An improved method for culturing mesenchymal stem cells and a culture medium used therefor

Abstract

The present invention provides an improved method for culturing mesenchymal stem cells and a culture medium used therefor, belonging to the technical field of cell culture. The improved mesenchymal stem cell culture medium provided by the present invention comprises 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; the proliferation promoter comprises paeoniflorin, total saponins of Panax notoginseng, wolfberry polysaccharide and alcohol extract of Saussurea involucrata. By using the above culture medium for culturing mesenchymal stem cells, the cell activity and proliferation ability of mesenchymal stem cells can be significantly increased, the cell viability is increased by more than 50%, and the cell proliferation ability is increased by more than 40%, having great application potential in the culture of mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to an improved method for culturing mesenchymal stem cells and a culture medium used therefor. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells, mainly existing in connective tissues and organ interstitium. They have the ability to differentiate into various cells such as muscle cells, liver cells, osteoblasts, adipocytes, chondrocytes, and stromal cells under suitable in vivo or in vitro environments. They have strong proliferation ability and multi-directional differentiation potential, and have an immunomodulatory function. Through cell-cell interactions and the production of cytokines, they inhibit the proliferation and immune response of T cells, thereby playing a role in immune reconstruction. They are convenient in source, easy to isolate, culture, amplify, and purify. They still have stem cell characteristics after multiple passages of amplification and do not have the characteristic 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 supplement serum. However, there are many unknown components in 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 likely to cause allergic reactions of the inoculator to the serum and cannot be applied to clinical research. Existing serum-free culture media usually use bovine serum albumin or human serum albumin, which have high costs, and the cell passage and proliferation abilities are also not ideal. It is hoped that the culture method of mesenchymal stem cells can be improved to enhance cell activity and proliferation ability. Summary of the Invention

[0004] The purpose of the present invention is to provide a method capable of enhancing the proliferation ability of mesenchymal stem cells.

[0005] In order to achieve the above-mentioned invention purpose, 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 promoter includes paeoniflorin, total saponins of Panax notoginseng, wolfberry polysaccharide, and ethanol extract of Saussurea involucrata.

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

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

[0014] The concentration of the 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 the sodium selenite is 0.2 - 0.8 μg / L;

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

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

[0020] The addition amount of the heparin is 2 - 5 IU / mL

[0021] The addition amount of the proliferation promoter is 20 - 25 μg / L.

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

[0023] Preferably, the ratio of paeoniflorin, total saponins of Panax notoginseng, wolfberry polysaccharide and ethanol extract of Saussurea involucrata is 1 - 3:1 - 3:1 - 2:3 - 5.

[0024] Preferably, the preparation method of the ethanol extract of Saussurea involucrata is as follows:

[0025] (1) After pulverizing Saussurea involucrata to 50 - 100 meshes, first soak and extract it with an ethanol solution with a volume fraction of 50 - 60% for 1 - 2 days, and perform solid-liquid separation to obtain solid 1;

[0026] (2) Add an ethanol solution with a volume fraction of 70 - 80% to solid 1, perform ultrasonic treatment every 5 - 8 hours, and extract for a total of 6 - 8 days, and perform solid-liquid separation to obtain extract 2.

[0027] (3) Take extract 2, place it under the condition of 70 - 75 °C, and volatilize ethanol until there is no obvious alcohol smell to obtain the ethanol extract of Saussurea involucrata.

[0028] Preferably, the addition amount of the ethanol solution in steps (1) and (2) is 1.2 to 1.5 times the volume of the saussurea involucrata, and the temperature during the extraction process is 40 to 50 °C;

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

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

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

[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, place it in DMEM medium for culture, change the medium after 3 to 5 days, remove the non-adherent cells, and obtain the primary mesenchymal stem cells;

[0033] (b) Culture the primary mesenchymal stem cells until the cell fusion reaches 70 to 80%, wash, digest, collect the single cell suspension, centrifuge, discard the supernatant, resuspend with the improved mesenchymal stem cell culture medium, and perform subculture;

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

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

[0036] The rotation speed of the first centrifugation is 1600 to 2000 r / min, and the time is 25 to 40 min;

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

[0038] The DMEM medium is a low-sugar DMEM medium, and the culture conditions are: 37 to 39 °C, 3 to 5% CO2.

