Aging mesenchymal stem cell as well as preparation method and application thereof
By adding 8 amino acids to the culture medium of mesenchymal stem cells and performing continuous replicative passages, the limitations and high cost of the chemically induced cell aging model in the prior art are solved, and a more realistic and economical cell Aging model.
Patent Information
- Application Number
- CN202411720086.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-02-18
AI Technical Summary
In the prior art, the chemically induced cell aging model has the problems of single mechanism, limited expression of aging markers and poor repetition, which cannot fully reflect the real aging process. At the same time, the number of human primary MSCs in vitro is limited, and they are prone to lose proliferation activity, and the cost is relatively high. high.
By adding 8 amino acids to the medium of mesenchymal stem cells, including tyrosine, leucine, isoleucine, phenylalanine, aspartic acid, threonine, serine and cysteine, Continuous replicative passages were performed to obtain aged mesenchymal stem cells.
This method can significantly improve the proliferation vitality of mesenchymal stem cells under the same culture algebra, extend iterable culture algebra, reduce costs, and show comprehensive multi-factor senescence characteristics in the later stage of culture, including the increase of cellular senescence markers and aging. Increase in related secretory phenotypes.
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Figure CN119464202A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of cell engineering and relates to aged mesenchymal stem cells and a preparation method and application thereof. Background Art
[0002] Aging is an inevitable process that occurs over time. It is a complex natural physiological phenomenon, which is manifested in the decline of body functions, weakened adaptability and resistance, etc. Aging has always been an important issue affecting the healthy development of the entire human society. The research on drugs to delay aging has become a hot topic and key issue in the field of life sciences.
[0003] Cellular senescence is an important manifestation of the overall aging of an organism, which is specifically manifested as the arrest of the cell cycle and the release of proinflammatory cytokines. At present, there are limited senescent cell models used for the evaluation of anti-aging drugs and the exploration of aging mechanisms, mainly using chemical drugs to induce aging. The specific method is to use sublethal doses of drugs such as doxorubicin, bleomycin and etoposide to induce cell DNA damage, thereby blocking the cell cycle and inducing cell senescence. There are many shortcomings in the chemically induced cell senescence model. The first is that the mechanism is single, and DNA damage is not the only driving factor of cell senescence. Secondly, there are limitations in the expression of aging markers. p16 and p21 are pathways that jointly mediate the process of cell aging and are independent of each other. Chemically induced aging models often cannot simultaneously reflect the activation of the two pathways of p16 and p21. Finally, the dosage and induction time of chemically induced drugs are often difficult to grasp, and are greatly affected by the state of the cells themselves, which leads to poor reproducibility of the model.
[0004] The inventors found that after multiple passages in vitro, the self-renewal ability of proliferation and differentiation of primary human mesenchymal stem cells (MSCs) will gradually be lost, and the widespread expression of aging markers will naturally appear. After multiple natural replication passages of MSCs in vitro, MSCs can show multi-factor driven aging characteristics. The natural aging of MSCs does not require drug induction, and the operation is simple and reproducible. Therefore, compared with chemically induced cell senescence, continuous replication and passage of human primary MSCs in vitro is a more ideal and realistic cell model for studying cell senescence. However, MSCs are limited in the number of passages in vitro and are prone to lose their proliferation activity. As a commonly used model for anti-aging drug screening and aging mechanism exploration, the cost is high and it is not convenient to prepare it into a commercial product. Summary of the invention
[0005] The object of the present invention is to provide a method for preparing aged mesenchymal stem cells in view of the above-mentioned deficiencies in the prior art.
[0006] Another object of the present invention is to provide aged mesenchymal stem cells prepared according to the method.
[0007] Another object of the present invention is to provide applications of the aged mesenchymal stem cells.
[0008] The purpose of the present invention can be achieved through the following technical solutions:
[0009] A method for preparing aged mesenchymal stem cells comprises the following steps: inoculating the mesenchymal stem cells in a modified D / F12 complete medium containing 10% fetal bovine serum and 100 mg / L penicillin and streptomycin, placing the cells in a CO2 cell culture box, culturing them at 5% CO2 and 37°C, digesting and passage the cells when the cell density reaches 90%, and obtaining the aged mesenchymal stem cells when the cells are cultured to the 12th generation; wherein the modified D / F12 complete medium is a D / F12 complete medium supplemented with composite amino acids, and the composite amino acids consist of 8 kinds of amino acids, including tyrosine, leucine, isoleucine, phenylalanine, aspartic acid, threonine, serine and cysteine.
