Method for culturing human umbilical cord mesenchymal stem cells

Abstract

The invention relates to the technical field of biology, in particular to a human umbilical cord mesenchymal stem cell culture method which comprises the following steps: pretreating a fresh umbilical cord, removing blood vessels and outer membranes, flushing and cutting into pieces; separating cells through double-enzyme digestion, screening, centrifuging, resuspending by using a serum-free culture medium A, and inoculating into a coating culture bottle; performing primary culture until the cell fusion degree is 80-90%; digesting the cells during passage, and inoculating the cells to a three-dimensional scaffold containing a culture medium B in proportion; the liquid is regularly changed in the three-dimensional culture, and the culture lasts for 4-6 generations. Efficient separation and adherence of stem cells are achieved through a double-enzyme gradient digestion technology and a three-dimensional sodium alginate-gelatin porous scaffold, a serum-free culture medium A containing human platelet lysate (HPL) and the like is adopted for primary culture, the proliferation efficiency is improved, and cell activity and high dryness are maintained; in the passage stage, a culture medium B containing umbilical cord blood serum substitute (UCB-SR) and the like is used, a low-oxygen environment and triaxial tensile stress stimulation are combined, apoptosis is inhibited, and the purity and dryness of stem cells are ensured.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for culturing human umbilical cord mesenchymal stem cells. Background Art

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) have shown significant potential in the treatment of immune diseases such as graft-versus-host disease (GVHD) due to their multipotential differentiation, immunomodulatory abilities, and low immunogenicity. However, their large-scale culture faces key technical bottlenecks, including low cell isolation efficiency, insufficient proliferation capacity, difficulty maintaining stemness, and easy loss of differentiation potential, which hinder the standardization and effectiveness of clinical applications.

[0003] In existing technologies, traditional two-dimensional culture systems lack the ability to simulate the in vivo microenvironment, resulting in poor cell adhesion and slow proliferation rates. Long-term culture can easily lead to senescence (e.g., increased β-galactosidase activity) and decreased differentiation efficiency (osteoblastic / adipogenic induction efficiency is often less than 80%). Furthermore, animal serum-based culture media carry the risk of contamination by exogenous pathogens, and the complex serum composition makes it difficult to precisely regulate cell status. Furthermore, traditional digestion methods (e.g., single enzyme digestion) do not adequately dissociate umbilical cord tissue, resulting in low cell yields. Conventional culture conditions (e.g., normoxic environment, lack of mechanical stimulation) cannot effectively maintain high expression of stem cell surface markers (e.g., CD73, CD90, CD105), and are prone to activating hematopoietic cell markers (e.g., CD34, CD45), affecting cell purity.

[0004] To address these issues, existing technologies have attempted to introduce three-dimensional scaffold culture. However, the pore size and porosity of the scaffold materials have not been optimized, resulting in insufficient cell infiltration. Furthermore, the single cytokine regulation system makes it difficult to simultaneously promote proliferation and regulate differentiation pathways. Therefore, a method for culturing human umbilical cord mesenchymal stem cells was developed to address these issues. Summary of the Invention

[0005] Technical problems solved In response to the above-mentioned shortcomings of the prior art, the present invention provides a method for culturing human umbilical cord mesenchymal stem cells. The present invention uses dual-enzyme gradient digestion technology (collagenase type IV 0.08%-0.12% + hyaluronidase 0.04%-0.06%) and a three-dimensional sodium alginate-gelatin porous scaffold (pore size 140-210μm, porosity 88%-92%) to achieve efficient separation and adhesion of stem cells. The primary culture adopts serum-free culture medium A containing human platelet lysate (HPL) and other substances to improve proliferation efficiency and maintain cell activity and high stemness. In the passage stage, culture medium B containing umbilical cord blood serum substitute (UCB-SR) and other substances is used, combined with a hypoxic environment and triaxial tensile stress stimulation to inhibit apoptosis and ensure the purity and stemness of stem cells. Its osteogenic / adipogenic differentiation efficiency is ≥92%.

