Stem cell three-dimensional hypoxia culture method for promoting VEGF high expression

Abstract

The invention discloses a stem cell three-dimensional hypoxia culture method for promoting VEGF high expression, and relates to the technical field of cell biology.The stem cell three-dimensional hypoxia culture method for promoting VEGF high expression comprises the following steps that S1, a stem cell culture medium is prepared; s2, mixing the mesenchymal stem cells with a stem cell culture medium, and inoculating the mixture into a three-dimensional bioreactor for intermittent inoculation; s3, after the cells are inoculated and cultured for 2-3 days, the dissolved oxygen of the three-dimensional bioreactor is reduced, and the mesenchymal stem cells continue to be cultured for 48-72 hours under the three-dimensional low-oxygen condition; s4, digesting and collecting the mesenchymal stem cells subjected to three-dimensional hypoxia culture; according to the method, the expression level of the vascular endothelial growth factor (VEGF) of the mesenchymal stem cells is improved by using a three-dimensional hypoxia culture system and adopting an intermittent cell inoculation mode, the method for clinically producing the mesenchymal stem cells on a large scale is established, the three-dimensional hypoxia stem cells are applied to promoting angiogenesis, and the method is high in operability and remarkable in effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology technology, and particularly to a stem cell three-dimensional hypoxic culture method for promoting high expression of VEGF. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) promote angiogenesis mainly through paracrine effects, differentiation potential, and immune regulation. MSCs can secrete various angiogenic factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and SDF-1, which can promote the proliferation, migration, and vascularization of endothelial cells.

[0003] In clinical applications, MSCs have been used to treat various ischemic diseases, including coronary artery disease, lower extremity ischemia, myocardial infarction, ischemic stroke, and femoral head necrosis. For example, in coronary artery disease, MSCs provide a new treatment option for patients who are not suitable for revascularization strategies by restoring endothelial integrity and inducing neovascularization. In myocardial infarction models, MSCs promote myocardial repair and angiogenesis by secreting factors such as VEGF and FGF. In ischemic stroke, MSCs and their exosomes significantly improve neurological function by promoting angiogenesis, inhibiting inflammatory responses, and promoting neural regeneration.

[0004] Three-dimensional hypoxic culture technology has shown significant application prospects in the field of stem cells. Studies have shown that three-dimensional culture can more closely mimic the in vivo environment and improve the proliferation and differentiation capacity of stem cells. In particular, low oxygen conditions have been shown to enhance the immune regulatory capacity of mesenchymal stem cells (MSCs) and the secretion of paracrine factors, thereby improving their therapeutic effect.

[0005] Existing technologies have not systematically integrated low oxygen pretreatment and three-dimensional culture to enhance the angiogenic capacity of mesenchymal stem cells (MSCs). The feasibility of clinical-level production is lacking, the process is complex, and it is difficult to meet the demand for the production of billions of cells. Existing technologies focus on low oxygen or three-dimensional single-dimensional optimization, but do not address the dynamic and precise regulation of three-dimensional hypoxic microenvironments and the standardization of clinical-level production. Therefore, in view of the current situation, there is an urgent need to develop a stem cell three-dimensional hypoxic culture method for promoting high expression of VEGF to meet the actual needs. SUMMARY

[0006] In view of the above, the present application aims at the existing problems in the prior art, and the main purpose is to provide a stem cell three-dimensional hypoxic culture method for promoting high expression of VEGF, which improves the expression level of mesenchymal stem cell vascular endothelial growth factor (VEGF) by using a three-dimensional hypoxic culture system and adopting intermittent cell inoculation, establishes a method that can be used for large-scale production of mesenchymal stem cells in clinical practice, and applies three-dimensional hypoxic stem cells to promote angiogenesis, which has strong operability and remarkable effect.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A stem cell three-dimensional hypoxic culture method for promoting high expression of VEGF, comprising the following steps:

[0009] S1, preparing a stem cell culture medium, which comprises an alpha MEM culture medium, 2%-8% human platelet lysate and 0.02%-0.08% shear protective agent;

[0010] S2, mixing mesenchymal stem cells with the stem cell culture medium and inoculating them in a three-dimensional bioreactor for intermittent inoculation;

[0011] S3, after cell inoculation culture for 2-3 days, continuously feeding nitrogen gas to reduce the dissolved oxygen (DO value) of the three-dimensional bioreactor, so that the mesenchymal stem cells continue to be cultured under three-dimensional hypoxic conditions for 48-72 hours;

[0012] S4, digesting and collecting the mesenchymal stem cells after three-dimensional hypoxic culture.

