Serum-free culture medium for maintaining dryness of mesenchymal stem cells as well as preparation method and application of serum-free culture medium

Abstract

The invention provides a serum-free medium for maintaining dryness of mesenchymal stem cells as well as a preparation method and application of the serum-free medium, and belongs to the technical field of biological medicines. The serum-free culture medium comprises a basic culture medium and an exogenous additive component, the basic culture medium is a DMEM / F12 culture medium, and the exogenous additive components comprise bFGF, EGF, PDGF-BB, recombinant human fibronectin, laminin, insulin, transferrin, sodium selenate, beta-mercaptoethanol and ascorbic acid. The serum-free culture medium provided by the invention is a special culture medium for the mesenchymal stem cells, which is completely free of animal-derived components, is suitable for large-scale amplification of clinical-grade cell treatment products, is relatively low in preparation cost and high in stem differentiation capability, and can remarkably improve the proliferation efficiency of the cultured mesenchymal stem cells.

Description

Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to a serum-free culture medium for maintaining the stemness of mesenchymal stem cells, its preparation method, and its application. Background Technology

[0003] Mesenchymal stem cell culture relies on fetal bovine serum (FBS), which has the following drawbacks: (1) batch-to-batch variability leads to unstable cell proliferation efficiency; (2) there is a risk of contamination from viruses and mycoplasma; (3) ethical and regulatory restrictions limit clinical translation; etc. Commercially available serum-free culture media, such as StemPro® MSC SFM, solve some of these problems, but they have drawbacks such as high cost (>$500 / L), insufficient maintenance of stemness, and decreased differentiation capacity after passage, which cause instability in the quality of cultured products.

[0004] In view of the shortcomings of the existing technology, there is an urgent need in the field for a special culture medium for mesenchymal stem cells (MSCs) that is completely free of animal-derived components, suitable for the large-scale expansion of clinical-grade cell therapy products, while solving the problems of high cost, decreased differentiation capacity after passage, and insufficient maintenance of stemness. Summary of the Invention

[0005] One of the objectives of this invention is to provide a serum-free culture medium for maintaining the stemness of mesenchymal stem cells. This culture medium is a mesenchymal stem cell-specific culture medium that is completely free of animal-derived components. It is suitable for the large-scale expansion of clinical-grade cell therapy products, has low preparation cost, and strong stemness differentiation ability.

[0006] The second objective of this invention is to provide a method for preparing a serum-free culture medium for maintaining the stemness of mesenchymal stem cells, thereby obtaining a serum-free culture medium that effectively maintains the cell proliferation efficiency of mesenchymal stem cells.

[0007] A third objective of this invention is to provide the application of the serum-free culture medium or the preparation method in the in vitro culture of mesenchymal stem cells.

[0008] The fourth objective of this invention is to provide the application of the serum-free culture medium or the preparation method in improving the proliferation efficiency of cultured mesenchymal stem cells.

[0009] The fifth objective of this invention is to provide a method for preparing mesenchymal stem cells based on the serum-free culture medium, thereby improving the proliferation efficiency of cultured mesenchymal stem cells.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a serum-free culture medium for maintaining the stemness of mesenchymal stem cells. The serum-free culture medium includes a basal culture medium and exogenous additives. The basal culture medium is DMEM / F12 medium, and the exogenous additives include bFGF, EGF, PDGF-BB, recombinant human fibronectin, laminin, insulin, transferrin, sodium selenate, β-mercaptoethanol, and ascorbic acid.

[0011] Preferably, the DMEM / F12 culture medium includes glucose at a concentration of 3.5~5.5 g / L and glutamine at a concentration of 2~4 mM; the mass ratio of bFGF, EGF and PDGF-BB in the exogenous added components is (7~9):(14~16):(9~11).

[0012] Preferably, the working concentrations of the exogenous additives in the serum-free culture medium are as follows: bFGF 7~9 ng / mL, EGF 14~16 ng / mL, PDGF-BB 9~11 ng / mL, recombinant human fibronectin 1~5 μg / mL, laminin 0.1~5 μg / mL, insulin 5~15 μg / mL, transferrin 5~15 μg / mL, sodium selenate 5~15 nM, β-mercaptoethanol 0.1~3 μM, and ascorbic acid 25~35 μM.

