3D culture medium for mesenchymal stem cells

Abstract

The invention belongs to the technical field of cell culture, and provides a mesenchymal stem cell 3D culture medium, which comprises a basic culture medium and an additive, the additive is composed of lipid, a metabolism regulator, adherin, a buffering agent and human platelet lysate. According to the mesenchymal stem cell 3D culture medium provided by the invention, through the synergistic effect of all the components, the attachment rate of cells on a microcarrier can be remarkably increased, formation and growth of cell three-dimensional aggregates are promoted, cell senescence can be remarkably reduced, and the proliferation efficiency is improved. In addition, the mesenchymal stem cells obtained through culture of the 3D culture medium can improve the treatment effect on ARDS. The culture medium is simple in component and good in culture effect, and can be widely applied to 3D culture of mesenchymal stem cells of various sources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and relates to a 3D culture medium for mesenchymal stem cells and application thereof in mesenchymal stem cell culture. BACKGROUND

[0002] The information disclosed in this background section is intended to increase an understanding of the general context of the present application and is not necessarily recognized in the prior art before the present application.

[0003] Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) have the potential of multi-directional differentiation and immunoregulation, and show great application potential in the fields of tissue engineering, regenerative medicine and cell therapy. Traditional stem cell culture is mainly based on two-dimensional (2D) culture. In this culture mode, cells only grow in one plane, which has many limitations. For example, the proliferation efficiency and space utilization rate of cells are low, which cannot meet the growing demand for large-dose cell applications in clinical practice. Moreover, the 2D culture environment is quite different from the real three-dimensional microenvironment of cells in vivo, which is difficult to maintain the physiological function and characteristics of cells. In the long-term culture process, cells are prone to problems such as weakened differentiation ability, slowed proliferation rate and cell aging.

[0004] Compared with 2D planar cell culture technology, three-dimensional (3D) culture technology co-cultures three-dimensional matrix and cells in vitro, so that cells can form three-dimensional cell aggregates and more truly simulate the living environment in the body. In the 3D culture system, cells can grow, differentiate and migrate in a three-dimensional manner, which is conducive to the proliferation, survival and maintenance of the characteristics of cells. However, the culture medium suitable for 3D culture of mesenchymal stem cells has many deficiencies. On the one hand, many culture media rely on the addition of serum, which not only increases the risk of contamination of exogenous viruses and pathogenic factors, but also leads to poor reproducibility of experimental results due to the complex composition of serum and large batch-to-batch differences. At the same time, residual serum can easily cause allergic reactions in inoculators, bringing great challenges to clinical research. On the other hand, existing commercial culture media have low cell adhesion rate (usually <60%) and limited expansion fold (about 5-8 times) in 3D microcarrier culture, so it is difficult to achieve efficient cell expansion and maintain the high-quality state of cells. SUMMARY

[0005] In view of the low cell adhesion rate and low expansion fold of the current 3D culture medium for mesenchymal stem cells in carrier culture, the present application provides a 3D culture medium for mesenchymal stem cells, which can significantly improve the low cell adhesion rate, expansion fold and inflammation inhibition function.

[0006] Another object of the present application is to provide a 3D culture method of mesenchymal stem cells.

[0007] To achieve the above object, the present application adopts the following technical solution.

[0008] A 3D culture medium of mesenchymal stem cells comprises a basic medium and an additive; the additive is composed of lipids, metabolic regulators, laminin, a buffer and human platelet lysate; The lipids are oleic acid, linoleic acid, docosahexaenoic acid (DHA) and cholesterol in a mass ratio of (1-2):(1-2):(0.5-1):(0.1-0.5); the concentration of the cholesterol in the 3D culture medium is 100 mg / L; The metabolic regulators are a combination of nicotinamide and L-carnitine; the concentration of the nicotinamide in the 3D culture medium is 5-10 mg / L, and the concentration of the L-carnitine in the 3D culture medium is 2-5 mg / L; The laminin is a combination of recombinant human vitronectin (vitronectin) and recombinant human fibronectin fragment (fibronectin); the concentration of the vitronectin in the 3D culture medium is 1-5 μg / mL, and the concentration of the fibronectin in the 3D culture medium is 3-8 μg / mL; The buffer is a combination of HEPES and sodium bicarbonate; the concentration of the HEPES in the 3D culture medium is 10-15 mM, and the concentration of the sodium bicarbonate in the 3D culture medium is 2.5-4.5 g / L; The addition amount of the human platelet lysate is 2-5% v / v of the basic medium.

