Mesenchymal stem cell culture medium without animal-derived components and application of mesenchymal stem cell culture medium

Abstract

The invention relates to the technical field of cell culture. The invention provides a mesenchymal stem cell culture medium without animal-derived components and application of the mesenchymal stem cell culture medium. The culture medium comprises a basic culture medium and an additive, the additives comprise FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, an HEPES buffer solution, sodium bicarbonate, SCF, TGF-beta, PDGF, ascorbic acid and transferrin. The mesenchymal stem cell culture medium disclosed by the invention is completely free of animal-derived components, and meets the requirements of FDA / EMA clinical-grade cell therapy products. The culture medium can support high-density amplification of mesenchymal stem cells, the activity of passage cells is effectively improved, and the amplification time is shortened. The method has remarkable significance in improving the amplification efficiency of the mesenchymal stem cells and promoting and applying factory growth.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a mesenchymal stem cell culture medium free of animal-derived components and application thereof. Background Art

[0002] Cell culture technology simulates the in vivo environment in vitro, allowing cells to survive, grow, reproduce, and maintain their primary structure and function. This allows for the acquisition of large numbers of cells, enabling the study of cell signal transduction, anabolism, growth, and proliferation. Of all in vitro cell cultures, the most challenging is the cultivation of human cells.

[0003] Mesenchymal stem cells (MSCs), a key member of the stem cell family, originate from the early developmental mesoderm and ectoderm. They are multipotent stem cells, originally discovered in the bone marrow. They possess multipotent differentiation potential, support hematopoiesis, promote stem cell engraftment, regulate immunity, and self-replicate. Under specific induction conditions in vivo or in vitro, MSCs can differentiate into a variety of tissue cells, including fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium. They retain their multipotential potential after continuous subculture and cryopreservation, making them ideal seed cells for repairing tissue and organ damage caused by aging and disease.

[0004] Most traditional mesenchymal stem cell culture media contain animal serum, which, on the one hand, causes contamination of fibroblasts, affects the growth of keratinocytes, and accelerates keratinocyte differentiation; on the other hand, the presence of serum and proteins of unknown composition interferes with basic research in cell biology, toxicology, pathology, etc.; at the same time, artificial tissue constructed with serum-added culture media for in vivo transplantation can cause the recipient to produce anti-bovine protein antibodies, causing an immune response, thereby leading to unsafe factors such as treatment failure, especially after repeated stem cell infusions.

[0005] Therefore, it is particularly important to develop a new mesenchymal stem cell culture medium that does not contain animal-derived components. Summary of the Invention

[0006] The purpose of the present invention is to provide a mesenchymal stem cell culture medium free of animal-derived components and its application, which is completely free of animal-derived components and meets the requirements of FDA / EMA clinical-grade cell therapy products.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a mesenchymal stem cell culture medium free of animal-derived components, the culture medium comprising a basal culture medium and additives;

[0009] The additives include FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, HEPES buffer, sodium bicarbonate, SCF, TGF-β, PDGF, ascorbic acid, and transferrin.

[0010] Preferably, the concentrations of the additives are: FGF-28-15 mg / L, IGF-115-25 mg / L, glutamine 1.2-1.4 mM, folic acid 0.3-0.5 mg / L, serotonin 8-12 mg / L, cholesterol 0.5-0.7 mg / L, arachidonic acid 0.05-0.1 mg / L, HEPES buffer 6-8 mM, sodium bicarbonate 1.7-1.9 g / L, SCF 60-70 μg / L, TGF-β 2-4 μg / L, PDGF 10-20 μg / L, ascorbic acid 12-16 mg / L, and transferrin 10-20 mg / L.

[0011] Preferably, the basal culture medium is DMEM culture medium, F12 culture medium, IMDM culture medium, MCDB 201 culture medium or MCDB 131 culture medium.

