CN107653225A - A kind of culture medium and its amplification method for being used to expand human mesenchymal stem cell - Google Patents
A kind of culture medium and its amplification method for being used to expand human mesenchymal stem cell Download PDFInfo
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Abstract
It is used to expanding the culture medium of human mesenchymal stem cell the invention discloses a kind of, including basal medium and the additive that makes an addition in culture medium, additive include the component of following final concentration：μ L/mL of linoleic acid 2 10, μ L/mL of people source AB types serum 5 15, the μ g/mL of human transferrin 5 15 etc..Human mesenchymal stem cell is expanded using the culture medium, comprised the following steps：(1) human mesenchymal stem cell is isolated from umbilical cord or placenta, or mononuclearcell is isolated from marrow blood, Cord blood or placental blood, be placed in culture medium and carry out isolation and purification culture；(2) cell after step (1) isolation and purification culture is placed in fresh culture medium again and carries out Secondary Culture.The present invention uses the culture medium and amplification method of specific components, can effectively accelerate cell amplification rate, shortens proliferation time, moreover it is possible to keeps the form of cell after passing on homogeneous, its differentiation is reduced, so as to ensure that the security of human mesenchymal stem cell.
Description
Technical field
The invention belongs to the culture technique field of mescenchymal stem cell, and in particular to one kind is dry thin for expanding human mesenchyme
The culture medium and its amplification method of born of the same parents.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is the important member of stem cell line, is derived from
The mesoderm and ectoderm of mesoderm growing early stage, belong to multipotential stem cell, in vivo or in vitro under specific inductive condition, can be divided into
The Various Tissues cell such as pancreas islet, nerve, blood vessel endothelium, bone, cartilage, muscle, liver, cardiac muscle.MSCs lacks immune because it is relative
Source property, culture technique is simple, and can freezen protective, therefore, it has also become cell and the seed cell of gene therapy research, have latent
Clinical value, it has also become the focus of stem-cell research.
The culture medium for being presently used for culture and the amplification of mescenchymal stem cell is mainly substantially adding in basal medium
Add certain density animal blood serum such as hyclone (FBS), animal blood serum contains foreign protei matter, using animal blood serum culture and
The autologous mescenchymal stem cell of amplification can cause to produce antibody in recipient's body, so as to cause immune response in clinical treatment.Cause
This, the culture medium without serum turns into the focus of people's research.But current visible mesenchymal stem cell serum-free culture technique
It is high in the prevalence of toxigenic capacity, expand limited amount, proliferation time length, the deficiencies of stem cell easily breaks up, directly affect clinic
The security of application.
The content of the invention
For above-mentioned deficiency of the prior art, the invention provides a kind of culture for being used to expand human mesenchymal stem cell
Base and its amplification method, can effectively solve existing amplification culture medium amplification rate is slow, and proliferation time is grown, and stem cell easily breaks up etc.
Problem.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
It is a kind of to be used to expanding the culture medium of human mesenchymal stem cell, including basal medium and make an addition to adding in culture medium
Add agent, the additive includes the component of following final concentration：Linoleic acid 2-10 μ L/mL, people source AB type serum 5-15 μ L/mL, people turn
Ferritin 5-15 μ g/mL, Glu 1.5-3mmol/mL, Catergen -5mg/mL, grape seed extract 0.5-1mg/
ML, rhodioside 1-5mg/mL, ginsenoside 5-10mg/mL, LBP-X 2-5mg/mL, ligustilide 0.2-0.6mg/
ML, platelet derived growth factor 0.1-0.2 μ L/mL and epithelical cell growth factor 0.1-0.2 μ L/mL.