[0010] As a preferred embodiment of the present invention, the final concentrations of the eight amino acids in D / F12 complete medium are: 44-47 mg / L tyrosine, 18-22 mg / L leucine, 2-3 mg / L isoleucine, 7-8 mg / L phenylalanine, 1-2 mg / L aspartic acid, 8-9 mg / L threonine, 5-6 mg / L serine and 6-7 mg / L cysteine.
[0011] As a further preferred embodiment of the present invention, the final concentrations of the eight amino acids in D / F12 complete medium are: 45.30 mg / L tyrosine, 19.68 mg / L leucine, 2.62 mg / L isoleucine, 7.09 mg / L phenylalanine, 1.11 mg / L aspartic acid, 8.02 mg / L threonine, 5.24 mg / L serine and 6.06 mg / L cysteine.
[0012] The aged mesenchymal stem cells prepared by the preparation method of the present invention.
[0013] The use of the aged mesenchymal stem cells of the present invention in preparing a mesenchymal stem cell aging model.
[0014] The senescent mesenchymal stem cells of the present invention are used as a mesenchymal stem cell aging model in screening drugs.
[0015] The senescent mesenchymal stem cells of the present invention are used as a mesenchymal stem cell aging model in screening therapeutic drugs for diseases caused by aging.
[0016] Beneficial effects:
[0017] At present, most of the commonly used senescent cell models are chemical drug-induced models. These models have the disadvantages of single mechanism, limited expression of senescent markers, poor repeatability, etc., and cannot fully reflect the real aging process. Compared with chemically induced cell senescence, human primary MSCs are continuously replicated and subcultured in vitro, which is a more ideal and realistic cell model for studying cell senescence. However, MSCs have a limited number of in vitro passages and are prone to lose proliferation activity, making them a common senescent cell model with high cost. In the present invention, it is found that mesenchymal stem cells containing 8 kinds of amino acids have higher proliferation activity than ordinary mesenchymal stem cells under the same culture generation, and can be iterated for more generations, which greatly reduces the culture cost of mesenchymal stem cells as senescent cell models, and can show comprehensive multi-factor aging characteristics in the later stage of culture, including the increase of cell senescence markers β-galactosidase, p53, p21, p16 and γH2Ax, the expression of Lamin B1 and pRb is reduced, the secretion of senescence-associated secretory phenotype (SASP) is increased, reactive oxygen species are accumulated, and mitochondrial membrane potential is reduced. In summary, the inventors proposed that mesenchymal stem cells containing 8 kinds of amino acids can be used as a new type of senescent cell model. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 .Effects of adding different compound amino acids after serial replicative passaging on the aging-associated secretory phenotype in mesenchymal stem cells.
[0019] Figure 2 .Changes in cell cycle markers in mesenchymal stem cells containing 8 kinds of amino acids and conventional mesenchymal stem cells that were continuously passaged under the same culture generations. A. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 7th generation. B. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 8th generation. C. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 9th generation. D. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 10th generation. E. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 11th generation. F. Western Blot detection of changes in aging markers p53 and p21 in AMSCs and MSCs of the 12th generation.
[0020] Figure 3 . Changes in senescence-related secretory phenotype and β-galactosidase in AMSCs of continuous passage. A. qPCR detection of senescence-related secretory phenotype content in AMSCs of continuous passage. B. Cell senescence β-galactosidase staining experiment detection of β-galactosidase content in AMSCs of continuous passage.
[0021] Figure 4 .Changes in reactive oxygen species and mitochondrial membrane potential in AMSCs during continuous passage. A. Detection of reactive oxygen species changes in AMSCs by live cell imaging. B. Quantitative graph of reactive oxygen species changes in AMSCs by live cell imaging. C. Detection of changes in mitochondrial membrane potential in AMSCs by JC-1 fluorescent probe.
