Culture method for hypoxia culture of umbilical cord mesenchymal stem cells

Abstract

The invention discloses a culture method for hypoxia culture of umbilical cord mesenchymal stem cells, which comprises the following steps: hypoxia pre-adaptive culture: inoculating umbilical cord mesenchymal stem cells in a culture medium containing bFGF (basic fibroblast growth factor), glutathione, astragalus polysaccharide and salidroside, and transferring the umbilical cord mesenchymal stem cells into a 5% O2 environment after the umbilical cord mesenchymal stem cells are preliminarily adhered to the wall in a normal oxygen environment; performing low-oxygen enrichment culture: replacing a second culture medium containing astragalus polysaccharide, salidroside, D-ribose, sodium pyruvate and D-glucose, and promoting efficient cell proliferation under 2% O2; performing function strengthening culture: adding tanshinone IIA and GSK-269962A, inducing cells to secrete VEGF under 1% O2, and maintaining high dryness. According to the method, through the synergistic effect of the three-stage gradient oxygen concentration and the specific serum-free culture medium, the cell proliferation multiple is remarkably increased, the total apoptosis rate is reduced, VEGF secretion is promoted, the multidirectional differentiation potential is reserved, the culture method is standardized in operation, the serum-free culture medium is definite in component, and a high-quality stem cell culture scheme is provided for the field of regenerative medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a culture method for culturing umbilical cord mesenchymal stem cells under hypoxia. Background Art

[0002] Mesenchymal stem cells (MSCs), a population of adult stem cells with self-renewal and multidirectional differentiation potential, are increasingly valuable in tissue engineering, regenerative medicine, and cell therapy. Umbilical cord mesenchymal stem cells (hUC-MSCs) have garnered widespread attention due to their low immunogenicity, ease of acquisition, robust proliferation, and minimal ethical controversy, making them a focus of scientific and medical research. Studies have shown that these cells can not only differentiate into functional cells such as osteoblasts, adipocytes, and chondrocytes, but also exert multiple functions, including immunomodulation, angiogenesis, and tissue repair, by secreting bioactive factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). These cells hold great promise for treating ischemic diseases, intervening in autoimmune diseases, and repairing organ damage.

[0003] In cell culture systems, precise regulation of the culture environment is crucial for maintaining the biological properties of umbilical cord mesenchymal stem cells. Studies have shown that a hypoxic microenvironment (typically defined as 1%-10% O₂) can regulate cellular energy metabolism, apoptosis, and stemness maintenance by activating signaling pathways such as hypoxia-inducible factor (HIF), thereby enhancing cell survival and functional activity. However, current mainstream hypoxic culture methods often utilize a constant oxygen concentration (e.g., 2%-5% O₂), failing to mimic the dynamic characteristics of the progressively hypoxic microenvironment in vivo. This single-concentration culture model presents significant technical bottlenecks. Firstly, constant hypoxia can easily lead to an imbalance in the intracellular redox balance, leading to accumulation of reactive oxygen species (ROS) and mitochondrial damage, which in turn exacerbates apoptosis. Secondly, disrupted energy metabolism (e.g., imbalance between glycolysis and oxidative phosphorylation) in hypoxic environments can lead to cell cycle arrest, inhibit proliferation efficiency, and compromise the expression of stem cell surface markers and the maintenance of multipotential differentiation potential.

[0004] Therefore, how to optimize hypoxic culture conditions and construct a culture method that can effectively reduce cell apoptosis rate, promote proliferation and maintain stem cell characteristics has become a key technical problem that urgently needs to be broken through in this field. It has important scientific significance and clinical value for promoting the large-scale application and standardized production of umbilical cord mesenchymal stem cells in regenerative medicine. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a culture method for culturing umbilical cord mesenchymal stem cells under hypoxia.

[0006] The present invention discloses a method for culturing umbilical cord mesenchymal stem cells in a low-oxygen gradient culture, which is characterized by comprising the following steps: Step 1: Hypoxia pre-adaptation culture: umbilical cord mesenchymal stem cells were cultured at 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / cm2 in a culture vessel containing the first culture medium, cultured in an incubator at 20% O2, 5% CO2, and 37°C for 8 hours, and then transferred to a hypoxic environment of 5% O2 for a further 28 hours to obtain a hypoxic pre-adapted cell system. The first culture medium is a serum-free medium containing bFGF, glutathione, astragalus polysaccharide, and salidroside. This step allows the cells to initially adhere to the wall in a normoxic environment. After a certain period of growth, the cells are transferred to a 5% O2 environment. The addition of bFGF, glutathione, astragalus polysaccharide, and salidroside activates related intracellular pathways, promotes cell adhesion and proliferation, and helps the cells initially adapt to the hypoxic environment, preparing them for subsequent culture.

