CN111235101B - Culture medium and culture method for human umbilical cord mesenchymal stem cells - Google Patents

Culture medium and culture method for human umbilical cord mesenchymal stem cells Download PDF

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CN111235101B
CN111235101B CN202010221952.1A CN202010221952A CN111235101B CN 111235101 B CN111235101 B CN 111235101B CN 202010221952 A CN202010221952 A CN 202010221952A CN 111235101 B CN111235101 B CN 111235101B
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于继莲
王长继
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Hainan Nanyi Anqi Biomedical Technology Co.,Ltd.
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Abstract

The invention discloses a culture medium for human umbilical cord mesenchymal stem cells, which comprises a basic culture medium and a composite additive added in the basic culture medium, wherein the composite additive comprises the following componentsThe final concentration meter comprises: 6.5-9.3 mug/L of silk fibroin, 5.4-7.2 mug/L of sodium carboxymethylcellulose, 10.0-14.0 mug/L of soybean oligopeptide, 10.5-14.8 mug/L of 3-picolinic acid and coenzyme Q108.0-12.0 mug/L, 15.0-20.0 mug/L inositol, 6.8-7.2ng/L sucrose fatty acid ester, 8.2-9.5 mug/L alpha-ketoglutaric acid and 22.0-26.0 mug/L microelement. The invention adopts the matching of sodium carboxymethylcellulose and silk fibroin to improve the adhesiveness of cells, is beneficial to cell fixation and promotes the growth of cells, and is also added with soybean oligopeptide, 3-picolinic acid and coenzyme Q10The alpha-ketoglutaric acid is used in a compounding way, so that the growth speed and the proliferation quantity of cells are improved, and the performance of secreting growth factors by the cells is improved. The trace elements of magnesium, iron, zinc and selenium are added, so that a better nutritional environment is provided for the growth of cells, and the proliferation of the cells is further promoted.

Description

Culture medium and culture method for human umbilical cord mesenchymal stem cells
Technical Field
The invention relates to the technical field of stem cells, in particular to a culture medium and a culture method for human umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent Stem Cells that have all the commonalities of Stem Cells, i.e., self-replicating ability and multipotent differentiation ability, and are widely available, for example, from bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue, adipose tissue, and the like. Mesenchymal stem cells belong to multipotent stem cells, and can be differentiated into various tissue cells such as pancreatic islets, nerves, vascular endothelium, bones, cartilages, muscles, livers, cardiac muscles and the like under specific induction conditions in vivo or in vitro. MSCs are relatively lack of immunogenicity, simple in culture technology and capable of being stored in a freezing mode, so that MSCs become seed cells for cell and gene therapy research, have potential clinical application values and become hot spots for stem cell research.
Human Umbilical Cord mesenchymal stem cells (HUC-MSCs) express the unique marks of various stem cells, and have the characteristics of large differentiation potential, strong proliferation capacity, low immunogenicity, convenient material taking, no ethical problem limitation, easy industrial preparation and the like. The culture supernatant of the human umbilical cord mesenchymal stem cells contains a large amount of nutrients such as cell growth factors, and the nutrients can effectively promote the metabolism of skin cells; promote the synthesis and secretion of macromolecules such as extracellular hyaluronic acid, glycoprotein and the like, and enhance the hydrophilicity of skin; promoting the growth, differentiation and repair of epidermal cells, and timely supplementing aged and dead cells; meanwhile, the repair of the cuticle can be accelerated, the replacement of new and old cells of the basement can be accelerated, and the skin can return to a young posture.
At present, the culture effect of a culture medium used in the conventional in vitro culture of HUC-MSCs is not ideal, and the technical defects of low cell proliferation speed, low expression, limited number of obtained and proliferated cells, less number and quality of secreted growth factors and the like exist, so that the search for a culture medium capable of promoting the growth of human umbilical cord mesenchymal stem cells is a technical problem to be solved urgently by technical personnel in the field.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a human umbilical cord mesenchymal stem cell culture medium which can promote the rapid and stable proliferation of cells and improve the activity of the cells.