[0022] Figure 5 .Changes in senescence markers in AMSCs of continuous passage. A. Western Blot detection of changes in senescence markers pRb, Lamin B1, p53, p21, and p16 in AMSCs of continuous passage. B. Quantitative graph of changes in senescence markers pRb, Lamin B1, p53, p21, and p16 in AMSCs of continuous passage detected by Western Blot. C. Immunofluorescence detection of changes in senescence marker γH2Ax in AMSCs of continuous passage. D. Quantitative graph of senescence marker γH2Ax in AMSCs of continuous passage detected by immunofluorescence. DETAILED DESCRIPTION
[0023] Example 1 Establishment of a mesenchymal stem cell continuous replication and passage aging model
[0024] MSC cells were cultured using D / F12 complete medium containing 10% fetal bovine serum and 100 mg / L penicillin and streptomycin to which multiple compound amino acids were added. The cells were cultured in a CO2 cell culture incubator (5% CO2, 37°C). When the cell density reached 90%, digestion and subculture were performed, and the cells were harvested at the 12th generation to obtain mesenchymal stem cells containing 6 amino acids, 8 amino acids, and 10 amino acids, respectively, for subsequent detection of aging markers. Among them, three different formulas of compound amino acids were added to D / F12 complete medium containing 10% fetal bovine serum and 100 mg / L penicillin and streptomycin, and replicated and subcultured synchronously with MSCs without the addition of amino acids.
[0025] Compound amino acid formula 1: Contains 6 kinds of amino acids. The final concentrations of the 6 kinds of amino acids in the culture medium are: 45.30mg / L tyrosine, 19.68mg / L leucine, 2.62mg / L isoleucine, 7.09mg / L phenylalanine, 1.11mg / L aspartic acid and 8.02mg / L threonine.
[0026] Compound amino acid formula 2: Contains 8 kinds of amino acids. The final concentrations of the 8 kinds of amino acids in the culture medium are: 45.30 mg / L tyrosine, 19.68 mg / L leucine, 2.62 mg / L isoleucine, 7.09 mg / L phenylalanine, 1.11 mg / L aspartic acid, 8.02 mg / L threonine, 5.24 mg / L serine and 6.06 mg / L cysteine.
[0027] Compound amino acid formula 3: contains 10 kinds of amino acids. The final concentrations of the 10 kinds of amino acids in the culture medium are: 45.30 mg / L tyrosine, 19.68 mg / L leucine, 2.62 mg / L isoleucine, 7.09 mg / L phenylalanine, 1.11 mg / L aspartic acid, 8.02 mg / L threonine, 5.24 mg / L serine, 6.06 mg / L cysteine, 1.875 mg / L glycine and 20.91 mg / L arginine.
[0028] Example 2 qRT-PCR experiment to detect SASP secreted by mesenchymal stem cell aging model
[0029] This experiment strictly followed the instructions of FastPure Cell / Tissue Total RNA Isolation Kit V2 to extract RNA. III RT SuperMix for qPCR and Taq Pro Universal SYBR qPCR Master Mix instructions for RNA reverse transcription and fluorescence quantification.
[0030] ① RNA extraction: cells were lysed with RNA isolater Total RNA Extraction Reagent, RNA was extracted by chloroform and isopropanol precipitation, washed with 75% ethanol, and then dissolved with DEPC water. RNA concentration and purity were detected by Nano drop and homogenized.
[0031] ② Reverse transcription: Prepare a mixed solution (20 μL system) in an RNase-free centrifuge tube, including template RNA (1 μg), 4×g DNA wiper Mix (4 μL), and RNase-free water (fixed to 16 μL), incubate at 42°C for 2 min, and add 4 μL III qRT SuperMix, mix by pipetting, incubate at 37°C for 15 min, and then at 85°C for 5 s.
[0032] ③ Fluorescence quantitative PCR reaction:
[0033] The fluorescence quantitative reaction system (20 μL) was prepared as shown in the following table.
[0034]
[0035] The qPCR reaction was performed according to the following procedure.
[0036]
[0037] ④ The results used the cell's own GAPDH as the internal reference and 2-△△Ct The relative expression level of the target gene in the cells was calculated by this method.
[0038] like Figure 1 As shown, the qPCR results showed that at the same culture generation in the later stage of MSCs culture, the pro-inflammatory factors Il1b, Ccl2, Tnf, and Il1a secreted by mesenchymal stem cells (AMSCs) containing 8 kinds of amino acids were significantly reduced.
[0039] Example 3 AOPI staining to detect cell viability of different stem cells at the same culture generation
[0040] Prepare cell suspensions from cells of different generations and dilute them appropriately to 1*10 6 ml, take an appropriate amount of cell suspension into a test tube, add AOPI dye, mix well, and use a cell counter to count live and dead cells respectively under the fluorescence channel to detect cell viability.