[0007] Step 2: Replace the culture medium in the hypoxia pre-adapted cell system obtained in step 1 with the second culture medium in full, place the culture container in an incubator at 2% O2, 5% CO2, and 37°C for 48 hours, and replace the second culture medium by half every 24 hours to obtain a hypoxic proliferating cell system. The second culture medium is a serum-free culture medium containing astragalus polysaccharide, salidroside, D-ribose, sodium pyruvate, and D-glucose. This step can inhibit cell apoptosis and switch the cell metabolism mode. The full replacement of the culture medium is to remove the metabolic waste generated by the first step of culture and provide fresh nutrients; the 2% O2 environment prompts the cells to further adapt to hypoxia and adjust their own metabolism; the half-volume replacement of the culture medium every 24 hours can continuously replenish nutrients to ensure the normal proliferation of cells in a hypoxic environment. At this time, the cells can use the added components in the serum-free culture medium for efficient proliferation. It should be noted that full replacement of the culture medium refers to removing all the old culture medium and adding an equal amount of fresh culture medium. Here, it specifically refers to removing all the first culture medium and adding an equal amount of fresh second culture medium. Half replacement refers to replacing only half of the culture medium and retaining the other half of the old culture medium. Here, it specifically refers to replacing half of the second culture medium every 24 hours.

[0008] Step 3: Functional enhancement culture: Add tanshinone IIA and GSK-269962A to the hypoxic proliferating cell system obtained in step 2, and then culture in an incubator with 1% O2, 5% CO2, and 37°C until the VEGF concentration is ≥1800pg / mL and the cell confluence reaches 80%~90%, thereby obtaining hypoxic cultured cells. The addition of tanshinone IIA in this step can target and inhibit the mitochondrial apoptosis pathway, and the addition of GSK-269962A forms a "dual-pathway anti-apoptosis" synergistic effect, inhibiting cell apoptosis and enhancing cell viability. The extremely low oxygen environment of 1% O2 can further induce adaptive changes in cells, prompting cells to secrete large amounts of functional factors such as VEGF. Data show that the VEGF concentration in the culture supernatant of P0 cells reached 2235pg / mL, and that of P5 cells reached 1952pg / mL, maintaining a high angiogenesis-promoting ability and a good growth state, meeting the needs of subsequent applications.

[0009] Preferably, the first culture medium is based on DMEM / F12, and the added components are calculated in final concentrations, including: 10ng / mL bFGF, 1mM glutathione, 10μg / mL astragalus polysaccharide and 1.0μg / mL salidroside. Among them, bFGF can activate the FGFR signaling pathway, promote cell adhesion and proliferation, and reserve cell number for hypoxic stress; glutathione, as an antioxidant, can remove excess reactive oxygen species produced by cells when oxygen concentration changes, and protect cell structure; astragalus polysaccharide can activate the AMPK signaling pathway, enhance mitochondrial function, and regulate cellular energy metabolism; salidroside can induce stable expression of HIF-1α, activate hypoxia response genes, and lay the foundation for subsequent adaptation to hypoxic environments. It is worth noting that the final concentration described in the present invention refers to: after the solution is prepared, the ratio of the mass or amount of each component in the solution to the total volume of the solution.

[0010] Preferably, the second culture medium is based on DMEM / F12 and contains the following components, calculated at final concentrations: 10 μg / mL astragalus polysaccharide, 1 μg / mL salidroside, 8 mM D-ribose, 0.2 mg / mL sodium pyruvate, and 1 g / L D-glucose. Astragalus polysaccharide and salidroside maintain their antioxidant and hypoxia adaptation effects; D-ribose participates in ATP salvage synthesis, rapidly replenishing energy consumption under hypoxia; sodium pyruvate, as a substrate for the tricarboxylic acid cycle, maintains cellular metabolic flux; and D-glucose provides a basal carbon source, working in conjunction with sodium pyruvate to regulate sugar metabolism and prevent lactic acid accumulation.

[0011] Preferably, in step 3, the added concentration of Tanshinone IIA is 0.5 μg / mL, and the added concentration of GSK-269962A is 1 μM.

[0012] Preferably, the umbilical cord mesenchymal stem cells are primary (P0) cells or passaged (Pn, n≥1) cells, wherein primary cells are non-passaged cells directly isolated from umbilical cord tissue, and passaged cells are primary cells that are digested with trypsin, centrifuged, and then inoculated and cultured.

[0013] Preferably, the culture container is a T25 cell culture flask, or other equivalent culture dishes.