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 human umbilical cord mesenchymal stem cells, which comprises a basic culture medium, and a composite additive added in the basic culture medium, wherein the composite additive comprises the following components in final concentration: 6.5-9.3 mug/L of silk fibroin, 5.4-7.2 mug/L of sodium carboxymethylcellulose, 10.0-14.0 mug/L of soybean oligopeptide, 10.5-14.8 mug/L of 3-picolinic acid and coenzyme Q108.0-12.0 mug/L, 15.0-20.0 mug/L inositol, 6.8-7.2ng/L sucrose fatty acid ester, 8.2-9.5 mug/L alpha-ketoglutaric acid and 22.0-26.0 mug/L microelement.
Further, the ingredients of the composite additive comprise, in final concentrations: 8.5 mu g/L of silk fibroin, 6.0 mu g/L of sodium carboxymethylcellulose, 12.0 mu g/L of soybean oligopeptide, 13.5 mu g/L of 3-picolinic acid, 1010.0 mu g/L of coenzyme Q, 18.0 mu g/L of inositol, 6.5ng/L of sucrose fatty acid ester, 8.7 mu g/L of alpha-ketoglutaric acid and 24.0 mu g/L of trace elements.
Further, the molecular weight of the silk fibroin is 1474-1531Da, and the average molecular weight of the soybean oligopeptide is less than 500 Da.
Further, the basic culture medium is LG-DMEM culture medium, and 10% fetal bovine serum is added into the basic culture medium.
Further, the trace elements are a mixture of magnesium, iron, zinc and selenium.
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 of the human umbilical cord mesenchymal stem cells for culture.
Further, the culture method of the human umbilical cord mesenchymal stem cells comprises the following steps: (1) adding 10% of fetal calf serum and a compound additive into a basic culture medium;
(2) inoculating primary human umbilical cord mesenchymal stem cells into the culture medium in the step (1), placing at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator, and changing the liquid every 3-4d until the cells are fully fused and passaged.
Further, the inoculation density in the step (2) is 5-6 × 104one/mL.
Compared with the prior art, the invention has the beneficial effects that:
1. the human umbilical cord mesenchymal stem cell culture medium adopts the matching of sodium carboxymethylcellulose and silk fibroin, wherein the sodium carboxymethylcellulose can promote the growth and the adhesion of cells, the silk fibroin contains various amino acids, and the silk fibroin with proper molecular weight has a promotion effect on the proliferation and the growth of the cells.
2. The culture medium of the human umbilical cord mesenchymal stem cells is also added with soybean oligopeptide, 3-picolinic acid and coenzyme Q10Alpha-ketoglutaric acid, wherein the soybean oligopeptide and coenzyme Q are used in a compounding way10Has good antioxidant effect, and can promote cell metabolism, improve intracellular enzyme activity, regulate protein metabolism, promote protein synthesis, and increase cell growth rate and proliferation numberAnd (4) increasing the secretion of growth factors by the cells.
3. The trace elements of magnesium, iron, zinc and selenium are added, so that a better nutritional environment is provided for the growth of cells, and the growth of the cells is further promoted. Increasing the activity of the cell.
Drawings
FIG. 1 is a graph showing growth curves of cells in example 2 of the present invention and comparative examples 1 to 6.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
Preparing primary human umbilical cord mesenchymal stem cells: collecting umbilical cord specimen of fetus born in due date in term, washing with D-Hank's solution, and shearing into pieces of about 1mm3The small blocks are digested by 0.1 percent collagenase II and 0.25 percent pancreatin for 30min respectively, and suspension cells are collected, namely the primary human umbilical cord mesenchymal stem cells.