[0009] The basic medium is DMEM / F12 or a combination of DMEM / F12 and MCDB131; preferably, the basic medium is a combination of DMEM / F12 and MCDB131 in a volume ratio of 3:1-2:1.

[0010] In the present application, the basic medium mixed by DMEM / F12 and MCDB131 in a certain ratio can provide more balanced amino acids, vitamins, trace elements and inorganic salts, and optimize the cell metabolic environment; the combination of various lipids can improve the formation of stem cell three-dimensional aggregates and maintain the stemness of stem cells; nicotinamide and L-carnitine as metabolic regulators can synergistically regulate cell energy metabolism and delay aging; vitronectin and fibronectin can significantly improve the adhesion efficiency of cells on the surface of microcarriers through synergistic effect; the double buffer and osmotic pressure regulation system composed of HEPES and sodium bicarbonate can stabilize the pH value and optimize the osmotic pressure, and adapt to the 3D dynamic culture environment.

[0011] The present application also provides a 3D culture method of mesenchymal stem cells, comprising the following steps: The mesenchymal stem cells are inoculated into the above 3D culture medium containing microcarriers, and after intermittent stirring culture, constant speed stirring culture.

[0012] The inoculation density of the mesenchymal stem cells is (1-5) x 10 4 / mL.

[0013] The concentration of the microcarriers is 1.5-3 mg / mL.

[0014] The intermittent stirring is 40-60 rpm for 5 min, 0 rpm for 25-35 min, and the cycle is 24 cycles.

[0015] The speed of the constant speed stirring is 40-60 rpm.

[0016] The end point of the above culture is that the glucose concentration is reduced to 3.0 mmol / L or below, and the glucose concentration is detected by glucose dehydrogenase method blood glucose test paper.

[0017] When the culture reaches the end point, the mesenchymal stem cells are dispersed and passaged or harvested.

[0018] A mesenchymal stem cell obtained by the above culture method.

[0019] The mesenchymal stem cell can be used for preparing a medicine for treating acute lung injury (ARDS).

[0020] The present application has the following advantages: The mesenchymal stem cell 3D culture medium provided by the present application can significantly improve the adhesion rate of cells on microcarriers, promote the formation and growth of three-dimensional cell aggregates, and also significantly reduce cell aging and improve proliferation efficiency through the synergistic effect between components. In addition, the mesenchymal stem cells obtained by using the 3D culture medium for culture can improve the treatment effect on ARDS. The components of the culture medium are simple, the culture effect is good, and it can be widely used for 3D culture of mesenchymal stem cells of various sources. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the influence of different concentrations of effective components on cell adhesion rate and three-dimensional cell aggregates in Example 1; Figure 2 is the influence of different concentrations of metabolic regulators on cell aging in Example 1; Figure 3 is the cell adhesion rate and proliferation fold under different culture conditions in Example 2; Figure 4 is the MSC phenotype detection result of Example 2 culture; Figure 5 is the MSC three-lineage differentiation detection result of Example 2 culture; Figure 6 is the effect of different culture conditions on MSC cell senescence in Example 3; Figure 7 is the comparison of MSC treatment of ARDS under different culture conditions; Figure 8 is the cell adhesion rate and proliferation fold of cells obtained in different culture media in Example 4; Figure 9 is the cell adhesion rate and proliferation fold of cells obtained in different culture media in Example 5; Figure 10 is the cell adhesion rate and proliferation fold of cells obtained in different culture media in Example 6. DETAILED DESCRIPTION

[0022] The present application will be further described in conjunction with the examples and drawings, but the present application is not limited by the following examples.