[0013] The present invention also provides a method for preparing the serum-free culture medium, wherein the preparation steps of the serum-free culture medium include dissolving the exogenous additive in the basal culture medium and mixing them evenly.

[0014] The present invention also provides the application of the serum-free culture medium or the preparation method in the in vitro culture of mesenchymal stem cells.

[0015] Preferably, the mesenchymal stem cells include human umbilical cord MSCs stem cells.

[0016] The present invention also provides the application of the serum-free culture medium or the preparation method in improving the proliferation efficiency of cultured mesenchymal stem cells.

[0017] Preferably, the mesenchymal stem cells include human umbilical cord MSCs stem cells.

[0018] The present invention also provides a method for preparing mesenchymal stem cells, wherein the mesenchymal stem cells are seeded into the serum-free culture medium for culture.

[0019] Preferably, the mesenchymal stem cells comprise human umbilical cord MSCs stem cells, and the seeding density of the human umbilical cord MSCs stem cells in the culture medium is 2 × 10⁻⁶. 4 ~2×10 5 / cm 2The culture temperature is 35~38℃, and the culture time is 12~48h.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a serum-free culture medium for maintaining the stemness of mesenchymal stem cells (MSCs). This medium is a completely animal-free culture medium specifically for MSCs, suitable for large-scale expansion of clinical-grade cell therapy products. It has low preparation cost, strong stemness differentiation capacity, and can significantly improve the proliferation efficiency of cultured MSCs. Optimized growth factor compatibility synergistically activates the PI3K / Akt pathway, thereby improving proliferation efficiency; and optimized glucose-glutamine ratio reduces lactate accumulation. The serum-free culture medium provided by this invention significantly improves the maintenance and proliferation capacity of MSCs. Furthermore, culturing MSCs using this serum-free culture medium for maintaining MSC stemness does not affect their osteogenic or adipogenic differentiation capacity; they maintain a normal karyotype after 10 passages, exhibiting good genomic stability; and are suitable for large-scale industrial production and in-depth development and utilization. Detailed Implementation

[0021] This invention provides a serum-free culture medium for maintaining the stemness of mesenchymal stem cells. The serum-free culture medium includes a basal culture medium and exogenous additives. The basal culture medium is DMEM / F12 medium, and the exogenous additives include bFGF, EGF, PDGF-BB, recombinant human fibronectin, laminin, insulin, transferrin, sodium selenate, β-mercaptoethanol, and ascorbic acid.

[0022] In this invention, the DMEM / F12 culture medium comprises glucose at a concentration of 3.5–5.5 g / L and glutamine at a concentration of 2–4 mM; preferably, the DMEM / F12 culture medium comprises glucose at a concentration of 4–5 g / L and glutamine at a concentration of 2.5–3.5 mM; more preferably, the DMEM / F12 culture medium comprises glucose at a concentration of 4.5 g / L and glutamine at a concentration of 3 mM. This invention optimizes the mass concentration ratio of glucose and glutamine, thereby reducing lactate accumulation and improving the proliferation efficiency of mesenchymal stem cells.

[0023] In this invention, the mass ratio of bFGF, EGF, and PDGF-BB in the exogenous additive components is (7~9):(14~16):(9~11). By optimizing the ratio of these growth factors, this invention achieves a synergistic effect, activating the PI3K / Akt pathway and significantly improving proliferation efficiency compared to a single growth factor.

