CN113151165B - Culture medium and culture method for human umbilical cord mesenchymal stem cell amplification - Google Patents
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Abstract
The invention discloses a culture medium for human umbilical cord mesenchymal stem cell amplification, which consists of a basic culture medium and the following components added in the basic culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin. The culture medium provided by the invention is not added with exogenous serum, has definite components, and the components have synergistic effect to promote the proliferation of umbilical cord mesenchymal stem cells and the secretion of cytokines. The added components such as eucalyptol, estradiol valerate and acetazolamide not only avoid the potential safety hazard caused by adding serum into the culture medium, but also can improve the proliferation efficiency of cells, obtain a large amount of umbilical cord mesenchymal stem cells in a short time and improve the content of cell factors in the culture solution. The invention also provides a culture method of the human umbilical cord mesenchymal stem cells, and the culture medium provided by the invention is safe and efficient, and the process is simple and convenient and is easy to operate.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a culture medium and a culture method for human umbilical cord mesenchymal stem cell amplification.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells of mesodermal origin with high self-renewal capacity and multipotent differentiation potential. At present, mesenchymal stem cells are mainly extracted from umbilical cords, bone marrow, adipose tissue, dental pulp, placenta. The mesenchymal stem cells still have multidirectional differentiation potential after continuous subculture and cryopreservation, and a new way is provided for clinical treatment of various diseases, such as nervous system diseases, nephropathy, autoimmune diseases, malignant tumors and the like.
The umbilical cord mesenchymal stem cell is a multifunctional stem cell existing in umbilical cord tissues of newborns, has the characteristics of convenient material acquisition, rich sources, stable biological characteristics and low immunogenicity, and becomes an ideal seed cell for the future stem cell in medical application. In addition, umbilical cord mesenchymal stem cells can secrete various stem cell factors in the culture process, including various growth factors such as Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF) and the like, the growth factors can promote cell growth and maintain cell dryness, and the umbilical cord mesenchymal stem cells are widely applied in the field of beauty and skin care, and the collection of conditioned medium is an effective method for obtaining the active ingredients.
At present, most of the formulas of culture media used for culturing the umbilical cord mesenchymal stem cells are DMEM/F12+ serum, or other nutrients required by cell growth are added on the basis of the DMEM/F12+ serum, so that the umbilical cord mesenchymal stem cells can be suitable for large-scale culture of the umbilical cord mesenchymal stem cells. However, the serum contains a large amount of unknown components, and may even contain some unknown allergens and pathogens, which cannot ensure the safety of cultured cells and cause obstacles to the clinical application of the cells. However, the serum-free culture medium used by the conventional umbilical cord mesenchymal stem cells has poor cell culture effect, has the problems of slow cell growth, poor activity, low content of cytokines in the culture medium and the like, and influences the popularization and application of umbilical cord mesenchymal stem cell biological products.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a culture medium for amplifying human umbilical cord mesenchymal stem cells, which is not added with serum, improves the culture efficiency of the umbilical cord mesenchymal stem cells and promotes the secretion of cytokines.
The invention also aims to provide a culture method of the human umbilical cord mesenchymal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a culture medium for amplifying human umbilical cord mesenchymal stem cells comprises a basic culture medium and the following components added in the basic culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin.
Further, the dosage of each component is as follows according to the final concentration: 12-16 mu g/mL of glutathione, 1-3 mu g/mL of alpha-tocopherol, 5.5-8.5ng/mL of cineole, 5-10ng/mL of estradiol valerate, 3.5-5ng/mL of acetazolamide, 5-10 mu g/mL of sodium selenite, 8-11 mu g/mL of vitamin C and 10-15 mu g/mL of insulin.
Further, the dosage of each component is as follows according to the final concentration: 14 mu g/mL of glutathione, 2 mu g/mL of alpha-tocopherol, 7.5ng/mL of cineole, 8ng/mL of estradiol valerate, 4.5ng/mL of acetazolamide, 7 mu g/mL of sodium selenite, 10 mu g/mL of vitamin C and 13 mu g/mL of insulin.
Further, the basic medium is DMEM/F12 medium.
The second purpose of the invention is realized by adopting the following technical scheme:
a culture method of human umbilical cord mesenchymal stem cells adopts the culture medium to carry out cell culture.
