CN105734011B - A kind of culture solution maintaining people's amnioic epithelium stem cell versatility - Google Patents
A kind of culture solution maintaining people's amnioic epithelium stem cell versatility Download PDFInfo
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Abstract
本发明公开了一种维持人羊膜上皮干细胞多能性的培养液，包括：基础培养液；血清替代品15%~25%；非必需氨基酸0.1%~2%；必需氨基酸1mmol/L~5mmol/L；二硫苏糖醇0.01mmol/L~1mmol/L；生长因子30ng/ml~120ng/ml；体系稳定因子0.1%~2%。本发明的培养液可以模拟干细胞在体内的生存环境，进而使得干细胞在体外培养时可以维持干细胞多能性，经过较多次数的传代培养后，依然可以获得具有高多能性的干细胞。
The invention discloses a culture solution for maintaining the pluripotency of human amniotic epithelial stem cells, comprising: a basic culture solution; 15%-25% of serum substitutes; 0.1%-2% of non-essential amino acids; and 1mmol/L-5mmol/L of essential amino acids. L; dithiothreitol 0.01mmol/L~1mmol/L; growth factor 30ng/ml~120ng/ml; system stability factor 0.1%~2%. The culture medium of the present invention can simulate the living environment of stem cells in vivo, so that stem cells can maintain stem cell pluripotency when cultured in vitro, and stem cells with high pluripotency can still be obtained after subculture for many times.
Description
Technical field
The present invention relates to technical field of cell culture, and in particular to a kind of maintenance people's amnioic epithelium stem cell versatility
Culture solution.
Background technique
Amnion is located on the inside of embryo villi film, be one layer without blood vessel, nerve, lymph, muscle transparent membrane, in development
Fetus connection it is close.The amnion-derived cell of people is mainly by two class cell compositions: human amnion membrane (human amnion
Epithelial cells, hAECs) and human amniotic mesenchymal cell (human amnion mesenchymecells,
hAMCs)。
Human amnion membrane (human amniotic epithelial cell, hAEC) has a variety of embryos of expression dry
The characteristics of cell marker and more comprehensive multi-lineage potential, the researchs such as Miki confirm that hAECs breaks up latent to triploblastica
Energy.Except expression embryonic stem cell surface antigen: SSEA-3 and SSEA-4, tumor resistance gene TRA 1-60 and TRA 1-81 it
Outside, it has recently found that hAECs also expresses the transcription factor of pluripotent stem cell, such as Oct-4, Sox-2, Nanog and REX-1 etc. and
Other antigens, for example, ABCG 2/BCRP of one of ATP combination magazine super families member of ATP efflux pump, CD9, CD24, on
Skin calcium mucin, integrin α6 and β, hepatocyte growth factor bind receptor (c-met) etc..CD34 (candidate stem cell is not expressed
And interior surface drying cell surface marker), SSEA-1, CD133 (candidate stem cell, endothelial cell and Malignant glioma cells surface mark
Note), weak expression c-kit (CD117) and CC-chemokine receptor (CRR4).
HAECs be it is a kind of can self duplication and the adult stem cell with multinomial differentiation potential, in different growth factors
Under adjusting, hepatic lineage, cardiac-like muscle cell, Deiter's cells, neuronal cell and islet-like cells etc. can be divided into, and
It can secrete the neurotrophic factors such as BDNF, NGF, these neurotrophic factors have to neuron repair, nutritional support
Effect, secondly, hAECs immunogenicity is low, is not easy to cause rejection, can be reduced inflammatory factor IFN γ and TNF after cell transplantation
Release, pass through and inhibit T lymphocyte proliferation, reduce the expression of pro-inflammatory cytokine IL-1 α and IL-1 β, there is immune adjust
The effect of section.Again, it is found from the treatment of hAECs Central nervous system injury model, it is thin that hAECs can be divided into neuron
Born of the same parents, astroglia substitution is damaged or dead cell, restores the function of being damaged region, improves the behavior of animal pattern
And pathological change.HAECs not only has great significance to neural restoration, all plays in the reparation of the tissues such as liver non-
Normal important role, and there is low immunogenicity and immune coordinate repression, while can avoid placenta stem-cell experiment and facing
Ethics problem in bed application, medically has broad application prospects in cell replacement therapy and regeneration.
Human amnion membrane is derived from placenta amnion tissue, although expression stem-cell marker molecule and related gene, not
Telomerase gene is expressed, amplification is restricted in vitro, can only be cultivated in vitro for 4 to 5 generations, and cell starts after a few days for culture ten
Atrophy, the versatility of cell also by strong influence, have reported related cultivating system, are all the concentration to growth factor EGF
It adjusts, but does not also take on a new look significantly to the maintenance of hAECs versatility, cell cannot equally maintain to cultivate over a long time.
