CN109777771A - The serum free medium and its application method of primary umbilical cord mesenchymal stem cells - Google Patents
The serum free medium and its application method of primary umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The present invention relates to the serum free medium of primary umbilical cord mesenchymal stem cells and its application method, the serum free medium of primary umbilical cord mesenchymal stem cells includes following components: α-MEM culture solution, AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.Fetal calf serum is not added in culture medium, so as to avoid immunological rejection issuable in clinical application, reduces the risk of microbiological contamination；FL can be cooperateed with to generate strong proliferation effect by cell factors such as addition IL-3, IL-6, G-CSF and SCF while inhibit the differentiation of umbilical cord mesenchymal stem cells；FGF-2 can be cooperateed with to promote umbilical cord mesenchymal stem cells Proliferation, Differentiation by adding EGF；The trophic factors broken up by the way that BMP-4 is added as stem cells hyperplasia, is able to maintain that and adjusts the normal vegetative state of umbilical cord mesenchymal stem cells.
Description
Technical field
The present invention relates to field of biotechnology, serum free medium more particularly to primary umbilical cord mesenchymal stem cells and
Its application method.
Background technique
Umbilical cord mesenchymal stem cells (MSCs) differentiation potential with higher, can be broken up to multiple directions.It bone,
There is wide potential applicability in clinical practice in terms of the organizational projects such as cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle.Navel
Band mescenchymal stem cell can be separated from people's umbilical cord, have the advantages of materials are convenient, and no ethics is disputed on.Umbilical cord mesenchyma
Stem cell have stronger immunoregulation effect, can promote Radiation in jury function and repair pathological tissues organ, organ transplant,
Autoimmune disease, leukaemia, bone and muscle degenerative disease etc., there is very high clinical value.
How primary umbilical cord mesenchymal stem cells culture, which realizes rapid amplifying and do not influence its Cell viability, is filled between umbilical cord
The key of matter stem cell culture, however culture umbilical cord mesenchymal stem cells technology is not mature enough in the prior art, shows tissue
The growth rate for the primary cell isolated is slow and Cell viability is lower.
Existing umbilical cord mesenchymal stem cells culture medium needs to add animal blood serum such as fetal calf serum, in this culture medium
Some unknown transformation or variation may occur for the stem cell of growth, cell interior structure, and animal blood serum also has initiation immune
The risk of rejection, and have be unfavorable for by the risk of the microbiological contaminations such as bacterium, virus stem cell clinical application and
Basic research.
The increment differentiation of umbilical cord mesenchymal stem cells, maturation process during fetal growth are considerably complicated, depend on various Hemopoietic factors
Adjusting, wherein cell stimulation factor play effect it is extremely crucial.
Summary of the invention
Based on this, the present invention provides the serum free medium and its application method of a kind of primary umbilical cord mesenchymal stem cells,
It realizes rapid amplifying and does not influence its Cell viability and avoid repelling and microbiological contamination wind caused by because of animal blood serum
Danger.
A kind of serum free medium of primary umbilical cord mesenchymal stem cells, including following components: α-MEM culture solution,
AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.
In one of the embodiments, the serum free medium of primary umbilical cord mesenchymal stem cells further include dexamethasone,
One or more of transferrins, arasaponin and combination of cytokines.
The concentration of dexamethasone is 0.1-0.5 μ g/ml in one of the embodiments,；The concentration of the transferrins is
0.01-0.1μg/ml；Arasaponin concentration is 5-15 μ g/ml.Dexamethasone combines AMD3100, and there is synergistic effect to promote navel
Band mescenchymal stem cell is mobilized from tissue, climbs out of and is proliferated.It is dry that traditional Chinese medicine ingredients arasaponin can speed up umbilical cord mesenchyma
The proliferation of cell.
Combination of cytokines includes IL-3, SCF, G-CSF, FL and EGF in one of the embodiments,；The IL-3,
The concentration of SCF, G-CSF, FL and EGF are respectively as follows: IL-3 10-20ng/ml；SCF 50-150ng/ml；G-CSF 1-100ng/
ml；FL 1-50ng/ml；EGF 5-15ng/ml.FL is a kind of key cytokines that can adjust early stage stem cell, with
The combination of the cell factors such as IL-3, IL-6, G-CSF and SCF generates strong proliferation effect to umbilical cord mesenchymal stem cells.
α-MEM culture solution is a kind of serum-free medium in one of the embodiments,.It can be stem cell growth and increasing
It grows and basic nutriment is provided.
The concentration of AMD3100 is 0.5-5 μ g/ml in one of the embodiments,.Promote the life of umbilical cord mesenchymal stem cells
Long and proliferation.