Example 2
A culture method of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) preparing a culture medium: adding 10% fetal calf serum and a compound additive into a basic culture medium LG-DMEM, wherein the compound additive comprises the following components in final concentration: silk fibroin 8.5 μ g/L with molecular weight of 1531Da, sodium carboxymethylcellulose 6.0 μ g/L, soybean oligopeptide 12.0 μ g/L, 3-picolinic acid 13.5 μ g/L, and coenzyme Q1010.0 mu g/L, inositol 18.0 mu g/L, sucrose fatty acid ester 6.5ng/L, alpha-ketoglutaric acid 8.7 mu g/L, and trace elements 24.0 mu g/L, wherein the trace elements are a mixture of magnesium, iron, zinc and selenium;
(2) taking primary human umbilical cord mesenchymal stem cells according to the ratio of 5 multiplied by 104The cells were seeded at a density of 15cm in a Petri dish and placed at 37 ℃ in 5% CO2Culturing in a saturated humidity incubator, changing the total amount of liquid every 3d, digesting with pancreatin when the primary human umbilical cord mesenchymal stem cells grow to 80% fusion, subculturing, and transferring to P7And (4) generation.
The cells of P7 generation are collected and detected by a flow cytometer, and the results are shown in Table 1, and the human umbilical cord mesenchymal stem cells obtained by the culture of the invention highly express CD105, CD44, CD13 and CD 29. The expression levels of CD106, CD166 and HLA-DR were less than 0.05%, indicating that the cultured cells have the basic characteristics of mesenchymal stem cells.
TABLE 1
Labeled antibodies Positive rate (%)
CD105 99.99
CD44 99.85
CD13 99.94
CD29 99.96
CD106 0.02
CD166 0.01
HLA-DR 0.01
Example 3
A culture method of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) preparing a culture medium: adding 10% fetal calf serum and a compound additive into a basic culture medium LG-DMEM, wherein the compound additive comprises the following components in final concentration: 6.5 mug/L silk fibroin with molecular weight of 1474Da, 5.4 mug/L sodium carboxymethylcellulose, 10.0 mug/L soybean oligopeptide, 10.5 mug/L3-picolinic acid and coenzyme Q108.0 mu g/L, 15.0 mu g/L inositol, 6.8ng/L sucrose fatty acid ester, 8.2 mu g/L alpha-ketoglutaric acid and 22.0 mu g/L trace elements, wherein the trace elements are a mixture of magnesium, iron, zinc and selenium;
(2) taking primary human umbilical cord mesenchymal stem cells according to the ratio of 5.5 multiplied by 104The cells were seeded at a density of 15cm in a Petri dish and placed at 37 ℃ in 5% CO2Culturing in a saturated humidity incubator, carrying out full liquid change every 4d, digesting by using pancreatin and then carrying out subculture when the primary human umbilical cord mesenchymal stem cells grow to 80% fusion, and transferring to P7 generation.
The cells of P7 generation are collected and detected by a flow cytometer, and the results are shown in Table 2, and the human umbilical cord mesenchymal stem cells obtained by the culture of the invention highly express CD105, CD44, CD13 and CD 29. The expression levels of CD106, CD166 and HLA-DR were less than 0.05%, indicating that the cultured cells have the basic characteristics of mesenchymal stem cells.
