CN112662624A - Serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells - Google Patents
Serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a serum-free medium for in vitro culture and amplification of mesenchymal stem cells, which is characterized by being an aqueous solution, comprising the following components in parts by weight: 20-40g/L of basal culture medium, 5-15ng/mL of growth factor, 8-22mg/mL of black tea fungus fermentation liquor, 3-20mg/mL of serum protein, 1-10ng/mL of selenomethionine, 0.1-0.4mg/mL of sodium ferulate, 3-8ng/mL of metformin, 5-12ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 150mg/L-550mg/L of L-glutamine, 30-110 mU M of compound vitamin and 12-24 mU M/L of insulin. The invention also provides a preparation method and application of the serum-free medium for the in vitro culture and amplification of the mesenchymal stem cells. The serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells disclosed by the invention has good biological safety, can effectively improve the amplification efficiency of the human mesenchymal stem cells, simultaneously keeps biological effect and multidirectional differentiation potential, and can timely and accurately provide the mesenchymal stem cells with relevant requirements for medical scientific research and teaching and clinical application.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are the main components of the bone marrow stroma, providing structural and functional support for the hematopoietic function of the bone marrow. The stem cell is an adult stem cell with the potential of self-replication and multi-directional differentiation. Under certain induction conditions, the cell has the capacity of differentiating into mesoderm cells such as osteoblasts, chondroblasts, myoblasts, tenocytes, adipocytes, stromal cells and the like; meanwhile, the MSCs can also be differentiated to the neuron cells of the ectoderm and the hepatic oval cells of the endoderm. The mesenchymal stem cells can promote the reconstruction of a hematopoietic system, have effective immunoregulation function, can inhibit the proliferation of T-lymphocytes in vitro and delay the immunological rejection after the tissue transplantation,it can also be used for preventing and treating graft-versus-host disease (GVHD) caused by Autologous Stem Cell Transplantation (ASCT), preventing immune rejection in organ transplantation, and treating autoimmune diseases such as rheumatic arthritis. However, bone marrow mesenchymal stem cells are present in bone marrow in very small amounts, about 10%4～105Since each bone marrow mononuclear cell only contains one bone marrow mesenchymal stem cell, the bone marrow mesenchymal stem cells can not meet clinical requirements only by autologous or allogeneic separation, and the bone marrow mesenchymal stem cells need to be actively cultured and amplified in vitro to meet the application requirements of people.
In vitro culture and amplification of the bone marrow mesenchymal stem cells are usually performed in a culture medium, and the formula of the culture medium has a great influence on the effect and efficiency of the in vitro culture and amplification of the bone marrow mesenchymal stem cells. Traditional media rely on animal serum, however, the biological safety of animal serum is in general question. The existing serum-free culture medium is prepared by adding purified proteins, hormones, growth factors, trace elements, vitamins and the like to a basic culture medium, and is characterized by definite chemical components, better maintenance of the growth of the mesenchymal stem cells and maintenance of the multidirectional differentiation potential of the mesenchymal stem cells. However, none of these serum-free media promotes bone marrow mesenchymal stem cell adherence. In the in vitro culture process, after the mesenchymal stem cells are completely attached to the wall in the serum-containing culture medium, the mesenchymal stem cells are replaced by the serum-free culture medium for culture, so that a certain amount of animal serum is remained in the culture medium, the true serum-free culture is not achieved in a strict sense, and great risk is possibly caused in the clinical application.
Disclosure of Invention
The invention aims to solve the problems and provides a serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells, the serum-free culture medium has good biological safety, can effectively improve the amplification efficiency of the human mesenchymal stem cells, simultaneously keeps biological effect and multidirectional differentiation potential, can timely and accurately provide the mesenchymal stem cells with relevant requirements for medical research and teaching and clinical application, inhibits senescence and apoptosis and keeps cell stability. The bone marrow mesenchymal stem cells obtained by adopting the culture medium for culture and amplification have strong viability after transplantation and sufficient dryness.