[0041] Conventional MSCs will grow slowly, age and stop growing after in vitro subculture. As shown in Table 1, under the same culture generation, the cell viability of AMSCs is higher than that of MSCs, especially after generation p10, indicating that AMSCs have higher proliferation activity and can be iteratively cultured for more generations.
[0042] Table 1. Cell viability of two mesenchymal stem cells at the same culture generation
[0043]
[0044] Note: AMSC represents the mesenchymal stem cells containing 8 kinds of amino acids prepared in Example 1, and MSC represents conventional mesenchymal stem cells; the same below.
[0045] Example 4 Western Blot detection of changes in senescence markers of mesenchymal stem cells at different generations ① Extraction of total cell protein: Digest, centrifuge, resuspend and count the cells in the logarithmic growth phase, and then use 1×10 6 The cells were inoculated into 6-well plates at a cell density of 100 cells / well. After 24 hours of complete cell adhesion, the old culture medium was removed on ice, and the cells were washed twice with pre-cooled PBS solution. Pre-cooled cell lysis solution containing proteasome inhibitors and phosphatase inhibitors was added to the wells. The cells were completely scraped off with a cell scraper, and the cell lysis solution was transferred to an EP tube and placed on ice for 30 minutes. The cells were vortexed every 15 minutes to complete the lysis. Centrifuge at 14000rpm / min and 4℃ for 30 minutes, and the supernatant was taken for subsequent experiments.
[0046] ②BCA protein quantification and protein denaturation: Use the BCA kit of Biyuntian for protein quantification, add different volumes of bovine serum albumin (BSA) or 2μL of the sample to be tested to a 96-well plate, and add PBS solution to make the final volume of each well 20μL. Prepare BCA working solution according to the volume ratio of BCA reagent A to BCA reagent B of 50:1, add 200μL of BCA working solution to each well, incubate at 60℃ for 30min, measure the absorbance at 562nm using an enzyme reader, draw a concentration-absorbance standard curve based on the absorbance of different concentrations of standard proteins, calculate the actual concentration of sample protein on the standard curve based on the absorbance of the sample to be tested, and calculate the loading volume of each sample based on the same protein loading amount. Take an appropriate amount of protein sample and add 5× Loading Buffer to mix evenly, then place the sample in 95℃ boiling water for denaturation for 10min, and load the sample after the sample returns to room temperature or place it in a -20℃ refrigerator for subsequent experiments.
[0047] ③ Gel preparation and electrophoresis: Prepare 6% concentrated gel and 8% separation gel according to the formula of the gel preparation kit, pour the solution into the glass plate, insert the comb teeth, and slowly pull out the comb teeth after the gel solidifies. Soak the gel in the electrophoresis solution for loading, add pre-stained standard protein Markers on both sides of the sample, and keep the sample loading amount consistent in each well. Constant voltage 65V concentrated electrophoresis for 30 minutes, adjust the voltage to 100V when bromophenol blue runs to the separation line between the concentrated gel and the separation gel, and continue electrophoresis for about 60 minutes until a clear target band appears in the gel (based on the Marker judgment), then stop the electrophoresis.
[0048] ④ Transfer and block: After the electrophoresis is completed, the protein bands separated by gel electrophoresis are transferred to the PVDF membrane by wet transfer. Cut the PVDF membrane of appropriate size and put it into methanol for activation. At the same time, soak the sponge pad and filter paper in the "sandwich" device in the transfer solution precooled at 4°C. Soak the gel in the transfer solution for a while, then lay the sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad on the negative plate in turn, cover the positive plate, install the "sandwich" device and put it into the transfer tank, put the entire transfer device in an ice water bath, turn on the power, constant current 300mA, transfer for 100min. After the transfer is completed, soak the PVDF membrane in 5% skim milk powder prepared in TBST, and slowly shake it on a shaker at room temperature for 1h to block it.
[0049] ⑤ Incubate primary and secondary antibodies: Remove the blocking solution, add specific primary antibodies diluted with 5% skim milk powder, and incubate overnight in a 4°C refrigerator. Recover the primary antibody, wash the PVDF membrane with TBST buffer at room temperature on a shaker for 5 min × 5 times. Add secondary antibodies diluted with 5% skim milk powder, incubate at 37°C for 1 hour, and then wash with TBST buffer at room temperature on a shaker for 5 min × 5 times.
