CN106635972B - Fibroblastic culture medium and preparation method thereof - Google Patents
Fibroblastic culture medium and preparation method thereof Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 42
- 230000003328 fibroblastic effect Effects 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 30
- 239000000654 additive Substances 0.000 claims abstract description 23
- 230000000996 additive effect Effects 0.000 claims abstract description 23
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 14
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 14
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 10
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 10
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims abstract description 8
- 102000002070 Transferrins Human genes 0.000 claims abstract description 7
- 108010015865 Transferrins Proteins 0.000 claims abstract description 7
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229960000890 hydrocortisone Drugs 0.000 claims abstract description 7
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 7
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 7
- 239000011781 sodium selenite Substances 0.000 claims abstract description 7
- 102000004877 Insulin Human genes 0.000 claims abstract description 5
- 108090001061 Insulin Proteins 0.000 claims abstract description 5
- 229940125396 insulin Drugs 0.000 claims abstract description 5
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims abstract description 4
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- 229930182833 estradiol Natural products 0.000 claims abstract description 4
- 239000000186 progesterone Substances 0.000 claims abstract description 4
- 229960003387 progesterone Drugs 0.000 claims abstract description 4
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims abstract description 3
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims abstract description 3
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 claims abstract description 3
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 claims abstract description 3
- 229940126864 fibroblast growth factor Drugs 0.000 claims abstract description 3
- 150000002308 glutamine derivatives Chemical class 0.000 claims abstract description 3
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
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Abstract
The present invention relates to a kind of inductions of inductive pluripotent stem cells into fibroblastic culture medium and preparation method thereof.The culture medium includes basic culture solution and additive, and the basic culture solution is the H-DMEM culture solution and F12 culture solution that volume ratio is 1:0.5~2；With the densimeter in the basic culture solution, the additive includes following component: nonessential amino acid, Glutamine Derivatives, insulin, hydrocortisone, transferrins, sodium selenite, fibroblast growth factor, estradiol, progesterone, 8- bromine adenosine cyclophosphate, human follicle stimulating hormone.The culture medium can be obtained compared with the higher fibroblast differentiation probability of the prior art, and passage can be stablized, improve the growth rate of cell, fibroblastic seed cell source is provided for skin tissue engineering, whole process does not use serum simultaneously, animal derived pathogen bring risk is effectively avoided, the safety of clinical application is improved.
Description
Technical field
The present invention relates to cell culture mediums, more particularly to a kind of fibroblastic culture medium and preparation method thereof.
Background technique
Skin tissue engineering is research hotspot in recent years.But the relevant cell of skin belongs to terminally differentiated cells,
It is difficult to carry out cultivating amplification, while the hair for carrying out source problem and also counteracting skin tissue engineering of seed cell in vitro on a large scale
Exhibition.
Due to self seed cell limited source, there are rejection, stem cells there are human relations again for allogeneic seed cell
Reason problem, therefore urine cell is collected from urine, urine cell is reprogrammed as inductive pluripotent stem cells (ips cell),
Ips cell is induced again as keratinocyte, fibroblast, sweat gland cells, hair papilla cell etc., and skin group can be solved
The seed source problem of weaver's journey, and cell is obtained from urine, it is convenient and efficient, do not have to obtain by wound, it is from a wealth of sources.
But the inducing ips cell of current use becomes fibroblastic culture medium, such as point of MEF culture medium
It is lower to change probability, it is difficult to meet actual demand, and contain serum, animal derived pathogen risk can be brought, influence clinical application
Safety.
Summary of the invention
Based on this, it is necessary to one kind is provided and is free of serum, it is highly-safe, and inductive pluripotent stem cells are improved at fiber
Fibroblastic culture medium of cell differentiation ratio.
A kind of fibroblastic culture medium, including basic culture solution and additive, the basic culture solution are volume ratio
For the H-DMEM culture solution and F12 culture solution of 1:0.5~2；With the addition meter in the basic culture solution, the additive
Including following component:
The additive includes following component in one of the embodiments:
The additive further includes antibiotic in one of the embodiments,.
