CN109777771B - Serum-free culture medium for primary umbilical cord mesenchymal stem cells and use method thereof - Google Patents
Serum-free culture medium for primary umbilical cord mesenchymal stem cells and use method thereof Download PDFInfo
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Abstract
The invention relates to a serum-free culture medium of primary umbilical cord mesenchymal stem cells and a use method thereof, wherein the serum-free culture medium of the primary umbilical cord mesenchymal stem cells comprises the following components: alpha-MEM culture, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF. Fetal calf serum is not added into the culture medium, so that immunological rejection possibly generated in clinical application is avoided, and the risk of pathogenic microorganism pollution is reduced; by adding cytokines such as IL-3, IL-6, G-CSF, SCF and the like, the strong proliferation effect can be generated by the cooperation with FL, and the differentiation of umbilical cord mesenchymal stem cells can be inhibited; the EGF is added to cooperate with the FGF-2 to promote the proliferation and differentiation of the umbilical cord mesenchymal stem cells; by adding BMP-4 as a nutritional factor for the proliferation and differentiation of stem cells, the normal proliferation state of umbilical cord mesenchymal stem cells can be maintained and regulated.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a serum-free culture medium of primary umbilical cord mesenchymal stem cells and a using method thereof.
Background
Umbilical cord Mesenchymal Stem Cells (MSCs) have a high differentiation potential and can be differentiated in multiple directions. It has wide clinical application prospect in the aspects of tissue engineering such as bones, cartilages, muscles, tendons, ligaments, nerves, livers, endothelia, cardiac muscles and the like. The umbilical cord mesenchymal stem cells can be separated from the human umbilical cord, and have the advantages of convenient material acquisition and no ethical dispute. The umbilical cord mesenchymal stem cells have stronger immunoregulation function, can promote hematopoietic recovery function and repair pathological tissue organs, and have high clinical application value in the aspects of organ transplantation, autoimmune diseases, leukemia, degenerative diseases of bones and muscles and the like.
How to realize rapid amplification of the primary umbilical cord mesenchymal stem cell culture without influencing the cell viability is the key of umbilical cord mesenchymal stem cell culture, however, the umbilical cord mesenchymal stem cell culture technology in the prior art is not mature enough, which shows that the proliferation speed of the primary cells separated from the tissues is slow and the cell viability is low.
The existing umbilical cord mesenchymal stem cell culture medium needs to be added with animal serum such as fetal bovine serum, the stem cells growing in the culture medium have the possibility of unknown transformation or variation of the internal structure of the cells, the animal serum also has the risk of causing immunological rejection reaction and the risk of being polluted by pathogenic microorganisms such as bacteria, viruses and the like, and the umbilical cord mesenchymal stem cell culture medium is not beneficial to clinical application and basic research of the stem cells.
The proliferation, differentiation, development and maturation process of umbilical cord mesenchymal stem cells is quite complex and depends on the regulation of various hematopoietic factors, wherein the role of cell stimulating factors is very critical.
Disclosure of Invention
Based on the serum-free culture medium, the invention provides the serum-free culture medium of the primary umbilical cord mesenchymal stem cells and the use method thereof, which realize rapid amplification without influencing the cell viability and avoid rejection and pathogenic microorganism pollution risks caused by animal serum.
A serum-free culture medium of primary umbilical cord mesenchymal stem cells comprises the following components: alpha-MEM culture, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF.
In one embodiment, the serum-free medium of the primary umbilical cord mesenchymal stem cells further comprises one or more of dexamethasone, transferrin, Panax notoginsenosides and a combination of cytokines.
In one embodiment, the concentration of dexamethasone is 0.1-0.5 μ g/ml; the concentration of the transferrin is 0.01-0.1 mug/ml; the concentration of Panax notoginsenosides is 5-15 μ g/ml. Dexamethasone in combination with AMD3100 had a synergistic effect in promoting mobilization, creeping out and proliferation of umbilical cord mesenchymal stem cells from the tissue. The Chinese medicinal component of the panax notoginseng saponins can accelerate the proliferation of umbilical cord mesenchymal stem cells.