[0012] The present invention also provides a method for preparing the culture medium, comprising the following steps:

[0013] (1) Add the additive to the basal culture medium and stir until completely dissolved to obtain a culture medium stock solution;

[0014] (2) Adjusting the pH value of the culture medium stock solution and filtering to obtain the culture medium.

[0015] Preferably, the pH value of the culture medium solution after adjustment in step (2) is 7.2 to 7.4.

[0016] Preferably, a filter with a specification of 0.22 μm is used during the filtration in step (2).

[0017] The present invention also provides application of the culture medium in culturing mesenchymal stem cells.

[0018] The present invention provides a mesenchymal stem cell culture medium free of animal-derived components and its application, wherein the culture medium comprises a basal culture medium and additives; the additives comprise FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, HEPES buffer, sodium bicarbonate, SCF, TGF-β, PDGF, ascorbic acid, and transferrin. The mesenchymal stem cell culture medium of the present invention is completely free of animal-derived components and meets the requirements of FDA / EMA clinical-grade cell therapy products. The culture medium can support high-density expansion of mesenchymal stem cells, effectively improves the activity of passaged cells, and shortens the expansion time. It has significant significance for improving the expansion efficiency of mesenchymal stem cells and promoting the application of factory-scale growth. DETAILED DESCRIPTION

[0019] The present invention provides a mesenchymal stem cell culture medium free of animal-derived components, the culture medium comprising a basal culture medium and additives;

[0020] The additives include FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, HEPES buffer, sodium bicarbonate, SCF, TGF-β, PDGF, ascorbic acid, and transferrin.

[0021] In the present invention, the concentrations of the additives are preferably: FGF-28-15 mg / L, IGF-115-25 mg / L, glutamine 1.2-1.4 mM, folic acid 0.3-0.5 mg / L, serotonin 8-12 mg / L, cholesterol 0.5-0.7 mg / L, arachidonic acid 0.05-0.1 mg / L, HEPES buffer 6-8 mM, sodium bicarbonate 1.7-1.9 g / L, SCF 60-70 μg / L, TGF-β 2-4 μg / L, PDGF 10-20 μg / L, ascorbic acid 12-16 mg / L, and transferrin 10-20 mg / L.

[0022] In the present invention, the concentration of FGF-2 is preferably 10 to 13 mg / L, more preferably 11 to 12 mg / L.

[0023] In the present invention, the concentration of IGF-1 is preferably 18 to 22 mg / L, more preferably 20 mg / L.

[0024] In the present invention, the concentration of glutamine is preferably 1.3 mM.

[0025] In the present invention, the concentration of folic acid is preferably 0.4 mg / L.

[0026] In the present invention, the concentration of serotonin is preferably 10 mg / L.

[0027] In the present invention, the concentration of cholesterol is preferably 0.6 mg / L.

[0028] In the present invention, the concentration of arachidonic acid is preferably 0.07 to 0.08 mg / L.

[0029] In the present invention, the concentration of the HEPES buffer is preferably 7 mM.

[0030] In the present invention, the concentration of sodium bicarbonate is preferably 1.8 g / L.

[0031] In the present invention, the concentration of SCF is preferably 65 μg / L.

[0032] In the present invention, the concentration of TGF-β is preferably 3 μg / L.

[0033] In the present invention, the concentration of PDGF is preferably 15 μg / L.

[0034] In the present invention, the concentration of ascorbic acid is preferably 14 mg / L.

[0035] In the present invention, the concentration of transferrin is preferably 15 mg / L.

[0036] In the present invention, the basal culture medium is preferably DMEM culture medium, F12 culture medium, IMDM culture medium, MCDB201 culture medium or MCDB 131 culture medium.

[0037] The present invention also provides a method for preparing the culture medium, comprising the following steps:

[0038] (1) Add the additive to the basal culture medium and stir until completely dissolved to obtain a culture medium stock solution;

[0039] (2) Adjusting the pH value of the culture medium stock solution and filtering to obtain the culture medium.