Further, it is a kind of to be used to expanding the culture medium of human mesenchymal stem cell, including basal medium and make an addition to training
The additive in base is supported, the additive includes the component of following final concentration：μ L/mL of linoleic acid 8, μ L/mL of people source AB types serum 10,
It is μ g/mL of human transferrin 10, Glu 2.2mmol/mL, vitamin C 3mg/mL, grape seed extract 0.8mg/mL, red
Red-spotted stonecrop glycosides 3mg/mL, ginsenoside 6mg/mL, LBP-X 3mg/mL, ligustilide 0.3mg/mL, platelet-derived life
The long μ L/mL of the factor 0.1 and epithelical cell growth factor 0.2 μ L/mL.
Further, rhodioside is prepared by the following method to obtain：By rhodiola root and water using solid-to-liquid ratio as 1-2：20-30
Mixing, soak at room temperature 0.5-1h, then decocts 0.5-1h, filters, then the water of 6-8 times of weight of the dregs of a decoction is added into the dregs of a decoction, continues
0.5-1h is decocted, filtering, merges filtrate twice, it is 1.1-1.2 to be then concentrated into relative density, and finally plus ethanol makes alcohol content
Percent by volume is 70-75%, is stirred, and is stood, and filtering, it is 1.1-1.2 that filtrate, which is concentrated into relative density, then dries and removes
Ethanol, it is made.
Further, basal medium is DMEM/F12 culture mediums.
The amplification method expanded using above-mentioned culture medium to mescenchymal stem cell, is comprised the following steps：
(1) human mesenchymal stem cell is primary is separately cultured
Human mesenchymal stem cell is isolated from umbilical cord or placenta, or list is isolated from marrow blood, Cord blood or placental blood
Individual nucleus, it is placed in above-mentioned culture medium and carries out isolation and purification culture；
(2) amplification cultivation
Cell after step (1) isolation and purification culture is placed in fresh above-mentioned culture medium again and carries out Secondary Culture.
Further, step (1) detailed process is as follows：
After 1. aseptically, umbilical cord or placenta are cleaned 2-3 times with PBS, chopping, tissue pieces are obtained,
Then tissue pieces are added in the enzyme liquid containing 0.05% pancreatin and 2mg/mL type Ⅳ collagenases, are placed in 37 DEG C of digestion overnight,
Mixed once every 3-5h during overnight；
2. removal step 1. middle supernatant, pancreatin is added into postdigestive tissue, 37 DEG C of digestion 5-10min are placed in, so
Culture medium is added afterwards and terminates digestion, is finally centrifuged, is abandoned supernatant；
3. by step, 2. gained precipitation is added in culture medium, 37 DEG C, 5%CO2Cultivated in incubator, one was changed every 3-4 days
Subculture, when cell fusion degree is 70-80%, with digestive juice vitellophag, it is then centrifuged for, collects cell.
Further, the volume ratio of 0.05% pancreatin and 2mg/mL type Ⅳ collagenases is 1：2-4.
Further, step (1) detailed process is as follows：
People's marrow blood, Cord blood or placental blood are gathered, is separated using lymphocyte separation medium and collects mononuclearcell, so
Nucleus is placed in above-mentioned culture medium afterwards, adjustment cell concentration is 1-3 × 106Individual/mL, it is subsequently placed in 37 DEG C, 5%CO2Condition
Adherent cell collecting after lower incubation 24h, the mescenchymal stem cell isolated and purified is made.
A kind of culture medium and its amplification method for being used to expand human mesenchymal stem cell provided by the invention, has with following
Beneficial effect：
(1) linoleic acid, people source AB types serum, human transferrin, platelet derived growth factor, epidermal growth because
Son, it can not only improve the purification effect of mescenchymal stem cell, moreover it is possible to keep the form of mescenchymal stem cell after passing on homogeneous, drop
Its low differentiation, so as to ensure that the security of human mesenchymal stem cell.
Human transferrin can also combine iron ion, reduce the toxicity that cell is subject to, and be utilized in time by stem cell, promote thin
Intracellular growth.