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

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

[0041] In the present invention, a relatively large number of highly pure and active mesenchymal stem cells are obtained by density gradient centrifugation combined with adherent method; the allicin selected in the present invention has an anti-inflammatory effect, can maintain cell stability, helps to form a more uniform cell layer, improves the efficiency and quality of cell culture, and at the same time acts together with proliferators such as paeoniflorin, total saponins of Panax notoginseng, lycium barbarum polysaccharide and ethanol extract of Saussurea involucrata, reduces the level of reactive oxygen species in the culture medium, provides antioxidant and anti-apoptosis protection for cells, and cooperates with other components synergistically to jointly enhance cell viability and proliferation ability. Specific embodiments

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

[0043] Preparation of bone marrow cells: Select a clean-grade 1-month-old female New Zealand white rabbit (weighing 1.2 kg), anesthetize the rabbit's ear marginal vein with 3% pentobarbital at 40 mg / kg, prepare the skin of the iliac part, disinfect with 5% povidone iodine solution, cover with a sterile drape, and perform iliac bone puncture with a No. 12 iliac puncture needle to extract bone marrow cells (1 mL of heparin, 3000 μ / mL, is pre-placed in the syringe).

[0044] The technical solutions provided by the present invention will be 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] An improved mesenchymal stem cell culture medium is composed of DMEM / F12 medium and exogenous additive components. Among them, the exogenous additive components are 8 mg / L of recombinant human serum albumin, 15 mg / L of recombinant transferrin, 12 μg / L of recombinant human epidermal growth factor, 10 μg / L of basic fibroblast growth factor, 8 μg / L of platelet-derived growth factor, 12 mg / L of insulin, 6 mg / L of L-glutamine, 6 mg / L of reduced glutathione, 0.3 mg / L of β-mercaptoethanol, 0.8 μg / L of sodium selenite, 1.0 mg / L of penicillin-streptomycin, 12 μg / L of allicin, 2 IU / mL of heparin and 25 μg / L of a proliferator (wherein, the proliferator is composed of paeoniflorin, total saponins of Panax notoginseng, lycium barbarum polysaccharide and ethanol extract of Saussurea involucrata according to a mass ratio of 1:2:2:5).

[0047] The preparation method of the ethanol extract of Saussurea involucrata is as follows:

[0048] (1) After pulverizing Saussurea involucrata to pass through a 50-mesh sieve, add an ethanol solution (volume fraction 55%) 1.2 times the volume of Saussurea involucrata, soak and extract for 2 days at an extraction temperature of 45°C, filter to obtain Solid 1;

[0049] (2) Add an ethanol solution (volume fraction 75%) 1.2 times the volume of Saussurea involucrata to Solid 1, perform ultrasonic treatment at 400 W for 40 min every 8 h, extract for a total of 8 days, centrifuge at 800 r / min for 10 min to obtain Extract 2;

[0050] (3) Place Extract 2 at 75°C and volatilize the ethanol until there is no obvious alcohol smell to obtain the ethanol extract of Saussurea involucrata.

[0051] Example 2

[0052] An improved mesenchymal stem cell culture medium is composed of DMEM / F12 medium and exogenous additive components. Among them, the exogenous additive components are recombinant human serum albumin at a concentration of 12 mg / L, recombinant transferrin at 10 mg / L, recombinant human epidermal growth factor at 15 μg / L, basic fibroblast growth factor at 6 μg / L, platelet-derived growth factor at 12 μg / L, insulin at 8 mg / L, L-glutamine at 8 mg / L, reduced glutathione at 4 mg / L, β-mercaptoethanol at 0.8 mg / L, sodium selenite at 0.2 μg / L, penicillin-streptomycin at 1.4 mg / L, allicin at 8 μg / L, heparin at 5 IU / mL, and a proliferation promoter (the proliferation promoter is composed of paeoniflorin, total saponins of Panax notoginseng, wolfberry polysaccharide, and the ethanol extract of Saussurea involucrata in a mass ratio of 2:2:2:4). The preparation method of the ethanol extract of Saussurea involucrata is the same as that in Example 1.