Although we can obtain more hAECs from people's amnion, can all occur for external research and transplantation treatment
Larger limitation, therefore one is stablized maintenance hAECs versatility cultivating system and is of great significance, it can be in external training for a long time
The stabilization of versatility is maintained in supporting, while also solving the problems, such as acute treatment cell requirements amount.
Summary of the invention
The object of the present invention is to provide a kind of stem cell mediums, can provide in analogue body for people amnioic epithelium stem cell
Culture environment, maintain culture after stem cell versatility.
In order to achieve the above object, providing a kind of maintenance people amnioic epithelium stem cell versatility in one embodiment of the present of invention
Culture solution, comprising:
Basic culture solution；
Serum replacement 15%~25%；
Nonessential amino acid 0.1%~2%；
Essential amino acid 1mmol/L~5mmol/L；
Dithiothreitol (DTT) 0.01mmol/L~1mmol/L；
Growth factor 30ng/ml~120ng/ml；
The stable system factor 0.1%~2%.
As optimal enforcement scheme of the invention, which includes:
Basic culture solution；
Serum replacement 20%；
Nonessential amino acid 1%；
Essential amino acid 2mmol/L；
Dithiothreitol (DTT) 0.05mmol/L；
Growth factor 4 8ng/ml；
The stable system factor 1%.
In prioritization scheme of the invention, it is necessary to which amino acid is L-Glutamine.
Wherein, growth factor includes:
Basic fibroblast growth factor 4ng/ml~10ng/ml；
Epithelical cell growth factor 10ng/ml~20ng/ml；
Hepatocyte growth factor 20ng/ml~40ng/ml；
- 2 10ng/ml of keratinocyte growth factor~20ng/ml.
Wherein, include in the stable system factor:
1 part~10 parts of β-aminoethanesulfonic acid；
1 part~10 parts of arginine；
1 part~10 parts of transforminggrowthfactor-α；
1 part~10 parts of para-insulin No.1 growth factor；
1 part~10 parts of granulocyte colony stimulating factor.
Wherein, basic culture solution DMEM/F12；The pH value of culture solution is 7.2~7.4.
For this purpose, the invention also discloses the preparation methods of the culture solution:
Basic culture solution is taken, serum replacement and nonessential amino acid are first added wherein, is uniformly mixed and steady to solution
After fixed, sequentially add essential amino acid, dithiothreitol (DTT), growth factor and the stable system factor and be uniformly mixed.
In conclusion the invention has the following advantages that
The present invention selects DMEM/F12 for basic culture medium, full of nutrition, is suitble to the requirement of a variety of stem cell cultures；
The present invention replaces serum, such as the product of Pall company, U.S. production using serum replacement.Blood serum substituting can
To reduce the risk of immunological rejection in cellular transplantation therapy；
Nonessential amino acid NEAA is common nonessential amino acid cultivate reagent in the present invention, can substitute and grow
The ingredient being depleted in journey, supplement nonessential amino acid can stimulating growth, extend the existence of cell；
L-Glutamine of the invention is the essential amino acid of cell growth, provides important energy for the cell of culture
Source.After taking off amino, L-Glutamine can be used as the energy source of culture cell, participate in the synthesis and nucleic acid metabolism of protein；
Dithiothreitol (DTT) active part of the invention is sulfydryl, and the compound of sulfur-bearing in serum is made to be reduced into gluathione
Peptide, can induce the proliferation of cell, while avoid damage of the peroxide to culture cell；
Basic fibroblast growth factor bFGF can effectively maintain the undifferentiated proliferation of human stem cell, promote stem cell
Self-renewing；Epithelical cell growth factor EGF can effectively facilitate the increment of stem cell and maintain the differentiation potential of stem cell；Liver
Porcine HGF HGF can promote the synthesis of cell DNA, cell division and cell movement；Keratinocyte growth factor-
2KGF-2 can promote cell Proliferation and cell-stimulating.
The stable system factor includes β-aminoethanesulfonic acid, arginine, transforminggrowthfactor-α TGF- α, para-insulin No.1
Growth factor IGF-1, granulocyte colony stimulating factor G-CSF can improve culture system in vitro simulated in vivo environment, promote dry
Cell Proliferation.
Cultivating system of the invention must be added when preparing in strict accordance with sequence, and serum replacement and nonessential ammonia is first added
Base acid, it is solution-stabilized (being indicated according to culture medium color change) to be mixed, essential amino acid, dithiothreitol (DTT) are sequentially added,
It is eventually adding growth factor and the stable system factor.