[0023] Example 1 Preparation of mesenchymal stem cell 3D culture medium 1. Prepare different 3D culture media according to the following formula: Formula 1: DMEM / F12 and MCDB131 are mixed in a volume ratio of 3:1 as the basic culture medium; then add oleic acid, linoleic acid, docosahexaenoic acid (DHA) and cholesterol in a mass ratio of 1:1:0.5:0.1, and make the cholesterol content 100 mg / L; nicotinamide concentration is 5 mg / L, L-carnitine concentration is 2 mg / L, vitronectin concentration is 1 μg / mL, fibronectin concentration is 3 μg / mL, HEPES concentration is 10 mM, sodium bicarbonate concentration is 2.5 g / L, and 5% (v / v) human platelet lysate is added to prepare mesenchymal stem cell 3D culture medium; Formula 2: Compared with Formula 1, the mass concentration ratio of oleic acid, linoleic acid, docosahexaenoic acid (DHA) and cholesterol is 0.5:0.5:0.2:0.05, and the cholesterol content is 500 mg / L; the rest of the ingredients remain unchanged; Formula 3: Compared with Formula 1, the concentration of nicotinamide is 3 mg / L, and the concentration of L-carnitine is 1 mg / L, and the rest of the ingredients remain unchanged; Formula 4: Compared with Formula 1, no vitronectin is added, and the rest of the ingredients remain unchanged; Formula 5: Compared with Formula 1, no fibronectin is added, and the rest of the ingredients remain unchanged.

[0024] 2. 3D culture of mesenchymal stem cells 5 125 mL shake flasks were silicified with a small amount of silicizing agent, and at the same time, 38 mL of the prepared complete medium was added respectively, and then 80 mg of microcarriers (3D TableTrix ®W01 (Huakan Biotechnology) was added to shake flasks and labeled as Group 1 (Formula 1), Group 2 (Formula 2), Group 3 (Formula 3), Group 4 (Formula 4), and Group 5 (Formula 5). The shake flasks were placed in an incubator and stirred at 40 rpm for 30 min. Simultaneously, the concentration of the resuscitated umbilical cord mesenchymal stem cells (hUC-MSCs) was adjusted to 1×10⁻⁶ cells / mL. 6 Add 2 mL (1×10⁻⁶ cells / mL) to the solution. 6 The prepared cell suspension (cells / mL) was placed in the reactor base and cultured in cycles of 40 rpm for 5 min, 0 rpm for 25 min, with intermittent stirring for 24 cycles. Then, it was cultured at a constant speed of 40 rpm for about 4 days until the glucose concentration decreased to about 3.0 mmol / L (glucose dehydrogenase method blood glucose test strip, Roche).

[0025] During cell harvesting, the microcarriers were first lysed using lysis buffer (R001-500, Huakan Biotechnology) at a final concentration of 1 mg / mL, at 37°C for 40 min, with the cells being pipetted 10 times every 10 min during this period. After complete lysis of the microcarriers, the cell suspension was removed, centrifuged at 600 × g for 5 min, and the supernatant was discarded to obtain the cell pellet.

[0026] 3. Adhesion efficiency 24 hours later, 2 mL of cell suspension was taken from each of groups 1, 4, and 5, and the microcarriers were lysed using lysis buffer at a final concentration of 1 mg / mL. Lysis was carried out at 37°C for 40 min, with the cells removed and pipetted 10 times every 10 min. After complete lysis of the microcarriers, the cell suspension was removed and centrifuged at 600 × g for 5 min. The supernatant was discarded, and the cells were resuspended in basal culture medium for counting. Cell adhesion rate was calculated simultaneously. Cell adhesion rate = (Number of cells 24 hours after inoculation / Number of cells at inoculation) × 100%.

[0027] The adhesion rates of groups 1, 4, and 5 are shown in the figure. Figure 1 As shown in Figure A, the adhesion rate of group 1 was significantly higher than that of groups 4 and 5, while there was no significant difference between groups 4 and 5. This indicates that fibronectin and hyalin, through synergistic effects, can significantly enhance the cell adhesion efficiency on the microcarrier surface.

[0028] 4. Formation of three-dimensional aggregates Cells harvested from groups 1 and 2 were used to adjust the cell density to 5 × 10⁶ cells / year using the corresponding culture medium. 4 Cells were counted at a density of 10 cells / mL to obtain a cell suspension. The cell suspension was then seeded into 6-well plates, with 2 mL of cell suspension inoculated into each well. After seeding, the 6-well plates were placed in a 37°C, 5% CO2 incubator for culture. The culture medium for each group was changed every 2 days to provide sufficient nutrients for cell growth, and the culture period was set to 7 days.