[0006] Technical Solution To achieve the above objectives, the present invention is implemented through the following technical solutions: In one aspect, a method for culturing human umbilical cord mesenchymal stem cells comprises the following steps: S100 umbilical cord pretreatment: fresh human umbilical cord tissue was obtained, blood vessels and the outer layer of Wharton's jelly were removed, and the tissue was rinsed three times with D-PBSA solution containing 1% to 2% double antibody by volume, each time for 4 to 6 minutes; the tissue was cut into 1 mm pieces. 3 Up to 3mm 3 tissue blocks; S200 enzymatic dissociation: Place the tissue block in DMEM / F12 solution containing 0.08% to 0.12% collagenase type IV and 0.04% to 0.06% hyaluronidase by mass volume, and digest at 37°C with shaking for 45 to 60 minutes; S300 primary inoculation: After terminating the digestion, pass through a 70 μm cell sieve, collect the cells by centrifugation, resuspend in a dedicated serum-free medium A, and culture at a density of 1.2×10 4 cells / cm 2 to 1.8×10 4 cells / cm 2 Seed in poly-lysine-coated cell culture flasks; Primary culture of S400 cells: Culture in a 37°C, 5% CO2, 95% humidity incubator, replacing 50% to 60% of medium A every 48 hours until the cell confluence reaches 80% to 90%. S500 passage expansion: digest with a mixture of 0.04% to 0.06% EDTA and 0.02% to 0.03% trypsin, and seed onto a three-dimensional alginate-gelatin scaffold containing medium B at a ratio of 1:2 to 1:4; S600 3D culture: Replace 40% to 50% of the volume of medium B every 72 hours and continue culturing to the 4th to 6th passage. Wherein, the culture medium A comprises: DMEM / F12 basal medium, 13% to 17% human platelet lysate HPL by volume, 0.8% to 1.2% insulin-transferrin-selenium complex ITS-X by volume, 8 ng / mL to 12 ng / mL epidermal growth factor EGF by volume, 4 ng / mL to 6 ng / mL platelet-derived growth factor PDGF-BB by volume, and 0.08 mM to 0.12 mM β-mercaptoethanol by volume; The culture medium B contains: α-MEM basal medium, umbilical cord blood serum substitute UCB-SR with a volume fraction of 7% to 9%, vitamin C phosphate magnesium with a mass volume concentration of 0.4% to 0.6%, basic fibroblast growth factor bFGF with a concentration of 18 ng / mL to 22 ng / mL, insulin-like growth factor-1 IGF-1 with a concentration of 13 ng / mL to 17 ng / mL, Rho-kinase inhibitor Y27632 with a concentration of 8 μM to 12 μM, and penicillin-streptomycin with a volume fraction of 0.8% to 1.2%.

[0007] Furthermore, the preparation method of the three-dimensional sodium alginate-gelatin scaffold in step S500 includes: dissolving sodium alginate and gelatin in deionized water at a mass ratio of 2.8:1 to 3.2:1 to form a mixed solution with a mass volume concentration of 3.5% to 4.5%, adding a CaCl2 cross-linking agent with a concentration of 8mM to 12mM, injecting it into a porous mold and freeze-drying it to form a porous scaffold with a pore size of 140μm to 210μm and a porosity of 88% to 92%.

[0008] Furthermore, the preparation method of human platelet lysate HPL in the culture medium A includes: extracting from human platelet concentrate, freezing and thawing at -80°C 2 to 4 times, centrifuging to remove debris, sterilizing with a 0.22 μm filter membrane, and adjusting the final concentration to 95 mg / mL to 105 mg / mL of total protein.

[0009] Furthermore, when replacing the culture medium in step S600, a cytokine enhancer is added simultaneously, wherein the composition includes: Wnt3a protein at a concentration of 4 ng / mL to 6 ng / mL, Notch signaling pathway inhibitor DAPT at a concentration of 0.4 μM to 0.6 μM, and sodium butyrate at a concentration of 2.5 mM to 3.5 mM, and the added amount is 4% to 6% of the volume of the replacement culture medium.

[0010] Furthermore, the digestion termination solution in step S500 is a DMEM / F12 solution containing 9% to 11% UCB-SR by volume and 0.08% to 0.12% soybean trypsin inhibitor by mass volume, and the digestion time is controlled at 3 minutes to 4 minutes.

[0011] Furthermore, the S500 and S600 steps are performed under hypoxic conditions with an oxygen concentration of 4.5% to 5.5%, and triaxial stretching with a frequency of 0.8 Hz to 1.2 Hz and a strain of 4% to 6% is applied every 24 hours, each lasting 25 minutes to 35 minutes.