Therefore, culture solution of the invention can simulate the living environment of stem cell in vivo, so that stem cell is in body
Stem cell versatility can be maintained when outer culture, after the secondary culture of more number, can still be obtained with high multipotency
The stem cell of property.
Detailed description of the invention
Fig. 1 is that stem cell carries out cell growth state when secondary culture to the 5th generation, the 15th generation, 20 generation.
Fig. 2 is the result that the 5th generation cell carries out cells pluripotency flow cytometer showed；
Fig. 3 is the result that the 15th generation cell carries out cells pluripotency flow cytometer showed；
Fig. 4 is the result that the 20th generation cell carries out cells pluripotency flow cytometer showed.
Specific embodiment
Embodiment 1
Basic culture solution DMEM/F12 is taken, serum replacement and nonessential amino acid are first added wherein, is uniformly mixed simultaneously
Essential amino acid L-Glutamine, dithiothreitol (DTT), growth factor and the stable system factor are sequentially added after solution-stabilized
It is uniformly mixed；It is indicated with volume fraction and substance molal volume ratio, the content of various composition in the culture solution of configuration are as follows:
Serum replacement 15%；PH value 7.4
Non-essential amino acid reagent 1%；
Essential amino acid 2mmol/L；
Dithiothreitol (DTT) 0.05mmol/L；
Basic fibroblast growth factor 5ng/ml；
Epithelical cell growth factor 15ng/ml；
Hepatocyte growth factor 30ng/ml；
- 2 10ng/ml of keratinocyte growth factor；
The stable system factor 1%.
Wherein, each group distribution ratio of the stable system factor are as follows:
β-aminoethanesulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Para-insulin No.1 growth factor 25%；
Granulocyte colony stimulating factor 30%.
Embodiment 2
Basic culture solution DMEM/F12 is taken, serum replacement and nonessential amino acid are first added wherein, is uniformly mixed simultaneously
Essential amino acid L-Glutamine, dithiothreitol (DTT), growth factor and the stable system factor are sequentially added after solution-stabilized
It is uniformly mixed；It is indicated with volume fraction and substance molal volume ratio, the content of various composition in the culture solution of configuration are as follows:
Serum replacement 20%
Non-essential amino acid reagent 0.8%；
Essential amino acid 3mmol/L；
Dithiothreitol (DTT) 0.08mmol/L；
Basic fibroblast growth factor 8ng/ml；
Epithelical cell growth factor 18ng/ml；
Hepatocyte growth factor 30ng/ml；
- 2 15ng/ml of keratinocyte growth factor；
The stable system factor 1.2%.
Wherein, each group distribution ratio of the stable system factor are as follows:
β-aminoethanesulfonic acid 15%；
Arginase 12 5%；
Transforminggrowthfactor-α 20%；
Para-insulin No.1 growth factor 20%；
Granulocyte colony stimulating factor 20%.
Embodiment 3
Basic culture solution DMEM/F12 is taken, serum replacement and nonessential amino acid are first added wherein, is uniformly mixed simultaneously
Essential amino acid L-Glutamine, dithiothreitol (DTT), growth factor and the stable system factor are sequentially added after solution-stabilized
It is uniformly mixed；It is indicated with volume fraction and substance molal volume ratio, the content of various composition in the culture solution of configuration are as follows:
Serum replacement 20%
Non-essential amino acid reagent 1%；
Essential amino acid L-Glutamine 2mmol/L；
Dithiothreitol (DTT) 0.05mmol/L；
Basic fibroblast growth factor 8ng/ml；
Epithelical cell growth factor 10ng/ml；
Hepatocyte growth factor 20ng/ml；
- 2 10ng/ml of keratinocyte growth factor；
The stable system factor 1.2%.
Wherein, each group distribution ratio of the stable system factor are as follows:
β-aminoethanesulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Para-insulin No.1 growth factor 25%；
Granulocyte colony stimulating factor 30%.
Embodiment 4
The preparation of 1L hAECs culture solution
Serum replacement 200mL, L-glutamine are added in 770mL DMEM/F12 (Gibco 11330) culture medium
(Gibco 25030) 10mL, 0.05mol/L dithiothreitol (DTT) (Sigma D9779) 1uL, it is slowly added to NEAA (Gibco
11140) 10mL, to culture medium stablize (culture medium colour stable) be then added bFGF (Gibco 13256-029) 2.5mL,
The KGF-II (Bio Vision 4060-25) of HGF (Bio Vision 4510-10) 200uL, 100ug/mL of 100ug/mL
EGF (Bio Vision 4022-100) 100uL, the stable system factor 10mL of 100uL, 0.1mg/mL, mix well, 4 DEG C of guarantors
It deposits.