TABLE 2
Labeled antibodies Positive rate (%)
CD105 99.98
CD44 99.92
CD13 99.95
CD29 99.93
CD106 0.01
CD166 0.02
HLA-DR 0.01
Example 4
A culture method of a culture medium of human umbilical cord mesenchymal stem cells comprises the following steps:
(1) preparing a culture medium: adding 10% fetal calf serum and a compound additive into a basic culture medium LG-DMEM, wherein the compound additive comprises the following components in final concentration: 9.3 mug/L of silk fibroin with the molecular weight of 1531Da, 7.2 mug/L of sodium carboxymethyl cellulose, 14.0 mug/L of soybean oligopeptide, 14.8 mug/L of 3-picolinic acid and coenzyme Q1012.0 mu g/L, 20.0 mu g/L inositol, 7.2ng/L sucrose fatty acid ester, 9.5 mu g/L alpha-ketoglutaric acid and 26.0 mu g/L trace elements, wherein the trace elements are a mixture of magnesium, iron, zinc and selenium;
(2) taking primary human umbilical cord mesenchymal stem cells according to the ratio of 6 multiplied by 104The cells were seeded at a density of 15cm in a Petri dish and placed at 37 ℃ in 5% CO2Culturing in a saturated humidity incubator, carrying out full liquid change every 4d, digesting by using pancreatin and then carrying out subculture when the primary human umbilical cord mesenchymal stem cells grow to 80% fusion, and transferring to P7 generation.
The cells of P7 generation are collected and detected by a flow cytometer, and the results are shown in Table 3, and the human umbilical cord mesenchymal stem cells obtained by the culture of the invention highly express CD105, CD44, CD13 and CD 29. The expression levels of CD106, CD166 and HLA-DR were less than 0.05%, indicating that the cultured cells have the basic characteristics of mesenchymal stem cells.
TABLE 3
Labeled antibodies Positive rate (%)
CD105 99.96
CD44 99.93
CD13 99.94
CD29 99.93
CD106 0.03
CD166 0.02
HLA-DR 0.01
Comparative example 1
Comparative example 1 provides a culture method of a culture medium of human umbilical cord mesenchymal stem cells, which is different from example 2 in the composition of a composite additive, and in comparative example 1, sodium carboxymethylcellulose is replaced with polyvinyl alcohol, and the rest is the same as in example 1.
Comparative example 2
Comparative example 2 provides a culture method of a culture medium of human umbilical cord mesenchymal stem cells, which is different from example 2 in the composition of the complex additive, silk fibroin is replaced with fibronectin in comparative example 2, and the rest is the same as in example 1.
Comparative example 3
Comparative example 3 provides a culture method of a culture medium of human umbilical cord mesenchymal stem cells, which is different from example 2 in the composition of the complex additive, in which 3-picolinic acid is replaced with vitamin E in comparative example 3, and the rest is the same as in example 1.
Comparative example 4
Comparative example 4 provides a culture method of a culture medium for human umbilical cord mesenchymal stem cells, which is different from example 2 in the composition of the complex additive, and in comparative example 4, alpha-ketoglutaric acid is replaced with linoleic acid, and the rest is the same as in example 1.
Comparative example 5
Comparative example 5 provides a culture method of human umbilical cord mesenchymal stem cell culture medium, which is different from that of example 2 in the composition of the complex additive, and the molecular weight of silk fibroin used in comparative example 5 is 1398Da, and the rest is the same as that of example 1.
Comparative example 6
Comparative example 6 provides a culture method of human umbilical cord mesenchymal stem cell culture medium, which is different from that of example 2 in the composition of the complex additive, and the molecular weight of silk fibroin used in comparative example 6 is 1588Da, and the rest is the same as that in example 1.
The culture solution in the culture flask when the human umbilical cord mesenchymal stem cells were cultured to P7 generation in example 1 and comparative examples 1 to 6, respectively, was collected, and the contents of Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), and transforming growth factor beta (TGF- β) in the cell broth were measured after filtration, and the results are shown in table 4.
TABLE 4
Figure BDA0002426390800000081
Figure BDA0002426390800000091
As can be seen from Table 4, the cell growth factor content in the culture broth of P7 generation cell obtained in example 2 of the present invention was significantly superior to that of comparative examples 1 to 6. In comparative example 1 and comparative example 2, sodium carboxymethylcellulose was replaced with polyvinyl alcohol, silk fibroin was replaced with fibronectin, 3-picolinic acid was replaced with vitamin E, and α -ketoglutaric acid was replaced with linoleic acid in comparative examples 3 and 4, respectively, and the content of cell growth factor in the obtained cell culture solution was reduced, indicating that soybean oligopeptide, 3-picolinic acid, coenzyme Q10And alpha-ketoglutaric acid are compounded, and the components have synergistic effect, so that the growth of cells is promoted, and the performance of secreting growth factors by the cells is improved. In comparative examples 5 to 6, the molecular weight of silk fibroin was adjusted, and the content of cell growth factors in the obtained cell culture solution was lower than that in example 1, which indicates that the secretion of cell growth factors can be better promoted only by using sodium carboxymethylcellulose in combination with silk fibroin having an appropriate molecular weight.