[0029] On the 7th day of culture, the formation of three-dimensional aggregates of cells in each group was observed under an inverted phase contrast microscope. To ensure the representativeness of the observation results, 3 different random fields of view were selected for each group, and photographs were taken by an image acquisition system equipped with the microscope to record the morphological changes of the three-dimensional aggregates of cells in each group at different time points. The photographs were processed with ImageJ image analysis software. The number of three-dimensional aggregates with a diameter of ≥50 μm in each field of view (aggregates with a diameter of <50 μm were considered as not forming effective aggregates and were not included in the statistical range) was counted, and the average number of effective aggregates in 3 fields of view of each group was calculated. At the same time, the diameter of each effective aggregate was measured using the software, and the average diameter of the effective aggregates in each group was calculated. By comparing the average number and average diameter of each group, the effects of different proportions of formulations on the number and size of three-dimensional aggregates of cells were analyzed.

[0030] By comparing the average number and average diameter of three-dimensional aggregates of cells in group 1 and group 2. The average number of effective aggregates in group 1 was significantly more than that in group 2, and the average diameter of group 1 was significantly larger than that of group 1 Figure 1 B).

[0031] 5. Aging markers The cells harvested from group 1 and group 3 were inoculated in 25 mL shake flasks (1×10 4 cells) respectively, and the corresponding culture medium was added. The flasks were placed in a 37°C, 5% CO2 incubator, and the medium was replaced every 3 days. The cells were cultured for 7 consecutive days.

[0032] On the 7th day, the cells were harvested and subjected to SA-β-Gal staining: according to the instructions of the kit, the proportion of positive cells (blue-stained cells / total cell number × 100%) was counted under a microscope. Five fields of view were randomly selected for each group for statistical analysis, and the average value was taken.

[0033] The results showed that the proportion of positive cells in group 1 was (5.12±1.1)%, and the proportion of positive cells in group 3 was (7.5±0.9)%. The aging of group 1 was significantly lower than that of group 3 Figure 2 ).

[0034] Example 2 3D culture of mesenchymal stem cells 1. Preparation of culture medium A commercially available 3D culture medium was purchased, and 5% (v / v) human platelet lysate was added to obtain a complete culture medium CT-3D.

[0035] Meanwhile, DMEM / F12 and MCDB131 are mixed in a volume ratio of 3:1 as a basic component; then oleic acid, linoleic acid, docosahexaenoic acid (DHA) and cholesterol with a mass ratio of 1:1:1:0.2 are added, wherein the content of cholesterol is 100 mg / L; the concentration of nicotinamide is 10 mg / L, the concentration of L-carnitine is 5 mg / L, the concentration of vitronectin is 2 μg / mL, the concentration of fibronectin is 5 μg / mL, the concentration of HEPES is 10 mM, the concentration of sodium bicarbonate is 2.5 g / L, and 5% (v / v) human platelet lysate is added to configure mesenchymal stem cell 3D culture medium ZY-3D.

[0036] The cells are cultured and the cell precipitate is harvested according to the method of 3D culture in Example 1.

[0037] 2. Proliferation effect detection After 24 hours of culture, 2 mL of cell suspension is taken, counted, and the cell adhesion rate is calculated.

[0038] The proliferation fold of the cells cultured for 4 days is calculated. Proliferation fold = amount of cells harvested on the 4th day / amount of inoculated cells; Meanwhile, the expression of CD31 (endothelial cell marker) of MSCs cultured in the two kinds of culture media is detected by flow cytometry.

[0039] The results show that, compared with the cells cultured in the commercialized 3D culture medium CT-3D, the proliferation fold of the cells obtained by ZY-3D medium is increased by 2.19 times, and the adhesion rate is increased by 1.74 times (A, B) after a certain period of culture. Figure 3 C). Figure 3

[0040] This shows that the 3D culture medium of the present application can better promote cell proliferation.

[0041] 3. Immunological phenotype The mesenchymal stem cells cultured by ZY-3D are prepared into a single cell suspension of 1×10 6 After washing once with PBS, the flow cytometry is used to detect the MSC phenotype after culture by using CDllb, CD19, CD34, CD45, CD44, HLA-DR, CD73, CD90 and CD105 antibodies.

[0042] The results are as follows: Figure 4 ​As shown: the negative markers CD11b, CD19, CD34, CD45, HLA-DR of mesenchymal stem cells cultured by the medium of the application are all expressed negative (≤2%), and the positive markers CD44, CD73, CD90, CD105 are all expressed positive (≥95%). This shows that the immunological phenotype of mesenchymal stem cells cultured by the medium of the application is not affected.