In order to achieve the above object, the present invention provides the following technical solution, a serum-free medium for in vitro culture and expansion of bone marrow mesenchymal stem cells, which is characterized in that the serum-free medium is an aqueous solution, and the components and the contents thereof are as follows: 20-40g/L of basal culture medium, 5-15ng/mL of growth factor, 8-22mg/mL of black tea fungus fermentation liquor, 3-20mg/mL of serum protein, 1-10ng/mL of selenomethionine, 0.1-0.4mg/mL of sodium ferulate, 3-8ng/mL of metformin, 5-12ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 150mg/L-550mg/L of L-glutamine, 30-110 mU M of compound vitamin and 12-24 mU M/L of insulin.
Preferably, the compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of (1-2) to 1 (0.8-1.2) to 2 (1-2).
Preferably, the basic medium is DMEM-F12.
Preferably, the DMEM-F12 culture solution is a mixture of F12 culture solution and DMEM culture solution in a volume ratio of 1 (2-4).
Preferably, the growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1 (1-3) to 1 (1-2) to 1 (2-3).
Preferably, the black tea fungus fermentation liquid is prepared according to the formula and the preparation method of the Chinese patent application No. 201711050756.7 in the patent example 1.
Another object of the present invention is to provide a method for preparing the serum-free medium for the in vitro culture and expansion of bone marrow mesenchymal stem cells, which is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The invention further aims to provide an application of the serum-free medium for the in vitro culture and amplification of the bone marrow mesenchymal stem cells.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
(1) the preparation method of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells, provided by the invention, has the advantages of simple process, no need of special equipment, convenience in operation, good repeatability, short preparation period and no environmental pollution.
(2) The serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells overcomes the defect that the traditional culture medium depends on animal serum, however, the biological safety of the animal serum is generally questioned by people; the existing serum-free culture medium is prepared by adding purified proteins, hormones, growth factors, trace elements, vitamins and other substances on a basic culture medium, and is characterized by definite chemical components, capability of better maintaining the growth of the mesenchymal stem cells and keeping the multidirectional differentiation potential of the mesenchymal stem cells; however, none of these serum-free media promotes bone marrow mesenchymal stem cell adherence. In the in vitro culture process, after the bone marrow mesenchymal stem cells are completely attached to the wall in a serum-containing culture medium, the bone marrow mesenchymal stem cells are replaced by a serum-free culture medium for culture, so that a certain amount of animal serum is remained in the culture medium, the true serum-free culture is not achieved in a strict sense, and the defect of great risk is still likely to exist in the clinical application; through the synergistic effect of the components, the prepared serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells has good biological safety, can effectively improve the amplification efficiency of the human mesenchymal stem cells, simultaneously keeps the biological effect and multidirectional differentiation potential, can timely and accurately provide the mesenchymal stem cells with relevant requirements for medical scientific research and teaching and clinical application, inhibits senescence and apoptosis and keeps the cell stability. The bone marrow mesenchymal stem cells obtained by adopting the culture medium for culture and amplification have strong viability after transplantation and sufficient dryness.
(3) The addition of the black tea fungus fermentation liquor can provide sufficient nutrition for the culture and amplification of the stem cells; the composition has synergistic effect with other components, and is beneficial to cell growth and proliferation, dryness maintenance, good stability of cells, and effective inhibition of apoptosis.
(4) The serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells, provided by the invention, has the synergistic effect of the addition of selenomethionine and sodium ferulate, can effectively improve the viability and the lumen forming capability of the human mesenchymal stem cells, can improve the curative effect of transplanting the human mesenchymal stem cells to repair tissue damage, and can improve the cell amplification rate and the cell dryness.
(5) The serum-free culture medium for in vitro culture and amplification of the bone marrow mesenchymal stem cells, provided by the invention, has the advantages that the addition of the metformin, the 3' -deoxynicotinamide adenine dinucleotide and the growth factor has a synergistic effect, the serum can be replaced, the requirement that the adherence is firstly carried out in the serum-containing culture medium in advance and then the serum-free culture medium is replaced is avoided, the residue of the serum is reduced to the greatest extent, the growth of the bone marrow mesenchymal stem cells is well maintained, and the multidirectional differentiation potential of the bone marrow mesenchymal stem cells is maintained.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the following provides a detailed description of the product of the present invention with reference to the examples.