[0012] Furthermore, the step S400 includes two selective liquid changes: From day 3 to day 5 after inoculation: replace the culture medium with medium A containing 4 μg / mL to 6 μg / mL fibronectin; When the cell confluence reaches 45% to 55%, replace the medium A with 0.8 μM to 1.2 μM TGF-β receptor inhibitor SB431542.

[0013] Furthermore, the obtained hUC-MSCs must meet the following requirements: CD73, CD90, and CD105 positivity rates ≥ 97.5%, CD34, CD45, and HLA-DR positivity rates ≤ 2.5%, cell doubling time ≤ 24 hours, senescence-related β-galactosidase activity ≤ 10%, and osteogenic / adipogenic induction differentiation efficiency ≥ 92%.

[0014] On the other hand, a method for culturing human umbilical cord mesenchymal stem cells is provided for preparing human umbilical cord mesenchymal stem cells for use in preparing a drug for treating graft-versus-host disease. After the cells are cultured in three dimensions for the second to fourth generations, they are resuspended in physiological saline containing 0.4 mg / mL to 0.6 mg / mL human albumin to a final concentration of 1.8×10 6 cells / mL to 2.2×10 6 cells / mL, and the intravenous dose was 0.8×10 6 cells / kg to 1.2×10 6 cells / kg body weight.

[0015] Beneficial effects Compared with the known public technology, the technical solution provided by the present invention has the following beneficial effects: 1. The present invention achieves efficient separation and adhesion of umbilical cord mesenchymal stem cells through dual-enzyme gradient digestion technology (collagenase type IV 0.08%-0.12% + hyaluronidase 0.04%-0.06%) and three-dimensional sodium alginate-gelatin porous scaffold (pore size 140-210μm, porosity 88%-92%). Combined with primary culture in serum-free medium A (containing factors such as HPL, EGF, PDGF-BB), the efficiency of cell proliferation is significantly improved, laying the foundation for subsequent passage and expansion. The three-dimensional culture system enhances the multidirectional differentiation ability of stem cells by simulating the in vivo microenvironment, meeting the high standard requirement of osteogenic / adipogenic induction differentiation efficiency ≥92%.

[0016] Second, the present invention introduces medium A and medium B, wherein medium A uses human platelet lysate (HPL) instead of serum to avoid the risk of animal-derived contamination. At the same time, the antioxidant combination of ITS-X complex and β-mercaptoethanol maintains the activity of primary cells. The selective medium exchange strategy (fibronectin and TGF-β inhibitor SB431542) regulates cell adhesion and differentiation pathways, ensuring that cells maintain high stemness even when the degree of confluence reaches 80%-90% during the primary culture stage. Culture medium B introduces umbilical cord blood serum substitute (UCB-SR) and Y27632 kinase inhibitor, combined with a hypoxic environment (4.5%-5.5% O2) and triaxial tensile stress stimulation, inhibiting cell apoptosis and maintaining a CD73 / CD90 / CD105 positivity rate ≥97.5% during the passage stage. At the same time, the positivity rate of hematopoietic cell markers such as CD34 / CD45 / HLA-DR is controlled at ≤2.5%, ensuring the purity and stemness of stem cells. DETAILED DESCRIPTION

[0017] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0018] The present invention will be further described below with reference to the embodiments.

[0019] Example 1: Orthogonal optimization experiment of umbilical cord pretreatment 1. Effect of flushing conditions on cell damage Orthogonal experiments were performed to optimize the concentration of the dual antibody in the rinsing solution (1%, 1.5%, 2%), rinsing time (4 min, 5 min, 6 min), and operating temperature (4°C, room temperature, 37°C). Cell viability (trypan blue rejection rate) was used as the evaluation indicator. The optimal combination: 1.5% double antibody, 5 minutes of washing, and 4°C operation, the cell viability reached 96.8% (see Table 1).

[0020] Table 1 Results of orthogonal test of umbilical cord flushing conditions:

[0021] 2. Effect of tissue size on enzymatic hydrolysis efficiency Cut the umbilical cord into 1mm pieces 3 , 2mm 3 , 3mm 3 Three specifications to test the cell yield after digestion: 2mm 3 The cell yield of group 1 was the highest (1.12×10 6 cells / g tissue), because too small tissue blocks may lead to over-digestion of cells, while too large blocks may lead to insufficient enzyme penetration (see Table 2).

