CN105734011A - Culture fluid for maintaining pluripotency of human amniotic epithelial stem cells - Google Patents
Culture fluid for maintaining pluripotency of human amniotic epithelial stem cells Download PDFInfo
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- CN105734011A CN105734011A CN201610134610.XA CN201610134610A CN105734011A CN 105734011 A CN105734011 A CN 105734011A CN 201610134610 A CN201610134610 A CN 201610134610A CN 105734011 A CN105734011 A CN 105734011A
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- culture fluid
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Abstract
The invention discloses culture fluid for maintaining the pluripotency of human amniotic epithelial stem cells. The culture fluid comprises base culture fluid, 15%-25% of serum substitutes, 0.1%-2% of non-essential amino acid, 1 mmol/L-5 mmol/L of essential amino acid, 0.01 mmol/L-1 mmol/L of dithiothreitol, 30 ng/ml-120 ng/ml of growth factors and 0.1%-2% of system stabilizing factors. The culture fluid has the advantages that in-vivo survival environments of the stem cells can be simulated by the culture fluid, accordingly, the pluripotency of the stem cells can be maintained when the stem cells are subjected to in-vitro culture, and stem cells with high pluripotency still can be obtained after repeated pass culture is carried out.
Description
Technical field
The present invention relates to technical field of cell culture, be specifically related to a kind of culture fluid maintaining people's amnioic epithelium stem cell versatility.
Background technology
Amniotic membrane is positioned at inside embryo villi film, be one layer without blood vessel, nerve, lymph, muscle transparent membrane, contact closely with developmental fetus.The amnion-derived cell of people is mainly by two class cellularities: human amnion membrane (humanamnionepithelialcells, hAECs) and human amniotic mesenchymal cell (humanamnionmesenchymecells, hAMCs).
The researchs such as human amnion membrane (humanamnioticepithelialcell, hAEC) has the feature and more comprehensive multi-lineage potential of expressing multiple embryonic stem cell marker, Miki confirm the hAECs potential broken up to triploblastica.Except expressing embryonic stem cell surface antigen: SSEA-3 and SSEA-4, outside tumor resistance gene TRA1-60 and TRA1-81, it have 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 antigen, for instance the ATP of ATP efflux pump is in conjunction with the ABCG2/BCRP of one of magazine super families member, CD9, CD24, E-cadherin, integrin α6 and β, hepatocyte growth factor bind receptor (c-met) etc..Do not express CD34 (hematopoietic stem cell and interior surface drying cell surface marker), SSEA-1, CD133 (hematopoietic stem cell, endotheliocyte and Malignant glioma cells surface markers), weak expression c-kit (CD117) and CC-chemokine receptor (CRR4).
HAECs be a kind of can self duplication and there is the adult stem cell of multinomial differentiation potential, under the adjustment of different somatomedin, hepatic lineage can be divided into, cardiac-like muscle cell, neurogliocyte, neuronal cell and islet-like cells etc., and it can secrete BDNF, the neurotrophic factors such as NGF, neuron is had reparation by these neurotrophic factors, the effect of nutritional support, secondly, hAECs immunogenicity is low, rejection is not easily caused after cell transplantation, the release of inflammatory factor IFN γ and TNF can be reduced, by suppressing T lymphopoiesis, reduce the expression of pro-inflammatory cytokine IL-1 α and IL-1 β, there is immunoregulatory effect.Again, find from the hAECs treatment to central nervous system's damage model, hAECs can be divided into the cell of neuronal cell, astrocyte replacement damaged or death, recovers the function in damaged region, improves behavioristics and the pathological change of animal pattern.CO2 laser weld is not only had great significance by hAECs, the reparation that liver etc. is organized all is played very important effect, and there is low immunogenicity and immunity coordinate repression, the ethical issues in placenta stem-cell experiment and clinical practice can be avoided simultaneously, medically have broad application prospects at cell replacement therapy and tissue regeneration.