In the embodiment, the required raw materials are all purchased commercially; the black tea fungus fermentation liquid is prepared according to the formula and the preparation method of the Chinese patent application No. 201711050756.7 in patent example 1.
Example 1
A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 20g/L of basal culture medium, 5ng/mL of growth factor, 8mg/mL of black tea fungus fermentation liquor, 3mg/mL of serum protein, 1ng/mL of selenomethionine, 0.1mg/mL of sodium ferulate, 3ng/mL of metformin, 5ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 150mg/L of L-glutamine, 30 mU M of compound vitamin and 12mU/L of insulin.
The compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of 1:1:0.8:2: 1.
The basic culture medium is DMEM-F12; the DMEM-F12 culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1:2.
The growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1:1:1:1: 2.
The preparation method of the serum-free medium for in vitro culture and amplification of the mesenchymal stem cells is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The application of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells in the in vitro culture and amplification of the mesenchymal stem cells.
Example 2
A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 25g/L of basal culture medium, 8ng/mL of growth factor, 12mg/mL of black tea fungus fermentation liquor, 8mg/mL of serum protein, 3ng/mL of selenomethionine, 0.2mg/mL of sodium ferulate, 4ng/mL of metformin, 7ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 250mg/L of L-glutamine, 50 mU M of compound vitamin and 17mU/L of insulin.
The compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of 1.3:1:0.9:2: 1.3.
The basic culture medium is DMEM-F12; the DMEM-F12 culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1: 2.5.
The growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1:1.5:1.3:1: 2.3.
The preparation method of the serum-free medium for in vitro culture and amplification of the mesenchymal stem cells is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The application of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells in the in vitro culture and amplification of the mesenchymal stem cells.
Example 3
A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 30g/L of basal culture medium, 10ng/mL of growth factor, 16mg/mL of black tea fungus fermentation liquor, 13mg/mL of serum protein, 6ng/mL of selenomethionine, 0.25mg/mL of sodium ferulate, 6ng/mL of metformin, 8ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 350mg/L of L-glutamine, 70 mU M of compound vitamin and 19mU/L of insulin.
The compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of 1.5:1:1:2: 1.5.
The basic culture medium is DMEM-F12; the DMEM-F12 culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1:3.
The growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1:2:1.5:1: 2.5.
The preparation method of the serum-free medium for in vitro culture and amplification of the mesenchymal stem cells is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The application of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells in the in vitro culture and amplification of the mesenchymal stem cells.
Example 4
A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 36g/L of basal culture medium, 13ng/mL of growth factor, 21mg/mL of black tea fungus fermentation liquor, 18mg/mL of serum protein, 9ng/mL of selenomethionine, 0.35mg/mL of sodium ferulate, 7ng/mL of metformin, 11ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 450mg/L of L-glutamine, 100 mU M of compound vitamin and 22mU/L of insulin.
The compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of 1.8:1:1.1:2: 1.8.
The basic culture medium is DMEM-F12; the DMEM-F12 culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1: 3.5.
The growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1:2.7:1.8:1: 2.8.
The preparation method of the serum-free medium for in vitro culture and amplification of the mesenchymal stem cells is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The application of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells in the in vitro culture and amplification of the mesenchymal stem cells.
Example 5
A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 40g/L of basal culture medium, 15ng/mL of growth factor, 22mg/mL of black tea fungus fermentation liquor, 20mg/mL of serum protein, 10ng/mL of selenomethionine, 0.4mg/mL of sodium ferulate, 8ng/mL of metformin, 12ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 550mg/L of L-glutamine, 110 mU M of compound vitamin and 24mU/L of insulin.
The compound vitamin is formed by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to the mass ratio of 2:1:1.2:2: 2.