In one of the embodiments, the antibiotic include in the basic culture solution additive amount be 0.05~
The penicillin of 0.2mg/mL and in the basic culture solution additive amount be 0.05~0.2mg/mL streptomysin.
The additive further includes free radical scavenger in one of the embodiments,.
In one of the embodiments, the free radical scavenger be in the basic culture solution additive amount be 0.05~
The beta -mercaptoethanol of 0.2mmol/L.
The present invention also provides the preparation methods of fibroblastic culture medium, include the following steps:
H-DMEM culture solution and F12 culture solution are mixed by the volume ratio, obtains the basic culture solution；
The insulin, hydrocortisone, transferrins, sodium selenite, nonessential amino acid, glutamine is derivative
Object, fibroblast growth factor, estradiol, progesterone, 8- bromine adenosine cyclophosphate, human follicle stimulating hormone are added to the basis training
In nutrient solution.
The present invention also provides application of the fibroblastic culture medium in Fibroblast cell-culture.
Inductive pluripotent stem cells are induced as fibroblastic method the present invention also provides a kind of, including are walked as follows
It is rapid:
Inductive pluripotent stem cells are cultivated using mTeSR1 culture medium, when convergence degree is 75~85%, digestion
It is unicellular；
It takes appropriate described unicellular, culture medium is formed with EB embryoid body and is cultivated, embryoid body is formed；
The embryoid body is seeded in the culture medium and is cultivated to get fibroblast.
The condition of culture of the embryoid body in one of the embodiments, are as follows: use CO2Concentration is 5~7%, and temperature is
37 DEG C of 24~50h of incubator culture.
Compared with prior art, the invention has the following advantages:
Fibroblastic culture medium of the invention adds specific addition on the basis of H-DMEM and F12 culture solution
Object, wherein bFGF is a kind of polypeptide and pancreas islet that mesoderm and neuroectodermal cells can be promoted to divide
Element, hydrocortisone, transferrins, sodium selenite, nonessential amino acid, Glutamine Derivatives, Desmocyte growth factor
It is mainly the effect for transmitting development signal that son, estradiol, progesterone, 8- bromine adenosine cyclophosphate, which reformulate a kind of composition, can be promoted
Mesoblastema into embryoid body is divided into fibroblast.Thus fibroblastic culture medium of the invention, which can be realized, mentions
The ratio that high inductive pluripotent stem cells break up to fibroblast, and passage can be stablized, the growth rate of cell is improved,
Fibroblastic seed cell source is provided for skin tissue engineering.Whole process does not use serum simultaneously, effectively keeps away
Exempt from animal derived pathogen bring risk, improves the safety of clinical application.
The culture medium can also effectively inhibit thin in incubation by adding specific antibiotic and free radical scavenger
The generation of bacterium and the oxygen radical for removing accumulation improve the survival rate of cell to maintain preferably culture environment.
Detailed description of the invention
Fig. 1 is that the experimental group of the embodiment of the present invention and contrast groups the comparison diagram of number of days needed for cells deformation occur；
Fig. 2 is the expression of the immunofluorescence type i collagen of microscopically observation experimental group and contrast groups；
Fig. 3 is that the fibroblast value-added speed in embodiment 1 in experimental group and contrast groups compares；
Fig. 4 is that the fibroblast value-added speed in embodiment 2 in experimental group and contrast groups compares；
Fig. 5 is that the fibroblast value-added speed in embodiment 3 in experimental group and contrast groups compares.
Specific embodiment
Fibroblastic culture medium of the invention and preparation method thereof is made below in conjunction with specific embodiment further detailed
Thin explanation.
H-DMEM culture solution, F12 culture solution, mTeSR1 culture medium, the EB embryoid body used in the embodiment of the present invention is formed
Culture medium is commercial product.