Embodiment 5
Basic culture solution DMEM/F12 is taken, serum replacement and nonessential amino acid are first added wherein, is uniformly mixed simultaneously
Essential amino acid L-Glutamine, dithiothreitol (DTT), growth factor and the stable system factor are sequentially added after solution-stabilized
It is uniformly mixed；It is indicated with volume fraction and substance molal volume ratio, the content of various composition in the culture solution of configuration are as follows:
Serum replacement 15%；PH value 7.4
Non-essential amino acid reagent 1%；
Essential amino acid 2mmol/L；
Dithiothreitol (DTT) 0.05mmol/L；
Basic fibroblast growth factor 5ng/ml；
Epithelical cell growth factor 15ng/ml；
Hepatocyte growth factor 30ng/ml；
- 2 10ng/ml of keratinocyte growth factor；
The stable system factor 1%.
Wherein, each group distribution ratio of the stable system factor are as follows:
β-aminoethanesulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Para-insulin No.1 growth factor 25%；
Granulocyte colony stimulating factor 20%~28%.
Vanillic aldehyde 1%~10%, preferably 2%.
Test example 1:hAECs secondary culture and cytomorphology detect
Primary hAECs is adherent through culture in 24 hours using the culture solution in embodiment 3, and quickly rises in value, and cellular morphology is just
Often, when cell confluency degree reaches 90%, 0.25% trypsase (sigma 110M7362V)/0.02%EDTA digestion is utilized
Liquid digests adherent hAECs, and cell is collected by centrifugation, can be with continuous passage culture p20, cell growth state according to 1:4 secondary culture
Normally, the cell growth state of different algebra such as Fig. 1.
The 5th generation of hAECs in vitro culture, cell growth state are normal；
The 15th generation of hAECs in vitro culture, cell growth state are normal；
The 20th generation of hAECs in vitro culture, cell growth state are normal；
Test example 2:hAECs cells pluripotency facs analysis
Use AccutaseTMEnzyme (eBioscience Cat.No.00-4555) digests adherent hAECs, is prepared into unicellular
Suspension, 1000rpm are centrifuged 5min, discard supernatant, and suitable streaming dye solution are added, cell is resuspended, adjustment cell concentration is
2×107/ mL, addition 20uL people FCR binding inhibitors (eBioscience Cat.No.14-9161) is incubated for before dyeing
20min adds 100uL dyeing liquor (eBioscience Cat.No.00-6993) according to 1:400 and is separately added into antibody SSEA-4
(Santa Curz sc-21704)、Tra-1-60(Santa Curz sc-21705)、SSEA-1(Santa Curz sc-
21702AF488), Tra-1-81 (Santa Curz sc-21706), flicks mixing.1mL is added to antibody SSEA-4 pipe
Foxp3Fixation/permeabilization (eBioscience Cat.No.00-5521) working solution is mediated fixed thin
Born of the same parents, room temperature or four degree, which are protected from light, is incubated for 30-60min, and 2mL1 × permeabilization buffer (eBioscience is added
Cat.No.00-8333) be resuspended cell 1000rpm room temperature be centrifuged 5min, abandon supernatant, again be added 2mL1 ×
Permeabilization buffer resuspension cell 1000rpm room temperature centrifugation 5min, abandoning supernatant, addition 100ul 1 ×
Secondary antibody goat anti-mouse IgM (Santa Curz sc- is added after cell is resuspended in permeabilization buffer
3767), room temperature, which is protected from light, is incubated for 20min, and 2mL1 × permeabilization buffer is washed once, the centrifugation of 1000rpm room temperature
5min abandons supernatant, is added 2mL streaming dyeing liquor (eBioscience Cat.No.00-4222), upper machine testing；To antibody Tra-
1mL Foxp3Fixation/permeabilization (eBioscience is added in 1-60, SSEA-1, Tra-1-81
Cat.No.00-5521) working solution, fixed cell of mediating, room temperature or four degree, which are protected from light, is incubated for 30-60min, and addition 2mL1 ×
The centrifugation of cell 1000rpm room temperature is resuspended in permeabilization buffer (eBioscience Cat.No.00-8333)
5min abandons supernatant, 2mL1 × permeabilization buffer is added again, cell 1000rpm room temperature centrifugation 5min is resuspended,
Abandon supernatant, be added 1 × permeabilization of 100ul buffer be resuspended cell after respectively to Tra-1-60, SSEA-1,
Secondary antibody goat anti-mouse IgM (Santa Curz sc-3768) is added in Tra-1-81 pipe, room temperature is protected from light incubation
20min, 2mL1 × permeabilization buffer are washed once, and 1000rpm room temperature is centrifuged 5min, abandon supernatant, and 2mL is added
Streaming dyeing liquor, upper machine testing.Cells pluripotency flow cytometer showed is learnt from Fig. 2-Fig. 4, when hAECs amplification in vitro is to p20,
It still is able to maintain its versatility, there is potential differentiation capability.