The concentration of BMP-4 is 0.01-0.1ng/ml in one of the embodiments,；The concentration of the insulin is 10-20
μg/ml；The concentration of the FGF-2 is 1-12ng/ml.FGF-2 and BMP-4 promotes the proliferation of umbilical cord mesenchymal stem cells.Pancreas islet
Element advantageously promotes absorption and utilization of the cell to glucose.
The concentration of IL-6 is 0.05-0.15 μ g/ml in one of the embodiments,；The concentration of the LIF is 0.01-0.1
μg/ml.LIF ELISA LIF is able to suppress stem cell differentiation, promotes stem cells hyperplasia.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, this is primary
For the serum free medium of umbilical cord mesenchymal stem cells for cultivating umbilical cord mesenchymal stem cells, the application method includes following step
It is rapid:
A, AMD3100, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF system is added by concentration in α-MEM culture solution
It is made culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, the original
It is used to cultivate umbilical cord mesenchymal stem cells for the serum free medium of umbilical cord mesenchymal stem cells, the application method includes following
Step:
A, in α-MEM culture solution by concentration be added AMD3100, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF,
Arasaponin is fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, the original
It is used to cultivate umbilical cord mesenchymal stem cells for the serum free medium of umbilical cord mesenchymal stem cells, the application method includes following
Step:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, the original
It is used to cultivate umbilical cord mesenchymal stem cells for the serum free medium of umbilical cord mesenchymal stem cells, the application method includes following
Step:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture to deactivate after 10 days.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, the original
It is used to cultivate umbilical cord mesenchymal stem cells for the serum free medium of umbilical cord mesenchymal stem cells, the application method includes following
Step:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF are added before culture to deactivate after 10 days, the 5th day addition FL of culture stops after 15 days
With.
The application method of the serum free medium of primary umbilical cord mesenchymal stem cells as described in any one of the above embodiments, the original
It is used to cultivate umbilical cord mesenchymal stem cells for the serum free medium of umbilical cord mesenchymal stem cells, the application method includes following
Step:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, EGF, G-CSF are added before culture to deactivate after 10 days, culture the 5th day addition SCF, FL stop after 15 days
With.
Supplementary explanation
IL-3 is Interleukin3 interleukin Ⅲ；
SCF is stem cell factor stem cell factor；
G-CSF is granulocyte colony-stimulating factor granulocyte colony stimulating factor；
FL is (flt3-ligand) III type tyrosine kinase receptors ligand；
EGF is Epidermal growth factor epithelical cell growth factor；
BMP-4 is Bone Morphogenetic Protein bone morphogenetic protein 4；
FGF-2 is 2 fibroblast growth factor 2 of Fibroblast Growth Factor；
IL-6 is Interleukin6 interleukin-6；
LIF is leukemia inhibitory factor LIF ELISA；
α-MEM is the primary section's minimum essential medium of modified form Dole；
FBS is fetal calf serum；
Magnificent Tong Shi glue refers to the gel filler between umbilical cord amniotic membrane and blood vessel, contains more umbilical cord mesenchymal stem cells；
Cell viability (%)=viable cell density/(viable cell density+dead cell density) × 100%.
Beneficial effects of the present invention:
1, by research and development serum free medium fetal calf serum is not added in culture medium, so as to avoid in clinical application in the present invention
Issuable immunological rejection reduces the risk of microbiological contamination；
2, the present invention can cooperate with FL to generate strong proliferation effect by cell factors such as addition IL-3, IL-6, G-CSF and SCF
Inhibit the differentiation of umbilical cord mesenchymal stem cells simultaneously；FGF-2 can be cooperateed with to promote umbilical cord mesenchymal stem cells proliferation by adding EGF
Differentiation；The trophic factors broken up by the way that BMP-4 is added as stem cells hyperplasia, is able to maintain that and adjusts umbilical cord mesenchymal stem cells
Normal vegetative state；There is proliferation function to umbilical cord mesenchymal stem cells by the way that traditional Chinese medicine ingredients arasaponin is added；It is added ground
Sai meter Song can cooperate with AMD3100 to have booster action to umbilical cord mesenchymal stem cells Proliferation, Differentiation；By changing adding for cell factor
Enter sequence, FL can be weakened to the value-added inhibiting effect of umbilical cord mesenchymal stem cells getting up early, while inhibiting middle and later periods umbilical cord mesenchyma
The differentiation of stem cell, and then the activity of umbilical cord mesenchymal stem cells Proliferation, Differentiation in vitro is improved, shorten proliferating cycle, improves thin
Born of the same parents' motility rate.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.But the present invention can be to be permitted
Mostly different form is realized, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes
It is more thorough and comprehensive to the understanding of the disclosure.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
The present invention provides a kind of serum free mediums of primary umbilical cord mesenchymal stem cells, including following components: α-MEM
Culture solution, AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.