In one embodiment, the cytokine combination comprises IL-3, SCF, G-CSF, FL, and EGF; the concentrations of IL-3, SCF, G-CSF, FL and EGF are: IL-310-20 ng/ml; SCF 50-150 ng/ml; G-CSF 1-100 ng/ml; FL 1-50 ng/ml; EGF 5-15 ng/ml. FL is a key cytokine capable of regulating early stem cells, and is combined with cytokines such as IL-3, IL-6, G-CSF, SCF and the like to generate strong proliferation effect on umbilical cord mesenchymal stem cells.
In one embodiment, the α -MEM medium is a serum-free medium. Can provide basic nutrients for the growth and proliferation of stem cells.
In one embodiment, the concentration of AMD3100 is 0.5-5 μ g/ml. Promoting the growth and proliferation of umbilical cord mesenchymal stem cells.
In one embodiment, BMP-4 is present at a concentration of 0.01 to 0.1 ng/ml; the concentration of the insulin is 10-20 mug/ml; the concentration of FGF-2 is 1-12 ng/ml. FGF-2 and BMP-4 promote proliferation of umbilical cord mesenchymal stem cells. Insulin better promotes the absorption and utilization of glucose by cells.
In one embodiment, the concentration of IL-6 is 0.05-0.15. mu.g/ml; the concentration of LIF is 0.01-0.1 μ g/ml. Leukemia inhibitory factor LIF can inhibit stem cell differentiation and promote stem cell proliferation.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods, for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6 and LIF into the alpha-MEM culture solution according to the concentration to prepare a culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 10 days later and were discontinued.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, SCF, EGF, G-CSF were added before culture until they were discontinued after 10 days, and FL was added at day 5 until they were discontinued after 15 days of culture.
Use of a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of the preceding methods for culturing umbilical cord mesenchymal stem cells, comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity;
c. IL-3, EGF and G-CSF were added before culture until they were discontinued after 10 days, and SCF and FL were added at day 5 until they were discontinued after 15 days.
Supplementary notes
IL-3 is Interleukin3 Interleukin 3;
SCF is stem cell factor;
G-CSF is a grandilocyte colony-stimulating factor;
FL is (flt 3-ligand) type III tyrosine kinase receptor ligand;
EGF is Epidermal growth factor;
BMP-4 is Bone Morphogenetic Protein 4 from Bone morphine;
FGF-2 is Fibroblast Growth Factor 2 of Fibrolast Growth Factor 2;
IL-6 is Interleukin6 from Interleukin 6;
LIF is a leukoinhibitory factor of the Leukemia inhibitor factor;
alpha-MEM is a modified Dulbecco's minimal essential medium;
FBS is fetal bovine serum;
the Wharton's jelly is a gelatinous filler between the amniotic membrane of the umbilical cord and blood vessels and contains more umbilical cord mesenchymal stem cells;
cell viability (%) = viable cell density/(viable cell density + dead cell density) × 100%.
The invention has the beneficial effects that:
1. according to the invention, a serum-free culture medium is developed, namely fetal calf serum is not added in the culture medium, so that possible immunological rejection reaction in clinical application is avoided, and the risk of pathogenic microorganism pollution is reduced;
2. according to the invention, by adding cytokines such as IL-3, IL-6, G-CSF and SCF, strong proliferation effect can be generated by cooperation with FL, and differentiation of umbilical cord mesenchymal stem cells can be inhibited; the EGF is added to cooperate with the FGF-2 to promote the proliferation and differentiation of the umbilical cord mesenchymal stem cells; by adding BMP-4 as a nutritional factor for proliferation and differentiation of stem cells, the normal proliferation state of umbilical cord mesenchymal stem cells can be maintained and regulated; the Chinese medicinal component of panax notoginseng saponins is added to proliferate umbilical cord mesenchymal stem cells; dexamethasone is added to cooperate with the AMD3100 to have auxiliary effect on proliferation and differentiation of the umbilical cord mesenchymal stem cells; by changing the addition sequence of the cell factors, the inhibition effect of FL on the early proliferation of the umbilical cord mesenchymal stem cells can be weakened, and the differentiation of the umbilical cord mesenchymal stem cells in the middle and later stages is inhibited, so that the in-vitro proliferation and differentiation activity of the umbilical cord mesenchymal stem cells is improved, the proliferation period is shortened, and the cell survival rate is improved.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The invention provides a serum-free culture medium of primary umbilical cord mesenchymal stem cells, which comprises the following components: alpha-MEM culture, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF.