[0022] Table 2 Relationship between tissue block size and cell yield:

[0023] Example 2: Kinetic mechanism of dual enzyme digestion 1. Effect of digestion time on cell activity Set the digestion time gradient (30min, 45min, 60min, 75min) to detect cell viability and DNA release: At 45 min, the DNA release reached a peak (11.2 μg / mL) and the cell viability was 95.3%. After 60 min, the viability dropped to 89.7%, indicating that the optimal digestion time was 45-60 min (Table 3).

[0024] Table 3. Digestion time and cell viability / DNA release.

[0025]

[0026] 2. Synergistic effect of dual enzyme concentration ratio The concentration of collagenase type IV was fixed at 0.1%, and the concentration of hyaluronidase was adjusted (0.04%, 0.05%, 0.06%). It was found that the highest cell yield was found at 0.05% (1.23×10 6 cells / g), which was 48% higher than that of the collagenase alone group (see Table 4).

[0027] Table 4 Effect of dual enzyme concentration ratio on cell yield:

[0028] Example 3: Optimization of biomimetic performance of three-dimensional scaffolds 1. Effect of pore size on cell infiltration Scaffolds with pore sizes of 140 μm, 175 μm, and 210 μm were prepared, and the invasion depth was measured 48 hours after cell inoculation: The cell infiltration depth of the 175μm pore size group reached 230μm, which was significantly higher than that of the 140μm group (180μm) and the 210μm group (205μm). This is because if the pore size is too small, the steric hindrance is large, and if it is too large, there is a lack of adhesion sites (see Table 5).

[0029] Table 5 Scaffold pore size and cell infiltration depth:

[0030] 2. Porosity and nutrient diffusion efficiency The glucose diffusion coefficient of scaffolds with different porosities (88%, 90%, 92%) was determined by dye diffusion experiments: The diffusion coefficient of the 90% porosity group reached 0.85×10 -6 cm 2 / s, close to natural cancellous bone (0.9×10 -6 cm 2 / s), ensuring deep cell survival (see Table 6).

[0031] Table 6 Porosity and glucose diffusion coefficient:

[0032] Example 4: Component screening experiment of culture medium A 1. Effect of HPL concentration gradient on proliferation Set the HPL volume fraction to 13%, 15%, and 17%, and detect the cell density after 3 days of culture: The cell density of the 15% group reached 2.8×10 4 cells / cm 2 , compared with the 13% group (2.2×10 4 ) increased by 27%, and there was no significant increase in the 17% group, suggesting that the optimal concentration was 15% (see Table 7).

[0033] Table 7 HPL concentration and cell proliferation efficiency:

[0034] 2. Effect of EGF on cell migration Transwell experiments showed that the number of migrating cells in the 10 ng / mL EGF group (420±35 cells / field) was twice that of the control group (210±22 cells), confirming that it significantly promoted cell spreading (see Table 8).

[0035] Table 8 EGF concentration and cell migration ability:

[0036] Example 5: Validation of the molecular mechanism of hypoxic culture 1. Effect of oxygen concentration on HIF-1α expression Western blot analysis showed that the expression of HIF-1α protein in the 5% O2 group (grayscale value 1.82±0.15) was 5.2 times that in the normoxia group (0.35±0.04), activating the stem cell stemness maintenance pathway (see Table 9).

[0037] Table 9 Effect of hypoxia on HIF-1α protein expression:

[0038] 2. Regulation of apoptosis-related genes by hypoxia qPCR detection showed that the Bcl-2 / Bax gene expression ratio in the 5% O2 group (2.35±0.21) was significantly higher than that in the normoxia group (1.12±0.13), inhibiting cell apoptosis (Table 10).

[0039] Table 10 Effects of hypoxia on apoptosis gene expression:

[0040] Example 6: Study of signaling pathways of mechanical stimulation 1. Effect of strain on FAK phosphorylation After stimulation with different strains (4%, 5%, and 6%) for 30 minutes, the phosphorylation level of p-FAKTyr397 in the 5% group (fluorescence intensity 285±22) increased by 185% compared with the static group (100±15), activating focal adhesion signaling (see Table 11).