(2) grape seed extract is the effective active nutritional ingredient extracted from natural grape seed, and it has efficient natural
Polyphenoils, unnecessary free radical can be removed, there is stronger antioxidation, moreover it is possible to suppress the growth of a variety of cancer cells,
In addition, grape seed extract is combined with vitamin C, rhodioside, ginsenoside, its effect of scavenging radical is strengthened, in conjunction with
Other materials, it can strengthen the cytoactive of mescenchymal stem cell.
Ligustilide can promote cell to breed by extraction by steam distillation, with rhodioside, ginsenoside,
LBP-X can have promotion and proliferation function to the human mesenchymal stem cell of in vitro culture, make these thin by specific proportioning
Born of the same parents continue to keep vegetative state, can accelerate its speed of growth, shorten the amplification cycle, while can also improve its cytoactive.
(3) DMEM/F12 culture mediums can provide basal nutrient for cell, ensure the survival rate of cell, while can also stablize thin
Osmotic pressure of the born of the same parents in separation and culture, whole process is set to be in a mild state.
(4) it the culture medium and amplification method prepared using specific components, can effectively accelerate cell amplification rate, shorten amplification
Time, moreover it is possible to keep the form of mescenchymal stem cell after passing on homogeneous, its differentiation is reduced, so as to ensure that human mesenchymal stem cell
Security.
Embodiment
Embodiment 1
It is a kind of to be used to expanding the culture medium of human mesenchymal stem cell, including basal medium and make an addition to adding in culture medium
Add agent, basal medium is DMEM/F12 culture mediums, and additive includes the component of following final concentration：μ L/mL of linoleic acid 2, Ren Yuan
μ L/mL of AB types serum 5, μ g/mL of human transferrin 5, Glu 1.5mmol/mL, vitamin C 2mg/mL, grape pip carry
Take thing 0.5mg/mL, rhodioside 1mg/mL, ginsenoside 5mg/mL, LBP-X 2mg/mL, ligustilide 0.2mg/
ML, the μ L/mL of platelet derived growth factor 0.1 and the μ L/mL of epithelical cell growth factor 0.1.
Wherein, rhodioside is prepared by the following method to obtain：By rhodiola root and water using solid-to-liquid ratio as 1：20 mixing, normal temperature
0.5h is soaked, then decocts 0.5h, is filtered, then the water of 6 times of weight of the dregs of a decoction is added into the dregs of a decoction, continues to decoct 0.5h, filters, close
And filtrate twice, it is 1.1-1.2 to be then concentrated into relative density, and finally plus ethanol makes the percent by volume of alcohol content be 70%,
Stirring, stand, filtering, it is 1.1-1.2 that filtrate, which is concentrated into relative density, then dries and removes ethanol, is made.
The amplification method expanded using above-mentioned culture medium to human mesenchymal stem cell, is comprised the following steps：
(1) human mesenchymal stem cell is primary is separately cultured
1. after aseptically, umbilical cord is cleaned 2-3 times with PBS, chopping, obtaining tissue pieces, then will
Tissue pieces are added in the enzyme liquid containing 0.05% pancreatin and 2mg/mL type Ⅳ collagenases, are placed in 37 DEG C of digestion overnight, overnight the phase
Between every 3h mix once；Wherein, the volume ratio of 0.05% pancreatin and 2mg/mL type Ⅳ collagenases is 1：2；
2. removal step 1. middle supernatant, pancreatin is added into postdigestive tissue, 37 DEG C of digestion 5min, Ran Houjia are placed in
Enter culture medium and terminate digestion, finally centrifuge, abandon supernatant；
3. by step, 2. gained precipitation is added in culture medium, 37 DEG C, 5%CO2Cultivate in incubator, changed once every 3 days
Culture medium, when cell fusion degree is 70-80%, with digestive juice vitellophag, it is then centrifuged for, collects cell.
(2) amplification cultivation
Cell after step (1) isolation and purification culture is placed in fresh culture medium again and carries out Secondary Culture.
