Improved mesenchymal stem cell culture method and culture medium used by same

Abstract

The invention provides an improved mesenchymal stem cell culture method and a culture medium used by the improved mesenchymal stem cell culture method, and belongs to the technical field of cell culture. The invention provides an improved mesenchymal stem cell culture medium. Comprising the following exogenous additive components: recombinant human serum albumin, recombinant transferrin, a recombinant human epidermal growth factor, a basic fibroblast growth factor, a platelet-derived growth factor, insulin, L-glutamine, reduced glutathione, beta-mercaptoethanol, sodium selenite, mycillin, allicin, heparin and a proliferation promoting agent. The proliferation promoting agent comprises paeoniflorin, panax notoginseng saponins, lycium barbarum polysaccharide and a snow lotus herb alcohol extract. When the culture medium is used for culturing the mesenchymal stem cells, the cell viability and multiplication capacity of the mesenchymal stem cells can be remarkably improved, the cell viability is improved by 50% or above, the cell multiplication capacity is improved by 40% or above, and the culture medium has great application potential in the aspect of culture of the mesenchymal stem cells.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to an improved mesenchymal stem cell culture method and a culture medium used therein. Background Art

[0002] Mesenchymal stem cells are a type of multipotent stem cells that are mainly found in connective tissue and organ stroma. They have the ability to differentiate into myocytes, hepatocytes, osteoblasts, adipocytes, chondrocytes, stromal cells and other cells in a suitable in vivo or in vitro environment. They have strong proliferation ability and multidirectional differentiation potential, and have immunoregulatory functions. They inhibit the proliferation of T cells and their immune response through cell-to-cell interactions and the production of cytokines, thereby playing the role of immune reconstruction. They are easy to obtain, isolate, culture, expand and purify. After multiple passages and expansions, they still have stem cell characteristics and do not have the characteristics of immune rejection. They have broad application prospects in clinical practice.

[0003] Most of the culture media used for in vitro culture of mesenchymal stem cells need to be supplemented with serum, but there are many unknown components in the serum, and the bioactive factors between different batches of serum are inconsistent. There may be contamination of exogenous viruses and pathogenic factors, resulting in poor reproducibility of products and experimental results. At the same time, the residual serum is also prone to cause allergic reactions to the serum in the vaccine recipients, and cannot be used in clinical research. The existing serum-free culture medium usually uses bovine serum albumin or human serum albumin, which is expensive, and the cell passage and proliferation ability are not ideal. It is hoped that the cell activity and proliferation ability can be improved by improving the culture method of mesenchymal stem cells. Summary of the invention

[0004] The object of the present invention is to provide a method for improving the proliferation ability of mesenchymal stem cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an improved mesenchymal stem cell culture medium, comprising the following exogenous additive components: recombinant human serum albumin, recombinant transferrin, recombinant human epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, insulin, L-glutamine, reduced glutathione, β-mercaptoethanol, sodium selenite, penicillin-streptomycin, allicin, heparin and a proliferation promoter;

[0007] The proliferation promoting agent comprises paeoniflorin, notoginseng total saponin, lycium barbarum polysaccharide and snow lotus ethanol extract.

[0008] Preferably, the concentration of the recombinant human serum albumin is 8 to 12 mg / L;

[0009] The concentration of the recombinant transferrin is 10-15 mg / L;

[0010] The concentration of the recombinant human epidermal growth factor is 12-15 μg / L;

[0011] The concentration of the basic fibroblast growth factor is 6-10 μg / L;

[0012] The concentration of the platelet-derived growth factor is 8 to 12 μg / L;

[0013] The concentration of the insulin is 8-12 mg / L;

[0014] The concentration of L-glutamine is 6-8 mg / L;

[0015] The concentration of the reduced glutathione is 4-6 mg / L;

[0016] The concentration of the β-mercaptoethanol is 0.3-0.8 mg / L;

[0017] The concentration of sodium selenite is 0.2-0.8 μg / L;

[0018] The concentration of penicillin-streptomycin is 1.0-1.4 mg / L;

[0019] The concentration of the allicin is 8-12 μg / L;

[0020] The amount of heparin added is 2 to 5 IU / mL

[0021] The added amount of the proliferation promoting agent is 20-25 μg / L.