In a preferred embodiment, the serum-free medium of the primary umbilical cord mesenchymal stem cells further comprises one or more of dexamethasone, transferrin, Panax notoginsenosides and a combination of cytokines. Different combinations of AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and panax notoginseng saponins are added into an alpha-MEM culture solution, meanwhile, a serum-free culture medium of primary umbilical cord mesenchymal stem cells is realized by adjusting the adding and stopping time of cytokines such as IL-3, SCF, EGF, G-CSF, FL and the like, fetal bovine serum is not added into the culture medium, so that the possible immune rejection reaction in clinical application is avoided, and the risk of pathogenic microorganism pollution is reduced.
Preferred 6 sets of embodiments are as follows:
example 1:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: alpha-MEM culture, AMD3100, BMP-4, insulin, FGF-2, IL-6, and LIF, the concentrations of each component being shown in Table 1.
TABLE 1
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
BMP-4 0.05ng/ml SCF 100ng/ml
Insulin 10 mug/ml EGF 7ng/ml
LIF 0.05μg/ml G-CSF 25ng/ml
FL 30ng/ml
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the final concentrations were determined in accordance with the table.
The serum-free culture medium of the primary umbilical cord mesenchymal stem cells is used for culturing the umbilical cord mesenchymal stem cells, and the culture method comprises the following steps:
a. preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6 and LIF into the alpha-MEM culture solution to prepare an umbilical cord mesenchymal stem cell culture solution;
b. culturing in T75 culture flask with 8ml culture solution, inoculating 2ml alpha-MEM suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2 full saturation humidity; changing the culture medium on 5 days, 10 days, 13 days and 15 days after the culture;
c. adding a cytokine: IL-3, SCF, EGF, G-CSF, FL were added to fresh medium at the concentrations of Table 1 until 15 days had elapsed.
Example 2:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, Panax notoginsenosides, the concentrations of each component are shown in Table 2.
TABLE 2
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
Transferrin 0.02 μ g/ml SCF 100ng/ml
BMP-4 0.05ng/ml EGF 7ng/ml
Insulin 10. mu.g/ml G-CSF 25ng/ml
IL-6 0.1μg/ml FL 30ng/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
Notoginseng radix total saponin 8 μ g/ml
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the final concentrations were determined in accordance with the table.
a. Preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginsenosides into the alpha-MEM culture solution according to the concentration to prepare an umbilical cord mesenchymal stem cell culture solution;
b. IL-3, SCF, EGF, G-CSF, FL were added to fresh medium at the concentrations shown in Table 2 until 15 days had elapsed.
Example 3:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides, and the concentrations of the components are shown in Table 3.
TABLE 3
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
Dexamethasone 0.2. mu.g/ml SCF 100ng/ml
Transferrin 0.02 mu g/ml EGF 7ng/ml
BMP-4 0.05ng/ml G-CSF 25ng/ml
Insulin 10. mu.g/ml FL 30ng/ml
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
Notoginseng radix total saponin 15 μ g/ml
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the final concentrations were determined in accordance with the table.
a. Preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginsenosides into the alpha-MEM culture solution according to the concentration to prepare an umbilical cord mesenchymal stem cell culture solution;
b. IL-3, SCF, EGF, G-CSF, FL were added to fresh medium at the concentrations of Table 3 until 15 days had elapsed.
Example 4:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: alpha-MEM culture solution, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and panax notoginseng saponins, wherein the concentration of each component is shown in Table 4.
TABLE 4
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
Dexamethasone 0.2. mu.g/ml SCF 100ng/ml
Transferrin 0.02 mu g/ml EGF 7ng/ml
BMP-4 0.05ng/ml G-CSF 25ng/ml
Insulin 10. mu.g/ml FL 30ng/ml
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
Notoginseng radix total saponin 5 μ g/ml
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the concentrations were determined in accordance with the table.
a. Preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginsenosides into the alpha-MEM culture solution according to the concentration to prepare an umbilical cord mesenchymal stem cell culture solution;
b. IL-3, SCF, EGF, G-CSF, FL were added to fresh medium at the concentrations shown in Table 4 before culture until 10 days later and were stopped.