One preferably in embodiment, the serum free medium of primary umbilical cord mesenchymal stem cells further include dexamethasone,
One or more of transferrins, arasaponin and combination of cytokines.By to α-MEM culture solution be added AMD3100,
Dexamethasone, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, the various combination in arasaponin, pass through simultaneously
It is dry that the time that the cell factors such as adjustment IL-3, SCF, EGF, G-CSF, FL are added and deactivate realizes a kind of primary umbilical cord mesenchyma
Fetal calf serum is not added in culture medium, so as to avoid immune row issuable in clinical application in the serum free medium of cell
Reprimand reaction, reduces the risk of microbiological contamination.
It is preferred that 6 groups of embodiments are as follows:
Embodiment 1:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: α-MEM culture solution,
The concentration of AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF, each component are as shown in table 1.
Table 1
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
BMP-4 0.05ng/ml SCF 100ng/ml
10 μ g/ml EGF 7ng/ml of insulin
LIF 0.05μg/ml G-CSF 25ng/ml
FL 30ng/ml
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, ultimate density is executed according to this table.
The serum free medium of above-mentioned primary umbilical cord mesenchymal stem cells is for cultivating umbilical cord mesenchymal stem cells, culture side
Method the following steps are included:
A, it prepares umbilical cord mesenchymal stem cells culture solution: AMD3100, transferrins, BMP-4, pancreas being added in α-MEM culture solution
Island element, FGF-2, IL-6, LIF are fabricated to umbilical cord mesenchymal stem cells culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；Culture start after the 5th day, 10 days, 13 days,
It carries out within 15 days changing liquid；
C, be added cell factor: culture before by 1 concentration of table be added into the fresh culture for change liquid IL-3, SCF, EGF, G-CSF,
FL terminated until 15 days.
Embodiment 2:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: AMD3100, turning iron egg
The concentration of white, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin, each component is as shown in table 2.
Table 2
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
0.02 μ g/ml SCF 100ng/ml of transferrins
BMP-4 0.05ng/ml EGF 7ng/ml
10 μ g/ml G-CSF 25ng/ml of insulin
IL-6 0.1μg/ml FL 30ng/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
8 μ g/ml of arasaponin
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, ultimate density is executed according to this table.
A, it prepares umbilical cord mesenchymal stem cells culture solution: AMD3100 is added by concentration in α-MEM culture solution, turns iron egg
White, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin are fabricated to umbilical cord mesenchymal stem cells culture solution；
B, IL-3, SCF, EGF, G-CSF, FL are added into the fresh culture for change liquid until 15 days tie by 2 concentration of table before culture
Beam.
Embodiment 3:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: AMD3100, fill in rice
The concentration of pine, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin, each component is as shown in table 3.
Table 3
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
0.2 μ g/ml SCF 100ng/ml of dexamethasone
0.02 μ g/ml EGF 7ng/ml of transferrins
BMP-4 0.05ng/ml G-CSF 25ng/ml
10 μ g/ml FL 30ng/ml of insulin
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
15 μ g/ml of arasaponin
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, ultimate density is executed according to this table.
A, prepare umbilical cord mesenchymal stem cells culture solution: in α-MEM culture solution by concentration be added AMD3100, fill in rice
Pine, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin are fabricated to umbilical cord mesenchymal stem cells culture
Liquid；
B, IL-3, SCF, EGF, G-CSF, FL are added into the fresh culture for change liquid until 15 days tie by 3 concentration of table before culture
Beam.
Embodiment 4:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: α-MEM culture solution,
AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin, the concentration of each component
As shown in table 4.
Table 4
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
0.2 μ g/ml SCF 100ng/ml of dexamethasone
0.02 μ g/ml EGF 7ng/ml of transferrins
BMP-4 0.05ng/ml G-CSF 25ng/ml
10 μ g/ml FL 30ng/ml of insulin
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
5 μ g/ml of arasaponin
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, concentration is executed according to this table.
A, prepare umbilical cord mesenchymal stem cells culture solution: in α-MEM culture solution by concentration be added AMD3100, fill in rice
Pine, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin are fabricated to umbilical cord mesenchymal stem cells culture
Liquid；
B, IL-3, SCF, EGF, G-CSF, FL are added into the fresh culture for change liquid after 10 days by 4 concentration of table before culture
It deactivates.