[0013] As a preferred solution: the shear protective agent in step S1 is poloxamer 188, and the stem cell culture medium is an alpha MEM culture medium, 5% human platelet lysate and 0.05% poloxamer 188.

[0014] As a preferred solution: the intermittent inoculation in step S2 is specifically as follows: after cell inoculation on D0, the rotation speed of the three-dimensional bioreactor is set to 30-40 rpm for continuous rotation for 5-10 minutes, 0 rpm for static standing for 20-30 minutes; then 30-40 rpm for rotation for 5-10 minutes, 0 rpm for static standing for 20-30 minutes, and so on for 20-30 intermittent cycles, and then constant rotation speed is 30-40 rpm; the rotation speed is gradually increased during later culture.

[0015] As a preferred scheme: the intermittent inoculation in the step S2 is specifically as follows: after the cell inoculation on D0 day, the rotation speed of the three-dimensional bioreactor is set to 35 rpm for continuous rotation for 5 minutes, 0 rpm for static state for 25 minutes; then 35 rpm for rotation for 5 minutes, 0 rpm for static state for 25 minutes, and so on for 24 intermittent cycles, and then constant rotation speed is 35 rpm; the rotation speed is gradually increased in the later culture, the rotation speed is adjusted to 40 rpm for continuous stirring for 24 hours on D3 day, and the rotation speed is adjusted to 45 rpm for continuous stirring for 24 hours on D4 day.

[0016] As a preferred scheme: the amount of the inoculated cells in the step S2 is 5*10 4 / mL, and the culture temperature is 37±0.5℃.

[0017] As a preferred scheme: the initial dissolved oxygen (DO value) in the step S2 is 80%-100%, and the pH is 7.2-7.4.

[0018] As a preferred scheme: the dissolved oxygen (DO value) after the cell inoculation culture for D2-3 days in the step S3 is 5%-10%, and the hypoxia time is 48 hours-72 hours.

[0019] As a preferred scheme: the dissolved oxygen in the step S3 is adjusted to 5% on D2 day, nitrogen gas is continuously introduced to maintain a hypoxic environment, and the hypoxia time is 72 hours.

[0020] As a preferred scheme: the mesenchymal stem cells collected after the digestion in the step S4 are detected for vascular endothelial growth factor (VEGF) expression level and HGF expression level, and the mesenchymal stem cells promoting high expression of VEGF are screened.

[0021] As a preferred scheme: the mesenchymal stem cells in the step S2 are from umbilical cord tissue, placenta tissue, adipose tissue, bone marrow tissue and dental pulp tissue.

[0022] Compared with the prior art, the present application has obvious advantages and beneficial effects, specifically, according to the above technical scheme, the present application adopts a three-dimensional hypoxic culture method for promoting high expression of VEGF of stem cells, uses a three-dimensional hypoxic culture system, adopts an intermittent cell inoculation mode, improves the vascular endothelial growth factor (VEGF) expression level of mesenchymal stem cells, establishes a method which can be used for large-scale production of mesenchymal stem cells in clinical practice, and applies the three-dimensional hypoxic stem cells to promoting angiogenesis, and the method has strong operability and remarkable effect.

[0023] In order to more clearly illustrate the structural features and effects of the present application, the following will be combined with the specific embodiments and the drawings to be described in detail. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A diagram for comparing the viability of mesenchymal stem cells between examples and comparative examples of the present application;

[0025] Figure 2 A diagram for comparing the differentiation potential of mesenchymal stem cells between examples and comparative examples of the present application;

[0026] Figure 3 A diagram for comparing the detection of surface markers of mesenchymal stem cells between examples and comparative examples of the present application;

[0027] Figure 4 A diagram for comparing the RNA relative expression of IDO of mesenchymal stem cells between examples and comparative examples of the present application;