[0024] The serum-free culture medium of the present invention further preferably contains the following working concentrations of the exogenous additives in the serum-free culture medium: bFGF 7~9 ng / mL, EGF 14~16 ng / mL, PDGF-BB 9~11 ng / mL, recombinant human fibronectin 1~5 μg / mL, laminin 0.1~5 μg / mL, insulin 5~15 μg / mL, transferrin 5~15 μg / mL, sodium selenate 5~15 nM, β-mercaptoethanol 0.1~3 μM, and ascorbic acid 25~35 μM; more preferably, bFGF 7.5~8.5 ng / mL, EGF 14.5~15.5 ng / mL, PDGF-BB 9.5~10.5 ng / mL, recombinant human fibronectin 2~4 μg / mL, laminin 0.5~3 μg / mL, insulin 6~10 μg / mL, and transferrin 6~10 μg / mL. The optimal composition is as follows: sodium selenate 6-10 nM, β-mercaptoethanol 0.1-1 μM, ascorbic acid 28-32 μM; most preferably, bFGF 8 ng / mL, EGF 15 ng / mL, PDGF-BB 10 ng / mL, recombinant human fibronectin 3 μg / mL, laminin 1 μg / mL, insulin 8 μg / mL, transferrin 8 μg / mL, sodium selenate 8 nM, β-mercaptoethanol 0.5 μM, and ascorbic acid 30 μM. In this invention, insulin, transferrin, and sodium selenate maintain energy homeostasis; β-mercaptoethanol and ascorbic acid scavenge ROS, protecting genomic stability; the synergistic effect of these components significantly improves the proliferation efficiency of mesenchymal stem cells in serum-free culture medium.

[0025] In this invention, the recombinant human fibronectin was purchased from Sigma (product number S5171), and the laminin was purchased from Thermo Fisher Scientific (product number A29249). It is primarily used to replace serum adhesion function, thereby improving the proliferation efficiency of mesenchymal stem cells in serum culture medium and ensuring the maintenance of stem differentiation capacity.

[0026] The present invention also provides a method for preparing the serum-free culture medium, wherein the preparation steps of the serum-free culture medium include dissolving the exogenous additive in the basal culture medium and mixing them evenly.

[0027] The present invention also provides the application of the serum-free culture medium or the preparation method in the in vitro culture of mesenchymal stem cells, wherein the mesenchymal stem cells preferably include human umbilical cord MSCs stem cells.

[0028] The present invention also provides the application of the serum-free culture medium or the preparation method in improving the proliferation efficiency of cultured mesenchymal stem cells, wherein the mesenchymal stem cells preferably include human umbilical cord MSCs stem cells.

[0029] The present invention also provides a method for preparing mesenchymal stem cells, wherein the mesenchymal stem cells are seeded into the serum-free culture medium for culture.

[0030] When preparing mesenchymal stem cells using the method described in this invention, the mesenchymal stem cells preferably include human umbilical cord MSCs stem cells, and the seeding density of the human umbilical cord MSCs stem cells in the culture medium is 2 × 10⁻⁶. 4 ~2×10 5 / cm 2 Further optimization of 2×10 4 / cm 2 The culture temperature is preferably 35~38℃, more preferably 36℃ or 37℃, and the culture time is preferably 12~48h, more preferably 18~36h, and more preferably any one of 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h or 35h.

[0031] This invention optimizes the combination of growth factors and synergistically activates the PI3K / Akt pathway, thereby improving proliferation efficiency to 31%. At the same time, the serum-free culture medium used in this invention to maintain the stemness of mesenchymal stem cells does not affect their osteogenic and adipogenic differentiation abilities; after 10 passages, they can still maintain a normal karyotype and have good genomic stability.

[0032] In the following examples, bFGF represents basic fibroblast growth factor, purchased from Sigma-Aldrich, catalog number GF003AF; EGF represents epidermal growth factor, purchased from Sigma-Aldrich, catalog number SRP3196; and PDGF-BB represents platelet-derived growth factor BB, purchased from Sigma-Aldrich, catalog number SRP3138.

[0033] In the examples below, the basal culture medium DMEM / F12 was purchased from Gibco, catalog number 111320033. Gibco™ StemFlex™ was purchased from Gibco, catalog number A3349401.

[0034] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0036] Example 1 A serum-free culture medium, wherein the components are accurately weighed and mixed according to the following proportions: Basic culture medium: DMEM / F12 (glucose concentration 4.5 g / L, glutamine concentration 3 mM), purchased from Gibco, catalog number 111320033; Exogenous added ingredients: bFGF 8 ng / mL, EGF 15 ng / mL, PDGF-BB 10 ng / mL, recombinant human fibronectin 3 μg / mL, laminin 1 μg / mL, insulin 8 μg / mL, transferrin 8 μg / mL, sodium selenate 8 nM, β-mercaptoethanol 0.5 μM, ascorbic acid 30 μM.