[0022] Preferably, the improved mesenchymal stem cell culture medium is obtained by adding the exogenous additive components to DMEM / F12 as the basic culture medium.

[0023] Preferably, the ratio of paeoniflorin, notoginseng total saponins, lycium barbarum polysaccharides and snow lotus ethanol extract is 1-3:1-3:1-2:3-5.

[0024] Preferably, the preparation method of the snow lotus ethanol extract is:

[0025] (1) After crushing the snow lotus into 50-100 meshes, soaking and extracting it in a 50-60% ethanol solution by volume for 1-2 days, and separating the solid and the liquid to obtain a solid 1;

[0026] (2) adding 70-80% ethanol solution by volume to solid 1, performing ultrasonic treatment every 5-8 hours, extracting for 6-8 days, and separating the solid from the liquid to obtain extract 2.

[0027] (3) Take extract 2, place it at 70-75° C., and allow the ethanol to evaporate until there is no obvious alcohol smell, thereby obtaining the snow lotus ethanol extract.

[0028] Preferably, the amount of ethanol solution added in steps (1) and (2) is 1.2 to 1.5 times the volume of the snow lotus, and the temperature of the extraction process is 40 to 50° C.;

[0029] The power of the ultrasonic treatment in step (2) is 300-450W, and the time is 30-40min.

[0030] The present invention also provides application of the improved mesenchymal stem cell culture medium in mesenchymal stem cell culture.

[0031] Preferably, the method for culturing mesenchymal stem cells is:

[0032] (a) Take the tissue to be extracted, add PBS solution and Percoll separation solution, perform the first centrifugation, take the middle mononuclear cell layer, centrifuge and wash, remove the supernatant, and culture in DMEM medium. After 3 to 5 days, replace the medium and remove the non-adherent cells to obtain primary mesenchymal stem cells;

[0033] (b) culturing the primary mesenchymal stem cells until the cell confluence is 70-80%, washing and digesting, collecting the single cell suspension, centrifuging, discarding the supernatant, adding the improved mesenchymal stem cell culture medium to resuspend, and subculturing;

[0034] (c) Isolation of mesenchymal stem cells.

[0035] Preferably, in step (a), the volume ratio of the tissue to be extracted, the PBS solution and the Percoll separation solution is 1:1-1.5:1-1.2;

[0036] The first centrifugation has a rotation speed of 1600-2000 r / min and a time of 25-40 min;

[0037] The rotation speed during the centrifugal washing is 1800-2200 r / min, the time is 5-10 min, and the washing solution is PBS solution;

[0038] The DMEM culture medium is a low-sugar DMEM culture medium, and the culture conditions are: 37-39° C., 3-5% CO 2 .

[0039] Preferably, the centrifugal speed in step (b) is 1400-1600 r / min, the time is 5 min, and during subculture, the initial subculture ratio is 1:1-2, and the subsequent subculture ratio is 1:3-6. The subculture conditions are: 37-39°C, 3-5% CO2.

[0040] The improved mesenchymal stem cell culture medium and the culture method thereof provided by the present invention can significantly improve cell viability and cell proliferation ability. Compared with the control example, the cell viability is improved by more than 50%, and the cell proliferation ability is improved by more than 40%. It has great application potential in the culture of mesenchymal stem cells.