[0028] Figure 5 A diagram for comparing the RNA relative expression of HGF of mesenchymal stem cells between examples and comparative examples of the present application;

[0029] Figure 6 A diagram for comparing the RNA relative expression of VEGF of mesenchymal stem cells between examples and comparative examples of the present application;

[0030] Figure 7 A diagram for comparing the intracellular VEGF concentration determination of mesenchymal stem cells between examples and comparative examples of the present application;

[0031] Figure 8 A diagram for comparing the HGF concentration determination in the culture supernatant of mesenchymal stem cells between examples and comparative examples of the present application;

[0032] Figure 9 A diagram for comparing the VEGF concentration determination in the culture supernatant of mesenchymal stem cells between examples and comparative examples of the present application. DETAILED DESCRIPTION

[0033] As shown in the present application, Figures 1 to 9 a three-dimensional hypoxic culture method for promoting VEGF high expression of stem cells, comprising the following steps:

[0034] S1, preparing a stem cell culture medium, the stem cell culture medium comprising αMEM culture medium, 2%-8% human platelet lysate and 0.02%-0.08% shear protective agent;

[0035] S2, mixing mesenchymal stem cells with the stem cell culture medium, inoculating in a three-dimensional bioreactor with commercially available microcarriers for intermittent inoculation;

[0036] S3, after 2-3 days of cell inoculation culture, continuously reducing the dissolved oxygen (DO value) of the three-dimensional bioreactor by continuously feeding nitrogen gas, so that the mesenchymal stem cells continue to be cultured under three-dimensional hypoxic conditions for 48-72 hours;

[0037] S4, digest and collect mesenchymal stem cells after three-dimensional hypoxic culture.

[0038] The shearing protective agent in step S1 is poloxamer 188, and the stem cell culture medium is αMEM medium, 5% human platelet lysate and 0.05% poloxamer 188; poloxamer 188 is a non-ionic surfactant used as a shearing protective agent in cell culture to reduce shear damage to cells in a three-dimensional bioreactor.

[0039] The intermittent inoculation in step S2 is specifically as follows: after cell inoculation on D0, the rotation speed of the three-dimensional bioreactor is set to 30-40 rpm for continuous rotation for 5-10 minutes, 0 rpm for static state for 20-30 minutes; then 30-40 rpm for rotation for 5-10 minutes, 0 rpm for static state for 20-30 minutes, and so on for 20-30 intermittent cycles, and then constant rotation speed of 30-40 rpm; the rotation speed is gradually increased during later culture.

[0040] The intermittent inoculation in step S2 is specifically as follows: after cell inoculation on D0, the rotation speed of the three-dimensional bioreactor is set to 35 rpm for continuous rotation for 5 minutes, 0 rpm for static state for 25 minutes; then 35 rpm for rotation for 5 minutes, 0 rpm for static state for 25 minutes, and so on for 24 intermittent cycles, and then constant rotation speed of 35 rpm; the rotation speed is gradually increased during later culture, and the rotation speed is adjusted to 40 rpm for continuous stirring for 24 hours on D3, and the rotation speed is adjusted to 45 rpm for continuous stirring for 24 hours on D4.

[0041] The amount of cells inoculated in step S2 is 5×10 4 The culture temperature is 37±0.5℃.

[0042] The initial dissolved oxygen (DO value) in step S2 is 80%-100%, and the pH is 7.2-7.4.

[0043] The dissolved oxygen (DO value) of the cells after inoculation and culture for 2-3 days in step S3 is 5%-10%, and the hypoxic time is 48-72 hours.

[0044] The dissolved oxygen of the cells after inoculation and culture for 2 days in step S3 is adjusted to 5%, and nitrogen gas is continuously introduced to maintain a hypoxic environment, and the hypoxic time is 72 hours.

[0045] In step S4, the mesenchymal stem cells after digestion and collection are detected for vascular endothelial growth factor (VEGF) expression level and HGF expression level, and mesenchymal stem cells promoting high expression of VEGF are screened; the mesenchymal stem cells after digestion and collection of three-dimensional hypoxic culture are detected for vascular endothelial growth factor (VEGF) expression level, and the mesenchymal stem cells with significantly increased expression level are the mesenchymal stem cells with the ability to promote angiogenesis.