[0037] The preparation method is as follows: Dissolve the above-mentioned exogenous additives at the above concentration in 1L of DMEM / F12 basic culture medium containing glucose and glutamine.

[0038] Example 2 A serum-free culture medium, differing from Example 1 in that it contains the following exogenous components: bFGF 7 ng / mL, EGF 14 ng / mL, PDGF-BB 9 ng / mL, recombinant human fibronectin 1 μg / mL, laminin 0.1 μg / mL, insulin 5 μg / mL, transferrin 5 μg / mL, sodium selenate 5 nM, β-mercaptoethanol 0.1 μM, and ascorbic acid 25 μM.

[0039] Example 3 A serum-free culture medium, differing from Example 1 in that it contains the following exogenous components: bFGF 9 ng / mL, EGF 16 ng / mL, PDGF-BB 11 ng / mL, recombinant human fibronectin 5 μg / mL, laminin 5 μg / mL, insulin 15 μg / mL, transferrin 15 μg / mL, sodium selenate 15 nM, β-mercaptoethanol 3 μM, and ascorbic acid 35 μM.

[0040] Example 4 A serum-free culture medium, differing from Example 1 in that it contains the following exogenous components: bFGF 7.5 ng / mL, EGF 14.5 ng / mL, PDGF-BB 9.5 ng / mL, recombinant human fibronectin 2 μg / mL, laminin 0.5 μg / mL, insulin 6 μg / mL, transferrin 6 μg / mL, sodium selenate 6 nM, β-mercaptoethanol 0.1 μM, and ascorbic acid 28 μM.

[0041] Example 5 A serum-free culture medium, differing from Example 1 in that it contains the following exogenous components: bFGF 8.5 ng / mL, EGF 15.5 ng / mL, PDGF-BB 10.5 ng / mL, recombinant human fibronectin 4 μg / mL, laminin 3 μg / mL, insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenate 10 nM, β-mercaptoethanol 1 μM, and ascorbic acid 32 μM.

[0042] Comparative Example 1 DMEM / F12, containing 10% FBS, purchased from Gibco, product number A566970.

[0043] Comparative Example 2 A serum-free culture medium, which differs from Example 1 in that the exogenous added growth factors are only bFGF, without EGF and PDGF-BB.

[0044] Comparative Example 3 A serum-free culture medium, which differs from Example 1 in that the exogenous added growth factors are only EGF, without bFGF and PDGF-BB.

[0045] Comparative Example 4 A serum-free culture medium, which differs from Example 1 in that the exogenous added growth factors are only PDGF-BB, without bFGF and EGF.

[0046] Application Example 1 Comparison of mesenchymal stem cell growth and proliferation efficiency in different culture media The cryopreserved tubes containing human umbilical cord MSCs (P3 generation) stem cells were thawed from liquid nitrogen, dispersed in DMEM / F12 basal medium, and centrifuged to remove the cryopreservation solution. The stem cells were then resuspended in the culture media of Example 1 and Comparative Example 1, respectively, at an inoculation rate of 5 × 10⁶ cells / mL. 5 The stem cells were seeded in culture flasks and cultured at 37°C to obtain proliferated mesenchymal stem cells. After 24 hours of culture, the adhesion rate of the stem cells was observed and calculated.

[0047] The cells were stained with 0.4% trypan blue, and the number of viable cells and survival rate were calculated. The results are shown in Table 1.

[0048] Table 1. Results of mesenchymal stem cell growth and proliferation efficiency in different serum-free culture media.

[0049] Experimental data showed that in cell proliferation experiments, stimulation with single bFGF (Comparative Example 2), EGF (Comparative Example 3), and PDGF-BB (Comparative Example 4) resulted in cell proliferation efficiencies of 10%, 12%, and 15%, respectively. However, when bFGF, EGF, and PDGF-BB acted synergistically in a ratio of 8:15:10 (Example 1), their activation effect on the PI-3K-Akt pathway was significantly enhanced, with a cell proliferation efficiency as high as 31%. Compared with single-factor stimulation, synergistic stimulation increased the proliferation efficiency by more than 20%, fully demonstrating the powerful promoting effect of the synergistic effect of the three growth factors on cell proliferation.