Human amnion membrane comes from placenta amnion tissue, although expressing stem-cell marker molecule and related gene, but not expressing telomerase gene, amplification is restricted in vitro, can only cultivate for 4 to 5 generations in vitro, cultivation ten cell after a few days starts atrophy, the versatility of cell is also affected by strong influence, has reported relevant cultivating system, is all that the concentration to somatomedin EGF adjusts, but to the maintenance of hAECs versatility also without obvious change, cell can not maintain cultivation equally over a long time.Although we can obtain more hAECs from people's amniotic membrane, but for external research and transplantation treatment, bigger restriction all can occur, therefore one stably maintain hAECs versatility cultivating system be significant, stablizing of versatility can be maintained, the problem simultaneously also solving acute treatment cell requirements amount in long-time In vitro culture.
Summary of the invention
It is an object of the invention to provide a kind of stem cell medium, it is possible to provide the culture environment in analogue body for people amnioic epithelium stem cell, maintain the stem cell versatility after cultivating.
For reaching above-mentioned purpose, one embodiment of the present of invention provides a kind of culture fluid maintaining people's amnioic epithelium stem cell versatility, including:
Basic culture solution；
Serum replacement 15%～25%；
Non essential amino acid 0.1%～2%；
Essential amino acids 1mmol/L～5mmol/L；
Dithiothreitol, DTT 0.01mmol/L～1mmol/L；
Somatomedin 30ng/ml～120ng/ml；
The stable system factor 0.1%～2%.
As the optimal enforcement scheme of the present invention, this culture fluid comprises:
Basic culture solution；
Serum replacement 20%；
Non essential amino acid 1%；
Essential amino acids 2mmol/L；
Dithiothreitol, DTT 0.05mmol/L；
Growth factor 4 8ng/ml；
The stable system factor 1%.
In the prioritization scheme of the present invention, it is necessary to aminoacid is L-glutaminate.
Wherein, somatomedin comprises:
Basic fibroblast growth factor 4ng/ml～10ng/ml；
Epithelical cell growth factor 10ng/ml～20ng/ml；
Hepatocyte growth factor 20ng/ml～40ng/ml；
Keratinocyte growth factor-210ng/ml～20ng/ml.
Wherein, the stable system factor comprises:
Beta-amino ethyl sulfonic acid 1 part～10 parts；
Arginine 1 part～10 parts；
Transforminggrowthfactor-α 1 part～10 parts；
Number growth factor of para-insulin 1 part～10 parts；
Granulocyte colony-stimulating factor 1 part～10 parts.
Wherein, basic culture solution is DMEM/F12；The pH value of culture fluid is 7.2～7.4.
For this, the preparation method that the invention also discloses this culture fluid:
Take basic culture solution, first add serum replacement and non essential amino acid wherein, mix homogeneously after solution-stabilized, sequentially add essential amino acids, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously.
In sum, the invention have the advantages that
The present invention selects culture medium based on DMEM/F12, and it is nutritious, is suitable for the requirement that multiple stem cell is cultivated；
The present invention adopts serum replacement to replace serum, for instance the product that Pall company of the U.S. produces.Blood serum substituting can reduce the risk of immunological rejection in cellular transplantation therapy；
In the present invention, non essential amino acid NEAA is that common non essential amino acid cultivates reagent, it is possible to substitutes composition depleted in growth course, supplementary non essential amino acid can stimulating growth, the existence of prolongation cell；
The L-glutaminate of the present invention is the essential amino acids of Growth of Cells, provides important energy source for cultured cells.After taking off amino, L-glutaminate can as cultivating the energy source of cell, the synthesis participating in protein and nucleic acid metabolism；
The dithiothreitol, DTT active part of the present invention is sulfydryl, makes the compound of sulfur-bearing in serum be reduced into glutathion, the propagation of energy inducing cell, avoids the peroxide infringement to cultivating cell simultaneously；
Basic fibroblast growth factor bFGF can effectively maintain the undifferentiated proliferation of human stem cell, promotes the self renewal of stem cell；Epithelical cell growth factor EGF can effectively facilitate the increment of stem cell and maintain the differentiation potential of stem cell；Hepatocyte growth factor 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 beta-amino ethyl sulfonic acid, arginine, transforminggrowthfactor-α TGF-α, number growth factor IGF-1 of para-insulin, granulocyte colony-stimulating factor G-CSF, culture system in vitro simulated in vivo environment can be improved, promote stem cells hyperplasia.