The basic culture medium is DMEM-F12; the DMEM-F12 culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1: 4.
The growth factor is formed by mixing transformation growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF) according to the mass ratio of 1:3:2:1: 3.
The preparation method of the serum-free medium for in vitro culture and amplification of the mesenchymal stem cells is characterized by comprising the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
The application of the serum-free culture medium for the in vitro culture and amplification of the mesenchymal stem cells in the in vitro culture and amplification of the mesenchymal stem cells.
Comparative example 1
The serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells has the same formula and preparation process as those in example 1 except that no black tea fungus fermented liquid is added.
Comparative example 2
The serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells has the same formula and preparation process as those in example 1 except that no selenomethionine and sodium ferulate are added.
Comparative example 3
A serum-free culture medium for in vitro culture and amplification of mesenchymal stem cells has the same formula and preparation method as those in example 1, except that metformin and 3' -deoxynicotinamide adenine dinucleotide are not added.
In order to further illustrate the beneficial technical effects of the invention, the serum-free cell culture solution suitable for enriching and culturing the tumor stem cells is tested for the effect, and the test method is as follows: tumor stem cells were seeded in 96-well plates at the same number of starting cells (cell seeding density 5X 10)4One/ml), placing at 37 deg.C and 5% CO2Culturing under the condition; after the cells adhere to the wall the next day, removing the stock solution and respectively changing the stock solution into each culture solution, wherein each group is provided with 5 repeat holes; after further culturing for 5 days, the MTT method measures the proliferation of tumor stem cells in each group. The results are shown in Table 1.
TABLE 1
As can be seen from table 1, the serum-free media for in vitro culture and expansion of mesenchymal stem cells in examples 1 to 5 have more excellent cell proliferation promoting ability than the comparative example, which is a result of the synergistic effect of the components.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; the present invention can be readily implemented by those of ordinary skill in the art in view of the foregoing description; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (7)
1. A serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells is characterized by being an aqueous solution, and comprising the following components in parts by weight: 20-40g/L of basal culture medium, 5-15ng/mL of growth factor, 8-22mg/mL of black tea fungus fermentation liquor, 3-20mg/mL of serum protein, 1-10ng/mL of selenomethionine, 0.1-0.4mg/mL of sodium ferulate, 3-8ng/mL of metformin, 5-12ng/mL of 3' -deoxynicotinamide adenine dinucleotide, 150mg/L-550mg/L of L-glutamine, 30-110 mU M of compound vitamin and 12-24 mU M/L of insulin.
2. The serum-free medium for the in vitro culture and amplification of the mesenchymal stem cells according to claim 1, wherein the vitamin complex is prepared by mixing vitamin A, vitamin B2, vitamin C, vitamin D and vitamin E according to a mass ratio of (1-2) to 1 (0.8-1.2) to 2 (1-2).
3. The serum-free medium for the in vitro culture and expansion of mesenchymal stem cells according to claim 1, wherein the basic medium is DMEM-F12.
4. The serum-free medium for the in vitro culture and expansion of the mesenchymal stem cells of the bone marrow according to claim 3, wherein the DMEM-F12 culture solution is a mixture of F12 culture solution and DMEM culture solution in a volume ratio of 1 (2-4).
5. The serum-free medium for the in vitro culture and amplification of the mesenchymal stem cells of the bone marrow according to claim 1, wherein the growth factors are transforming growth factor beta 1 (TGF-beta 1), heparin binding epidermal growth factor (HB-beta), insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and Epidermal Growth Factor (EGF) mixed according to a mass ratio of 1 (1-3) to (1-2) to 1 (2-3).
6. A method for preparing the serum-free culture medium for the in vitro culture and expansion of the mesenchymal stem cells according to any one of claims 1 to 5, which comprises the following steps: the components are uniformly mixed according to the parts by weight, and then are filtered and sterilized to obtain the serum-free culture medium for in vitro culture and amplification of the mesenchymal stem cells.
7. Use of the serum-free medium for the in vitro culture and expansion of mesenchymal stem cells according to any one of claims 1-5.
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