[0043] 4. Tri-lineage differentiation ability The mesenchymal stem cells cultured by ZY-3D were inoculated into a 6-well plate at a density of 2×10 5 cells / well, and after the confluence rate reached more than 80%, the bone or adipogenic induction culture solution was replaced, the liquid was replaced twice a week, the adipogenic induction culture was performed for 14 days, and the osteogenic induction culture was performed for 21 days. Alizarin red S was used to stain the induced osteoblasts, and oil red O was used to stain the induced adipocytes and take photos.

[0044] The mesenchymal stem cells cultured by ZY-3D were adjusted to 8×10 6 cells / mL, and then dropped into a 6-well plate. Two hours later, chondrogenic induction culture solution was added, the liquid was replaced once a week, and the induction culture was performed for 21 days. The cartilage balls were taken out and fixed in 4% paraformaldehyde, and after paraffin embedding, sectioning, and staining with safranin O and alcian blue, photos were taken.

[0045] As Figure 5 The results show that after the mesenchymal stem cells cultured by the medium of the application are induced and cultured by the osteogenic induction medium, calcium nodules stained red by alizarin red S can be detected, after the mesenchymal stem cells are induced and cultured by the adipogenic induction medium, oil droplets stained red by oil red O can be detected, and after the mesenchymal stem cells are induced and cultured by the chondrogenic induction medium, safranin O and alcian blue staining are respectively dyed red and blue, indicating that the differentiation ability is not affected.

[0046] Example 3 3D culture of mesenchymal stem cells 1. Grouping and culture Experimental group: MSCs were inoculated in a 125 mL culture flask (1×10 4 cells), and according to the method of Example 2, microcarriers and ZY-3D medium in Example 2 were added, and the culture was placed in a 37℃, 5% CO2 incubator, and the liquid was replaced every 3 days; Control group 1: a commercially available ordinary 2D MSCs culture medium was used, and 5% (v / v) human platelet lysate was added to prepare a complete culture medium. MSCs were inoculated in a culture dish at a density of 1×10 4 cells / cm 2 , and the liquid was replaced every 3 days; Control group 2: CT-3D medium in Example 2 was used, and the culture was performed in the same way as the experimental group.

[0047] 2. Senescence marker detection The number of SA-β-Gal staining positive cells was detected at the 7th day and the 14th day of culture: according to the kit instructions, the proportion of positive cells (blue-stained cells / total cell number x 100%) was counted under a microscope, 5 fields of view were randomly selected for each group, and the average value was taken.

[0048] The SA-β-Gal staining statistical results are shown in Table 1: Figure 6 At the 7th day of culture, the proportion of positive cells in the experimental group was (8.7±1.1)%, that in the control group 1 was (26.4±1.3)%, and that in the control group 2 was (18.9±1.0)%; at the 14th day of culture, the proportion of positive cells in the experimental group was (21.9±1.7)%, that in the control group 1 was (53.8±2.9)%, and that in the control group 2 was (37.8±2.0)%.

[0049] The above results show that the proportion of senescent cells in the experimental group is significantly lower than that in the two control groups as the culture time is prolonged, and there is a significant difference at the 7th day and the 14th day, p<0.001.

[0050] Application Example 1 MSCs for treating acute lung injury (ARDS) 1. Experimental grouping MSCs obtained by different methods in Example 3 were used for experiments, and according to the culture medium, the MSCs were named as ZY-3D-MSCs, CT-2D-MSCs and CT-3D-MSCs.

[0051] 60 C57BL / 6 mice were randomly divided into 5 groups, 10 mice in each group, and were grouped according to the following treatment methods: Normal control group (Normal group): the mice were not treated, but were only raised in the same feeding environment; ARDS model group (Model group): the ARDS model was established by tracheal administration of LPS, and the same amount of normal saline was injected into the tail vein after modeling; 2D traditional culture medium-MSCs treatment group (CT-2D-MSCs group): after LPS modeling, CT-2D-MSCs (5x10 6 individuals / kg) were injected into the tail vein; 3D market culture medium-MSCs treatment group (CT-3D-MSCs group): after LPS modeling, CT-3D-MSCs (5x10 6 individuals / kg) were injected into the tail vein; 3D special culture medium-MSCs treatment group (ZY-3D-MSCs group): after LPS modeling, ZY-3D-MSCs (5x10 6 individuals / kg) were injected into the tail vein.