[0041] The present invention obtains a large number of mesenchymal stem cells with high purity and activity by combining density gradient centrifugation with adhesion method; the allicin selected by the present invention has anti-inflammatory effect, can maintain cell stability, helps to form a more uniform cell layer, and improves the efficiency and quality of cell culture, and also acts together with proliferation promoting ingredients such as paeoniflorin, notoginseng total saponins, lycium barbarum polysaccharides and snow lotus ethanol extract to reduce the level of active oxygen in the culture medium, provide anti-oxidation and anti-apoptosis protection for cells, and cooperate with other ingredients to jointly enhance cell vitality and proliferation ability. DETAILED DESCRIPTION

[0042] In the present invention, paeoniflorin, notoginseng total saponins, lycium barbarum polysaccharides, salidroside and icariin were purchased from Beijing Kangruina Biotechnology Co., Ltd.; DMEM / F12 culture medium and low-glucose DMEM culture medium were purchased from Hyclone Company, USA.

[0043] Preparation of bone marrow cells: One-month-old female New Zealand white rabbits (weighing 1.2 kg) of clean grade were selected, and the rabbits were anesthetized through the ear vein with 3% pentobarbital 40 mg / kg. The iliac skin was prepared, disinfected with 5% povidone-iodine solution, and covered with sterile drapes. The iliac bone was punctured with a No. 12 iliac needle to extract bone marrow cells (1 mL of heparin, 3000 μ / mL, was pre-placed in the syringe).

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] A modified mesenchymal stem cell culture medium, consisting of a DMEM / F12 culture medium and exogenous additive components, wherein the exogenous additive components are 8 mg / L recombinant human serum albumin, 15 mg / L recombinant transferrin, 12 μg / L recombinant human epidermal growth factor, 10 μg / L basic fibroblast growth factor, 8 μg / L platelet-derived growth factor, 12 mg / L insulin, 6 mg / L L-glutamine, 6 mg / L reduced glutathione, 0.3 mg / L β-mercaptoethanol, 0.8 μg / L sodium selenite, 1.0 mg / L penicillin-streptomycin, 12 μg / L allicin, 2 IU / mL heparin and 25 μg / L proliferation promoter (wherein the proliferation promoter is composed of paeoniflorin, notoginseng total saponins, wolfberry polysaccharides and snow lotus ethanol extract in a mass ratio of 1:2:2:5).

[0047] The preparation method of the snow lotus ethanol extract is:

[0048] (1) After crushing the snow lotus into a 50-mesh sieve, adding an ethanol solution (volume fraction of 55%) with a volume of 1.2 times that of the snow lotus, soaking and extracting for 2 days at an extraction temperature of 45° C., filtering, and obtaining a solid 1;

[0049] (2) adding 1.2 times the volume of snow lotus in ethanol solution (volume fraction of 75%) to solid 1, ultrasonically treating at 400W for 40min every 8h, for a total of 8 days, and centrifuging at 800r / min for 10min to obtain extract 2;

[0050] (3) The extract 2 is placed at 75° C. to evaporate the ethanol until there is no obvious alcohol smell, thereby obtaining the snow lotus ethanol extract.

[0051] Example 2

[0052] A modified mesenchymal stem cell culture medium, consisting of a DMEM / F12 culture medium and exogenous additive components, wherein the exogenous additive components are 12 mg / L recombinant human serum albumin, 10 mg / L recombinant transferrin, 15 μg / L recombinant human epidermal growth factor, 6 μg / L basic fibroblast growth factor, 12 μg / L platelet-derived growth factor, 8 mg / L insulin, 8 mg / L L-glutamine, 4 mg / L reduced glutathione, 0.8 mg / L β-mercaptoethanol, 0.2 μg / L sodium selenite, 1.4 mg / L penicillin, 8 μg / L allicin, 5 IU / mL heparin and 20 μg / L proliferation promoter (the proliferation promoter is composed of paeoniflorin, notoginseng total saponins, lycium barbarum polysaccharide and snow lotus ethanol extract according to a mass ratio of 2:2:2:4). The preparation method of the snow lotus ethanol extract is the same as that of Example 1.