[0014] Preferably, both the first culture medium and the second culture medium do not contain animal serum, and are sterilized by filtration through a 0.22 μm filter membrane before use.

[0015] In summary, the beneficial effects of the present invention are: (1) The present invention adopts a low-oxygen gradient culture method, sets up three stages of low-oxygen pre-adaptation culture, low-oxygen proliferation culture and function enhancement culture, simulates the progressive hypoxic microenvironment in the body, and realizes precise regulation of the growth process of umbilical cord mesenchymal stem cells. In the pre-adaptation stage, the cells gradually adapt to the low-oxygen microenvironment by gradually transitioning from normoxia to a low-oxygen environment of 5% O2; in the proliferation stage, the oxygen concentration is further reduced to 2% O2, and a second culture medium with specific ingredients is used to promote efficient cell proliferation; in the function enhancement stage, in an extremely low-oxygen environment of 1% O2, specific additives are combined to enhance the therapeutic function of the cells. This method effectively solves the problems of poor cell growth, poor adaptability and weak function in traditional single low-oxygen culture methods, and improves the quality and application value of the cells.

[0016] (2) The present invention optimizes the hypoxic pre-adaptation of umbilical cord mesenchymal stem cells by adding bFGF, glutathione, astragalus polysaccharide, and salidroside to the first culture medium. bFGF promotes cell adhesion and proliferation, glutathione eliminates excess reactive oxygen species produced by cells when oxygen concentration changes, astragalus polysaccharide activates the AMPK signaling pathway to enhance mitochondrial function, and salidroside induces stable expression of HIF-1α. The synergistic effect of multiple components helps cells quickly adapt to the hypoxic environment during the pre-adaptation stage, reduces stress damage, lays a good foundation for subsequent culture, and solves the problem that cells are easily damaged and difficult to adapt to the hypoxic environment in the initial stage.

[0017] (3) The present invention achieves efficient support for cell metabolism in the hypoxic proliferation stage by adding astragalus polysaccharide, salidroside, D-ribose, sodium pyruvate and D-glucose to the second culture medium. Astragalus polysaccharide and salidroside continue the antioxidant and hypoxia adaptation regulatory effects, D-ribose participates in ATP rescue synthesis to quickly replenish energy, sodium pyruvate serves as a substrate for the tricarboxylic acid cycle to maintain metabolic flux, and D-glucose cooperates to regulate sugar metabolism. These components cooperate with each other to provide sufficient nutrition and energy for cell proliferation in a hypoxic environment, inhibit cell apoptosis, switch the cell metabolism mode, and solve the problems of slow cell proliferation, insufficient energy supply, and metabolic imbalance in a hypoxic environment.

[0018] (4) The present invention achieves a significant improvement in the cell's anti-apoptosis ability and functional factor secretion by adding tanshinone IIA and GSK-269962A during the functional enhancement culture stage and combining it with an extremely low oxygen environment of 1% O2. Tanshinone IIA targets and inhibits the mitochondrial apoptosis pathway, forming a "dual-pathway anti-apoptosis" synergistic effect with GSK-269962A. At the same time, the extremely low oxygen environment further induces adaptive changes in cells, prompting them to secrete a large amount of functional factors such as VEGF. The setting of this stage enables cells to maintain a good growth state and high angiogenesis-promoting ability in an extremely low oxygen environment, solving the problems of severe cell apoptosis and insufficient secretion of functional factors in traditional extremely low oxygen culture.

[0019] (5) The serum-free culture medium based on DMEM / F12 used in the present invention has a clear composition, stable properties, is serum-free, and has no batch-to-batch variability, meeting the requirements of standardized production. The first and second culture media are specifically supplemented with different components based on the needs of cells at different culture stages. This is closely integrated with the hypoxic gradient culture method, facilitating the industrial production and widespread application of the culture medium, and resolving the problem that existing culture systems are unable to meet the needs of large-scale, standardized production. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

[0021] In the present invention, the components and reagents involved are all conventional commercial products or can be obtained by conventional technical means in the art. Unless otherwise specified, the materials, methods and examples of the present invention are only illustrative and not restrictive.

[0022] Example 1: Serum-free culture medium for low-oxygen gradient culture of umbilical cord mesenchymal stem cells and its preparation This example provides serum-free culture media for hypoxic gradient culture of mesenchymal stem cells. The first culture medium is based on DMEM / F12 (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) supplemented with 10 ng / mL bFGF, 1 mM glutathione, 10 μg / mL astragalus polysaccharide, and 1.0 μg / mL salidroside. The culture medium is sterilized by filtration through a 0.22 μm filter and stored at 4°C. The second culture medium is based on DMEM / F12 (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) supplemented with 10 μg / mL astragalus polysaccharide, 1.0 μg / mL salidroside, 8 mM D-ribose, 0.2 mg / mL sodium pyruvate, and 1 g / L D-glucose. The culture medium is sterilized by filtration through a 0.22 μm filter and stored at 4°C.