Cultivation results analysis are as follows:
When being tested using the method and reagent of embodiment 3, hAECs in vitro culture 5-20 generation, cells pluripotency
Label expression is normal, has stable differentiation potential.In addition, the Examples 1 to 5 by other 4 embodiments all has preferably
Test effect, wherein preferably with embodiment 5 and embodiment 3, embodiment 5 is good compared with 1 effect of embodiment.Illustrate training of the invention
Nutrient solution is suitable for the continuous passage culture of hAECs, can maintain its versatility and potential differentiation capability very well, overcome it
The shortcomings that no telomerase activity.
Claims (6)
1. a kind of culture solution for maintaining people's amnioic epithelium stem cell versatility, it is characterised in that: consist of the following compositions:
Basic culture solution；
Serum replacement 15%~25%；
Nonessential amino acid 0.1%~2%, manufacturer are Gibco company, article No. 11140；
L-Glutamine 1mmol/L~5mmol/L；
Dithiothreitol (DTT) 0.01mmol/L~1mmol/L；
Growth factor 30ng/m L~120ng/m L；
The stable system factor 0.1%~2%；
The growth factor consists of the following compositions:
Basic fibroblast growth factor 4ng/m L~10ng/m L；
Epithelical cell growth factor 10ng/m L~20ng/m L；
Hepatocyte growth factor 20ng/m L~40ng/m L；
- 2 10ng/m L~20ng/m L of keratinocyte growth factor；
The stable system factor consists of the following compositions:
1 part~10 parts of β-aminoethanesulfonic acid；
1 part~10 parts of arginine；
1 part~10 parts of transforminggrowthfactor-α；
1 part~10 parts of para-insulin No.1 growth factor；
1 part~10 parts of granulocyte colony stimulating factor.
2. culture solution as described in claim 1, it is characterised in that: consist of the following compositions:
Basic culture solution；
Serum replacement 20%；
Nonessential amino acid 1%；
L-Glutamine 2mmol/L；
Dithiothreitol (DTT) 0.05mmol/L；
Growth factor 4 8ng/m L；
The stable system factor 1%.
3. culture solution as described in claim 1, it is characterised in that: the growth factor consists of the following compositions:
Basic fibroblast growth factor 8ng/m L；
Epithelical cell growth factor 10ng/m L；
Hepatocyte growth factor 20ng/m L；
- 2 10ng/m L of keratinocyte growth factor.
4. culture solution as described in claim 1, it is characterised in that: the basic culture solution is DMEM/F12.
5. culture solution as described in claim 1, it is characterised in that: the pH value of the culture solution is 7.2~7.4.
6. the method for preparing any culture solution in Claims 1 to 5, steps of the method are:
It takes basic culture solution, serum replacement and nonessential amino acid is first added wherein, be uniformly mixed and after solution-stabilized,
L-Glutamine, dithiothreitol (DTT), growth factor and the stable system factor is sequentially added to be uniformly mixed.
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| EP1775341A1 (en) * | 2004-06-28 | 2007-04-18 | National University of Corporation Hiroshima University | Method of inducing the differentiation of amnion-origin cells and utilization of the same |
| CN101765657A (en) * | 2007-11-09 | 2010-06-30 | Rnl生物技术株式会社 | Method for isolating and culturing adult stem cells derived from human amniotic epithelium |
| CN104371970A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Culture medium for culturing human amniotic epithelial cells |
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|---|---|---|---|---|
| EP1775341A1 (en) * | 2004-06-28 | 2007-04-18 | National University of Corporation Hiroshima University | Method of inducing the differentiation of amnion-origin cells and utilization of the same |
| CN101765657A (en) * | 2007-11-09 | 2010-06-30 | Rnl生物技术株式会社 | Method for isolating and culturing adult stem cells derived from human amniotic epithelium |
| CN104371970A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Culture medium for culturing human amniotic epithelial cells |
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| 人羊膜上皮细胞的分离、培养及鉴定;方宁等;《遵义医学院学报》;20090430;第32卷(第2期);第121-124页 |
| 羊膜上皮细胞体外培养条件的优化及其干细胞标志的表达;陈宥艺等;《中国实验血液学杂志》;20111231;第19卷(第2期);第464-468页 |
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