Further, the culture method of the human umbilical cord mesenchymal stem cells comprises the following steps:
(1) taking a basal culture medium, adding glutathione, alpha-tocopherol, eucalyptol, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin, and uniformly mixing;
(2) inoculating the human umbilical cord mesenchymal stem cells to be cultured in the culture medium of the step (1), and culturing at 37 ℃ and 5% CO2The amplification culture was carried out under the conditions of (1), and the continuous culture was completed for 7 days.
Further, the density of the human umbilical cord mesenchymal stem cells inoculated in the culture medium is 1-5X 104one/mL.
Further, the culture medium was changed every 2 days during the culture.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a culture medium for human umbilical cord mesenchymal stem cell amplification, which is free from exogenous serum, has definite components and synergistic effect of the components, and promotes the proliferation of umbilical cord mesenchymal stem cells and the secretion of cytokines.
2. The eucalyptol, estradiol valerate, acetazolamide and other components added into the culture medium not only avoid the potential safety hazard existing in the serum added into the culture medium, but also can improve the proliferation efficiency of the umbilical cord mesenchymal stem cells, can obtain a large amount of umbilical cord mesenchymal stem cells in a short time, improve the content of cytokines in the culture solution and provide guarantee for the safe application of the umbilical cord mesenchymal stem cells.
3. The invention also provides a culture method of the human umbilical cord mesenchymal stem cells, and the culture medium provided by the invention is safe and efficient, and the process is simple and convenient and is easy to operate.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Preparing and amplifying the human umbilical cord mesenchymal stem cells:
(1) cleaning fresh healthy umbilical cord with PBS, removing blood vessel with forceps, and cutting separated Wharton's jelly into 1-2mm pieces3The tissue block was spread on the bottom of a T75 flask, and the medium was incubated at 37 ℃ in 5% CO with alpha-MEM containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin2Culturing in an incubator;
(2) and when the umbilical cord mesenchymal stem cells appear at the periphery of the tissue block after the culture for 7 days, continuously culturing, discarding the umbilical cord tissue block and the original culture solution when the umbilical cord mesenchymal stem cells are fused to 80 percent, washing with normal saline, adding 0.25 percent of pancreatin for digestion, and using the digested cells in the following amplification culture.
Example 1
A culture medium for expanding human umbilical cord mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin; the dosage of each component is as follows according to the final concentration: 14 mu g/mL of glutathione, 2 mu g/mL of alpha-tocopherol, 7.5ng/mL of cineole, 8ng/mL of estradiol valerate, 4.5ng/mL of acetazolamide, 7 mu g/mL of sodium selenite, 10 mu g/mL of vitamin C and 13 mu g/mL of insulin.
A culture method of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) taking a DMEM/F12 culture medium, adding glutathione, alpha-tocopherol, eucalyptol, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin, and uniformly mixing;
(2) inoculating the human umbilical cord mesenchymal stem cells to be cultured in the culture medium of the step (1), wherein the density of the inoculated human umbilical cord mesenchymal stem cells in the culture medium is 3 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is used for amplification culture, the culture medium is replaced every 2 days in the culture process, and continuous culture is completed for 7 days.
Example 2
A culture medium for expanding human umbilical cord mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin; the dosage of each component is as follows according to the final concentration: 12 mu g/mL of glutathione, 1 mu g/mL of alpha-tocopherol, 5.5ng/mL of cineole, 5ng/mL of estradiol valerate, 3.5 ng/mL of acetazolamide, 5 mu g/mL of sodium selenite, 8 mu g/mL of vitamin C and 10 mu g/mL of insulin.
A culture method of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) taking a DMEM/F12 culture medium, adding glutathione, alpha-tocopherol, eucalyptol, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin, and uniformly mixing;
(2) inoculating the human umbilical cord mesenchymal stem cells to be cultured in the culture medium of the step (1), wherein the density of the human umbilical cord mesenchymal stem cells inoculated in the culture medium is 1 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is used for amplification culture, the culture medium is replaced every 2 days in the culture process, and continuous culture is completed for 7 days.
Example 3
A culture medium for expanding human umbilical cord mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin; the dosage of each component is as follows according to the final concentration: 16 mug/mL of glutathione, 3 mug/mL of alpha-tocopherol, 8.5ng/mL of cineole, 10ng/mL of estradiol valerate, 5ng/mL of acetazolamide, 10 mug/mL of sodium selenite, 11 mug/mL of vitamin C and 15 mug/mL of insulin.
A culture method of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) taking a DMEM/F12 culture medium, adding glutathione, alpha-tocopherol, eucalyptol, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin, and uniformly mixing;
(2) inoculating the human umbilical cord mesenchymal stem cells to be cultured in the culture medium of the step (1), wherein the density of the human umbilical cord mesenchymal stem cells inoculated in the culture medium is 5 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is used for amplification culture, the culture medium is replaced every 2 days in the culture process, and continuous culture is completed for 7 days.