[0050] Application Example 2 I. Detection of mesenchymal stem cell stemness maintenance capacity: Identification of surface markers in mesenchymal stem cells Stem cells cultured to 90% confluence in the culture medium of Example 1 and Comparative Example 1 were used to prepare a single-cell suspension of mesenchymal stem cells with a density of 5 × 10⁻⁶ cells / mL. 6 mL; antibodies CD73 / CD90 / CD105 were diluted 1:100 with FACS buffer containing 1% BSA; the following different groups were set up: Control group: 190 μL FACS buffer + 10 μL mesenchymal stem cell suspension; Single-stain group: CD73 Single Staining Group: Take 10 μL of cell suspension, add CD73 fluorescently labeled antibody diluted 1:100 with FACS buffer containing 1% BSA, and mix thoroughly. This group uses only CD73 antibody for staining to determine the staining effect of CD73 antibody on cells and the distribution characteristics of its fluorescence signal in flow cytometry detection, which will be used for compensation regulation in subsequent multi-antibody staining and analysis of single marker expression.

[0051] CD90 single staining group: Take 10 μL of cell suspension, add CD90 fluorescently labeled antibody diluted according to the above ratio, and mix well. This group of experiments can clarify the staining characteristics of CD90 antibody, eliminate mutual interference of fluorescence signals between different antibodies, and provide a basis for accurately analyzing the expression of CD90 markers on the cell surface.

[0052] CD105 single staining group: Take 10 μL of cell suspension and add CD105 fluorescently labeled antibody diluted proportionally. This group is used to evaluate the staining effect and fluorescence signal distribution of CD105 antibody, which helps to accurately determine the expression of CD105 markers on the cell surface.

[0053] Experimental group (co-stained with three surface fluorescently labeled antibodies): 10 μL of cell suspension was taken, and CD73, CD90, and CD105 fluorescently labeled antibodies diluted 1:100 with FACS buffer containing 1% BSA were added and mixed thoroughly. The aim was to detect the simultaneous expression of these three mesenchymal stem cell marker antigens (CD73, CD90, and CD105) on the cell surface, in order to comprehensively identify whether the cells are mesenchymal stem cells.

[0054] Each group was incubated on ice with shaking in the dark for 45 min, centrifuged at 1000 rpm for 5 min, washed twice with PBS, resuspended in pre-cooled buffer at 4°C, and then flow cytometry sorted. Target cells were transported on ice in medium containing 4% FBS, centrifuged at 1500 rpm, and then replaced with serum-free medium as in Example 1. The cells were then inoculated and cultured in a 37°C, 5% CO2 incubator.

[0055] The results of surface marker detection are shown in Table 2: The positive rates of CD73, CD90 and CD105 in human umbilical cord MSCs obtained by serum-free culture medium in Example 1 were ≥98% by flow cytometry.

[0056] Table 2 Positive rates of CD73, CD90, and CD105

[0057] II. Identification of multi-lineage differentiation of mesenchymal stem cells: Adipogenic differentiation was detected using an adipogenic differentiation kit (Gibco, catalog number A1007001), and the specific procedures were performed according to its instructions as follows: Human umbilical cord mesenchymal stem cells obtained from serum-free culture in Example 1 were cultured at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of [specific density] in 6-well plates and cultured until 100% confluence. The culture medium was discarded, and 2 mL of adipogenic differentiation induction medium A was added. After 3 days of induction, 2 mL of adipogenic differentiation induction medium B was added. After 24 hours of induction, the medium was replaced with solution A. This process of alternating between solutions A and B was repeated 3-5 times (12-20 days). Solution B was used to maintain the cells for 4-7 days until the lipid droplets became sufficiently large and round. During the maintenance culture period using solution B, fresh solution B was used every 2-3 days. After 7 days of adipogenic differentiation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and the fixative was discarded. After washing three times with PBS, 1 mL of Oil Red O working solution was added and stained for 30 minutes. The staining solution was discarded, the cells were washed three times with PBS, and observed under a microscope.