Must add in strict accordance with order when the cultivating system of the present invention is prepared, it is initially charged serum replacement and non essential amino acid, solution-stabilized (according to the change instruction of culture medium color) to be mixed, sequentially add essential amino acids, dithiothreitol, DTT, be eventually adding somatomedin and the stable system factor.
Therefore, the culture fluid of the present invention can simulate stem cell living environment in vivo, and then can maintain stem cell versatility when stem cell is cultivated in vitro, after the Secondary Culture of more number of times, still can obtain the stem cell with high versatility.
Accompanying drawing explanation
Fig. 1 is stem cell cell growth state when carrying out 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.
Detailed description of the invention
Embodiment 1
Taking basic culture solution DMEM/F12, first add serum replacement and non essential amino acid wherein, mix homogeneously also sequentially adds essential amino acids L-glutaminate, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously after solution-stabilized；Representing with volume fraction and material molal volume ratio, in the culture fluid of configuration, the content of various compositions is:
Serum replacement 15%；PH value 7.4
Non essential amino acid reagent 1%；
Essential amino acids 2mmol/L；
Dithiothreitol, DTT 0.05mmol/L；
Basic fibroblast growth factor 5ng/ml；
Epithelical cell growth factor 15ng/ml；
Hepatocyte growth factor 30ng/ml；
Keratinocyte growth factor-210ng/ml；
The stable system factor 1%.
Wherein, each component proportion of this stable system factor is:
Beta-amino ethyl sulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Number growth factor 25% of para-insulin；
Granulocyte colony-stimulating factor 30%.
Embodiment 2
Taking basic culture solution DMEM/F12, first add serum replacement and non essential amino acid wherein, mix homogeneously also sequentially adds essential amino acids L-glutaminate, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously after solution-stabilized；Representing with volume fraction and material molal volume ratio, in the culture fluid of configuration, the content of various compositions is:
Serum replacement 20%
Non essential amino acid reagent 0.8%；
Essential amino acids 3mmol/L；
Dithiothreitol, DTT 0.08mmol/L；
Basic fibroblast growth factor 8ng/ml；
Epithelical cell growth factor 18ng/ml；
Hepatocyte growth factor 30ng/ml；
Keratinocyte growth factor-215ng/ml；
The stable system factor 1.2%.
Wherein, each component proportion of this stable system factor is:
Beta-amino ethyl sulfonic acid 15%；
Arginase 12 5%；
Transforminggrowthfactor-α 20%；
Number growth factor 20% of para-insulin；
Granulocyte colony-stimulating factor 20%.
Embodiment 3
Taking basic culture solution DMEM/F12, first add serum replacement and non essential amino acid wherein, mix homogeneously also sequentially adds essential amino acids L-glutaminate, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously after solution-stabilized；Representing with volume fraction and material molal volume ratio, in the culture fluid of configuration, the content of various compositions is:
Serum replacement 20%
Non essential amino acid reagent 1%；
Essential amino acids L-glutaminate 2mmol/L；
Dithiothreitol, DTT 0.05mmol/L；
Basic fibroblast growth factor 8ng/ml；
Epithelical cell growth factor 10ng/ml；
Hepatocyte growth factor 20ng/ml；
Keratinocyte growth factor-210ng/ml；
The stable system factor 1.2%.
Wherein, each component proportion of this stable system factor is:
Beta-amino ethyl sulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Number growth factor 25% of para-insulin；
Granulocyte colony-stimulating factor 30%.
Embodiment 4
The preparation of 1LhAECs culture fluid
null770mLDMEM/F12 (Gibco11330) culture medium adds serum replacement 200mL、L-glutamine(Gibco25030)10mL、0.05mol/L dithiothreitol, DTT (SigmaD9779) 1uL、It is slowly added to NEAA (Gibco11140) 10mL，Treat that culture medium stable (culture medium colour stable) then adds bFGF (Gibco13256-029) 2.5mL、HGF (BioVision4510-10) 200uL of 100ug/mL、KGF-II (BioVision4060-25) 100uL of 100ug/mL、EGF (BioVision4022-100) 100uL of 0.1mg/mL、Stable system factor 10mL，Fully mixing，4 DEG C of preservations.