[0046] The mesenchymal stem cells in step S2 are derived from umbilical cord tissue, placental tissue, adipose tissue, bone marrow tissue, and dental pulp tissue.

[0047] Embodiment:

[0048] A three-dimensional hypoxic culture method for promoting VEGF high expression of stem cells, comprising cell inoculation and three-dimensional hypoxic culture process; specifically comprising the following steps:

[0049] S1, prepare a stem cell culture medium, which comprises αMEM culture medium + 5% human platelet lysate + 0.05% poloxamer 188.

[0050] S2, resuscitate 1 tube of P2 generation umbilical cord mesenchymal stem cells from the cell bank, and define the resuscitation day as day 0. The resuscitation method is as follows: open the water bath, adjust the temperature to 37℃; place the P2 generation umbilical cord mesenchymal stem cells in the water bath for rapid rewarming, and the rewarming time is 2 minutes; after the cells are completely dissolved, the resuscitation is completed.

[0051] After resuscitation, the umbilical cord mesenchymal stem cells are taken out, mixed with the stem cell culture medium (αMEM culture medium + 5% human platelet lysate + 0.05% poloxamer 188), inoculated in a three-dimensional bioreactor, and the inoculation cell amount is 5×10 4 / mL, the inoculation volume is 2L, the weight of the inoculated microcarriers is 4g, the culture temperature is set to 37±0.5℃, the initial dissolved oxygen (DO value) is 80-100%, the dissolved oxygen is automatically fed with O2 when it is lower than the set value, the pH is 7.2-7.4, and the automatic CO2 feeding is set to adjust the pH. The rotation speed of the three-dimensional bioreactor is set to 35rpm for 5 minutes, 0rpm for 25 minutes; then 35rpm for 5 minutes, 0rpm for 25 minutes, and so on for 24 intermittent cycles, and then constant rotation speed is 35rpm for continuous stirring. In the later culture, the rotation speed is increased day by day, the rotation speed is adjusted to 40rpm for continuous stirring for 24 hours on day 3, and the rotation speed is adjusted to 45rpm for continuous stirring for 24 hours on day 4.

[0052] On day 1, 1L of stem cell culture medium (αMEM culture medium + 5% human platelet lysate + 0.05% poloxamer 188) is supplemented into the three-dimensional bioreactor.

[0053] On day 2, the dissolved oxygen is adjusted to 5%, and nitrogen gas is continuously fed to maintain a hypoxic environment for 72 hours.

[0054] On day 3, the rotation speed is adjusted to 40rpm for continuous stirring for 24 hours.

[0055] On day 4, the rotation speed is adjusted to 45rpm for continuous stirring for 24 hours.

[0056] S4, the third day after the three-dimensional hypoxic culture, the mesenchymal stem cells were collected, and the P3 generation cells were obtained. The mesenchymal stem cells were resuspended with a cryopreservation solution containing 5% DMSO, cooled by a programmed cooling instrument, and then stored in a liquid nitrogen tank.

[0057] After one week, the cryopreserved P3 generation mesenchymal stem cells were recovered, and the cell viability, differentiation potential, surface markers, VEGF and HGF expression levels were detected.

[0058] Comparative Example:

[0059] One tube of P2 generation mesenchymal stem cells from the same donor as in the example was recovered from the cell bank. The recovery method was as follows: the water bath was opened, and the temperature was adjusted to 37°C; the P2 generation mesenchymal stem cells were placed in the water bath for rapid warming, and the warming time was 3 minutes; after the cells were completely dissolved, the recovery was completed.

[0060] After recovery, the mesenchymal stem cells were mixed with the stem cell culture medium (αMEM medium + 5% human platelet lysate + 0.05% poloxamer 188), inoculated in 75cm 2 culture bottles, and the inoculation density was 5000-6000 cells / cm 2 . After 3-5 days of culture, the confluence reached 85-95%, and the cells were harvested to obtain P3 generation cells. The cells were resuspended with a cryopreservation solution containing 5% DMSO, cooled by a programmed cooling instrument, and then stored in a liquid nitrogen tank.