Embodiment 1
One, contrived experiment group and control group:
1. experimental group culture medium:
Experimental group culture medium includes basic culture solution and additive, and the basic culture solution is the H- that volume ratio is 1:1
DMEM culture solution and F12 culture solution, total volume 500mL；With the addition meter in the basic culture solution, the additive
Including following component:
Preparation method: Insulin 3 0mg, hydrocortisone are added in 500mL basic culture solution according to aforementioned proportion
0.2mg, transferrins 50mg, 5 μ g of sodium selenite, beta -mercaptoethanol 0.05mmoL, NEAA 5mL, GlutaMAX 5mL, mould
7.5 μ g, E2 5nmoL, the P4 bromo- cAMP 0.5mmoL of 0.5mmoL, 8- of plain 50mg, streptomysin 50mg, bFGF, human follicle stimulation
Plain FSH 50nmoL.
2. control group culture medium
Control group uses existing MEF culture solution 500mL, and 10mLFBS is added wherein.
Two, external evoked ips breaks up to fibroblast
(1) recovery cell: selecting the ips cell of P30 to recover, then cultivated with mTeSR1 culture medium, when reaching
When to 80% convergence degree, be with the digestion of Accutase digestive juice it is unicellular, count.
(2) 1 × 10 the formation of simple embryoid body: is added in each AggreWellTM800 cultivation plate hole6It is a above-mentioned slender
Then born of the same parents are added EB enrichment liquid and form culture medium, by product using operation manual, by AggreWellTM800 culture plate low speed from
The heart 700r/min, 5min, are put into 5%CO2, cultivate in 37 DEG C of incubators, form simple embryoid body after cultivating 48h.
(3) induction differentiation: will form simple embryoid body and be transferred in 24 orifice plate of low adsorption, and 24 orifice plates are divided into two groups, experiment
Group and control group, every group of 12 holes, experimental group experimental group culture medium culture, control group control group culture medium culture are changed every other day
Liquid records that deformation occurs in which day cell (result is as shown in Figure 1).Choose ameboid cell group, with immunofluorescence method identification at
Fibrocyte determines that the cell of deformation is continuous culture again after fibroblast, and passage all carries out cell count every time, thinner
The growth rate of born of the same parents.
Three, fibroblastic identification
With the antigen presentation of identified by immunofluorescence fibroblast type i collagen:
1. experimental group, control group respectively discard culture medium, are washed three times with PBS, each 5min after induction 15 days.
2. the fixed 1h of 4% paraformaldehyde 1mL is added in every hole, washed three times with PBS, each 5min.
3.0.1%Triton rupture of membranes 10min is washed three times, each 5min with PBS.
4. discarding PBS, experimental group, control group are separately added into 500 μ l of rabbit anti-human type-collagen (1:50), and 4 DEG C of wet box were incubated for
Night.
5. being washed three times with PBS, each 5min.Experimental group, control group are separately added into secondary antibody goat-anti rabbit-anti IgG (1:200) 500
μ L, room temperature, which is protected from light, is incubated for 1h.
6. being washed three times with PBS, each 5min.
7.50% glycerol mounting, fluorescence microscopy under the microscope, as a result as shown in Fig. 2, simultaneously to issue fluorescence cell into
Row counts, as a result as shown in Figure 3.
Embodiment 2
A kind of inductive pluripotent stem cells induction of the present embodiment is at fibroblastic method, and method is the same as in embodiment 1
The step of two, difference is: the culture medium of use includes basic culture solution and additive, and the basic culture solution is that volume ratio is
The H-DMEM culture solution and F12 culture solution of 1:2, total volume 500mL；It is described with the densimeter in the basic culture solution
Additive includes following component:
| Components Name | Working concentration |
| NEAA | 0.5%v/v |
| GlutaMAX | 2%v/v |
| Insulin | 1μg/mL |
| Hydrocortisone | 1μg/mL |
| Transferrins | 150μg/mL |
| Sodium selenite | 5ng/mL |
| Beta -mercaptoethanol | 0.2mmol/L |
| Penicillin | 0.2mg/mL |
| Streptomysin | 0.05mg/mL |
| b FGF | 10ng/mL |
| E2 | 5nmol/L |
| P4 | 0.5mmol/L |
| The bromo- cAMP of 8- | 0.5mmol/L |
| FSH | 150nmol/L |
The fibroblastic identification method of culture gained is the same as the step three in embodiment 1, fluorescence microscopy microscopic observation result
It is similar with Fig. 2, the cell for issuing fluorescence is counted, as a result as shown in Figure 4.