[0052] 2. Modeling and dosing After weighing, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), fixed on the operating table, and the trachea was exposed. LPS solution was slowly dropped into the trachea at a dose of 7 mg / kg using a microsyringe, and then the mice were rotated vertically to ensure uniform distribution of LPS in the lungs. The normal control group was given the same amount of normal saline by tracheal instillation. After modeling, the mice were given drugs by tail vein injection, twice a day.

[0053] 3. Inflammatory factor level detection The state of the mice was observed and recorded daily.

[0054] After drug treatment, the mice were sacrificed by cervical dislocation 24 hours later. The skin at the mouse's neck was peeled off, the thymus was removed, and the fascia around the trachea was cut off. A small hole was cut in the upper part of the trachea with scissors, and a lung lavage tube was inserted into the hole and tied with surgical thread to prevent air leakage. The entire lung was lavaged with 0.6 mL of pre-cooled normal saline, repeated 5 times, and the recovery rate was greater than 80% to indicate that the recovery was qualified. More than 2.4 mL of lung lavage fluid was obtained. Centrifugation at 1500 rpm for 5 min collected the supernatant of the lung lavage fluid. The level of inflammatory cell change was detected by a blood cell counter.

[0055] Compared with the Normal group, the mice in the Model group showed obvious symptoms such as listlessness, reduced activity, and rapid breathing, as shown in Table 1, and the WBC and other inflammatory cells in BALF were significantly increased, indicating that the modeling was successful. Compared with the Model group, the above indicators of each treatment group were improved, but the improvement of the ZY-3D-MSCs group was more significant. Figure 7

[0056] Example 4 Preparation of mesenchymal stem cell 3D culture medium DMEM / F12 and MCDB131 were mixed in a volume ratio of 3:1 as the basic culture medium; then oleic acid, linoleic acid, docosahexaenoic acid (DHA), and cholesterol were added in a mass ratio of 1:1:1:0.5 to make the cholesterol content 100 mg / L; the concentration of nicotinamide was 10 mg / L, the concentration of L-carnitine was 5 mg / L, the concentration of vitronectin was 5 μg / mL, the concentration of fibronectin was 8 μg / mL, the concentration of HEPES was 15 mM, the concentration of sodium bicarbonate was 4.5 g / L, and 5% (v / v) human platelet lysate was added to prepare the mesenchymal stem cell 3D culture medium.

[0057] According to the inoculation density of 1×10 4 ​The umbilical cord-derived MSCs (passage 6) were subjected to 3D culture by the method of Example 2 with 60 rpm for 5 min, 0 rpm for 35 min for 24 cycles of intermittent stirring, and then constant stirring at 60 rpm. The cell adhesion rate was detected and calculated after 24 h. The cells were cultured for 4 days, and the glucose concentration in the culture medium was detected by blood glucose test paper (glucose dehydrogenase method, Roche) until it was below 3 mmol / L. The proliferation fold was detected and calculated, and the medium was replaced or the cells were harvested according to the above method. The results showed that the cell adhesion rate was 96%, and the proliferation fold was 11.2, which was significantly higher than that of the commercially available 3D culture medium CT-3D (P < 0.05). Figure 8 ).

[0058] Example 5 Preparation of mesenchymal stem cell 3D culture medium DMEM / F12 and MCDB131 were mixed at a volume ratio of 2:1 as the basic culture medium. Then, oleic acid, linoleic acid, docosahexaenoic acid (DHA), and cholesterol were added at a mass ratio of 1.5:1:0.8:0.1 to make the cholesterol content 100 mg / L, the nicotinamide concentration 5 mg / L, the L-carnitine concentration 2 mg / L, the vitronectin concentration 1 μg / mL, the fibronectin concentration 3 μg / mL, the HEPES concentration 10 mM, and the sodium bicarbonate concentration 2.5 g / L. At the same time, 5% (v / v) human platelet lysate was added to configure the mesenchymal stem cell 3D culture medium.

[0059] The adipose-derived MSCs (passage 6) were subjected to 3D culture by the method of Example 2 with 50 rpm for 5 min, 0 rpm for 30 min for 24 cycles of intermittent stirring, and then constant stirring at 50 rpm. The cell adhesion rate was detected and calculated after 24 h. The cells were cultured for 4 days, and the glucose concentration in the culture medium was detected by blood glucose test paper (glucose dehydrogenase method, Roche) until it was below 3 mmol / L. The proliferation fold was detected and calculated, and the medium was replaced or the cells were harvested according to the above method. The results showed that the cell adhesion rate was 100%, and the proliferation fold was 11.5, which was significantly higher than that of the commercially available 3D culture medium CT-3D (P < 0.05). Figure 9 ).