[0058] Osteogenic differentiation induction assay was performed using an osteogenic differentiation kit (Gibco, catalog number A1007201) following the instructions: Human umbilical cord mesenchymal stem cells cultured in serum-free culture as described in Example 1 were cultured at a rate of 5 × 10⁻⁶. 5 The cells were seeded at a density of 6-well plates and cultured until the cell confluence reached 60-70%. The complete mesenchymal stem cell culture medium (i.e., serum-free medium) was discarded, and 2 mL of osteogenic induction differentiation medium was added. Fresh complete mesenchymal stem cell osteogenic induction differentiation medium (preheated to 37℃) was added every 3 days, and alizarin staining was performed after 12 hours. Seven days after osteogenic induction differentiation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, the fixative was discarded, the cells were washed three times with PBS, and 1 mL of alizarin staining solution was added. The cells were incubated at room temperature for 3-5 min, the staining solution was discarded, the cells were washed three times with PBS, and observed under a microscope.

[0059] Based on the test results: Osteogenesis induction: The number of calcium nodules stained with alizarin red was 15% higher than that in the FBS group; Adipogenic induction: Two human umbilical cord MSCs stem cell samples obtained from serum-free culture medium in Example 1 were randomly divided into an experimental group and a control group, with three samples in each group. The experimental group underwent adipogenic induction differentiation treatment, while the control group did not. After adipogenic induction and Oil Red O staining, the lipid droplet area in the cells of the experimental and control groups was measured using image analysis software. Statistical software was used to analyze the measured lipid droplet area data, calculate the mean and standard deviation, and perform independent samples analysis. t The test was used to determine the statistical difference between the two groups.

[0060] Table 3 Results of lipogenic induction treatment

[0061] As shown in Table 3, the experimental results indicate that the oil red O staining lipid droplet area increased by 20% in the experimental group (Example 1) compared to the control group. "*" indicates a statistically significant difference compared to the control group. P <0.05) means that this increase in lipid droplet area is not caused by random factors, but is a real effect of the adipogenic induction treatment.

[0062] The results showed that culturing mesenchymal stem cells in the serum-free culture medium of the present invention, which maintains the stemness of mesenchymal stem cells, did not affect their osteogenic and adipogenic differentiation abilities.

[0063] III. Genomic stability testing: To verify the genomic stability of serum-free human umbilical cord mesenchymal stem cells passaged to P10 in Example 1, we conducted the following chromosome karyotype analysis system experiment after cell proliferation and passage.

[0064] Thaw the cryopreservation tubes containing human umbilical cord MSCs (P3 generation) stem cells from liquid nitrogen, disperse them in DMEM / F12 basal medium, centrifuge to remove the cryopreservation solution, and resuspend the stem cells in the medium described in Example 1 above, with an inoculum size of 5 × 10⁻⁶. 5 Inoculate into culture flasks containing serum-free culture medium from Example 1 and incubate at 37°C in a 5% CO2 incubator.

[0065] First, cell proliferation and passage are performed, starting from the initial culture (P0). After the cells grow to 90% confluence, they are passaged at a ratio of 1:3 (the volume ratio of cell suspension to cell suspension, specifically, the cell suspension in the original culture container is divided into 3 equal parts and passaged into 3 culture containers. The volume ratio of the new serum-free culture medium to the cell suspension is 1:10, until P10.

[0066] The yield of mesenchymal stem cells from different passages was recorded during passage, with specific data as follows: the yield of cells harvested from passage P0 was approximately 1.5 × 10⁻⁶. 6 The number of cells harvested from the P2 generation is approximately 3.2 × 10⁶. 6 The number of cells harvested from the P4 generation is approximately 6.8 × 10⁶. 6 The number of cells harvested from P6 generation is approximately 1.2 × 10⁶. 7 The number of cells harvested from the P8 generation is approximately 2.0 × 10⁶. 7 The number of cells harvested from passage P10 is approximately 3.5 × 10⁶. 7 The number of cells harvested generally increased with the number of passages, indicating that the cells had good proliferative capacity during passage.