Embodiment 3
A kind of inductive pluripotent stem cells induction of the present embodiment is at fibroblastic method, and method is the same as in embodiment 1
The step of two, difference is: the culture medium of use includes basic culture solution and additive, and the basic culture solution is that volume ratio is
The H-DMEM culture solution and F12 culture solution of 1:0.5, total volume 500mL；With the densimeter in the basic culture solution, institute
Stating additive includes following component:
| Components Name | Working concentration |
| NEAA | 2%v/v |
| GlutaMAX | 0.5%v/v |
| Insulin | 10μg/mL |
| Hydrocortisone | 0.1μg/mL |
| Transferrins | 50μg/mL |
| Sodium selenite | 15ng/mL |
| Beta -mercaptoethanol | 0.05mmol/L |
| Penicillin | 0.05mg/mL |
| Streptomysin | 0.2mg/mL |
| b FGF | 20ng/mL |
| E2 | 15nmol/L |
| P4 | 2mmol/L |
| The bromo- cAMP of 8- | 2mmol/L |
| FSH | 50nmol/L |
The fibroblastic identification method of culture gained is the same as the step three in embodiment 1, fluorescence microscopy microscopic observation result
It is similar with Fig. 2, the cell for issuing fluorescence is counted, as a result as shown in Figure 5.
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 (10)
1. a kind of induce inductive pluripotent stem cells for fibroblastic culture medium, which is characterized in that by basic culture solution
It is formed with additive；The basic culture solution is the H-DMEM culture solution and F12 culture solution that volume ratio is 1:0.5~2；With institute
The addition meter in basic culture solution is stated, the additive is grouped as by following group:
2. culture medium according to claim 1, which is characterized in that with the addition meter in the basic culture solution, institute
Additive is stated to be grouped as by following group:
3. culture medium according to claim 1 or 2, which is characterized in that the additive further includes antibiotic.
4. culture medium according to claim 3, which is characterized in that the antibiotic, which is included in the basic culture solution, to be added
The penicillin that dosage is 0.05~0.2mg/mL and the chain that additive amount is 0.05~0.2mg/mL in the basic culture solution
Mycin.
5. culture medium according to claim 1 or 2, which is characterized in that the additive further includes free radical scavenger.
6. culture medium according to claim 5, which is characterized in that the free radical scavenger is in the basic culture solution
Middle additive amount is the beta -mercaptoethanol of 0.05~0.2mmol/L.
7. the preparation method of culture medium described in any one of claims 1-6, which comprises the steps of:
H-DMEM culture solution and F12 culture solution are mixed by the volume ratio, obtains the basic culture solution；
By the insulin, hydrocortisone, transferrins, sodium selenite, nonessential amino acid, Glutamine Derivatives, at
Fibroblast growth factor, estradiol, progesterone, 8- bromine adenosine cyclophosphate, human follicle stimulating hormone are added to the basic culture solution
In.
8. application of the culture medium described in any one of claims 1-6 in Fibroblast cell-culture.
9. a kind of induce inductive pluripotent stem cells for fibroblastic method, which comprises the steps of:
Inductive pluripotent stem cells are cultivated using mTeSR1 culture medium, when convergence degree is 75~85%, are digested for list
Cell；
It takes appropriate described unicellular, culture medium is formed with EB embryoid body and is cultivated, embryoid body is formed；
The embryoid body is seeded in any one of claim 1-6 culture medium and is cultivated to get fibroblast.
10. according to claim 9 induce inductive pluripotent stem cells for fibroblastic method, feature exists
In the condition of culture of the embryoid body are as follows: use CO2Concentration is 5~7%, 24~50h of incubator culture that temperature is 37 DEG C.
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