[0060] Example 6 Preparation of mesenchymal stem cell 3D culture medium DMEM / F12 and MCDB131 were mixed in a volume ratio of 2.5:1 as a basic medium; then oleic acid, linoleic acid, docosahexaenoic acid (DHA) and cholesterol were added in a mass ratio of 2:2:0.5:0.1, so that the cholesterol content was 100 mg / L, the nicotinamide concentration was 8 mg / L, the L-carnitine concentration was 3 mg / L, the vitronectin concentration was 3 μg / mL, the fibronectin concentration was 5 μg / mL, the HEPES concentration was 12 mM, the sodium bicarbonate concentration was 3.5 g / L, and 5% (v / v) human platelet lysate was added to configure a mesenchymal stem cell 3D culture medium.

[0061] The dental pulp-derived MSCs (6th generation) were subjected to 3D culture according to the method of Example 2, and the cell adhesion rate was detected and calculated after 24 h, and the culture was continued for 4 days. When the glucose concentration in the culture medium was below 3 mmol / L, the glucose concentration was detected by using a blood glucose test paper (glucose dehydrogenase method, Roche), and the proliferation fold was calculated. Then the medium was replaced and the culture was continued according to the above method; the results showed that the cell adhesion rate was 108%, and the proliferation fold was 12.1, which was significantly higher than that of the commercially available 3D culture medium CT-3D (P<0.05). Figure 10 ).

[0062] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. A 3D culture medium for mesenchymal stem cells, comprising a basal culture medium and additives, characterized in that, The additive consists of lipids, metabolic regulators, adhesion proteins, buffers, and human platelet lysates; The lipids are oleic acid, linoleic acid, docosahexaenoic acid and cholesterol in a mass ratio of (1-2): (1-2): (0.5-1): (0.1-0.5); the cholesterol concentration in the 3D culture medium is 100 mg / L. The metabolic regulator is a combination of nicotinamide and L-carnitine, with the concentration of nicotinamide in the 3D culture medium being 5-10 mg / L and the concentration of L-carnitine in the 3D culture medium being 2-5 mg / L. The adhesion protein is a combination of hydrin and fibronectin, with the concentration of hydrin in 3D medium being 1-5 μg / mL and the concentration of fibronectin in 3D medium being 3-8 μg / mL. The buffer is a combination of HEPES and sodium bicarbonate, with HEPES at a concentration of 10-15 mM and sodium bicarbonate at a concentration of 2.5-4.5 g / L in the 3D medium. The amount of human platelet lysate added is 2-5% v / v of the basal culture medium.

2. The 3D culture medium according to claim 1, characterized in that, The basal culture medium is DMEM / F12 or a combination of DMEM / F12 and MCDB131.

3. The 3D culture medium according to claim 1, characterized in that, The basal culture medium was a combination of DMEM / F12 and MCDB131 in a volume ratio of 3:1 to 2:

1.

4. A method for 3D culture of mesenchymal stem cells, characterized in that, Includes the following steps: Mesenchymal stem cells were seeded into 3D culture medium containing microcarriers as described in any one of claims 1-3, and cultured with intermittent stirring followed by constant-speed stirring.

5. The method according to claim 4, characterized in that, The seeding density of mesenchymal stem cells is (1-5)×10⁻⁶. 4 cells / mL; The concentration of the microcarriers was 1.5-3 mg / mL; Intermittent stirring was performed at 40-60 rpm for 5 minutes, followed by 0 rpm for 25-35 minutes, for a total of 24 cycles. The constant stirring speed is 40-60 rpm.

6. The method according to claim 4, characterized in that, The culture endpoint was a reduction in glucose concentration to 3.0 mmol / L or below, which was detected using glucose dehydrogenase test strips. At the end of the culture period, the mesenchymal stem cells are dispersed and passaged or harvested.

7. A mesenchymal stem cell obtained by the method as described in any one of claims 4-6.

8. The application of the mesenchymal stem cells as described in claim 7 in the preparation of a drug for treating acute lung injury, and the preparation of such a drug for treating acute lung injury.

9. An acute lung injury treatment drug prepared from mesenchymal stem cells as described in claim 7.

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