CN110923196B - Serum-free medium, preparation method thereof and mesenchymal stem cell culture method - Google Patents
Serum-free medium, preparation method thereof and mesenchymal stem cell culture method Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture, in particular to a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells. The culture medium consists of the following components: nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine, sodium selenite and basal medium. The invention discovers that polyvinyl alcohol can replace the carrier effect of serum albumin in a serum-free culture medium, effectively improves the proliferation capacity of mesenchymal stem cells, has more stable performance than the serum albumin, and can effectively solve the problem of the proliferation and the passage capacity reduction of the stem cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells.
Background
Research in the field of regenerative medicine represented by stem cells has continuously made a major technical breakthrough in the last two decades, and has led to high attention on industrialization and clinical application of stem cell technology. By 2019, 16 stem cell products are on the market worldwide, and the intermediate stem cells (MSCs) become the most product and most rapidly developed stem cell types due to various advantages. Mesenchymal stem cells are derived from mesoderm in early development, and are a type of non-hematopoietic stem cells widely existing in bone marrow, subcutaneous fat, periosteum, muscle, synovium, synovial fluid, liver, peripheral tissues, umbilical cord blood, placenta and other tissues. The MSCs have high self-renewal capacity and multidirectional differentiation potential, can be cultured and amplified in vitro, can support the growth of hematopoietic stem cells, and also has the function of immune regulation; under different induction conditions, the cells can be differentiated into bones, cartilages, muscles, nerves, cardiac muscles, endothelia, fat and the like in vitro, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. Therefore, the MSCs have wide clinical application prospect, are the first choice seed cells for cell replacement therapy and tissue engineering, and are the research hotspots in the field of transplantation and the treatment of autoimmune diseases. At present, the demand for the mesenchymal stem cell related technology and products which are large-scale, standardized and industrialized and can be clinically applied is increasingly called for in the industry.
In studies in the field of biomedical engineering, the acquisition of large numbers of seed cells is often achieved by in vitro culture and harvesting of stem cells. At present, a serum culture medium or a serum-free culture medium is used for mesenchymal stem cell culture. Serum media mostly contain animal serum, such as the most common Fetal Bovine Serum (FBS). FBS is complex in composition, contains heterogeneous proteins, and is likely to carry viruses or to be infected with mycoplasma. On the other hand, the FBS has large batch difference and unstable source, and has large influence on the process of amplifying the MSCs in vitro on a large scale. Research shows that MSCs phagocytose protein in culture medium during culture, and the culture medium contains bovine serum albumin, so that anti-bovine albumin antibody produced in a recipient can cause immune response, and the MSCs are ineffective or especially ineffective after repeated infusion treatment. Therefore, more and more researchers and enterprises are beginning to develop alternatives to FBSs.
Serum-Free Medium (SFM), as the name implies, means that it is not necessary to add animal or human Serum to the cell culture. However, in order to meet the requirement of cell growth, materials for replacing serum functions, mainly including binding proteins, growth factors, adhesion factors, hormones, trace elements, and the like, are usually added to the culture medium. The development of SFM applied to mesenchymal stem cells has gone through four major stages. The first generation is serum-free culture medium in general meaning, adopts various biological materials capable of replacing serum function, contains a large amount of animal-derived proteins and unidentified components, such as animal or human-derived platelet lysate and the like; compared with a serum-containing culture medium, the method has the advantages of high experimental accuracy, repeatability and stability; however, since the chemical composition of the additive substance is not clear, most of the additive substances contain a large amount of animal-derived proteins, which is not favorable for the separation and purification of target proteins and has high cost. The second generation is serum-free and animal protein-free culture medium, and the added components of the culture medium do not use animal protein at all, but replace various recombinant proteins or animal and plant protein hydrolysates; the method has the advantages that the stability is improved compared with the first generation, the cost is reduced, and the effect is correspondingly improved; but the cost is extremely high, and the method is only suitable for scientific research users with small demand