[0023] Example 2: Hypoxia Gradient Culture of P5 Umbilical Cord Mesenchymal Stem Cells In this example, P5 umbilical cord mesenchymal stem cells were cultured under hypoxic gradient conditions. The specific operations were as follows: Step 1: Hypoxia pre-adaptation culture: cryopreserved P5 umbilical cord mesenchymal stem cells were rapidly revived in a 37°C water bath, washed 1-2 times with PBS to remove the cryopreservation solution, and cultured at 2×10 4 pieces / cm 2 Cells were seeded at a low density in a T25 dish containing the first culture medium and cultured in a 20% O₂, 5% CO₂, 37°C incubator for 8 hours. After 8 hours, the culture flasks were transferred to a 5% O₂, 5% CO₂, 37°C incubator for a further 28 hours. During this time, cell attachment and growth were observed to establish a hypoxia-preconditioned cell system.

[0024] Step 2: Hypoxic proliferation culture: Discard the culture medium in the hypoxic pre-adapted cell system obtained in step 1, wash twice with PBS, add the full amount of the second culture medium, and then culture the cells in a 2% O2, 5% CO2, 37°C incubator for 48 hours. During this period, half of the second culture medium was replaced every 24 hours to obtain a hypoxic proliferating cell system.

[0025] Step 3: Functional enhancement culture: Add 0.5 μg / mL tanshinone IIA and 1 μM GSK-269962A to the hypoxic proliferating cell system obtained in step 2, and then culture in an incubator with 1% O2, 5% CO2, and 37°C. Detect VEGF concentration and cell morphology every 12 hours during the culture period. Terminate the culture when the VEGF concentration is ≥1800 pg / mL and the cell confluence reaches 80%~90% to obtain hypoxic cultured cells.

[0026] Comparative Example 1: Normoxia Culture of Umbilical Cord Mesenchymal Stem Cells This comparative example provides a method for culturing umbilical cord mesenchymal stem cells in normoxia. The specific operation is as follows: the P5 umbilical cord mesenchymal stem cells are revived in step 1 of Example 1, and then 2×104 pieces / cm 2 The cells were seeded at a density of 100 μg / ml in a T25 culture flask using DMEM / F12 medium containing 10% fetal bovine serum. The cells were cultured in an incubator with 20% O2, 5% CO2, and 37°C. The medium was changed every 2 to 3 days during the culture process. The cell morphology was observed regularly under an inverted phase contrast microscope. The culture was terminated when the cell confluence reached 80% to 90%.

[0027] Comparative Example 2: Culture of umbilical cord mesenchymal stem cells using a single culture medium in the pre-adaptation phase and constant hypoxia (5% O2) This comparative example provides a method for culturing umbilical cord mesenchymal stem cells using a single culture medium in the pre-adaptation stage and constant hypoxia. The specific operation is as follows: the P5 umbilical cord mesenchymal stem cells are revived in the same manner as in step 1 of Example 1, and then 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / cm2 in a T25 culture flask, cultured using the first culture medium in Example 1, and placed in an incubator with 5% O2, 5% CO2, and 37°C. The culture medium was changed every 2 to 3 days during the culture process, and the cell morphology was regularly observed under an inverted phase contrast microscope. When the cell confluence reached 80% to 90%, the culture was terminated.

[0028] Comparative Example 3: Culture of umbilical cord mesenchymal stem cells using a single culture medium in the proliferation phase and constant hypoxia (2% O2) This comparative example provides a method for culturing umbilical cord mesenchymal stem cells using a single culture medium in the proliferation stage and constant hypoxia. The specific operation is as follows: the P5 umbilical cord mesenchymal stem cells are revived in the same manner as in step 1 of Example 1, and then 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / cm2 in a T25 culture flask, cultured using the second culture medium in Example 1, and placed in an incubator with 2% O2, 5% CO2, and 37°C. The culture medium was changed every 2 to 3 days during the culture process, and the cell morphology was regularly observed under an inverted phase contrast microscope. When the cell confluence reached 80% to 90%, the culture was terminated.

[0029] Example 3: Hypoxia Gradient Culture of P0 Umbilical Cord Mesenchymal Stem Cells This embodiment provides a method for culturing P0 umbilical cord mesenchymal stem cells in a hypoxic gradient. The specific operation is the same as that in Example 1, except that P0 umbilical cord mesenchymal stem cells obtained from fresh umbilical cord tissue are used in step 1.

