight of the Artemisia argyi powder.

8. The adipose-derived stem cell culture medium as described in claim 4, characterized in that, In step 5, the amount of xylanase added is 5-6% of the precipitate weight.

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