[0040] In the present invention, the pH value of the culture medium solution after adjustment in step (2) is preferably 7.2 to 7.4, and more preferably 7.3.

[0041] In the present invention, the filtration in step (2) preferably uses a filter with a specification of 0.22 μm.

[0042] The present invention also provides application of the culture medium in culturing mesenchymal stem cells.

[0043] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] A method for preparing a mesenchymal stem cell culture medium free of animal-derived ingredients comprises the following steps:

[0046] (1) Adding additives to the IMDM medium to obtain the following concentrations: FGF-28 mg / L, IGF-125 mg / L, glutamine 1.2 mM, folic acid 0.5 mg / L, serotonin 8 mg / L, cholesterol 0.7 mg / L, arachidonic acid 0.05 mg / L, HEPES buffer 8 mM, sodium bicarbonate 1.9 g / L, SCF 60 μg / L, TGF-β 2 μg / L, PDGF 20 μg / L, ascorbic acid 16 mg / L, and transferrin 10 mg / L, stirring until completely dissolved to obtain a stock medium solution;

[0047] (2) The pH value of the culture medium stock solution was adjusted to 7.4, and the culture medium was obtained by filtering the culture medium using a filter with a specification of 0.22 μm.

[0048] Example 2

[0049] A method for preparing a mesenchymal stem cell culture medium free of animal-derived ingredients comprises the following steps:

[0050] (1) Adding additives to F12 medium to adjust the concentrations of the additives to 15 mg / L FGF-2, 15 mg / L IGF-1, 1.4 mM glutamine, 0.3 mg / L folic acid, 12 mg / L serotonin, 0.5 mg / L cholesterol, 0.1 mg / L arachidonic acid, 6 mM HEPES buffer, 1.7 g / L sodium bicarbonate, 70 μg / L SCF, 4 μg / L TGF-β, 10 μg / L PDGF, 12 mg / L ascorbic acid, and 20 mg / L transferrin, and stirring until completely dissolved to obtain a stock medium solution;

[0051] (2) The pH value of the culture medium stock solution was adjusted to 7.2, and the culture medium was obtained by filtering the solution through a filter with a specification of 0.22 μm.

[0052] Example 3

[0053] A method for preparing a mesenchymal stem cell culture medium free of animal-derived ingredients comprises the following steps:

[0054] (1) Adding additives to the DMEM medium to adjust the concentration of the additives to 12 mg / L FGF-2, 120 mg / L IGF-1, 1.3 mM glutamine, 0.4 mg / L folic acid, 10 mg / L serotonin, 0.6 mg / L cholesterol, 0.08 mg / L arachidonic acid, 7 mM HEPES buffer, 1.8 g / L sodium bicarbonate, 65 μg / L SCF, 3 μg / L TGF-β, 15 μg / L PDGF, 14 mg / L ascorbic acid, and 15 mg / L transferrin, and stirring until completely dissolved to obtain a culture medium stock solution;

[0055] (2) The pH value of the culture medium stock solution was adjusted to 7.3, and the culture medium was obtained by filtering the culture medium using a filter with a specification of 0.22 μm.

[0056] Comparative Example 1

[0057] Cholesterol and arachidonic acid were removed from the additives, and the remaining steps and formula were consistent with those in Example 3.

[0058] Comparative Example 2

[0059] Folic acid and ascorbic acid were removed from the additives, and the remaining steps and formula were consistent with those in Example 3.

[0060] Comparative Example 3

[0061] PDGF was removed from the additives, and the remaining steps and formula were consistent with those in Example 3.

[0062] Test example

[0063] 1. Cell Acquisition

[0064] The adipose tissue was washed with physiological saline to remove fascia and blood vessels, then minced and digested with an equal volume of 0.1% collagenase solution at 37°C for 30 minutes. The cells were then centrifuged at 280g for 5 minutes, resuspended in DMEM-F12 medium containing 5% serum replacement (Thermo Fisher Scientific), passed through a 40μM cell sieve, and centrifuged and resuspended once more to obtain isolated cells.