[0053] Example 3

[0054] A modified mesenchymal stem cell culture medium, consisting of a DMEM / F12 culture medium and exogenous additive components, wherein the exogenous additive components are 10 mg / L recombinant human serum albumin, 10 mg / L recombinant transferrin, 12 μg / L recombinant human epidermal growth factor, 10 μg / L basic fibroblast growth factor, 10 μg / L platelet-derived growth factor, 10 mg / L insulin, 8 mg / L L-glutamine, 5 mg / L reduced glutathione, 0.5 mg / L β-mercaptoethanol, 0.5 μg / L sodium selenite, 1.0 mg / L penicillin, 10 μg / L allicin, 5 IU / mL heparin and 24 μg / L proliferation promoter (the proliferation promoter is composed of paeoniflorin, notoginseng total saponins, lycium barbarum polysaccharide and snow lotus ethanol extract according to a mass ratio of 3:3:1:5). The preparation method of the snow lotus ethanol extract is the same as that of Example 1.

[0055] Example 4

[0056] An improved method for culturing mesenchymal stem cells is:

[0057] (a) Take 6 mL of bone marrow cells, add equal volumes of PBS solution and Percoll separation solution, centrifuge at 1800 r / min for 40 min, take the middle mononuclear cell layer, add equal volume of PBS solution, centrifuge at 2000 r / min for 5 min, remove the supernatant, and then centrifuge at 2.0×10 8 The cells were inoculated at a density of 100 μg / L in a low-glucose DMEM medium containing 10% FBS, 100 μg / L penicillin, and 100 mg / L streptomycin. After culturing for 3 days at 38°C, 5% CO2, and saturated humidity, the low-glucose DMEM medium was replaced to remove non-adherent cells and obtain primary mesenchymal stem cells.

[0058] (b) The primary mesenchymal stem cells were cultured until the cells were 80% confluent, washed with PBS solution, and digested for 3 min by adding 2 mL of 0.25% trypsin solution to remove trypsin, and then 3 times the volume of trypsin was added to terminate the digestion by adding low-glucose DMEM medium, collecting the cell suspension, centrifuging at 1600 r / min for 5 min, discarding the supernatant, and then resuspending in the improved mesenchymal stem cell medium described in Example 3 to adjust the cell density to 2×10 5 / mL, and then subcultured at 38°C, 5% CO2 and saturated humidity. The initial subculture ratio was 1:1. After 3 days of culture, when the cell fusion was 70%, subcultured at a ratio of 1:5.

[0059] (c) Isolation of mesenchymal stem cells: The culture medium was changed every three days. When the cell fusion degree reached 80%, the mesenchymal stem cells were collected after digestion, neutralization and washing according to the requirements of passaging.

[0060] Comparative Example 1

[0061] Different from Example 4, the proliferation promoting agent is composed of notoginseng total saponins, salidroside, lycium barbarum polysaccharide and icariin in a mass ratio of 3:3:1:5.

[0062] Comparative Example 2

[0063] Different from Example 4, the ratio of paeoniflorin, notoginseng total saponins, wolfberry polysaccharides and snow lotus ethanol extract is 3:3:4:1.

[0064] Comparative Example 3

[0065] Different from Example 4, the commercially available mesenchymal stem cell (MSC) serum-free culture medium purchased from Beijing Kelin Biotechnology Co., Ltd. was used to resuspend the cells in step (b).

[0066] Comparative Example 4

[0067] A method for culturing mesenchymal stem cells:

[0068] (a) Take bone marrow cells, add low-glucose DMEM medium containing 10% FBS, 100μ / L penicillin, and 100mg / L streptomycin, and gently pipette to make a cell suspension, inoculate it into a 25mL culture bottle, and culture it in a saturated humidity incubator with a volume fraction of 5% CO2 and 37℃ for 3 days, then replace the low-glucose DMEM medium to remove non-adherent cells. Thereafter, change the medium once every three days to obtain primary cells.