[0067] Subsequently, chromosome karyotype analysis was performed on P0 and P10 cells, with the following specific steps: Cells in metaphase were treated with 0.1 µg / mL colchicine at 37°C. P0 and P10 generation cells, after reaching the logarithmic growth phase and with approximately 90% confluence, were generally seeded or passaged for 2-3 days. Colchicine was then added for 3 hours to induce cell accumulation in metaphase. After adding colchicine, the cells were gently shaken and incubated at 37°C with 5% CO2 for another 3 hours. Cells were then swelled by hypotonic treatment and fixed three times using methanol-glacial acetic acid (3:1 v / v): 20 minutes for the first fixation, 30 minutes for the second, and 40 minutes for the third. Cell slides were prepared after multiple fixations, with a cell suspension concentration of 1 × 10⁶ cells / mL at the time of slide preparation. 5To ensure clear chromosome dispersion and easy observation, the cell suspension was placed 10 cm above the slide using a dropper, allowing it to fall naturally onto the pre-soaked ice water slide. This facilitates rapid cell spread and chromosome dispersion. After placement, the cells were stained with Giemsa stain (a 1:9 volume ratio of Giemsa stock solution and phosphate buffer) for 10 minutes.

[0068] The results showed that, in P0, 95 out of 100 cells had normal karyotypes, representing 95% of the cells; in P10, 92 out of 100 cells had normal karyotypes, representing 92% of the cells. No obvious abnormalities such as chromosomal deletions, duplications, or translocations were found in either P0 or P10. This clearly demonstrates that the cell line can maintain a normal karyotype after 10 passages, exhibiting good genomic stability.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A serum-free medium for maintaining the stemness of mesenchymal stem cells, characterized by, The serum-free culture medium comprises a basic culture medium and exogenous added components; the basic culture medium is DMEM / F12 culture medium, and the exogenous added components comprise bFGF, EGF, PDGF-BB, recombinant human fibronectin, laminin, insulin, transferrin, sodium selenate, β-mercaptoethanol and ascorbic acid.

2. The serum-free medium according to claim 1, characterized in that, The DMEM / F12 culture medium comprises glucose at a concentration of 3.5-5.5 g / L and glutamine at a concentration of 2-4 mM; in the exogenous added components, the mass ratio of bFGF, EGF and PDGF-BB is (7-9):(14-16):(9-11).

3. The serum-free medium according to claim 1, characterized in that, The working concentrations of the exogenous added components in the serum-free culture medium are as follows: bFGF 7-9 ng / mL, EGF 14-16 ng / mL, PDGF-BB 9-11 ng / mL, recombinant human fibronectin 1-5 μg / mL, laminin 0.1-5 μg / mL, insulin 5-15 μg / mL, transferrin 5-15 μg / mL, sodium selenate 5-15 nM, β-mercaptoethanol 0.1-3 μM, and ascorbic acid 25-35 μM.

4. The method for preparing serum-free culture medium according to any one of claims 1 to 3, characterized in that, The preparation step of the serum-free culture medium comprises dissolving the exogenous added components in the basic culture medium and mixing uniformly to obtain.

5. Application of the serum-free culture medium according to any one of claims 1-3 or the preparation method according to claim 4 in culturing mesenchymal stem cells in vitro.

6. Use according to claim 5, characterized in that, The mesenchymal stem cells comprise human umbilical cord MSCs stem cells.

7. Application of the serum-free culture medium according to any one of claims 1-3 or the preparation method according to claim 4 in improving the proliferation efficiency of cultured mesenchymal stem cells.

8. Use according to claim 7, characterized in that, The mesenchymal stem cells comprise human umbilical cord MSCs stem cells.

9. A method of preparing mesenchymal stem cells, characterized by, Mesenchymal stem cells are inoculated into the serum-free culture medium according to any one of claims 1-3 for culture.

10. The method of claim 9, wherein, The mesenchymal stem cells include human umbilical cord MSCs stem cells, the seeding density of the human umbilical cord MSCs stem cells in the culture medium is 2x10 4 ~2x10 5 / cm 2 ; the culture temperature is 35~38℃, and the culture time is 12~48h.

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