and is difficult to be used by enterprises developing large-scale production. At present, the performance of the product on the market is stable, and most of scientific research users use the second generation SFM products. The third generation is a component-limited medium, also known as a double-no medium; the added components are completely free of serum, free of protein or extremely low in protein content, and the contained protein is definite in components; the third-generation SFM has obvious advantages, the cell culture and the production are easy to be constant, the separation and the purification of target protein are easier, and the cost is greatly reduced; however, at the present stage, the products all have the defects of insufficient cell generation and expansion capacity, which is also a bottleneck to be broken through by SFM research and development enterprises at present; and has high specificity to cultured cells, so that the cell lines suitable for culture are few, and the product development difficulty is great. The fourth generation is a chemical component limited culture medium, the added components do not contain serum or any protein, unstable protein substances are mainly replaced by compounds, and the medium can be sterilized at high temperature and is suitable for a totipotent culture medium for the growth of various cells; at present, no such products are on the market and still in the development stage. Serum-free media are currently on the market, in which bovine serum albumin or human serum albumin is almost an essential raw material in the base. The serum albumin has a large adding proportion in a serum-free culture medium, and has the functions of not only serving as a transport protein, but also increasing the viscosity of the culture medium and protecting cells from mechanical damage. Research shows that bovine or human serum albumin may promote stem cell differentiation, resulting in reduction of stem cell proliferation and passability.
Disclosure of Invention
In view of the above, the invention provides a serum-free medium, a preparation method thereof and a culture method of mesenchymal stem cells. The serum-free culture medium can effectively solve the problems of stem cell proliferation and passage capacity reduction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a serum-free culture medium, which consists of the following components:
the invention selects a high molecular organic compound-polyvinyl alcohol (PVA), which can replace the carrier effect of serum albumin in a culture medium, effectively improves the proliferation capacity of mesenchymal stem cells, and has more stable performance than the serum albumin. Industrial grade PVA is an important chemical raw material for the manufacture of polyvinyl acetal, gasoline resistant pipelines and vinylon synthetic fibers, fabric treating agents, emulsifiers, paper coatings, adhesives, glues, and the like. The medical PVA is a safe polymer organic matter, has no toxicity to human body, no side effect and excellent biocompatibility, is especially widely used in medical treatment, such as water gel in ophthalmology, wound dressing and artificial joint, and is also used in medicinal film, artificial kidney film and other fields. The safety can be seen from the use in wound skin repair, and eye drop products. Some of these types are also commonly used in cosmetic masks, facial cleansers, lotions and lotions, as a safe film former. The latest international research finds that polyvinyl alcohol contained in common glue can be used for a culture solution of hematopoietic stem cells, so that the culture cost of the hematopoietic stem cells is expected to be greatly reduced, and the glue helps to treat diseases such as leukemia.
The invention provides a high molecular organic compound polyvinyl alcohol which replaces the common serum albumin in the existing serum-free medium product and can effectively improve the defects of insufficient passage and amplification capacity of mesenchymal stem cells.
Preferably, the amounts of the components in the culture medium are as follows:
preferably, the amounts of the components in the culture medium are as follows:
more preferably, the media is prepared with the following components:
preferably, the basal medium is one of DMEM/F12, high-sugar DMEM, and a-MEM.
Preferably, penicillin and streptomycin can also be added into the serum-free medium.
The invention also provides a preparation method of the serum-free culture medium, which comprises the steps of dissolving non-essential amino acid, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine and sodium selenite in a basal culture medium, filtering and sterilizing.
Preferably, the pore size of the filter used for the filter sterilization is not more than 0.22. mu.m.
Preferably, the pore size of the filter used for the filter sterilization is 0.22. mu.m.
The invention also provides a culture method of the mesenchymal stem cells, and the serum-free culture medium is adopted to culture the mesenchymal stem cells.
Preferably, the mesenchymal stem cell is one of umbilical cord mesenchymal stem cell, bone marrow mesenchymal stem cell, adipose mesenchymal stem cell, amniotic fluid mesenchymal stem cell and placenta mesenchymal stem cell.