[0069] (b) When the primary cells grew to 80%, the culture flask containing the adherent cells was taken out, the culture medium was aspirated, the cells were rinsed twice with PBS buffer, and digested with 2 mL of 0.25% trypsin at 37°C for 5 min. When the cells began to shrink and round, the digestion was terminated with an equal amount of low-glucose DMEM culture medium, centrifuged at 1600 r / min for 5 min, the supernatant was discarded, and the improved mesenchymal stem cell culture medium described in Example 3 was added. The cells were repeatedly pipetted gently without bubbles to prepare a single cell suspension, and the cell density was adjusted to 2×10 5 / mL, and then subcultured at 38°C, 5% CO2 and saturated humidity. The initial subculture ratio was 1:1. After 3 days of culture, when the cell fusion was 70%, subcultured at a ratio of 1:5.

[0070] (c) Same as step (c) of Example 4.

[0071] Test Example 1

[0072] Evaluation of mesenchymal stem cell activity: P5 cells obtained by the methods of Example 4 and Comparative Examples 1 to 4 were divided into 5 groups and 4×10 4The cells were inoculated in a 96-well plate at a density of 100 μL / mL, and 100 μL of the culture medium used for resuspension in the corresponding embodiment and comparative example was added to each well. Each group of cells in the embodiment was inoculated with 5 parallel wells. After the cells adhered for 72 hours, 10 μL MTS reagent was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 4 hours, and then the OD value at 490 nm was detected using a microplate reader. The OD value is used to evaluate the vitality of mesenchymal stem cells. The larger the OD value, the better the vitality of the mesenchymal stem cells, and the smaller the OD value, the worse the vitality of the mesenchymal stem cells.

[0073] Table 1 Cell viability

[0074] Group Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 <![CDATA[OD 490nm Value]]> 2.458 1.914 1.976 1.484 1.781

[0075] As can be seen from Table 1, the viability of MSC cells cultured in Example 4 is significantly better than that in the comparative example, indicating that the improved mesenchymal stem cell culture medium and culture method thereof provided in the present application can significantly improve cell viability.

[0076] Test Example 2

[0077] Evaluation of cell proliferation ability: P5 cells obtained by the methods of Example 4 and Comparative Examples 1 to 4 were taken and 2×10 4 The cells were seeded at a density of 1 / mL in a 12-well plate and divided into 5 groups, each with 12 wells. 1 mL of the improved mesenchymal stem cell culture medium used in the resuspension step (b) of the corresponding embodiment or comparative example was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator. After 5 days of culture, 3 wells were taken from each well for cell counting by trypan blue staining and the average value was taken. The technical results are shown in Table 2.

[0078] Table 2 Cell counting results (×10 4 indivual)

[0079] Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 <![CDATA[Counting result (×10 4 pieces)]]> 47.6 36.5 37.4 26.8 34.7

[0080] As can be seen from Table 2, the proliferation ability of MSC cells cultured in Example 4 is much higher than that of other comparative examples, indicating that the improved mesenchymal stem cell culture medium and culture method provided in the present application can significantly improve the cell proliferation ability and has great application potential in the culture of mesenchymal stem cells.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An improved mesenchymal stem cell culture medium, characterized in that: The exogenous additives include the following: recombinant human serum albumin, recombinant transferrin, recombinant human epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, insulin, L-glutamine, reduced glutathione, β-mercaptoethanol, sodium selenite, penicillin-streptomycin, allicin, heparin and proliferation promoter; The proliferation promoting agent comprises paeoniflorin, notoginseng total saponin, lycium barbarum polysaccharide and snow lotus ethanol extract.