Embodiment 5:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: α-MEM culture solution,
AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin, the concentration of each component
As shown in table 5.
Table 5
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
0.2 μ g/ml SCF 100ng/ml of dexamethasone
0.02 μ g/ml EGF 7ng/ml of transferrins
BMP-4 0.05ng/ml G-CSF 25ng/ml
10 μ g/ml FL 30ng/ml of insulin
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
7 μ g/ml of arasaponin
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, ultimate density is executed according to this table.
A, prepare umbilical cord mesenchymal stem cells culture solution: in α-MEM culture solution by concentration be added AMD3100, fill in rice
Pine, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin are fabricated to umbilical cord mesenchymal stem cells culture
Liquid；
B, IL-3, SCF, EGF, G-CSF are added into the fresh culture for change liquid by 5 concentration of table before culture to stop after 10 days
With the 5th day addition FL of culture is deactivated after 15 days.
Embodiment 6:
The serum free medium of the primary umbilical cord mesenchymal stem cells of the present embodiment includes following components: α-MEM culture solution,
AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin, the concentration of each component
As shown in table 6.
Table 6
Medium component concentrations of cells factor concentration
AMD3100 1μg/ml IL-3 10ng/ml
0.2 μ g/ml SCF 100ng/ml of dexamethasone
0.02 μ g/ml EGF 7ng/ml of transferrins
BMP-4 0.05ng/ml G-CSF 25ng/ml
10 μ g/ml FL 30ng/ml of insulin
IL-6 0.1μg/ml
FGF-2 5ng/ml
LIF 0.05μg/ml
12 μ g/ml of arasaponin
Remarks: being basic culture solution with α-MEM, according to the addition type requirements of embodiment each stage heterogeneity and cell factor
Culture solution is prepared, ultimate density is executed according to this table.
A, prepare umbilical cord mesenchymal stem cells culture solution: in α-MEM culture solution by concentration be added AMD3100, fill in rice
Pine, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF, arasaponin are fabricated to umbilical cord mesenchymal stem cells culture
Liquid；
B, before culture by 6 concentration of table be added into the fresh culture for change liquid IL-3, EGF, G-CSF after 10 days deactivate, training
The 5th day addition SCF, FL is supported to deactivate after 15 days.
Control group: it prepares umbilical cord mesenchymal stem cells culture solution: 15% FBS being added in α-MEM culture solution.
The umbilical cord mesenchymal stem cells culture solution of embodiment 1 to 6 and control group is tested, cell count and detection are thin
Born of the same parents' survival rate.
Cell count and Cell viability detection method:
1, after cultivating 10 days, the convergence degree of cell is detected daily, when convergence degree is in 85%-90%, culture bottle is taken out, in cleaning
It is rocked in workbench, tissue block is made to fall off；
2, tissue block and culture solution pour into 50ml centrifuge tube, and 800g is centrifuged 5min, and first time supernatant is spare as terminate liquid；
3, each culture bottle is gently rinsed cell 2 times with 10ml physiological saline, the salt water after outwelling washing；
4,5ml pancreatin digestive juice is added in each culture bottle, shakes up digestion to most of adherent spindle cell and starts shedding off in circle
Shape adrift can digest about 4-6min (with observing under inverted microscope), and 6ml is then added into each culture bottle and terminates
Liquid terminates digestion；
5, cell and suspension are filtered together into 50ml centrifuge tube, with 10ml brine culture bottle, then filtering is fallen
Enter in the same centrifuge tube, 300g is centrifuged 10min, is then settled to 50ml with physiological saline resuspension, mixes, carries out cell count
It is detected with Cell viability.
6, Cell viability calculates:
Cell viability is exactly the ratio of living cells, and the present invention uses trypan exclusion stain living cell counting:
1) it, takes 20ul cell suspension to be added in 80ul trypan blue solution, dilutes 5 times.The outstanding oscillation in whirlpool, the cell sample of mixed diluting
This；
2) 10ul dilution, is drawn, is counted using hemacytometer；
3), dead cell dyes the dead cell of blue, and living cells is transparent refractive power (being unstained).
4), cell density calculation method:
Total viable cell average value × extension rate × 10=viable count/mL in each grid.
5), cell quantity calculation method is as follows:
(viable cell density+dead cell density) × 5(dilutes 5 times) × 50(constant volume 50ml)=cell quantity
6), Cell viability calculation method is as follows:
Viable cell density/(viable cell density+dead cell density) × 100%=Cell viability.