[0050] ⑥ Chemiluminescence: Mix ECL luminescent solution A and solution B in a 1:1 ratio, evenly drip onto the PVDF membrane, and place it in a chemical imager for exposure. Set the exposure time according to the exposure requirements of the target protein, and collect and save the image.
[0051] ⑦ Protein band image analysis: Image J software was used to perform semi-quantitative analysis on the grayscale value of protein bands, and GraphPad Prism 8 was used to draw graphs.
[0052] During stem cell aging, the expression of cell cycle-related proteins increases, eventually leading to cell cycle arrest and growth stagnation. Figure 2 As shown in A-2F, Western Blot results show that the expression of aging markers p53 and p21 in AMSCs is lower than that in conventional mesenchymal stem cells starting from the 9th generation, indicating that AMSCs have higher proliferation activity and can be cultured for more generations.
[0053] Example 5
[0054] Studies have reported that during cell aging, the secretion of the senescence-associated secretory phenotype (SASP) increases, and the accumulation of senescence-associated β-galactosidase (SA-β-Gal) in cells increases.
[0055] 5.1 The SASP secreted by the mesenchymal stem cell aging model was detected according to the qRT-PCR experimental method of Example 2.
[0056] 5.2 Cell senescence β-galactosidase staining experiment to detect the β-galactosidase content in the mesenchymal stem cell aging model
[0057] The cells were inoculated into a total of 6-well plates. After 24 hours of complete attachment of the cells, the cell culture medium was removed, and the cells were washed once with PBS or HBSS. 1 ml of β-galactosidase staining fixative was added and fixed at room temperature for 15 minutes. The cell fixative was removed and the cells were washed 3 times with PBS or HBSS for 3 minutes each time. The PBS or HBSS was removed and 1 ml of β-galactosidase staining working solution was added to each well. Incubate at 37°C overnight. The 6-well plate can be sealed with plastic wrap to prevent evaporation. Anti-fluorescence quenching agent was added, and then the intracellular β-galactosidase content was recorded with an optical microscope. Three areas were randomly selected from each well for photography (20X).
[0058] like Figure 3 As shown in A, the qPCR results showed that at the 12th generation, the secretion of pro-inflammatory factors Il1b, Ccl2, and Tnf by AMSCs increased significantly, while the immunosuppressive / anti-inflammatory cytokine Tgfb1 decreased significantly. Figure 3As shown in B, the cell senescence β-galactosidase staining experiment showed that the β-galactosidase level in AMSCs increased significantly at the 12th generation.
[0059] Example 6
[0060] 6.1 Detection of ROS content in mesenchymal stem cell aging model by live cell imaging
[0061] The cells were inoculated into a total of 6-well plates. After 24 hours of complete cell adhesion, the cell culture medium was removed, 1 mL of diluted DCFH-DA probe was added, and the probe was loaded in situ in a cell culture incubator at 37°C for 20 minutes. The cells were then washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells, and the cells were immediately observed and photographed under a fluorescence microscope, and 3 areas were taken for photography (20X).
[0062] 6.2 Detection of mitochondrial membrane potential in mesenchymal stem cell aging model using JC-1 fluorescent probe staining in living cells
[0063] The cells were seeded into a total of 6-well plates. After 24 hours of complete cell adhesion, the cell culture medium was removed, and 1 ml of JC-1 staining working solution was added and mixed thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes. Add 1 mL of diluted DCFH-DA probe and incubate in a cell culture incubator at 37°C for 20 minutes to load the probe in situ. Then remove the supernatant and wash twice with JC-1 staining buffer. Add 2 ml of cell culture medium, which may contain serum and phenol red. Immediately observe and take pictures under a fluorescence microscope, and take 3 areas for shooting (20X).
[0064] Studies have shown that during the process of cell aging, the level of intracellular oxidative stress increases, the release of reactive oxygen species (ROS) increases, mitochondrial dysfunction occurs, and membrane potential decreases. Figure 4 A and Figure 4 As shown in B, the live cell imaging results showed that the level of ROS in the cytoplasm of AMSCs increased at passage 12. Figure 4 C shows that the JC-1 fluorescent probe staining results showed that the mitochondrial membrane potential of AMSCs decreased at the 12th generation.