[0030] To verify the effectiveness of the method for culturing mesenchymal stem cells in a hypoxic gradient culture according to the present invention, the umbilical cord mesenchymal stem cells cultured in Examples 2 to 3 and Comparative Examples 1 to 3 were tested as follows: Experiment 1: Cell proliferation ability detection On days 3, 5, and 7 of culture, pipette an appropriate amount of cell suspension from each group into a centrifuge tube, centrifuge, and discard the supernatant. Mix a small amount of cell suspension with trypan blue at a ratio of 1:1 and count using a Thermo Fisher Scientific Countess3 automated cell counter to calculate cell density and cell proliferation rate. Three replicates were set up for each sample. The calculation formula is as follows: Cell proliferation fold = (cell density at the detection time point / cell density at the time of inoculation) × 100%.

[0031] Table 1 Cell proliferation times As shown in Table 1, the cell proliferation results for Examples 2 and 3 were significantly higher than those for the normoxia group (Comparative Example 1) and the constant hypoxia group (Comparative Examples 2 / 3) at all sampling points, demonstrating the stimulatory effect of hypoxic gradient culture on cell proliferation. The proliferation of P0 primary cells, due to their greater stemness, was superior to that of P5 passaged cells, resulting in the highest proliferation rate in Example 3. Data on day 7 showed that the proliferation rates of the gradient culture groups were 2.5 and 2.0 times those of the normoxia group, respectively, demonstrating the synergistic optimization effect of the hypoxic gradient and staged culture methods of the present invention on cell proliferation. Comparative Example 3 exhibited superior proliferation capacity to Comparative Example 2 due to the inclusion of energy metabolism-supporting components such as D-ribose and sodium pyruvate in the culture medium, indicating that the second culture medium is more adapted to the metabolic demands of the hypoxic proliferation phase. The proliferation capacity of Comparative Example 2 under constant 5% O₂ was intermediate between the normoxia group and the hypoxic gradient group, reflecting the stimulatory effect of pre-adaptation components (such as bFGF and salidroside) on initial hypoxia proliferation. However, the lack of subsequent gradient regulation resulted in insufficient proliferation momentum in the later stages.

[0032] Experiment 2: Cell apoptosis rate detection Umbilical cord mesenchymal stem cells from each group were cultured to the endpoint, the supernatant discarded, and the cells digested with an appropriate amount of trypsin. After microscopic observation, cells began to round up and the intercellular spaces increased. The suspension was then dispersed to form a single-cell suspension and transferred to a centrifuge tube. The suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded. The cells were washed twice with pre-chilled PBS, centrifuged and the supernatant discarded. The cells were then resuspended in 500 μL of Binding Buffer. Then, 5 μL of Annexin V-FITC and 5 μL of PI were added to each group, gently mixed, and incubated at room temperature in the dark for 15–20 minutes to complete staining. The stained cell suspension was transferred to a flow cytometer and immediately analyzed by flow cytometry. Cell apoptosis data were recorded. The apoptotic rate was calculated according to the following formula. Three replicates were performed for each sample.

[0033] Early apoptotic cell rate (%) = number of early apoptotic cells / total number of cells × 100%.

[0034] Late apoptotic cell rate (%) = number of late apoptotic cells / total number of cells × 100%.

[0035] Total cell apoptosis rate (%) = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%.

[0036] As shown in Table 2, the total apoptosis rates of cells in Examples 2 and 3 were 9.0% and 6.4%, respectively, significantly lower than those in the normoxia group (Comparative Example 1, total apoptosis rate 21.0%), indicating that hypoxic gradient culture reduces cell apoptosis through gradual adaptation to the hypoxic environment and synergistically reducing anti-apoptotic components. Furthermore, due to their stronger stemness, the total apoptosis rate of P0 primary cells was approximately 29% lower than that of P5, demonstrating the influence of generation on cell anti-apoptosis ability. The total apoptosis rate in Comparative Example 3 was 12.7%, lower than that in Comparative Example 2 (total apoptosis rate of 16.3%), indicating that components such as D-ribose and sodium pyruvate in the second culture medium reduce cell apoptosis by optimizing energy metabolism. The higher apoptosis rate in Comparative Example 2 may be due to the lack of anti-apoptotic components (such as tanshinone IIA) in the first culture medium, and 5% O2 is not the optimal hypoxia protection concentration.