Preferably, the culture conditions are 5% CO2At 37 ℃, the density inoculation is (0.1-10) multiplied by 104one/mL.
The invention provides a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells. The culture medium consists of the following components: nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine, sodium selenite and basal medium. The invention has the technical effects that:
according to the invention, the polyvinyl alcohol is added into the serum-free culture medium, and the polyvinyl alcohol can replace the carrier effect of serum albumin in the culture medium, so that the proliferation capacity of the mesenchymal stem cells is effectively improved, and the performance of the mesenchymal stem cells is more stable than that of the serum albumin.
The serum-free culture medium provided by the invention can effectively solve the problems of stem cell proliferation and passage capacity reduction.
Drawings
FIG. 1 shows the morphology of UC-MSCs in each group (100X);
FIG. 2 shows growth curves for various groups of UC-MSCs;
FIG. 3 shows the adipogenic differentiation effect of UC-MSCs in each group (400X);
FIG. 4 is a graph showing the osteogenic differentiation effect of UC-MSCs in each group (40X).
Detailed Description
The invention discloses a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells, and the method can be realized by appropriately improving process parameters by taking the contents into reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the invention, the components and reagents are conventional commercial products. Such as DMEM/F12 basal medium (cat 11330), an optional amino acid solution (cat 11140-. Other components are commercially available from sigma, MP, Gibco, and the like.
The invention is further illustrated by the following examples:
example 1 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 2g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68：500mg/L
Tween
22mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Example 2 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 4g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68：500mg/L
Tween
22mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Example 3 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 10g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68：500mg/L
Tween
22mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Comparative example 1 serum-free medium preparation:
compared with the example 1, the comparison example adopts the recombinant human serum albumin to replace the polyvinyl alcohol, and the serum-free culture medium of the comparison example comprises the following components:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Recombinant human serum albumin: 2g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68：500mg/L
Tween
22mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Comparative example 2 serum-free medium preparation:
compared with the comparative example 1 and the examples, the comparative example does not add the recombinant human serum albumin or the polyvinyl alcohol, and the serum-free culture medium of the comparative example comprises the following components:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68：500mg/L
Tween
22mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Test examples Effect detection
The following experiments were carried out with the serum-free media of examples 1 to 3 of the present invention set as experimental group 1 to 3, the complete medium containing 10% FBS set as control group 1, the medium of comparative example 1 set as control group 2, and the medium of comparative example 2 set as control group 3.
(I) UC-MSCs morphology observation and activity detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104/cm2The density was inoculated in T25 flasks, with 3 replicates per group set up. Placing in 5% CO2The culture was carried out in an incubator at 37 ℃. UC-MSCs images were collected after 48h of culture, and the results are shown in FIG. 1. Each group of UC-MSCs grows in a monolayer adherent manner, and most cells are in a long fusiform shape and have irregular shapes.
After 72h of culture, digesting and collecting UC-MSCs in 0.05% trypsin solution, calculating the cell number and cell viability of each group, and showing that the UC-MSCs activity of the experimental group is higher than that of the three control groups (Table 1).
TABLE 1 UC-MSCs Activity assay results for each group
(II) UC-MSCs proliferation rate detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104one/mL of the seed was inoculated in a 24-well plate and charged with 5% CO2The culture was carried out in an incubator at 37 ℃. Cells were collected daily for cell counting, 3 wells were counted for each random collection, and cell growth curves were plotted for 7 consecutive days, with the results shown in tables 2-3, FIG. 2. As can be seen from the results in tables 2-3 and FIG. 2, the UC-MSCs cultured in the human serum-free medium of the present invention have higher proliferation activity than those in the control group 1 (containing serum), the control group 2 and the control group 3. After three days of culture, the proliferation fold of each experimental group is significantly higher than that of the control group 1, the control group 2 and the control group 3.
According to the multiplication time calculation formula: DT ═ t × lg2/(lgNt-lgNo) ], where t is the incubation time; no is the number of cells counted for the first time; nt is the number of cells after t time.