The cells subcultured in example 2 and comparative examples 1 to 6 were each digested with 0.25% trypsin and then cultured at 2X 104One well, seeded in 24-well plates, 3 wells digested every 24 hours, cells collected and viable cells counted with 0.4% trypan blue, growth curves were plotted, and the results are shown in fig. 1.
As can be seen from FIG. 1, the cells of example 2 proliferated rapidly and in large amounts. The cell proliferation speed of comparative examples 1 to 6 is lower than that of example 2, which shows that the invention adopts the combination of adding sodium carboxymethyl cellulose and silk fibroin in the culture medium to promote the cell growth, soybean oligopeptide, 3-picolinic acid and coenzyme Q10The alpha-ketoglutaric acid is compounded to regulate the metabolism of protein, promote the synthesis of the protein and improve the growth speed and the proliferation quantity of cells.
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 (7)

1. A culture medium for human umbilical cord mesenchymal stem cells, which is characterized by comprising a basic culture medium and a composite additive added in the basic culture medium, wherein the composite additive comprises the following components in final concentration: 6.5-9.3 mug/L of silk fibroin, 5.4-7.2 mug/L of sodium carboxymethylcellulose, 10.0-14.0 mug/L of soybean oligopeptide, 10.5-14.8 mug/L of 3-picolinic acid and coenzyme Q108.0-12.0 mug/L, 15.0-20.0 mug/L inositol, 6.8-7.2ng/L sucrose fatty acid ester, 8.2-9.5 mug/L alpha-ketoglutaric acid and 22.0-26.0 mug/L microelement;
the molecular weight of the silk fibroin is 1474-1531Da, and the average molecular weight of the soybean oligopeptide is less than 500 Da.
2. The human umbilical cord mesenchymal stem cell culture medium of claim 1, wherein the composition of the composite additive comprises, in final concentration: 8.5 mu g/L of silk fibroin, 6.0 mu g/L of sodium carboxymethylcellulose, 12.0 mu g/L of soybean oligopeptide, 13.5 mu g/L of 3-picolinic acid and coenzyme Q1010.0 mu g/L, inositol 18.0 mu g/L, sucrose fatty acid ester 6.5ng/L, alpha-ketoglutaric acid 8.7 mu g/L, and trace elements 24.0 mu g/L.
3. The human umbilical cord mesenchymal stem cell culture medium of claim 1, wherein the basal medium is LG-DMEM medium, and 10% fetal bovine serum is added to the basal medium.
4. The human umbilical cord mesenchymal stem cell culture medium of claim 1, wherein the trace element is a mixture of magnesium, iron, zinc and selenium.
5. A culture method of human umbilical cord mesenchymal stem cells, which is characterized in that the culture medium of the human umbilical cord mesenchymal stem cells of claim 3 is adopted for culture.
6. The method for culturing human umbilical cord mesenchymal stem cells according to claim 5, comprising the steps of:
(1) adding 10% of fetal calf serum and a compound additive into a basic culture medium;
(2) inoculating primary human umbilical cord mesenchymal stem cells into the culture medium in the step (1), placing at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator, and changing the liquid every 3-4d until the cells are fully fused and passaged.
7. The method for culturing human umbilical cord mesenchymal stem cells according to claim 6, wherein the seeding density in the step (2) is 5 to 6 x 104one/mL.
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