[0037] Table 2 Cell apoptosis rate Experiment 3: Cell function factor detection Umbilical cord mesenchymal stem cells from each group were cultured to the endpoint, centrifuged to remove cell debris, and VEGF cytokine concentrations were measured using an ELISA kit. The specific procedure was to first prepare the ELISA kit, set up standard wells, sample wells, and blank wells in a 96-well plate, and sequentially add the standard, treated supernatant, and related reagents. After incubation at 37°C and washing the plate (repeated 3-5 times), the detection antibody and enzyme-labeled secondary antibody were added and incubated and washed. The substrate solution was then added to develop color in the dark. Finally, the reaction was terminated with stop solution. The absorbance at 450 nm (OD value) was measured using a microplate reader. The cytokine concentration was calculated according to the standard curve. The data were statistically analyzed to assess VEGF cytokine secretion.

[0038] Table 3 VEGF cytokine secretion The results, as shown in Table 3, show that VEGF concentrations in Example 3 and Example 2 were 2235 pg / mL and 1952 pg / mL, respectively, both exceeding the functional enhancement target set by the invention (≥1800 pg / mL) and significantly higher than those in the normoxia group (Comparative Example 1, VEGF concentration of 896 pg / mL), confirming the enhanced effect of hypoxic gradient culture on angiogenic factor secretion. P0 cells, due to their greater differentiation potential and hypoxia response, secreted approximately 14% more VEGF than P5 cells. The higher VEGF concentration in Comparative Example 3 than in Comparative Example 2 reflects the enhanced metabolism and factor secretion induced by lower oxygen concentrations and the proliferation phase culture medium components. Comparative Example 2, due to the use of pre-adaptation medium and the lack of anti-apoptotic components used in the functional enhancement phase, exhibited lower VEGF secretion than Examples 2 and 3. The lowest VEGF concentration was observed in the normoxia group (Comparative Example 1), confirming the mechanism by which hypoxia induces VEGF secretion and demonstrating that Examples 2 and 3 cultured umbilical cord mesenchymal stem cells, representative of hypoxic umbilical cord mesenchymal stem cells.

[0039] Experiment 4: Positive rate of cell stemness gene expression Umbilical cord mesenchymal stem cells cultured to the end point in each group were taken, digested with an appropriate amount of trypsin, centrifuged and discarded the supernatant, washed twice with PBS, and centrifuged and discarded the supernatant. Add an appropriate amount of 4% paraformaldehyde solution to the cell pellet, fix at room temperature for 15-20 minutes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash the cells twice with PBS. Add PBS solution containing 0.1% TritonX-100 and permeabilize at room temperature for 10-15 minutes. The permeabilized cells were resuspended in PBS solutions containing Oct4 primary antibody, Sox2 primary antibody and HIF-1α primary antibody, and incubated overnight at 4°C in the dark. The next day, centrifuged and discarded the supernatant, washed 3 times with PBS, and then added the AlexaFluor488 fluorescent-labeled secondary antibody corresponding to the primary antibody, incubated at room temperature in the dark for 1-2 hours, washed 3 times with PBS, and resuspended in an appropriate amount of PBS to adjust the cell concentration to 1×10 6 ~1×10 7 Cells / mL were transferred to a sample tube dedicated to the flow cytometer. Detection was performed using a flow cytometer, with the excitation wavelength set to 488 nm and the emission wavelength set to 519 nm, collecting fluorescence signals with a wavelength of 510-530 nm. The fluorescence intensity of each cell was analyzed and recorded by flow cytometry, and unstained cells were set as negative controls. The number of positive cells and the total number of detected cells were counted by the flow cytometer software, and the positive cell rate was calculated according to the following formula: Oct4 positive rate (%) = number of cells expressing Oct4 gene positive / total number of cells detected × 100% Sox2 positive rate (%) = number of Sox2 gene positive cells / total number of cells detected × 100% HIF-1α positive rate (%) = number of HIF-1α gene expression positive cells / total number of detected cells × 100% Table 4 Positive rate of cell stemness gene expression The results are shown in Table 4. The positive rates for Oct4, Sox2, and HIF-1α in Example 3 were 89.2%, 84.7%, and 92.6%, respectively, significantly higher than those in the normoxia group (Comparative Example 1), indicating that hypoxic gradient culture and anti-apoptotic components synergistically activate stemness regulatory pathways. The higher expression of stemness genes in Example 3 compared to Example 2 is due to the enhanced self-renewal capacity of primary cells. In particular, the HIF-1α positive rate, as high as 92.6%, reflects the efficient induction of hypoxia-responsive genes in an extremely hypoxic environment. The overall positive rate of stemness genes in Comparative Example 3 was higher than in Comparative Example 2, indicating that lower oxygen concentrations and proliferation medium components can more effectively maintain cell stemness.