7, cell flow cytometer detection
All cells that the present invention is cultivated all carry out flow cytometer detection, can guarantee that umbilical cord mesenchymal stem cells purity does not occur
Larger change.
8, experimental method used in following embodiments is conventional method unless otherwise specified.
9, the materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
As a result statistics and analysis:
Group convergence degree reaching standard time (d) cell quantity Cell viability
Embodiment 1 14 0.653 × 107 83.2%
Embodiment 2 13 0.678 × 107 82.9%
Embodiment 3 12 0.659 × 107 87.1%
Embodiment 4 12 0.67 × 107 91.6%
Embodiment 5 12 0.67 × 107 95.8%
Embodiment 6 11 0.683 × 107 98.3%
Control group 15 0.627 × 107 93.6%
Interpretation of result: cell amplification aspect, embodiment 1-6 cell quantity reaching standard time are below control group.In terms of Cell viability
Illustrate that embodiment 5-6 Cell viability is superior to control group.Comparison obtains: embodiment 5 and 6 proliferation time of embodiment are short (most short
Amplification reaches target quantity in time) and cell activity height, it is superior to control group.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (14)
1. a kind of serum free medium of primary umbilical cord mesenchymal stem cells, which is characterized in that including following components: α-MEM training
Nutrient solution, AMD3100, BMP-4, insulin, FGF-2, IL-6 and LIF.
2. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that the original
Serum free medium for umbilical cord mesenchymal stem cells further includes dexamethasone, transferrins, arasaponin and cell factor
One or more of combination.
3. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 2, which is characterized in that describedly
The concentration of Sai meter Song is 0.1-0.5 μ g/ml；The concentration of the transferrins is 0.01-0.1 μ g/ml；The arasaponin is dense
Degree is 5-15 μ g/ml.
4. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 2, which is characterized in that described
Combination of cytokines includes IL-3, SCF, G-CSF, FL and EGF；The concentration of described IL-3, SCF, G-CSF, FL and EGF are distinguished
Are as follows: IL-3 10-20ng/ml；SCF 50-150ng/ml；G-CSF 1-100ng/ml；FL 1-50ng/ml；EGF 5-15ng/
ml。
5. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that the α-
MEM culture solution is a kind of serum-free medium.
6. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that described
The concentration of AMD3100 is 0.5-5 μ g/ml.
7. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 1 or 2, which is characterized in that institute
The concentration for stating BMP-4 is 0.01-0.1ng/ml；The concentration of the insulin is 10-20 μ g/ml；The concentration of the FGF-2 is 1-
12ng/ml。
8. the serum free medium of primary umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that described
The concentration of IL-6 is 0.05-0.15 μ g/ml；The concentration of the LIF is 0.01-0.1 μ g/ml.
9. the application method of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8,
It is characterized in that, the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells, institute
State application method the following steps are included:
A, AMD3100, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF system is added by concentration in α-MEM culture solution
It is made culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
10. the user of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8
Method, which is characterized in that the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells,
The application method the following steps are included:
A, in α-MEM culture solution by concentration be added AMD3100, transferrins, BMP-4, insulin, FGF-2, IL-6, LIF,
Arasaponin is fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
11. the user of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8
Method, which is characterized in that the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells,
The application method the following steps are included:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture terminated until 15 days.
12. the user of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8
Method, which is characterized in that the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells,
The application method the following steps are included:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate；
C, IL-3, SCF, EGF, G-CSF, FL are added before culture to deactivate after 10 days.
13. the user of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8
Method, which is characterized in that the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells,
The application method the following steps are included:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, SCF, EGF, G-CSF are added before culture to deactivate after 10 days, the 5th day addition FL of culture stops after 15 days
With.
14. the user of the serum free medium of primary umbilical cord mesenchymal stem cells as claimed in any one of claims 1 to 8
Method, which is characterized in that the serum free medium of the primary umbilical cord mesenchymal stem cells is used to cultivate umbilical cord mesenchymal stem cells,
The application method the following steps are included:
A, in α-MEM culture solution by concentration be added AMD3100, dexamethasone, transferrins, BMP-4, insulin, FGF-2,
IL-6, LIF, arasaponin are fabricated to culture solution；
B, it is cultivated in T75 culture bottle, every bottle of 8ml culture solution, is inoculated with the suspension of the α-MEM culture solution of 2ml China Tong Shi glue,
Density is 0.25g/ml, is cultivated under 37 DEG C, the full saturated humidity of 5%CO2；
C, IL-3, EGF, G-CSF are added before culture to deactivate after 10 days, culture the 5th day addition SCF, FL stop after 15 days
With.
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