Example 5:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: alpha-MEM culture solution, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and panax notoginseng saponins, wherein the concentration of each component is shown in Table 5.
TABLE 5
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
Dexamethasone 0.2. mu.g/ml SCF 100ng/ml
Transferrin 0.02 mu g/ml EGF 7ng/ml
BMP-4 0.05ng/ml G-CSF 25ng/ml
Insulin 10. mu.g/ml FL 30ng/ml
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
7 microgram/ml of panax notoginseng saponins
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the final concentrations were determined in accordance with the table.
a. Preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginsenosides into the alpha-MEM culture solution according to the concentration to prepare an umbilical cord mesenchymal stem cell culture solution;
b. IL-3, SCF, EGF, G-CSF were added to fresh medium at the concentrations of Table 5 before culture until 10 days later and discontinued after culture on day 5 with FL added until 15 days later.
Example 6:
the serum-free culture medium of the primary umbilical cord mesenchymal stem cells of the present example comprises the following components: alpha-MEM culture solution, AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and panax notoginseng saponins, wherein the concentration of each component is shown in Table 6.
TABLE 6
Concentration of Medium Components cytokine concentration
AMD3100 1μg/ml IL-3 10ng/ml
Dexamethasone 0.2. mu.g/ml SCF 100ng/ml
Transferrin 0.02 mu g/ml EGF 7ng/ml
BMP-4 0.05ng/ml G-CSF 25ng/ml
Insulin 10. mu.g/ml FL 30ng/ml
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
Panax notoginsenosides 12 μ g/ml
Remarking: the culture medium was prepared from α -MEM as a base medium according to the requirements of the various components and the types of cytokines added at the respective stages of the examples, and the final concentrations were determined in accordance with the table.
a. Preparing an umbilical cord mesenchymal stem cell culture solution: adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF and Panax notoginsenosides into the alpha-MEM culture solution according to the concentration to prepare an umbilical cord mesenchymal stem cell culture solution;
b. before the culture, IL-3, EGF and G-CSF were added to fresh medium at the concentrations shown in Table 6 until they were stopped after 10 days, and SCF and FL were added at 5 days until they were stopped after 15 days.
Preparing an umbilical cord mesenchymal stem cell culture solution: to the α -MEM culture medium was added 15% FBS.
The culture solutions of umbilical cord mesenchymal stem cells of examples 1 to 6 and the control group were tested, counted and tested for cell survival.
Cell counting and cell viability detection methods:
1. after culturing for 10 days, detecting the confluence degree of the cells every day, taking out the culture bottle when the confluence degree is 85% -90%, and shaking the culture bottle in a clean workbench to enable the tissue blocks to fall off;
2. pouring the tissue blocks and the culture solution into a 50ml centrifuge tube, centrifuging for 5min at 800g, and taking the supernatant liquid for the first time as a stop solution for later use;
3. each culture bottle is gently washed for 2 times by 10ml of physiological saline, and the washed saline is poured off;
4. adding 5ml of pancreatin digestive juice into each culture bottle, shaking up and digesting until most adherent fusiform cells begin to fall off and float circularly (observing under an inverted microscope), digesting for about 4-6min, and then adding 6ml of stop solution into each culture bottle to stop digestion;
5. filtering the cells and the suspension together into a 50ml centrifuge tube, washing the culture flask with 10ml of normal saline, then filtering and pouring into the same centrifuge tube, centrifuging for 10min at 300g, then re-suspending with normal saline to 50ml, mixing uniformly, and carrying out cell counting and cell viability detection.
6. Calculating the cell viability:
the cell viability rate is the proportion of viable cells, and the invention counts the viable cells by trypan blue exclusion:
1) 20ul of the cell suspension was added to 80ul of trypan blue solution and diluted 5-fold. Vortex suspension oscillation is carried out, and diluted cell samples are mixed;
2) 10ul of the dilution was aspirated and counted using a hemocytometer;
3) dead cells stained blue, live cells were clear with refraction (unstained).
4) And a cell density calculation method:
the average of the total number of viable cells in each square x dilution factor x 10= viable cell number/mL.