2. The improved mesenchymal stem cell culture medium according to claim 1, characterized in that The concentration of the recombinant human serum albumin is 8-12 mg / L; The concentration of the recombinant transferrin is 10-15 mg / L; The concentration of the recombinant human epidermal growth factor is 12-15 μg / L; The concentration of the basic fibroblast growth factor is 6-10 μg / L; The concentration of the platelet-derived growth factor is 8 to 12 μg / L; The concentration of the insulin is 8-12 mg / L; The concentration of L-glutamine is 6-8 mg / L; The concentration of the reduced glutathione is 4-6 mg / L; The concentration of the β-mercaptoethanol is 0.3-0.8 mg / L; The concentration of sodium selenite is 0.2-0.8 μg / L; The concentration of penicillin-streptomycin is 1.0-1.4 mg / L; The concentration of the allicin is 8-12 μg / L; The amount of heparin added is 2 to 5 IU / mL The added amount of the proliferation promoting agent is 20-25 μg / L.

3. The improved mesenchymal stem cell culture medium according to claim 1, characterized in that The improved mesenchymal stem cell culture medium is obtained by adding the exogenous additive components to DMEM / F12 as the basic culture medium.

4. The improved mesenchymal stem cell culture medium according to claim 3, characterized in that: The ratio of the paeoniflorin, notoginseng total saponin, wolfberry polysaccharide and snow lotus ethanol extract is 1-3:1-3:1-2:3-5.

5. The improved mesenchymal stem cell culture medium according to claim 4, characterized in that The preparation method of the snow lotus ethanol extract is: (1) After crushing the snow lotus into 50-100 meshes, soaking and extracting it in a 50-60% ethanol solution by volume for 1-2 days, and separating the solid and the liquid to obtain a solid 1; (2) adding 70-80% ethanol solution by volume to solid 1, performing ultrasonic treatment every 5-8 hours, extracting for 6-8 days, and separating the solid from the liquid to obtain extract 2. (3) Take extract 2, place it at 70-75° C., and allow the ethanol to evaporate until there is no obvious alcohol smell, thereby obtaining the snow lotus ethanol extract.

6. The improved mesenchymal stem cell culture medium according to claim 5, characterized in that In steps (1) and (2), the amount of ethanol solution added is 1.2 to 1.5 times the volume of snow lotus, and the temperature during the extraction process is 40 to 50° C.; The power of the ultrasonic treatment in step (2) is 300-450W, and the time is 30-40min.

7. Use of the improved mesenchymal stem cell culture medium according to any one of claims 1 to 6 in culturing mesenchymal stem cells.

8. The use according to claim 7, characterized in that The method for culturing mesenchymal stem cells is: (a) Take the tissue to be extracted, add PBS solution and Percoll separation solution, perform the first centrifugation, take the middle mononuclear cell layer, centrifuge and wash, remove the supernatant, and culture in DMEM medium. After 3 to 5 days, replace the medium and remove the non-adherent cells to obtain primary mesenchymal stem cells; (b) culturing the primary mesenchymal stem cells until the cell confluence is 70-80%, washing and digesting, collecting the single cell suspension, centrifuging, discarding the supernatant, adding the improved mesenchymal stem cell culture medium to resuspend, and subculturing; (c) Isolation of mesenchymal stem cells.

9. The use according to claim 8, characterized in that In step (a), the volume ratio of the tissue to be extracted, the PBS solution and the Percoll separation solution is 1:1-1.5:1-1.2; The first centrifugation has a rotation speed of 1600-2000 r / min and a time of 25-40 min; The rotation speed during the centrifugal washing is 1800-2200 r / min, the time is 5-10 min, and the washing solution is PBS solution; The DMEM culture medium is a low-sugar DMEM culture medium, and the culture conditions are: 37-39° C., 3-5% CO 2 .

10. The use according to claim 9, characterized in that: The centrifugal speed in step (b) is 1400-1600 r / min, the time is 5 min, and during subculture, the initial subculture ratio is 1:1-2, and the subsequent subculture ratio is 1:3-6. The subculture conditions are: 37-39° C., 3-5% CO2.