Embodiment 2
It is a kind of to be used to expanding the culture medium of human mesenchymal stem cell, including basal medium and make an addition to adding in culture medium
Add agent, basal medium is DMEM/F12 culture mediums, and additive includes the component of following final concentration：μ L/mL of linoleic acid 10, Ren Yuan
μ L/mL of AB types serum 15, μ g/mL of human transferrin 15, Glu 3mmol/mL, vitamin C 5mg/mL, grape pip carry
Take thing 1mg/mL, rhodioside 5mg/mL, ginsenoside 10mg/mL, LBP-X 5mg/mL, ligustilide 0.6mg/
ML, the μ L/mL of platelet derived growth factor 0.2 and the μ L/mL of epithelical cell growth factor 0.2.
Wherein, rhodioside is prepared by the following method to obtain：By rhodiola root and water using solid-to-liquid ratio as 2：20 mixing, normal temperature
1h is soaked, then decocts 1h, is filtered, then the water of 8 times of weight of the dregs of a decoction is added into the dregs of a decoction, continues to decoct 0.5h, filtering, merges two
Secondary filtrate, it is 1.1-1.2 to be then concentrated into relative density, and finally plus ethanol makes the percent by volume of alcohol content be 75%, stirring,
Stand, filtering, it is 1.1-1.2 that filtrate, which is concentrated into relative density, then dries and removes ethanol, is made.
The amplification method expanded using above-mentioned culture medium to human mesenchymal stem cell, is comprised the following steps：
(1) human mesenchymal stem cell is primary is separately cultured
Cord blood is gathered, is separated using lymphocyte separation medium and collects mononuclearcell, nucleus is then placed in training
Support in base, adjustment cell concentration is 1-3 × 106Individual/mL, it is subsequently placed in 37 DEG C, 5%CO2Under the conditions of be incubated 24h after collect it is adherent
Cell, the mescenchymal stem cell isolated and purified is made.
(2) amplification cultivation
Cell after step (1) isolation and purification culture is placed in fresh culture medium again and carries out Secondary Culture.
Embodiment 3
It is a kind of to be used to expanding the culture medium of human mesenchymal stem cell, including basal medium and make an addition to adding in culture medium
Add agent, basal medium is DMEM/F12 culture mediums, and additive includes the component of following final concentration：μ L/mL of linoleic acid 8, Ren Yuan
μ L/mL of AB types serum 10, μ g/mL of human transferrin 10, Glu 2.2mmol/mL, vitamin C 3mg/mL, grape pip
Extract 0.8mg/mL, rhodioside 3mg/mL, ginsenoside 6mg/mL, LBP-X 3mg/mL, ligustilide
0.3mg/mL, the μ L/mL of platelet derived growth factor 0.1 and the μ L/mL of epithelical cell growth factor 0.2.
Wherein, rhodioside is prepared by the following method to obtain：By rhodiola root and water using solid-to-liquid ratio as 2：20 mixing, normal temperature
1h is soaked, then decocts 1h, is filtered, then the water of 8 times of weight of the dregs of a decoction is added into the dregs of a decoction, continues to decoct 0.5h, filtering, merges two
Secondary filtrate, it is 1.1-1.2 to be then concentrated into relative density, and finally plus ethanol makes the percent by volume of alcohol content be 75%, stirring,
Stand, filtering, it is 1.1-1.2 that filtrate, which is concentrated into relative density, then dries and removes ethanol, is made.
The amplification method expanded using above-mentioned culture medium to human mesenchymal stem cell, is comprised the following steps：
(1) human mesenchymal stem cell is primary is separately cultured
1. after aseptically, umbilical cord is cleaned 2-3 times with PBS, chopping, obtaining tissue pieces, then will
Tissue pieces are added in the enzyme liquid containing 0.05% pancreatin and 2mg/mL type Ⅳ collagenases, are placed in 37 DEG C of digestion overnight, overnight the phase
Between every 3h mix once；Wherein, the volume ratio of 0.05% pancreatin and 2mg/mL type Ⅳ collagenases is 1：3；
2. removal step 1. middle supernatant, pancreatin is added into postdigestive tissue, 37 DEG C of digestion 10min are placed in, then
Add culture medium and terminate digestion, finally centrifuge, abandon supernatant；
3. by step, 2. gained precipitation is added in culture medium, 37 DEG C, 5%CO2Cultivate in incubator, changed once every 3 days
Culture medium, when cell fusion degree is 70-80%, with digestive juice vitellophag, it is then centrifuged for, collects cell.