Comparative example 1
Comparative example 1 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: eucalyptol was omitted and the procedure in example 1 was repeated.
Comparative example 2
Comparative example 2 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: the eucalyptol was replaced with 1, 8-cineole, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: estradiol valerate was omitted and the procedure was the same as in example 1.
Comparative example 4
Comparative example 4 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: the acetazolamide was omitted and the procedure was as in example 1.
Comparative example 5
Comparative example 5 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: eucalyptol and acetazolamide were omitted and replaced with the same amount of fetal bovine serum, and the remainder was the same as in example 1.
Comparative example 6
Comparative example 6 provides a medium for amplifying human umbilical cord mesenchymal stem cells, which is different from comparative example 1 in that: eucalyptol, estradiol valerate and acetazolamide were omitted and replaced with the same amount of fetal calf serum, and the rest was the same as in example 1.
The cell staining was performed using 0.4% trypan blue, and the proliferation times and cell viability of umbilical cord mesenchymal stem cells in examples 1 to 3 and comparative examples 1 to 6 were counted after cell counting, and the results are shown in table 1.
Proliferation fold = total number of cells counted in 7 days of culture/initial number of inoculations;
cell viability = number of viable cells/total number of cells x 100%.
TABLE 1
| Group of | Fold of proliferation | Rate of cell viability |
| Example 1 | 52.47 | 99.35% |
| Example 2 | 50.89 | 98.32% |
| Example 3 | 51.63 | 98.47% |
| Comparative example 1 | 25.76 | 81.56% |
| Comparative example 2 | 28.35 | 84.79% |
| Comparative example 3 | 31.65 | 88.52% |
| Comparative example 4 | 33.07 | 85.49% |
| Comparative example 5 | 35.47 | 90.92% |
| Comparative example 6 | 24.59 | 84.83% |
As can be seen from Table 1, the cell proliferation fold in examples 1 to 3 was higher than that in comparative examples 1 to 6, the cell proliferation activity was better, and the proliferation efficiency and the cell viability rate were better than those in comparative examples 1 to 6.
In comparative examples 1 to 2, eucalyptol is omitted or replaced by 1, 8-cineole, and the proliferation activity of the cells is obviously reduced compared with that of example 1, which shows that the eucalyptol added in the invention is helpful for improving the proliferation activity of umbilical cord mesenchymal stem cells and improving the amplification efficiency of the cells.
The method comprises the following steps of respectively omitting estradiol valerate and acetazolamide in a comparative example 3 and a comparative example 4, reducing proliferation activity and activity rate of umbilical cord mesenchymal stem cells to different degrees, omitting eucalyptol and acetazolamide in a comparative example 5, and replacing the eucalyptol, estradiol valerate and acetazolamide with equivalent fetal bovine serum in a comparative example 6, wherein proliferation multiple and activity rate of the umbilical cord mesenchymal stem cells are not as good as those of example 1.
The culture media of examples 1 to 3 and comparative examples 1 to 6 were added to 12-well plates, respectively, 3 wells were provided per each set of culture media, umbilical cord mesenchymal stem cells were seeded at a cell density of 1 × 104Per mL, 5% CO at 37 ℃2The culture medium of examples 1 to 3 and comparative examples 1 to 6 were cultured for 48 hours, and cytokines were detected in the culture solutions of examples 1 to 3 and comparative examples 1 to 6 by enzyme-linked immunosorbent assay (ELISA): the contents of Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF) are shown in table 2.