[0040] Compared with the data from Experiment 3, HIF-1α positivity in each group showed a strong positive correlation with VEGF secretion, further validating HIF-1α's regulatory role in angiogenesis. Compared with the data from Experiment 2, groups with high Oct4 / Sox2 positivity had lower apoptosis rates, demonstrating a synergistic effect between stemness maintenance and anti-apoptosis.

[0041] Experiment 5: Cell differentiation ability detection Osteogenic differentiation ability test: The osteogenic induction medium was based on DMEM / F12 medium, and 1×10 -7 mol / L dexamethasone, 10mmol / L sodium β-glycerophosphate, 50μg / mL vitamin C, 100U / mL penicillin, and 100μg / mL streptomycin. The umbilical cord mesenchymal stem cells were cultured using osteogenic induction medium. The umbilical cord mesenchymal stem cells cultured to the end point in each group were taken and 1×10 4 pieces / cm 2 Cells were seeded in a 6-well plate at a density of 100 μg / mL, and 2 mL of osteogenic induction medium was added to each well. The cells were induced and cultured for 21 days, with the medium replaced every 3 days. After induction, the culture medium was discarded, the cells were washed twice with PBS, and then 4% paraformaldehyde was added, fixed for 30 minutes, and washed three times with PBS. Alizarin red stain was then added and stained for 30 minutes at room temperature in the dark. The cells were washed several times with distilled water to remove excess stain. The cells were then washed several times with 60% isopropanol to remove excess stain. The 6-well plate was observed under an inverted microscope, and multiple fields of view were selected for counting. The cell differentiation efficiency was calculated according to the formula. Three replicate experimental groups were set up for each sample.

[0042] Osteoblast differentiation efficiency = (total number of mineralized nodule cells / total number of cells) × 100%.

[0043] Adipogenic differentiation ability test: Adipogenic induction medium was based on DMEM / F12 medium, and 1×10 -6 mol / L dexamethasone, 0.5mmol / L 3-isobutyl-1-methylxanthine (IBMX), 10μg / mL insulin, 200μmol / L indomethacin, 100U / mL penicillin, and 100μg / mL streptomycin. Umbilical cord mesenchymal stem cells were cultured using adipogenic induction medium. Umbilical cord mesenchymal stem cells cultured to the endpoint in each group were taken and 1×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a 6-well plate, and 2 mL of adipogenic induction medium was added to each well. The cells were cultured for 14 days and the induction medium was replaced every 3 days. After induction, the culture medium was discarded, the cells were washed twice with PBS, and then 4% paraformaldehyde was added for fixation for 30 minutes, and then washed three times with PBS. Oil red O stain was then added and stained at room temperature in the dark for 60 minutes, then washed several times with 60% isopropanol to remove excess stain. The 6-well plate was observed under an inverted microscope, multiple fields of view were selected for counting, and the cell differentiation efficiency was calculated according to the formula. Three replicate experimental groups were set up for each sample.

[0044] Adipocyte differentiation efficiency = (total number of adipocytes stained / total number of cells) × 100%.

[0045] Table 5 Cell osteogenic differentiation efficiency The results of cell osteogenic differentiation efficiency are shown in Table 5. Example 2 achieved an osteogenic differentiation efficiency of 75.2%, a 44% increase compared to the normoxia group (Comparative Example 1, with an osteogenic differentiation efficiency of 52.1%), indicating that the osteogenic differentiation ability of umbilical cord mesenchymal stem cells cultured in a hypoxic gradient was less impaired. Example 3 achieved an even higher osteogenic differentiation efficiency than Example 2, demonstrating the enhanced osteogenic potential of primary cells under a hypoxic gradient. This demonstrates that a hypoxic environment promotes the expression of osteogenic-related genes such as Runx2 and Osterix by activating the HIF-1α pathway, and that, combined with the inductive effects of sodium β-glycerophosphate and vitamin C in the culture medium, it significantly enhances the efficiency of mineralized nodule formation. The results of Comparative Example 3 were slightly higher than those of Comparative Example 2, indicating that lower oxygen concentrations and the components of the culture medium during the proliferation phase have a synergistic effect on osteogenic differentiation.

[0046] Table 6 Cell adipogenic differentiation efficiency The results of cell adipogenic differentiation efficiency are shown in Table 6. Example 2 achieved an adipogenic differentiation efficiency of 70.3%, a 35% increase compared to the normoxia group (Comparative Example 1, osteogenic differentiation efficiency was 51.9%). This indicates that hypoxic gradient culture did not impair the multidirectional differentiation ability of stem cells, but rather preserved their adipogenic differentiation potential by maintaining stemness. The adipogenic inducer effectively induced lipid droplet formation, and the gradient culture group had a high cell survival rate and a more complete differentiation process. Example 3 had an osteogenic differentiation efficiency (78.5%) higher than Example 2, consistent with the characteristic that increased stem cell passage number leads to a slight decrease in differentiation ability, but still significantly superior to Comparative Examples 2 and 3.