(2) amplification cultivation
Cell after step (1) isolation and purification culture is placed in fresh culture medium again and carries out Secondary Culture.
Comparative example 1
Compared with Example 3, difference is comparative example 1：Grape seed extract is not contained in culture medium, remaining and implementation
Example 3 is identical.
Comparative example 2
Compared with Example 3, difference is comparative example 2：Do not contained in culture medium grape seed extract, rhodioside,
Ginsenoside and LBP-X, remaining is same as Example 3.
Comparative example 3
Compared with Example 3, difference is comparative example 3：Vitamin C, grape seed extract, red is not contained in culture medium
Red-spotted stonecrop glycosides, ginsenoside, LBP-X and ligustilide, remaining is same as Example 3.
Experimental example
1st, the cell number harvested when original cuiture time and primary passage is compared
When as shown in Table 1, using medium culture human mesenchymal stem cell of the present invention, its incubation time is significantly shorter than control
Group, when culture was to the 7th day, the cell quantity of 3 groups of embodiment has reached passage condition, and far more than control group.
2nd, ability of cell proliferation is compared
Embodiment 3 and comparative example 1-3 groups cell are reached into P15 generations always：
Embodiment 3：P15 generation group cells remain in that good multiplication capacity, 2-3 days fusion rates still can reach 85% with
On, it is still more uniform to P30 generations, its cellular morphology to continue culture.
Since P10 generations, cell proliferation rate substantially slows down comparative example 1, and cell becomes big, while have also appeared old
Change.
Comparative example 2 and the aging speed of comparative example 3 are fast compared with comparative example 1, and the aging speed of comparative example 3 is most fast.
3rd, cellular morphology is observed
Embodiment 3 and comparative example 1-3 cells are reached into P15 generations always, observe the form of its human mesenchymal stem cell.
The P15 of embodiment 3 is fusiformis for cellular morphology, and individual is smaller, and comparative example 1-3 P15 becomes for cell individual
Greatly, there is obvious aging in cell.
4th, flow cytometer detection
Embodiment 3 and comparative example 1-3 cells are reached into P15 generations always, take 50,000 cells to do flow cytometer detection respectively, by examining
Result to be surveyed to understand, the still altimeter of embodiment 3 reaches mescenchymal stem cell mark antigen, and CD34, CD45, HLA-DR are negative, CD90,
CD105, CD73 are positive, and cell still keeps cells and characteristic of stem, and comparative example 1-3 is then mutually far short of what is expected.
In summary, the culture medium and amplification method that the present invention is prepared using specific components, cell amplification can effectively be accelerated
Speed, shorten proliferation time, improve ability of cell proliferation, moreover it is possible to keep the form of mescenchymal stem cell after passing on homogeneous, reduce
It breaks up, so as to ensure that the security of human mesenchymal stem cell.
Claims (8)
1. a kind of culture medium for being used to expand human mesenchymal stem cell, it is characterised in that including basal medium and make an addition to training
The additive in base is supported, the additive includes the component of following final concentration：Linoleic acid 2-10 μ L/mL, people source AB type serum 5-
15 μ L/mL, human transferrin 5-15 μ g/mL, Glu 1.5-3mmol/mL, vitamin C 2-5mg/mL, grape pip carry
Take thing 0.5-1mg/mL, rhodioside 1-5mg/mL, ginsenoside 5-10mg/mL, LBP-X 2-5mg/mL, in Angelica acutiloba phthalein
Ester 0.2-0.6mg/mL, platelet derived growth factor 0.1-0.2 μ L/mL and epithelical cell growth factor 0.1-0.2 μ L/mL.