[0065] The separated cells were cultured at 37°C and 5% CO2. After the cells adhered to the wall and grew for 48 hours, the medium was changed to remove the non-adherent cells. Thereafter, half the volume of the medium was changed every 3 days. When the cells reached 80% confluence, they were digested with 0.25% trypsin and passaged at a ratio of 1:4. The third generation cells obtained were used for three-dimensional culture.

[0066] 2. Cell Expansion

[0067] The third generation cells obtained in step 1 were cultured at a rate of 5 × 10 4The standard of cells / mL was inoculated into the culture medium prepared in Examples 1 to 3 and Comparative Examples 1 to 3 at 37°C, and the cells were cultured according to the same environment and standards. After 3 days of culture, the number of cells in each group was detected and the proliferation times were calculated. The results are shown in Table 1.

[0068] Table 1

[0069] Group Example 1 Example 2 Example 3 Control group 1 Control group 2 Control group 3 proliferation multiples 5.27 5.09 5.31 2.09 3.57 3.66

[0070] Conclusion: The culture medium prepared in Examples 1 to 3 of the present invention can support high-density expansion of mesenchymal stem cells and shorten the expansion time.

[0071] As can be seen from the above embodiments, the present invention provides a mesenchymal stem cell culture medium without animal-derived components and its application, wherein the culture medium includes a basal culture medium and additives; the additives include FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, HEPES buffer, sodium bicarbonate, SCF, TGF-β, PDGF, ascorbic acid, and transferrin. The mesenchymal stem cell culture medium of the present invention is completely free of animal-derived components and meets the requirements of FDA / EMA clinical-grade cell therapy products. The culture medium can support high-density expansion of mesenchymal stem cells, effectively improves the activity of passaged cells, and shortens the expansion time. It has significant significance for improving the expansion efficiency of mesenchymal stem cells and promoting the application of factory-based growth.

[0072] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell culture medium free of animal-derived ingredients, characterized in that: The culture medium includes a basal culture medium and additives; The additives include FGF-2, IGF-1, glutamine, folic acid, serotonin, cholesterol, arachidonic acid, HEPES buffer, sodium bicarbonate, SCF, TGF-β, PDGF, ascorbic acid, and transferrin.

2. The culture medium according to claim 1, characterized in that The concentrations of the additives are: FGF-28-15 mg / L, IGF-115-25 mg / L, glutamine 1.2-1.4 mM, folic acid 0.3-0.5 mg / L, serotonin 8-12 mg / L, cholesterol 0.5-0.7 mg / L, arachidonic acid 0.05-0.1 mg / L, HEPES buffer 6-8 mM, sodium bicarbonate 1.7-1.9 g / L, SCF 60-70 μg / L, TGF-β 2-4 μg / L, PDGF 10-20 μg / L, ascorbic acid 12-16 mg / L, and transferrin 10-20 mg / L.

3. The culture medium according to claim 1 or 2, characterized in that The basal culture medium is DMEM culture medium, F12 culture medium, IMDM culture medium, MCDB 201 culture medium or MCDB 131 culture medium.

4. The method for preparing the culture medium according to any one of claims 1 to 3, characterized in that: The steps include: (1) Add the additive to the basal culture medium and stir until completely dissolved to obtain a culture medium stock solution; (2) Adjusting the pH value of the culture medium stock solution and filtering to obtain the culture medium.

5. The preparation method according to claim 4, characterized in that The pH value of the culture medium solution after adjustment in step (2) is 7.2-7.

4.

6. The preparation method according to claim 4 or 5, characterized in that The filtration in step (2) uses a filter with a specification of 0.22 μm.

7. Use of the culture medium according to any one of claims 1 to 3 in culturing mesenchymal stem cells.