[0041] Table 11 Strain stress and FAK phosphorylation level:

[0042] 2. Effect of Frequency on Calcium Signaling Calcium imaging showed that [Ca 2+ ]i oscillation amplitude (ΔF / F0=0.68±0.05) was significantly higher than that of the 0.5Hz (0.32±0.03) and 1.5Hz (0.45±0.04) groups, optimizing the mechanical transduction efficiency (see Table 12).

[0043] Table 12 Stimulation frequency and calcium signal intensity:

[0044] Example 7: Standardized Process for Stem Cell Quality Control 1. Flow Cytometry Gating Strategy for Surface Marker Detection CD73-PE / CD90-FITC / CD105-APC three-color labeling was used and analyzed using FlowJo software: Live cell gate (DAPI - ) accounted for ≥95%, CD73 + CD90 + CD105 + Cell ratio ≥98%, CD34 - CD45 - HLA-DR - The cell ratio was ≤2% (see Table 13).

[0045] Table 13 Surface Marker Flow Test Standards:

[0046] 2. Dual-Indicator Threshold for Aging Detection The percentage of cells positive for β-galactosidase staining was ≤10%, and the relative telomere length (T / S value) was ≥0.8, which significantly delayed senescence compared with two-dimensional culture (T / S=0.65) (see Table 14).

[0047] Table 14 Comparison of aging detection indicators:

[0048] Example 8: Dose optimization experiment for GVHD treatment 1. Effect of cell concentration on therapeutic efficacy Set the cell resuspension concentration to 1.0 × 10 6 cells / mL, 2.0×10 6 cells / mL、3.0×10 6 cells / mL, found 2.0×10 6 The GVHD remission rate was the highest in the 40 cells / mL group (92.5%), and excessively high concentrations led to pulmonary vascular microembolism (see Table 15).

[0049] Table 15 Cell concentration and GVHD remission rate:

[0050] 2. Effect of administration timing on survival rate Drugs were administered on the 1st, 3rd, and 5th day after modeling. The survival rate of the group administered on the 3rd day reached 88.7%. At this time, the inflammatory factor (IL-6) was at its peak, and the stem cell immunoregulatory effect was the best (see Table 16).

[0051] Table 16: Dosage timing and mouse survival rate:

[0052] Example 9: Application Expansion - Bone Tissue Engineering After the 4th generation hUC-MSCs were combined with the three-dimensional scaffold, osteogenic induction medium (containing 10 mM sodium β-glycerophosphate and 50 μg / mL vitamin C) was added. After culturing for 21 days: Quantitative analysis of Alizarin red staining showed that the absorbance of calcium nodules (A562 = 1.32 ± 0.10) was 69% higher than that of two-dimensional culture (0.78 ± 0.08) (see Table 17).

[0053] Table 17 Comparison of osteogenic differentiation efficiency:

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for culturing human umbilical cord mesenchymal stem cells, characterized in that: The method comprises the following steps: S100 umbilical cord pretreatment: fresh human umbilical cord tissue was obtained, blood vessels and the outer layer of Wharton's jelly were removed, and the tissue was rinsed three times with D-PBSA solution containing 1% to 2% double antibody by volume, each time for 4 to 6 minutes; the tissue was cut into 1 mm pieces. 3 Up to 3mm 3 tissue blocks; S200 enzymatic dissociation: Place the tissue block in DMEM / F12 solution containing 0.08% to 0.12% collagenase type IV and 0.04% to 0.06% hyaluronidase by mass volume, and digest at 37°C with shaking for 45 to 60 minutes; S300 primary inoculation: After terminating the digestion, pass through a 70 μm cell sieve, collect the cells by centrifugation, resuspend in a dedicated serum-free medium A, and culture at a density of 1.2×10 4 cells / cm 2 to 1.8×10 4 cells / cm 2 Seed in poly-lysine-coated cell culture flasks; Primary culture of S400 cells: Culture in a 37°C, 5% CO2, 95% humidity incubator, replacing 50% to 60% of medium A every 48 hours until the cell confluence reaches 80% to 90%. S500 passage expansion: digest with a mixture of 0.04% to 0.06% EDTA and 0.02% to 0.03% trypsin, and seed onto a three-dimensional alginate-gelatin scaffold containing medium B at a ratio of 1:2 to 1:4; S600 3D culture: Replace 40% to 50% of the volume of medium B every 72 hours and continue culturing to the 4th to 6th passage. Wherein, the culture medium A comprises: DMEM / F12 basal medium, 13% to 17% human platelet lysate HPL by volume, 0.8% to 1.2% insulin-transferrin-selenium complex ITS-X by volume, 8 ng / mL to 12 ng / mL epidermal growth factor EGF by volume, 4 ng / mL to 6 ng / mL platelet-derived growth factor PDGF-BB by volume, and 0.08 mM to 0.12 mM β-mercaptoethanol by volume; The culture medium B contains: α-MEM basal medium, umbilical cord blood serum substitute UCB-SR with a volume fraction of 7% to 9%, vitamin C phosphate magnesium with a mass volume concentration of 0.4% to 0.6%, basic fibroblast growth factor bFGF with a concentration of 18 ng / mL to 22 ng / mL, insulin-like growth factor-1 IGF-1 with a concentration of 13 ng / mL to 17 ng / mL, Rho-kinase inhibitor Y27632 with a concentration of 8 μM to 12 μM, and penicillin-streptomycin with a volume fraction of 0.8% to 1.2%.

2. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The method for preparing the three-dimensional sodium alginate-gelatin scaffold in step S500 includes: dissolving sodium alginate and gelatin in deionized water at a mass ratio of 2.8:1 to 3.2:1 to form a mixed solution with a mass volume concentration of 3.5% to 4.5%, adding a CaCl2 cross-linking agent with a concentration of 8mM to 12mM, injecting the solution into a porous mold and freeze-drying the solution to form a porous scaffold with a pore size of 140μm to 210μm and a porosity of 88% to 92%.

3. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The preparation method of human platelet lysate HPL in the culture medium A comprises: extracting from human platelet concentrate, freezing and thawing at -80°C for 2 to 4 times, centrifuging to remove debris, sterilizing with a 0.22 μm filter membrane, and adjusting the final concentration to 95 mg / mL to 105 mg / mL of total protein.

4. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: When replacing the culture medium in the S600 step, a cytokine enhancer is added simultaneously, wherein the cytokine enhancer comprises: Wnt3a protein at a concentration of 4 ng / mL to 6 ng / mL, Notch signaling pathway inhibitor DAPT at a concentration of 0.4 μM to 0.6 μM, and sodium butyrate at a concentration of 2.5 mM to 3.5 mM, with the added amount being 4% to 6% of the volume of the replacement culture medium.

5. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The digestion termination solution in step S500 is a DMEM / F12 solution containing 9% to 11% UCB-SR by volume and 0.08% to 0.12% soybean trypsin inhibitor by mass volume, and the digestion time is controlled at 3 minutes to 4 minutes.

6. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The S500 and S600 steps are performed under hypoxic conditions with an oxygen concentration of 4.5% to 5.5%, and triaxial stretching with a frequency of 0.8 Hz to 1.2 Hz and a strain of 4% to 6% is applied every 24 hours, each lasting 25 minutes to 35 minutes.

7. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The S400 step includes two selective fluid changes: From day 3 to day 5 after inoculation: replace the culture medium with medium A containing 4 μg / mL to 6 μg / mL fibronectin; When the cell confluence reaches 45% to 55%, replace the medium A with 0.8 μM to 1.2 μM TGF-β receptor inhibitor SB431542.

8. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein: The obtained hUC-MSCs must meet the following requirements: CD73, CD90, and CD105 positivity rates ≥ 97.5%, CD34, CD45, and HLA-DR positivity rates ≤ 2.5%, cell doubling time ≤ 24 hours, senescence-related β-galactosidase activity ≤ 10%, and osteogenic / adipogenic differentiation efficiency ≥ 92%.

9. Use of human umbilical cord mesenchymal stem cells prepared by the method for culturing human umbilical cord mesenchymal stem cells according to any one of claims 1 to 8 in preparing a drug for treating graft-versus-host disease, characterized in that: After the cells were cultured in three-dimensional culture for the second to fourth generations, they were resuspended in physiological saline containing 0.4 mg / mL to 0.6 mg / mL human albumin to a final concentration of 1.8 × 10 6 cells / mL to 2.2×10 6 cells / mL, and the intravenous dose was 0.8×10 6 cells / kg to 1.2×10 6 cells / kg body weight.

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