2. the culture medium according to claim 1 for being used to expand human mesenchymal stem cell, it is characterised in that the additive
Include the component of following final concentration：μ L/mL of linoleic acid 8, μ L/mL of people source AB types serum 10, μ g/mL of human transferrin 10, L- paddy
Glutamine 2.2mmol/mL, vitamin C 3mg/mL, grape seed extract 0.8mg/mL, rhodioside 3mg/mL, ginsenoside
6mg/mL, LBP-X 3mg/mL, ligustilide 0.3mg/mL, the μ L/mL of platelet derived growth factor 0.1 and epidermis are thin
The μ L/mL of the intracellular growth factor 0.2.
3. the culture medium according to claim 1 or 2 for being used to expand human mesenchymal stem cell, it is characterised in that the base
Basal culture medium is DMEM/F12 culture mediums.
4. the culture medium according to claim 1 or 2 for being used to expand human mesenchymal stem cell, it is characterised in that described red
Red-spotted stonecrop glycosides is prepared by the following method to obtain：By rhodiola root and water using solid-to-liquid ratio as 1-2：20-30 is mixed, soak at room temperature 0.5-
1h, 0.5-1h is then decocted, filtered, then the water of 6-8 times of weight of the dregs of a decoction is added into the dregs of a decoction, continued to decoct 0.5-1h, filter, close
And filtrate twice, it is 1.1-1.2 to be then concentrated into relative density, and finally plus ethanol makes the percent by volume of alcohol content be 70-
75%, stir, stand, filtering, it is 1.1-1.2 that filtrate, which is concentrated into relative density, then dries and removes ethanol, is made.
5. the amplification method expanded using the culture medium described in claim any one of 1-4 to human mesenchymal stem cell, its
It is characterised by, comprises the following steps：
(1) human mesenchymal stem cell is primary is separately cultured
Human mesenchymal stem cell is isolated from umbilical cord or placenta, or single core is isolated from marrow blood, Cord blood or placental blood
Cell, it is placed in culture medium and carries out isolation and purification culture；
(2) amplification cultivation
Cell after step (1) isolation and purification culture is placed in fresh culture medium again and carries out Secondary Culture.
6. the amplification method of human mesenchymal stem cell according to claim 5, it is characterised in that step (1) detailed process
It is as follows：
After 1. aseptically, umbilical cord or placenta are cleaned 2-3 times with PBS, chopping, tissue pieces are obtained, then
Tissue pieces are added in the enzyme liquid containing 0.05% pancreatin and 2mg/mL type Ⅳ collagenases, are placed in 37 DEG C of digestion overnight, overnight
Period mixes once every 3-5h；
2. removal step 1. middle supernatant, pancreatin is added into postdigestive tissue, 37 DEG C of digestion 5-10min, Ran Houjia are placed in
Enter culture medium and terminate digestion, finally centrifuge, abandon supernatant；
3. by step, 2. gained precipitation is added in culture medium, 37 DEG C, 5%CO2Cultivated in incubator, changed every 3-4 days and once cultivate
Base, when cell fusion degree is 70-80%, with digestive juice vitellophag, it is then centrifuged for, collects cell.
7. the amplification method of human mesenchymal stem cell according to claim 6, it is characterised in that 0.05% pancreatin and 2mg/
The volume ratio of mL type Ⅳ collagenases is 1：2-4.
8. the amplification method of human mesenchymal stem cell according to claim 5, it is characterised in that step (1) detailed process
It is as follows：
People's marrow blood, Cord blood or placental blood are gathered, is separated using lymphocyte separation medium and collects mononuclearcell, then will
Nucleus is placed in culture medium, and adjustment cell concentration is 1-3 × 106Individual/mL, it is subsequently placed in 37 DEG C, 5%CO2Under the conditions of be incubated
Adherent cell collecting after 24h, the mescenchymal stem cell isolated and purified is made.
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