[0053] Example 3

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

[0055] Example 4

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

[0057] (a) Take 6 mL of bone marrow cells, add an equal volume of PBS solution and Percoll separation solution, centrifuge at 1800 r / min for 40 min, take the middle mononuclear cell layer, add an equal volume of PBS solution, centrifuge and wash at 2000 r / min for 5 min, discard the supernatant, and then inoculate at a density of 2.0×10 8 cells / L into a low-glucose DMEM medium containing 10% FBS, 100 μ / L of penicillin, and 100 mg / L of streptomycin. After culturing for 3 d at 38 °C under 5% CO2 and saturated humidity conditions, replace the low-glucose DMEM medium, remove the non-adherent cells, and obtain primary mesenchymal stem cells.

[0058] (b) Culture the primary mesenchymal stem cells until the cell confluence reaches 80%, wash with PBS solution, add 2 mL of 0.25% trypsin solution to digest for 3 min, remove the trypsin, and then add a low-glucose DMEM medium 3 times the volume of the trypsin to terminate digestion. Collect the cell suspension, centrifuge at 1600 r / min for 5 min, discard the supernatant, and resuspend with the improved mesenchymal stem cell culture medium described in Example 3. Adjust the cell density to 2×10 5 cells / mL, and then perform subculture at 38 °C under 5% CO2 and saturated humidity conditions. The initial subculture ratio is 1:1. After culturing for 3 d, when the cell confluence reaches 70%, perform subculture at a ratio of 1:5.

[0059] (c) Isolation of mesenchymal stem cells: Change the culture medium every three days. When the cell confluence reaches 80%, after digestion, neutralization, and washing according to the requirements of subculture, collect the mesenchymal stem cells.

[0060] Comparative Example 1

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

[0062] Comparative Example 2

[0063] Different from Example 4, the ratio of paeoniflorin, total saponins of Panax notoginseng, lycium barbarum polysaccharide and alcohol extract of Saussurea involucrata is 3:3:4:1.

[0064] Comparative Example 3

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

[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, 100 μ / L penicillin, and 100 mg / L streptomycin, gently pipette to make a cell suspension, inoculate it into a 25 mL culture flask, place it in an incubator with 5% CO2 volume fraction, 37 °C saturated humidity for 3 days, then replace the low-glucose DMEM medium and remove the non-adherent cells. Thereafter, change the medium once every three days to obtain primary cells.

[0069] (b) When the primary cells grow to 80%, take out the culture flask containing adherent cells, aspirate the medium, rinse twice with PBS buffer, digest with 2 mL of 0.25% trypsin at 37 °C for 5 min. When the cell morphology begins to shrink and become round, terminate the digestion with an equal volume of low-glucose DMEM medium, centrifuge at 1600 r / min for 5 min, discard the supernatant, add the improved mesenchymal stem cell medium described in Example 3, and pipette gently without air bubbles repeatedly to make a single-cell suspension. Adjust the cell density to 2×10 5 cells / mL, and then perform subculture under the conditions of 38 °C, 5% CO2 and saturated humidity. The initial subculture ratio is 1:1. After 3 days of culture, when the cell fusion reaches 70%, subculture at a ratio of 1:5.

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

[0071] Test Example 1

[0072] Evaluation of mesenchymal stem cell viability: Take the P5 generation cells obtained by treating with the methods corresponding to Example 4 and Comparative Examples 1-4, divide them into 5 groups, and use 4×10 4Inoculate at a density of

[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 the MSC cells obtained by culturing in Example 4 is significantly better than that of the comparative examples, indicating that the improved mesenchymal stem cell culture medium and its culture method provided in this application can significantly enhance cell viability.