[0061] After one week, the cryopreserved P3 generation cells were recovered, and the cell viability, differentiation potential, surface markers, VEGF and HGF expression levels were detected.

[0062] Table 1: Comparison of mesenchymal stem cell flow expression rates between the example and the comparative example

[0063] Group CD90 CD73 CD105 CD29 CD45 CD34 CD79a CD14 HLA-DR Comparative Example 99.99％ 98.67％ 99.64％ 99.98％ 1.32％ 2.00％ 0.03％ 0.45％ 0.32％ Example 99.97％ 99.10％ 99.92％ 100.0％ 1.00％ 1.21％ 0.69％ 0.67％ 0.25％

[0064] The results in Table 1 show that the cells cultured in the three-dimensional hypoxic system were detected by flow cytometry, and the positive expression rates of mesenchymal stem cell specific markers CD90, CD73, CD105, and CD29 were all more than 95%, while the expression rates of negative markers CD45, CD34, CD79a, CD14, and HLA-DR were all less than 2%. The results confirm that the prepared cell population has very high purity and good uniformity, which meets the typical phenotypic characteristics of mesenchymal stem cells.

[0065] Figure 1 The results show that the survival rate of cells cultured in the three-dimensional hypoxic condition is still maintained at more than 90%, which reflects a good cell activity state; Figure 2The results show that the cells cultured under three-dimensional hypoxic conditions can differentiate into osteoblasts, adipocytes and chondrocytes, and have the typical three-line differentiation potential of mesenchymal stem cells. Figure 3 The results show that the positive expression rates of mesenchymal stem cell specific markers CD90, CD73, CD105 and CD29 detected by flow cytometry are all more than 95%, and the expression rates of negative markers CD45, CD34, CD79a, CD14 and HLA-DR are all less than 2%. The results confirm that the prepared cell population has very high purity and good uniformity, and meets the typical phenotypic characteristics of mesenchymal stem cells; Figure 4 The results show that the RNA expression level of IDO, i.e. indoleamine-2,3-dioxygenase (ido), in the cells cultured under three-dimensional hypoxic conditions is significantly higher than that in the control group. IDO is a key immunosuppressive molecule that mediates the regulation of T cells by mesenchymal stem cells. The increase in the RNA expression of IDO under three-dimensional hypoxic culture conditions indicates that the immunoregulatory ability of mesenchymal stem cells is enhanced; Figure 5 The results show that the RNA expression level of HGF in the cells cultured under three-dimensional hypoxic conditions is significantly higher than that in the control group, which is more than 6 times that of the control. High expression of HGF and VEGF in stem cells synergistically enhances the pro-angiogenic effect, mainly including promoting ischemia improvement, promoting repair, functional recovery, etc. Figure 6 The results show that the RNA expression level of VEGF in the cells cultured under three-dimensional hypoxic conditions is significantly higher than that in the control group, indicating that the transcription or expression level of VEGF gene is increased, which means that the synthesis of VEGF in the cells is increased, and the related functions can be enhanced; Figure 7 The results show that the concentration of VEGF in the cells cultured under three-dimensional hypoxic conditions is 3 times that of the control group, indicating that the expression level of VEGF protein in the cells cultured under three-dimensional hypoxic conditions is increased; Figure 8 The results show that the concentration of HGF in the culture supernatant of cells cultured under three-dimensional hypoxic conditions is 1.3 times that of the control group, indicating that more HGF protein is secreted by the cells into the culture medium; Figure 9 The results show that the concentration of VEGF in the culture supernatant of cells cultured under three-dimensional hypoxic conditions is 65 times that of the control group, indicating that more VEGF protein is secreted by the cells into the culture medium. The cells can secrete VEGF, and the concentration of VEGF in the supernatant is significantly increased. This indicates that the cells not only express VEGF in the cells, but also secrete it outside the cells, which can be used for surrounding tissues or in vitro applications.

[0066] The design of the present application focuses on the present application by adopting a three-dimensional hypoxic culture method for promoting high expression of VEGF, using a three-dimensional hypoxic culture system, adopting an intermittent cell inoculation method, detecting the expression of VEGF gene of cells, the expression level of VEGF protein in cells and the level of VEGF protein secreted outside cells, determining that the three-dimensional hypoxic culture method improves the expression level of mesenchymal stem cell vascular endothelial growth factor (VEGF), and establishing a method which can be used for large-scale production of mesenchymal stem cells in clinical practice, and applying three-dimensional hypoxic stem cells to promote angiogenesis, the method has strong operability and the effect is improved by 65 times.