Embodiment 5
Taking basic culture solution DMEM/F12, first add serum replacement and non essential amino acid wherein, mix homogeneously also sequentially adds essential amino acids L-glutaminate, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously after solution-stabilized；Representing with volume fraction and material molal volume ratio, in the culture fluid of configuration, the content of various compositions is:
Serum replacement 15%；PH value 7.4
Non essential amino acid reagent 1%；
Essential amino acids 2mmol/L；
Dithiothreitol, DTT 0.05mmol/L；
Basic fibroblast growth factor 5ng/ml；
Epithelical cell growth factor 15ng/ml；
Hepatocyte growth factor 30ng/ml；
Keratinocyte growth factor-210ng/ml；
The stable system factor 1%.
Wherein, each component proportion of this stable system factor is:
Beta-amino ethyl sulfonic acid 10%；
Arginase 12 0%；
Transforminggrowthfactor-α 15%；
Number growth factor 25% of para-insulin；
Granulocyte colony-stimulating factor 20%～28%.
Vanillin 1%～10%, it is preferable that 2%.
Test example 1:hAECs Secondary Culture and morphocytology detection
Primary hAECs adopts the culture fluid in embodiment 3 to cultivate adherent through 24 hours, and quickly rise in value, cellular morphology is normal, when cell confluency degree reaches 90%, utilize the trypsin sigma110M7362V of 0.25%)/0.02%EDTA Digestive system adherent the hAECs of digestion, centrifugal collecting cell, according to 1:4 Secondary Culture, continuous passage can cultivating p20, cell growth state is normal, cell growth state such as Fig. 1 of different algebraically.
In hAECs In vitro culture the 5th generation, cell growth state is normal；
In hAECs In vitro culture the 15th generation, cell growth state is normal；
In hAECs In vitro culture the 20th generation, cell growth state is normal；
Test example 2:hAECs cells pluripotency facs analysis
Use AccutaseTMEnzyme (eBioscienceCat.No.00-4555) digests adherent hAECs, prepares into single cell suspension, and 1000rpm is centrifuged 5min, supernatant discarded, adds appropriate streaming dye solution re-suspended cell, and adjusting cell concentration is 2 × 107/ mL, add 20uL people's FCR binding inhibitors (eBioscienceCat.No.14-9161) before dyeing and hatch 20min, add 100uL dyeing liquor (eBioscienceCat.No.00-6993) and be separately added into antibody SSEA-4 (SantaCurzsc-21704), Tra-1-60 (SantaCurzsc-21705), SSEA-1 (SantaCurzsc-21702AF488), Tra-1-81 (SantaCurzsc-21706) according to 1:400, flick mixing.null1mLFoxp3Fixation/permeabilization (eBioscienceCat.No.00-5521) working solution is added to antibody SSEA-4 pipe，Mediate and fix cell，Room temperature or four degree of lucifuges hatch 30-60min，Add the centrifugal 5min of 2mL1 × permeabilizationbuffer (eBioscienceCat.No.00-8333) re-suspended cell 1000rpm room temperature，Abandon supernatant，Again add the centrifugal 5min of 2mL1 × permeabilizationbuffer re-suspended cell 1000rpm room temperature，Abandon supernatant，Two anti-goatanti-mouseIgM (SantaCurzsc-3767) are added after adding 100ul1 × permeabilizationbuffer re-suspended cell，Room temperature lucifuge hatches 20min，2mL1 × permeabilizationbuffer washes once，1000rpm room temperature is centrifuged 5min，Abandon supernatant，Add 2mL streaming dyeing liquor (eBioscienceCat.No.00-4222)，Upper machine testing；nullTo antibody Tra-1-60、SSEA-1、Tra-1-81 adds 1mLFoxp3Fixation/permeabilization (eBioscienceCat.No.00-5521) working solution，Mediate and fix cell，Room temperature or four degree of lucifuges hatch 30-60min，Add the centrifugal 5min of 2mL1 × permeabilizationbuffer (eBioscienceCat.No.00-8333) re-suspended cell 1000rpm room temperature，Abandon supernatant，Again add the centrifugal 5min of 2mL1 × permeabilizationbuffer re-suspended cell 1000rpm room temperature，Abandon supernatant，Respectively to Tra-1-60 after addition 100ul1 × permeabilizationbuffer re-suspended cell、SSEA-1、Tra-1-81 pipe adds two anti-goatanti-mouseIgM (SantaCurzsc-3768)，Room temperature lucifuge hatches 20min，2mL1 × permeabilizationbuffer washes once，1000rpm room temperature is centrifuged 5min，Abandon supernatant，Add 2mL streaming dyeing liquor，Upper machine testing.From Fig. 2, cells pluripotency flow cytometer showed is learnt, when hAECs amplification in vitro to p20, still is able to maintain its versatility, has potential differentiation capability.