[0065] Example 7 Immunofluorescence detection of γH2AX content, a senescence marker in mesenchymal stem cell aging models
[0066] The cells were inoculated into confocal culture dishes. After 24 hours, the cells were completely attached to the wall and the culture medium was removed. The cells were washed with pre-cooled PBS at room temperature for 5 min × 3 times on a shaker. A 4% paraformaldehyde fixative was added to each dish and the cells were fixed on a shaker at room temperature for 30 min. The fixative was removed and the cells were washed with pre-cooled PBS at room temperature for 5 min × 3 times on a shaker. A PBS solution containing 0.5% Triton X-100 was added to each dish to permeabilize the cell membrane and the cells were permeabilized on a shaker at room temperature for 20 min. A 3% BSA prepared in PBS was added to each dish for blocking and the cells were blocked on a shaker at room temperature for 1 h. A primary antibody diluted in BSA was added to each dish and incubated overnight at 4°C. The primary antibody was removed and the cells were washed with PBS at room temperature for 5 min × 3 times on a shaker. A BSA-diluted Alexa Fluor 500 was added to each dish in the dark. 594 goat anti-rabbit secondary antibody was incubated on a shaker at room temperature in the dark for 1 hour; the secondary antibody was removed and the samples were washed with PBS at room temperature for 5 minutes × 3 times on a shaker; DAPI staining solution was added to each dish and incubated on a shaker at room temperature in the dark for 20 minutes; DAPI was removed and the samples were washed with PBS at room temperature for 5 minutes × 3 times on a shaker; anti-fluorescence quencher was added, and then the intracellular protein expression was observed with a confocal microscope, and 3 areas were taken for photography (63X).
[0067] During cell senescence, the expression of cell cycle-related proteins p53, p21, and p16 increases, while the expression of pRb decreases, ultimately leading to cell cycle arrest, thinning of the nuclear membrane of senescent cells, decreased expression of Lamin B1, and the appearance of heterochromatin γH2AX, a marker of DNA damage. Figure 5 As shown in A and 5B, Western Blot results showed that at the 12th passage, the expressions of senescence markers p53, p21, and p16 were upregulated, while pRb and Lamin B1 were significantly downregulated. Figure 5 As shown in C and 5D, immunofluorescence results showed that the expression of senescence marker γH2Ax was upregulated and the cells had DNA damage at passage 12.
Claims
1. A method for preparing aged mesenchymal stem cells, characterized in that: Mesenchymal stem cells are inoculated in a modified D / F12 complete medium containing 10% fetal bovine serum and 100 mg / L penicillin and streptomycin, placed in a CO2 cell culture incubator, cultured at 5% CO2 and 37°C, and digested and passaged when the cell density reaches 90%. The aged mesenchymal stem cells are obtained when the culture reaches the 12th generation; wherein the modified D / F12 complete medium is a D / F12 complete medium supplemented with complex amino acids, and the complex amino acids consist of 8 kinds of amino acids, including tyrosine, leucine, isoleucine, phenylalanine, aspartic acid, threonine, serine and cysteine.
2. The preparation method according to claim 1, characterized in that: The final concentrations of the eight amino acids in the D / F12 complete medium are: 44-47 mg / L tyrosine, 18-22 mg / L leucine, 2-3 mg / L isoleucine, 7-8 mg / L phenylalanine, 1-2 mg / L aspartic acid, 8-9 mg / L threonine, 5-6 mg / L serine and 6-7 mg / L cysteine.
3. The preparation method according to claim 2, characterized in that: The final concentrations of the eight amino acids in the D / F12 complete medium are: 45.30 mg / L tyrosine, 19.68 mg / L leucine, 2.62 mg / L isoleucine, 7.09 mg / L phenylalanine, 1.11 mg / L aspartic acid, 8.02 mg / L threonine, 5.24 mg / L serine and 6.06 mg / L cysteine.
4. Aged mesenchymal stem cells prepared according to the preparation method according to any one of claims 1 to 3.
5. Use of the aged mesenchymal stem cells according to claim 4 in preparing a mesenchymal stem cell aging model.
6. Use of the aged mesenchymal stem cells according to claim 4 as a mesenchymal stem cell aging model in screening drugs.
7. Use of the aged mesenchymal stem cells according to claim 4 as a mesenchymal stem cell aging model in screening therapeutic drugs for diseases caused by aging.