The result shows that the doubling time of the UC-MSCs in the experimental group is shorter than that of the control group 1, the control group 2 and the control group 3, which indicates that the proliferation rate of the UC-MSCs cultured by the serum-free culture medium is higher.
TABLE 2 7-day cell count results for UC-MSCs in each group
TABLE 3 UC-MSCs doubling time results for each group
| Experimental groups | Control group 1 | Control group 2 | Control group 3 | Experimental group 1 | Experimental group 2 | Experimental group 3 |
| Doubling time (h) | 29.18 | 32.79 | 34.27 | 27.12 | 22.72 | 26.24 |
(III) UC-MSCs surface marker detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104/cm2Inoculating into 10cm culture dish at density, and placing in 5% CO2The culture was carried out in an incubator at 37 ℃. After 3 days, each group of UC-MSCs is digested by 0.05% trypsin solution and detected by flow cytometry on the surface markers such as CD105, CD73, CD90, CD34, CD45, HLA-DR and the like. The results are shown in Table 4. The detection result shows that the UC-MSCs of the experimental group, the control group 1 and the control group 2 have surface markerCD105, CD73 and CD90 positive expression (the expression rate is more than or equal to 95 percent), and have negative expression (the expression rate is less than or equal to 2 percent) of CD34, CD45 and HLA-DR, and the groups have no significant difference. The expression rates of CD105 and CD34 in control group 3 did not meet the standard. The result shows that the UC-MSCs cultured by the serum-free culture medium does not influence the expression of the surface markers.
TABLE 4 detection results of UC-MSCs surface marker of each group
(IV) detection of UC-MSCs multidirectional differentiation potential
Selecting UC-MSCs of P3 generation for experiment, culturing UC-MSCs of experiment group 2, control group 1 and control group 2 to passage of P5 generation at 1 × 105The cells were seeded in 6-well plates at a density of one mL/mL and 5% CO was added2The culture was carried out in an incubator at 37 ℃. When the fusion degree of UC-MSCs in each group reaches more than 80%, a control hole and an induction hole are respectively arranged to induce the UC-MSCs to form bone and fat differentiation. And (3) carrying out oil red O staining on the cells in the adipogenic differentiation experiment group after 14 days, and carrying out alizarin red staining on the cells in the osteogenic differentiation experiment group after 21 days. The experimental result shows that the UC-MSCs cultured by the serum-free culture medium does not influence the osteogenic differentiation potential of the adipogenic bone marrow and maintains the dryness (figure 3 and figure 4).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A method for culturing mesenchymal stem cells is characterized in that a serum-free culture medium is adopted to culture the mesenchymal stem cells;
the mesenchymal stem cells are umbilical cord mesenchymal stem cells;
the serum-free culture medium consists of the following components:
non-essential amino acids: 1vt% of
Glutamine (b): 2mM
Hydrocortisone: 50 mug/L
Dexamethasone: 10 microgram/L
Polyvinyl alcohol: 2 to 10g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 microgram/L
Recombinant human basic fibroblast growth factor: 20 microgram/L
Recombinant human Wnt-3a protein: 20 microgram/L
Recombinant human fibronectin: 50 mug/L
Recombinant human laminin: 20 microgram/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta-mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68： 500 mg/L
Tween 80： 22 mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
Basic culture medium: and (4) the balance.
2. The culture method according to claim 1, wherein the basal medium is one of DMEM/F12, high-sugar DMEM, and a-MEM.
3. The culture method according to claim 1 or 2, wherein the serum-free medium is prepared by: dissolving nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine and sodium selenite in a basal medium, and filtering and sterilizing.
4. The culture method according to claim 3, wherein the filter used for the filter sterilization has a pore size of 0.22 μm.
5. The method according to claim 1 or 2, wherein the culturing is performed under conditions of 5% CO2At 37 ℃, the density inoculation is (0.1-10) multiplied by 104one/mL.
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| CN111621476B (en) * | 2020-05-12 | 2021-03-09 | 天信和(苏州)生物科技有限公司 | Serum-free culture medium for mesenchymal stem cells and preparation method thereof |
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