Cultivation results analysis is:
When the method and reagent that use embodiment 3 are tested, in hAECs In vitro culture 5-20 generation, cells pluripotency marker expression is normal, has stable differentiation potential.Additionally, be respectively provided with good test effect through the embodiment 1～5 of other 4 embodiments, wherein with embodiment 5 and embodiment 3 preferably, embodiment 5 is effective compared with embodiment 1.The culture fluid describing the present invention is suitable for the continuous passage cultivation of hAECs, it is possible to maintain its versatility and potential differentiation capability, the shortcoming overcoming its no telomerase activity very well.
Claims (9)
1. maintain a culture fluid for people's amnioic epithelium stem cell versatility, including:
Basic culture solution；
Serum replacement 15% ~ 25%；
Non essential amino acid 0.1% ~ 2%；
Essential amino acids 1mmol/L ~ 5mmol/L；
Dithiothreitol, DTT 0.01mmol/L ~ 1mmol/L；
Somatomedin 30ng/ml ~ 120ng/ml；
The stable system factor 0.1% ~ 2%.
2. culture fluid as claimed in claim 1, it is characterised in that including:
Basic culture solution；
Serum replacement 20%；
Non essential amino acid 1%；
Essential amino acids 2mmol/L；
Dithiothreitol, DTT 0.05mmol/L；
Growth factor 4 8ng/ml；
The stable system factor 1%.
3. culture fluid as claimed in claim 1, it is characterised in that: described essential amino acids is L-glutaminate.
4. culture fluid as claimed in claim 1, it is characterised in that: described somatomedin comprises:
Basic fibroblast growth factor 4ng/ml ~ 10ng/ml；
Epithelical cell growth factor 10ng/ml ~ 20ng/ml；
Hepatocyte growth factor 20ng/ml ~ 40ng/ml；
Keratinocyte growth factor-210ng/ml ~ 20ng/ml.
5. culture fluid as claimed in claim 1, it is characterised in that: described somatomedin comprises:
Basic fibroblast growth factor 8ng/ml；
Epithelical cell growth factor 10ng/ml；
Hepatocyte growth factor 20ng/ml；
Keratinocyte growth factor-210ng/ml.
6. culture fluid as claimed in claim 1, it is characterised in that: the described stable system factor comprises:
Beta-amino ethyl sulfonic acid 1 part ~ 10 parts；
Arginine 1 part ~ 10 parts；
Transforminggrowthfactor-α 1 part ~ 10 parts；
Number growth factor of para-insulin 1 part ~ 10 parts；
Granulocyte colony-stimulating factor 1 part ~ 10 parts.
7. culture fluid as claimed in claim 1, it is characterised in that: described basic culture solution is DMEM/F12.
8. culture fluid as claimed in claim 1, it is characterised in that: the pH value of described culture fluid is 7.2 ~ 7.4.
9. the method for arbitrary described culture fluid in preparation claim 1 ~ 8, steps of the method are:
Take basic culture solution, first add serum replacement and non essential amino acid wherein, mix homogeneously after solution-stabilized, sequentially add essential amino acids, dithiothreitol, DTT, somatomedin and stable system factor mix homogeneously.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2016
- 2016-03-09 CN CN201610134610.XA patent/CN105734011B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (5)
| Title |
|---|
| 储炬等: "《现代生物工艺学》", 30 September 2007, 华东理工大学出版社 * |
| 刘雯: "《医学细胞与遗传学实验教程》", 31 October 2008, 复旦大学出版社 * |
| 方宁等: "人羊膜上皮细胞的分离、培养及鉴定", 《遵义医学院学报》 * |
| 王建等: "人羊膜上皮细胞干细胞特性及向胰岛素分泌细胞分化的研究", 《南方医科大学学报》 * |
| 陈宥艺等: "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", 《中国实验血液学杂志》 * |
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