[0047] In summary, the present invention constructs an efficient culture system that simulates the progressive hypoxic microenvironment in vivo through the coordinated design of a low-oxygen gradient culture method and a staged serum-free culture medium. The pre-adaptation stage enables cells to adapt smoothly to the low-oxygen environment, the proliferation stage significantly improves the efficiency of cell proliferation and inhibits apoptosis, and the function enhancement stage prompts cells to secrete a large amount of functional factors such as VEGF, while maintaining high stemness gene expression and multidirectional differentiation potential. Experimental data show that this method can increase the cell proliferation multiple by nearly 1 times compared with normoxic culture, reduce the total apoptosis rate by more than 60%, and achieve a VEGF concentration of more than 1800pg / mL. The osteogenic / adipogenic differentiation efficiency is increased by 44% and 35% respectively, and the serum-free culture medium has clear ingredients and controllable risks, which solves the problems of poor cell adaptability, insufficient function and limited standardized production in traditional hypoxic culture, and provides a high-quality umbilical cord mesenchymal stem cell culture solution for the field of regenerative medicine.

[0048] At the same time, the present invention forms a significantly innovative technical solution through precise gradient oxygen concentration control and the sequential addition of specific functional components. Any equivalent improvements or parameter optimizations based on the core principles of the present invention, including but not limited to equivalent replacement of culture medium components, subtle adjustments to the oxygen concentration gradient, and reasonable separation of culture stages, should be included in the scope of protection of the present invention to fully guarantee the technological innovation and application value of the low-oxygen gradient culture method in the field of stem cell culture.

Claims

1. A method for culturing umbilical cord mesenchymal stem cells in a low oxygen gradient culture, characterized in that: The steps include: Step 1: Hypoxia pre-adaptation culture: umbilical cord mesenchymal stem cells were cultured at 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / cm2 in a culture container containing a first culture medium, cultured in an incubator at 20% O2, 5% CO2, and 37°C for 8 hours, and then transferred to a hypoxic environment at 5% O2 and cultured for 28 hours to obtain a hypoxic pre-adapted cell system, wherein the first culture medium is a serum-free culture medium containing bFGF, glutathione, astragalus polysaccharide, and salidroside; Step 2, hypoxic proliferation culture: The culture medium in the hypoxic pre-adapted cell system obtained in step 1 is fully replaced with a second culture medium, and the culture container is placed in an incubator at 2% O2, 5% CO2, and 37°C for 48 hours. During this period, half of the second culture medium is replaced every 24 hours to obtain a hypoxic proliferative cell system, wherein the second culture medium is a serum-free medium containing astragalus polysaccharide, salidroside, D-ribose, sodium pyruvate, and D-glucose; Step 3: Functional enhancement culture: Add tanshinone IIA and GSK-269962A to the hypoxic proliferating cell system obtained in step 2, and then culture in an incubator with 1% O2, 5% CO2, and 37°C until the VEGF concentration is ≥1800 pg / mL and the cell confluence reaches 80%~90%, thereby obtaining hypoxic cultured cells.

2. The culture method according to claim 1, wherein The first culture medium is based on DMEM / F12, and the added components include, by final concentration, 10 ng / mL bFGF, 1 mM glutathione, 10 μg / mL astragalus polysaccharide, and 1.0 μg / mL salidroside.

3. The culture method according to claim 1, wherein The second culture medium is based on DMEM / F12, and the added components, calculated by final concentration, include: 10 μg / mL astragalus polysaccharide, 1 μg / mL salidroside, 8 mM D-ribose, 0.2 mg / mL sodium pyruvate and 1 g / L D-glucose.

4. The culture method according to claim 1, wherein In step 3, the added concentration of Tanshinone IIA was 0.5 μg / mL, and the added concentration of GSK-269962A was 1 μM.

5. The culture method according to claim 1, wherein The umbilical cord mesenchymal stem cells are primary cells or passage cells, wherein the primary cells are non-passage cells directly separated from umbilical cord tissue, and the passage cells are cells obtained by inoculating and culturing the primary cells after trypsin digestion and centrifugation.

6. The culture method according to claim 1, wherein The culture container is a T25 cell culture flask.

7. The culture method according to any one of claims 1 to 6, characterized in that The first culture medium and the second culture medium do not contain animal serum and are both sterilized by filtration through a 0.22 μm filter membrane before use.

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