TABLE 2
| Sample (I) | EGF（ng/mL） | VEGF（ng/mL） | FGF（ng/mL） |
| Example 1 | 62.98 | 53.54 | 120.26 |
| Example 2 | 60.37 | 54.78 | 118.47 |
| Example 3 | 61.49 | 53.61 | 119.25 |
| Comparative example 1 | 25.36 | 23.96 | 57.44 |
| Comparative example 2 | 28.94 | 29.22 | 62.67 |
| Comparative example 3 | 31.73 | 35.39 | 68.15 |
| Comparative example 4 | 35.64 | 34.36 | 72.39 |
| Comparative example 5 | 41.81 | 39.91 | 80.28 |
| Comparative example 6 | 26.39 | 30.31 | 65.90 |
As can be seen from table 2, the cytokine content in examples 1 to 3 is higher than that in comparative examples 1 to 6 because the composition of the culture medium is adjusted in comparative examples 1 to 6, eucalyptol is omitted or eucalyptol is replaced by 1, 8-cineole in comparative examples 1 to 2, estradiol valerate and acetazolamide are omitted in comparative examples 3 and 4, eucalyptol and acetazolamide are omitted in comparative example 5 and are replaced by equal amounts of fetal calf serum, eucalyptol, estradiol valerate and acetazolamide are omitted in comparative example 6 and are replaced by equal amounts of fetal calf serum, and the content of cytokines in the culture solution is reduced to different degrees, which shows that the synergistic effect of eucalyptol, estradiol valerate and acetazolamide in the components of the culture medium of the present invention effectively promotes the secretion of cytokines by mesenchymal stem cells between umbilical cords.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (6)
1. A culture medium for amplifying human umbilical cord mesenchymal stem cells is characterized by comprising a basic culture medium and the following components added in the basic culture medium: glutathione, alpha-tocopherol, cineole, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin; the basic culture medium is DMEM/F12 culture medium;
the dosage of each component is as follows according to the final concentration: 12-16 mug/mL of glutathione, 1-3 mug/mL of alpha-tocopherol, 5.5-8.5ng/mL of cineole, 5-10ng/mL of estradiol valerate, 3.5-5ng/mL of acetazolamide, 5-10 mug/mL of sodium selenite, 8-11 mug/mL of vitamin C and 10-15 mug/mL of insulin.
2. The medium for amplifying the human umbilical cord mesenchymal stem cells according to claim 1, wherein the medium comprises the following components in terms of final concentration: 14 mu g/mL of glutathione, 2 mu g/mL of alpha-tocopherol, 7.5ng/mL of cineole, 8ng/mL of estradiol valerate, 4.5ng/mL of acetazolamide, 7 mu g/mL of sodium selenite, 10 mu g/mL of vitamin C and 13 mu g/mL of insulin.
3. A method for culturing human umbilical cord mesenchymal stem cells, which comprises culturing the cells in the medium of any one of claims 1 to 2.
4. The method for culturing human umbilical cord mesenchymal stem cells according to claim 3, comprising the steps of:
(1) taking a basal culture medium, adding glutathione, alpha-tocopherol, eucalyptol, estradiol valerate, acetazolamide, sodium selenite, vitamin C and insulin, and uniformly mixing;
(2) inoculating the human umbilical cord mesenchymal stem cells to be cultured in the culture medium of the step (1), and culturing at 37 ℃ and 5% CO2The amplification culture was carried out under the conditions of (1), and the continuous culture was completed for 7 days.
5. The method for culturing human umbilical cord mesenchymal stem cells according to claim 4, wherein the density of the human umbilical cord mesenchymal stem cells seeded in the culture medium is 1-5 x 104one/mL.
6. The method for culturing human umbilical cord mesenchymal stem cells according to claim 4, wherein the culture medium is replaced every 2 days during the culture.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567635A (en) * | 2016-02-21 | 2016-05-11 | 中国医科大学附属第一医院 | Improved Neurobasal B27 culture medium, preparing method and application |
| CN110317779A (en) * | 2019-06-06 | 2019-10-11 | 深圳市艾一生命科技有限公司 | The umbilical cord mesenchymal stem cells culture medium of serum-free |
| CN112592892A (en) * | 2020-12-25 | 2021-04-02 | 夏爽 | Culture medium and culture method for inducing secretion of umbilical cord mesenchymal stem cell factors |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567635A (en) * | 2016-02-21 | 2016-05-11 | 中国医科大学附属第一医院 | Improved Neurobasal B27 culture medium, preparing method and application |
| CN110317779A (en) * | 2019-06-06 | 2019-10-11 | 深圳市艾一生命科技有限公司 | The umbilical cord mesenchymal stem cells culture medium of serum-free |
| CN112592892A (en) * | 2020-12-25 | 2021-04-02 | 夏爽 | Culture medium and culture method for inducing secretion of umbilical cord mesenchymal stem cell factors |
Non-Patent Citations (2)
| Title |
|---|
| Eudesmin exerts antitumor effects by down-regulating EZH2 expression in nasopharyngeal carcinoma cells;Min Yu等;《Chem Biol Interact》;20190701;第307卷;第51-57页 * |
| 脐血间充质干细胞的无血清培养;李剑等;《中华血液学杂志》;20060630;第27卷(第6期);第409-410页 * |
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