[0067] The above is only a preferred embodiment of the present application, not any limitation on the technical scope of the present application, so any slight modification, equivalent change and modification made on the above embodiment according to the technical essence of the present application still belongs to the scope of the technical solution of the present application.

Claims

1. A method for promoting VEGF high expression in stem cells in a three-dimensional hypoxic culture, characterized by: It comprises the following steps: S1, preparing a stem cell culture medium, which comprises an alpha MEM culture medium, 2%-8% human platelet lysate and 0.02%-0.08% shear protective agent; S2, mixing mesenchymal stem cells with the stem cell culture medium and inoculating in a three-dimensional bioreactor for intermittent inoculation; S3, after the cell inoculation culture for 2-3 days, continuously inputting nitrogen gas to reduce the dissolved oxygen (DO value) of the three-dimensional bioreactor, so that the mesenchymal stem cells continue to be cultured under three-dimensional low-oxygen conditions for 48-72 hours; S4, digesting and collecting the mesenchymal stem cells after three-dimensional low-oxygen culture.

2. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 1, characterized in that: The shear protective agent in the step S1 is poloxamer 188, and the stem cell culture medium is an alpha MEM culture medium, 5% human platelet lysate and 0.05% poloxamer 188.

3. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 1, characterized in that: The intermittent inoculation in the step S2 is specifically as follows: after the cell inoculation on D0, the rotation speed of the three-dimensional bioreactor is set to 30-40 rpm for continuous rotation for 5-10 minutes, 0 rpm for static standing for 20-30 minutes; then 30-40 rpm for rotation for 5-10 minutes, 0 rpm for static standing for 20-30 minutes, and so on for 20-30 intermittent cycles, and then constant rotation speed is 30-40 rpm; and the rotation speed is gradually increased in the later culture.

4. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 3, characterized in that: The intermittent inoculation in the step S2 is specifically as follows: after the cell inoculation on D0, the rotation speed of the three-dimensional bioreactor is set to 35 rpm for continuous rotation for 5 minutes, 0 rpm for static standing for 25 minutes; then 35 rpm for rotation for 5 minutes, 0 rpm for static standing for 25 minutes, and so on for 24 intermittent cycles, and then constant rotation speed is 35 rpm; and the rotation speed is gradually increased in the later culture, the rotation speed is adjusted to 40 rpm for continuous stirring for 24 hours on D3, and the rotation speed is adjusted to 45 rpm for continuous stirring for 24 hours on D4.

5. The method of claim 1, wherein the stem cells are cultured in a three-dimensional low-oxygen culture system. The amount of cells inoculated in the step S2 is 5 x 10 4 / mL, and the culture temperature is 37 ± 0.5°C.

6. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 1, characterized in that: The initial dissolved oxygen (DO value) of the inoculation in the step S2 is 80%-100%, and the pH is 7.2-7.

4.

7. The method of claim 1, wherein the stem cells are cultured in a three-dimensional low oxygen environment to promote high expression of VEGF. The dissolved oxygen (DO value) of the cell inoculation culture after D2-3 days in the step S3 is 5%-10%, and the low-oxygen time is 48 hours-72 hours.

8. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 7, characterized in that: The dissolved oxygen is adjusted to 5% on D2 in the step S3, nitrogen gas is continuously inputted to maintain a low-oxygen environment, and the low-oxygen time is 72 hours.

9. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 1, characterized in that: The mesenchymal stem cells after digestion and collection in the step S4 are detected for vascular endothelial growth factor (VEGF) expression level and HGF expression level, and mesenchymal stem cells promoting high expression of VEGF are screened.

10. The method for promoting VEGF high expression in three-dimensional hypoxic stem cell culture according to claim 1, characterized in that: The mesenchymal stem cells in the step S2 are derived from umbilical cord tissue, placenta tissue, adipose tissue, bone marrow tissue and dental pulp tissue.

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