5) The cell number calculation method comprises the following steps:
(viable cell density + dead cell density) × 5 (dilution 5 times) × 50 (constant volume 50 ml) = number of cells
6) The cell viability calculation method comprises the following steps:
viable cell density/(viable cell density + dead cell density) × 100% = cell viability.
7. Cell flow assay
All cells cultured by the method are subjected to flow detection, so that the purity of the umbilical cord mesenchymal stem cells is not changed greatly.
8. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
9. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
And (3) result statistics and analysis:
group confluence time to reach standard (d) cell number and cell viability
Example 1140.653X 10783.2%
Example 2130.678X 10782.9%
Example 3120.659X 10787.1%
Example 4120.67X 10791.6%
Example 5120.67X 10795.8%
Example 6110.683X 10798.3%
Control 150.627X 10793.6%
And (4) analyzing results: in terms of cell expansion, the time for reaching the standard of the cell number is lower in examples 1-6 than that in the control group. The aspect of cell viability indicates that the cell viability of the samples 5-6 is better than that of the control group. The comparison results in that: examples 5 and 6 had short amplification times (the number reached by amplification in the shortest time) and high cell activities, which were superior to those of the control group.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A serum-free culture medium of primary umbilical cord mesenchymal stem cells is characterized by comprising the following components: alpha-MEM culture solution, AMD3100, BMP-4, insulin, FGF-2, IL-6, LIF, panax notoginseng saponins and cytokine combination, wherein the concentration of the AMD3100 is 0.5-5 mu g/ml, and the concentration of the BMP-4 is 0.01-0.1 ng/ml; the concentration of the insulin is 10-20 mug/ml; the concentration of the FGF-2 is 1-12ng/ml, and the concentration of the IL-6 is 0.05-0.15 mu g/ml; the LIF concentration is 0.01-0.1 μ G/ml, the Panax notoginsenosides concentration is 5-15 μ G/ml, the cytokine combination comprises IL-3, SCF, G-CSF, FL and EGF; the concentrations of IL-3, SCF, G-CSF, FL and EGF are: IL-310-20 ng/ml; SCF 50-150 ng/ml; G-CSF 1-100 ng/ml; FL 1-50 ng/ml; EGF 5-15 ng/ml.
2. The serum-free culture medium of primary umbilical cord mesenchymal stem cells according to claim 1, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells further comprises one or more of dexamethasone, transferrin.
3. The serum-free culture medium of primary umbilical cord mesenchymal stem cells according to claim 2, wherein the concentration of dexamethasone is 0.1-0.5 μ g/ml; the concentration of the transferrin is 0.01-0.1 mug/ml.
4. The serum-free culture medium of primary umbilical cord mesenchymal stem cells according to claim 1, wherein the alpha-MEM culture medium is a serum-free culture medium.
5. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6 and LIF into the alpha-MEM culture solution according to the concentration to prepare a culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Culturing under the full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
6. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Culturing under the full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
7. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Is fully saturatedCulturing under humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 15 days were over.
8. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Culturing under the full saturation humidity;
c. IL-3, SCF, EGF, G-CSF, FL were added before culture until 10 days later and were discontinued.
9. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Culturing under the full saturation humidity;
c. IL-3, SCF, EGF, G-CSF were added before culture until they were discontinued after 10 days, and FL was added at day 5 until they were discontinued after 15 days of culture.
10. The method of using a serum-free culture medium of primary umbilical cord mesenchymal stem cells according to any of claims 1 to 4, wherein the serum-free culture medium of primary umbilical cord mesenchymal stem cells is used for culturing umbilical cord mesenchymal stem cells, the method of use comprising the steps of:
a. adding AMD3100, dexamethasone, transferrin, BMP-4, insulin, FGF-2, IL-6, LIF, and Panax notoginsenosides into alpha-MEM culture solution according to concentration to obtain culture solution;
b. culturing in T75 culture flask, inoculating 8ml culture medium into 2ml α -MEM culture medium suspension of Wharton jelly with density of 0.25g/ml, and culturing at 37 deg.C and 5% CO2Culturing under the full saturation humidity;
c. IL-3, EGF and G-CSF were added before culture until they were discontinued after 10 days, and SCF and FL were added at day 5 until they were discontinued after 15 days.
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