[0076] Test Example 2

[0077] Evaluation of cell proliferation ability: Take the P5-generation cells obtained by treating with the methods corresponding to Example 4 and Comparative Examples 1-4, and inoculate them in a 12-well plate at a density of 2×10 4 cells / mL. Divide them into 5 groups, with 12 wells in each group. Add 1 mL of the improved mesenchymal stem cell culture medium used for resuspension in step (b) of the corresponding example or comparative example to each well, and place them in an incubator at 37°C and 5% CO2 for culture. When culturing for 5 days, take 3 wells from each group for cell counting with trypan blue staining, and take the average value. The technical results are shown in Table 2,

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

[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 the MSC cells obtained by culturing 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 this application can significantly improve cell proliferation ability and have great application potential in the culture of mesenchymal stem cells.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An improved mesenchymal stem cell culture medium, characterized in that, The improved mesenchymal stem cell culture medium is obtained by using DMEM / F12 as the basal medium and adding exogenous additive components. The exogenous additive components are composed of the following 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; Among them, 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 - 12 μg / L; The concentration of the insulin is 8 - 12 mg / L; The concentration of the 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 the sodium selenite is 0.2 - 0.8 μg / L; The concentration of the penicillin-streptomycin is 1.0 - 1.4 mg / L; The concentration of the allicin is 8 - 12 μg / L; The addition amount of the heparin is 2 - 5 IU / mL; The addition amount of the proliferation promoter is 20 - 25 μg / L; The proliferation promoter is paeoniflorin, total saponins of Panax notoginseng, lycium barbarum polysaccharide, and ethanol extract of Saussurea involucrata; The ratio of paeoniflorin, total saponins of Panax notoginseng, lycium barbarum polysaccharide, and ethanol extract of Saussurea involucrata is 3:3:1:5; The preparation method of the ethanol extract of Saussurea involucrata is as follows: (1) After pulverizing Saussurea involucrata to 50 - 100 meshes, first soak and extract it with an ethanol solution with a volume fraction of 50 - 60% for 1 - 2 days, perform solid-liquid separation to obtain solid 1; (2) Add an ethanol solution with a volume fraction of 70 - 80% to solid 1, perform ultrasonic treatment every 5 - 8 hours, and extract for a total of 6 - 8 days, then perform solid-liquid separation to obtain extract 2; (3) Take extract 2, place it under the condition of 70 - 75 °C, and let it volatilize ethanol until there is no obvious alcohol smell to obtain the ethanol extract of Saussurea involucrata; In steps (1) and (2), the addition amount of the ethanol solution is 1.2 - 1.5 times the volume of Saussurea involucrata, and the temperature during the extraction process is 40 - 50 °C; In step (2), the power of the ultrasonic treatment is 300 - 450 W, and the time is 30 - 40 min.

2. The application of the improved mesenchymal stem cell culture medium according to claim 1 in the culture of mesenchymal stem cells.

3. The application according to claim 2, characterized in that, The method for culturing mesenchymal stem cells is as follows: (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 place it in DMEM medium for culture. After 3 - 5 days, change the medium, remove the non-adherent cells to obtain primary mesenchymal stem cells; (b)Cultivate the primary mesenchymal stem cells until the cell confluence reaches 70 - 80%, wash, digest, collect the single - cell suspension, centrifuge, discard the supernatant, resuspend with the modified mesenchymal stem cell medium, and perform sub - culture; (c)Isolation of mesenchymal stem cells.

4. The application according to claim 3, wherein, In step (a), the volume ratio of the tissue to be extracted, PBS solution and Percoll separation solution is 1:1 - 1.5:1 - 1.2; The rotation speed of the first centrifugation is 1600 - 2000 r / min, and the time is 25 - 40 min; When centrifuging and washing, the rotation speed is 1800 - 2200 r / min, the time is 5 - 10 min, and the washing solution is PBS solution; The DMEM medium is low - glucose DMEM medium, and the culture conditions are: 37 - 39 °C, 3 - 5% CO2.

5. The application according to claim 4, wherein, In step (b), the rotation speed of centrifugation is 1400 - 1600 r / min, the time is 5 min. When performing sub - culture, the initial sub - culture ratio is 1:1 - 2, and the subsequent sub - culture ratio is 1:3 - 6. The sub - culture conditions are: 37 - 39 °C, 3 - 5% CO2.